Reproductive Genetics

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Great progress has been made in the field of genetics within the past decade. This, combined with our growing knowledge, has impacted on this important area with interesting consequences. The ability to identify genetic defects before implantation, to diagnose fetal abnormalities and to introduce screening programmes means that genetic testing now has a major role in preventive medicine. These topics are discussed in detail in the book. In parallel with these advances, other aspects that cannot be ignored, such as education of the public and the potential ethical dilemmas that may arise by virtue of these new methodologies, are raised and discussed in this volume, which is based on the 57th RCOG Study Group and includes a set of consensus views from the expert participants.

Reproductive Genetics

This is a unique book, covering areas not available elsewhere. Within its pages, the authors discuss many diverse areas relating to reproduction and genetics.

12.5 mm Spine Width

This book provides topical and essential information for practising clinicians, researchers and other healthcare professionals interested in these fields of study.

Edited by Sean Kehoe, Lyn Chitty and Tessa Homfray

RCOG Press Royal College of Obstetricians and Gynaecologists 27 Sussex Place, Regent’s Park, London NW1 4RG

www.rcog.org.uk

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Reproductive Genetics Edited by

Sean Kehoe Lyn Chitty and Tessa Homfray

Reproductive genetics

Since 1973 the Royal College of Obstetricians and Gynaecologists has regularly convened Study Groups to address important growth areas within obstetrics and gynaecology. An international group of eminent clinicians and scientists from various disciplines is invited to present the results of recent research and to take part in in-depth discussions. The resulting volume, containing enhanced versions of the papers presented, is published within a few months of the meeting and provides a summary of the subject that is both authoritative and up to date.

SOME PREVIOUS STUDY GROUP PUBLICATIONS AVAILABLE The Placenta: Basic Science and Implantation and Early Clinical Practice Development Edited by JCP Kingdom, ERM Jauniaux Edited by Hilary Critchley, Iain and PMS O’Brien Cameron and Stephen Smith Disorders of the Menstrual Cycle Edited by PMS O’Brien, IT Cameron and AB MacLean Infection and Pregnancy Edited by AB MacLean, L Regan and D Carrington

Contraception and Contraceptive Use Edited by Anna Glasier, Kaye Wellings and Hilary Critchley Multiple Pregnancy Edited by Mark Kilby, Phil Baker, Hilary Critchley and David Field

Pain in Obstetrics and Gynaecology Edited by A MacLean, R Stones and Heart Disease and Pregnancy S Thornton Edited by Philip J Steer, Michael A Gatzoulis and Philip Baker Incontinence in Women Edited by AB MacLean and L Cardozo Teenage Pregnancy and Reproductive Health Maternal Morbidity and Mortality Edited by Philip Baker, Kate Guthrie, Edited by AB MacLean and J Neilson Cindy Hutchinson, Roslyn Kane and Kaye Wellings Lower Genital Tract Neoplasia Edited by Allan B MacLean, Albert Obesity and Reproductive Health Singer and Hilary Critchley Edited by Philip Baker, Adam Balen, Lucilla Poston and Naveed Sattar Pre-eclampsia Edited by Hilary Critchley, Allan Renal Disease in Pregnancy MacLean, Lucilla Poston and James Edited by John M Davison, Catherine Walker Nelson-Piercy, Sean Kehoe and Philip Baker Preterm Birth Edited by Hilary Critchley, Phillip Cancer and Reproductive Health Bennett and Steven Thornton Edited by Sean Kehoe, Eric Jauniaux, Pierre Martin-Hirsch and Philip Savage Menopause and Hormone Replacement Reproductive Ageing Edited by Hilary Critchley, Ailsa Gebbie Edited by Susan Bewley, William and Valerie Beral Ledger and Dimitrios Nikolaou

Reproductive genetics Edited by

Sean Kehoe, Lyn Chitty and Tessa Homfray

Sean Kehoe MD FRCOG Convenor of Study Groups, Lead Consultant in Gynaecological Oncology, Oxford Gynaecological Cancer Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU Lyn Chitty PhD MRCOG Professor of Genetics and Fetal Medicine, Clinical Molecular Genetics Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH and Consultant in Genetics and Fetal Medicine, University College London Hospitals NHS Foundation Trust, London Tessa Homfray FRCP Consultant, Medical Genetics, Department of Genetics, St George’s University of London, Cranmer Terrace, London SW17 0RE

Published by the RCOG Press at the Royal College of Obstetricians and Gynaecologists, 27 Sussex Place, Regent’s Park, London NW1 4RG www.rcog.org.uk Registered charity no. 213280 First published 2009 © 2009 The Royal College of Obstetricians and Gynaecologists No part of this publication may be reproduced, stored or transmitted in any form or by any means, without the prior written permission of the publisher or, in the case of reprographic reproduction, in accordance with the terms of licences issued by the Copyright Licensing Agency in the UK [www.cla.co.uk]. Enquiries concerning reproduction outside the terms stated here should be sent to the publisher at the UK address printed on this page. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant laws and regulations and therefore for general use. While every effort has been made to ensure the accuracy of the information contained within this publication, the publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check current indications and accuracy by consulting other pharmaceutical literature and following the guidelines laid down by the manufacturers of specific products and the relevant authorities in the country in which they are practising. The rights of Sean Kehoe, Lyn Chitty and Tessa Homfray to be identified as Editors of this work has been asserted by them in accordance with the Copyright, Designs and Patents Act 1988. ISBN 978-1-906985-16-5 A machine-readable catalogue record for this publication can be obtained from the British Library [www.bl.uk/catalogue/listings.html] Cover image: Control of new synthesised DNA helix © medicalpicture GmbH (image number 16874270) RCOG Editor: Andrew Welsh Original design by Karl Harrington, FiSH Books, London Typesetting by Andrew Welsh Index by Liza Furnival, Medical Indexing Ltd Printed by Henry Ling Ltd, The Dorset Press, Dorchester DT1 1HD

Contents

Participants

vii

Declarations of personal interest

x

Preface

xi

1

Genetic aetiology of infertility Anu Bashamboo, Celia Ravel and Ken McElreavey

2

Disorders of sex development Lin Lin and John C Achermann

15

3

Preimplantation genetic diagnosis: current practice and future possibilities Alison Lashwood and Tarek El-Toukhy

35

Ethical aspects of saviour siblings: procreative reasons and the treatment of children Mark Sheehan

59

4

1

5

Epigenetics, assisted reproductive technologies and growth restriction Jennifer M Frost, Sayeda Abu-Amero, Caroline Daelemans and Gudrun E Moore 71

6

Fetal stem cell therapy Jennifer Ryan, Michael Ting and Nicholas Fisk

7

Prenatal gene therapy Khalil Abi-Nader and Anna David

101

8

Ethical aspects of stem cell therapy and gene therapy Søren Holm

123

9

Fetal dysmorphology: the role of the geneticist in the fetal medicine unit in targeting diagnostic tests Tessa Homfray

131

10 Fetal karyotyping: what should we be offering and how? John Crolla 11

147

Non-invasive prenatal diagnosis: the future of prenatal genetic diagnosis? Lyn Chitty, Gail Norbury and Helen White 159

12 Non-invasive prenatal diagnosis for fetal blood group status Geoff Daniels, Kirstin Finning, Peter Martin and Edwin Massey 13

83

173

Selective termination of pregnancy and preimplantation genetic diagnosis: some ethical issues in the interpretation of the legal criteria Rosamund Scott 183

vi  |  CONTENTS

14 Implementation and auditing of new genetics and tests: translating genetic tests into practice in the NHS Rob Elles and Ian Frayling

193

15 New advances in prenatal genetic testing: the parent perspective Jane Fisher

199

16 Informed consent: what should we be doing? Jenny Hewison and Louise Bryant

205

17 Consensus views arising from the 57th Study Group: Reproductive Genetics

217

Index

221

Participants John C Achermann

Wellcome Trust Senior Research Fellow in Clinical Science, Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

Lyn Chitty

Professor of Genetics and Fetal Medicine, Clinical Molecular Genetics Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK and Consultant in Genetics and Fetal Medicine, University College London Hospitals NHS Foundation Trust, London.

John Crolla

Consultant Clinical Scientist, Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury SP2 8BJ, UK.

Geoff Daniels

Head of Molecular Diagnostics, International Blood Group Reference Laboratory, NHS Blood and Transplant, 500 Northway, Filton, Bristol BS34 7QH, UK.

Anna David

Senior Lecturer and Honorary Consultant in Obstetrics and Maternal/Fetal Medicine, Institute for Women’s Health, University College London, 86–96 Chenies Mews, London WC1 6HX, UK.

Jane Fisher

Director, Antenatal Results and Choices (ARC), 73 Charlotte Street, London W1T 4PN, UK.

Nicholas Fisk

Director, University of Queensland Centre for Clinical Research, Royal Women’s and Brisbane Hospital campus, Building 71/918, Herston 4029, Brisbane, Queensland, Australia.

Ian Frayling

Laboratory Director, All-Wales Medical Genetics Service; Consultant in Genetic Pathology and Clinical Genetics; Honorary Senior Research Fellow, Cardiff University; Institute of Medical Genetics, University Hospital of Wales, Cardiff CF14 4XW, UK.

Jenny Hewison

Professor of the Psychology of Healthcare, Institute of Health Sciences, University of Leeds, Charles Thackrah Building, 101 Clarendon Road, Leeds LS2 9LJ, UK.

Søren Holm

Professor of Bioethics, CSEP, School of Law, University of Manchester, Oxford Road, Manchester M13 9PL, UK.

Tessa Homfray

Consultant, Medical Genetics, Department of Genetics, St George’s University of London, Cranmer Terrace, London SW17 0RE, UK.

Sean Kehoe

Convenor of Study Groups, Lead Consultant in Gynaecological Oncology, Oxford Gynaecological Cancer Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.

Alison Lashwood

Consultant Nurse in Genetics and PGD, Clinical Genetics, 7th Floor, Borough Wing, Guy’s Hospital, London SE1 9RT, UK.

viii  |  PARTICIPANTS

Ken McElreavey

Head, Human Developmental Genetics Unit, Institut Pasteur, 25 rue du Dr Roux, Paris 75724, France.

Gudrun E Moore

Professor of Clinical and Molecular Genetics, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

Rosamund Scott

Professor of Medical Law and Ethics, Centre of Medical Law and Ethics and School of Law, King’s College London, Strand, London WC2R 2LS, UK.

Mark Sheehan

Oxford BRC Ethics Fellow and James Martin Research Fellow, Program on the Ethics of the New Biosciences, Suite 8, Littlegate House, 16/17 St Ebbe’s Street, Oxford OX1 1PT, UK.

Additional contributors Khalil Abi-Nader

Clinical Academic Research Fellow, Academic Department of Maternal Fetal Medicine, Institute for Women’s Health, University College London, 86–96 Chenies Mews, London WC1E 6HX, UK.

Sayeda Abu-Amero

Senior Research Fellow, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

Anu Bashamboo

Chief Scientist, Human Developmental Genetics Unit, Institut Pasteur, 25 rue du Dr Roux, Paris 75724, France.

Louise Bryant

Lecturer in the Psychology of Healthcare, University of Leeds, Leeds Institute of Health Sciences, Room 1.12, Charles Thackrah Building, 101 Clarendon Road, Woodhouse, Leeds LS2 9LJ, UK.

Caroline Daelemans

Wellbeing of Women Research Fellow, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

Rob Elles

Director of Molecular Genetics, Regional Molecular Genetics Service, Genetic Medicine (6th Floor), St Mary’s Hospital, Oxford Road, Manchester M13 9WL, UK.

Tarek El-Toukhy

Consultant Gynaecologist and Subspecialist in Reproductive Medicine, Assisted Conception Unit, 11th Floor, Tower Wing, Guy’s Hospital, London SE1 9RT, UK.

Kirstin Finning

Clinical Scientist, International Blood Group Reference Laboratory, NHS Blood and Transplant, 500 Northway, Filton, Bristol BS34 7QH, UK.

Jane Fisher

Director, ARC (Antenatal Results and Choices), 73 Charlotte Street, London W1T 4PN, UK.

Jennifer M Frost

MRC PhD student, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

PARTICIPANTS  |  ix

Lin Lin

Postdoctoral Research Associate, Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

Peter Martin

Clinical Scientist, International Blood Group Reference Laboratory, NHS Blood and Transplant, 500 Northway, Filton, Bristol BS34 7QH, UK.

Edwin Massey

Consultant Haematologist, NHS Blood and Transplant, 500 Northway, Filton, Bristol BS34 7QH, UK.

Gail Norbury

Director, North East Thames Regional Molecular Genetics Laboratory, Great Ormond Street Hospital, L6 York House, 37 Queen Square, London WC1N 3BH, UK.

Celia Ravel

University Clinical Lecturer, Human Developmental Genetics Unit, Institut Pasteur, 25 rue du Dr Roux, Paris 75724, France.

Jennifer Ryan

Postdoctoral Fellow, University of Queensland Centre for Clinical Research, Royal Women’s and Brisbane Hospital campus, Building 71/918, Herston 4029, Brisbane, Queensland, Australia.

Michael Ting

Postdoctoral Research Fellow, University of Queensland Centre for Clinical Research, Royal Women’s and Brisbane Hospital campus, Building 71/918, Herston 4029, Brisbane, Queensland, Australia.

Helen White

Senior Scientist, National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Salisbury, Wiltshire, SP2 8BJ, UK.

DECLARATIONS OF PERSONAL INTEREST All contributors to the Study Group were invited to make a specific Declaration of Interest in relation to the subject of the Study Group. This was undertaken and all contributors complied with this request. Khalil Abi-Nader’s department receives financial support from Ark Therapeutics Ltd, UK. John Achermann is a member of the annual meeting steering committee of the Endocrine Society. He has received reimbursement of approximately £300 each for chapters on sex development in books on internal medicine and on endocrinology. Lyn Chitty is a member of the National Screening Committee and the RCOG Ethics Committee. She is an editor of Prenatal Diagnosis and receives travel costs and honoraria. Geoff Daniels is President of the British Blood Transfusion Society. Rob Elles is a former chairman of the British Society for Human Genetics. He has received minimal editorial fees from Springer for the book Molecular Diagnosis of Genetic Diseases. Tarek El-Toukhy is a member of the Royal College of Obstetricians and Gynaecologists, the British Fertility Society and Infertility Network UK. Jane Fisher is a trustee of the Genetic Interest Group (GIG) and the Director of Antenatal Results and Choices (ARC). ARC is a member of the pro-choice coalition Voice for Choice. Nicholas Fisk acts as a medical legal consultant in the UK, USA and Australia. He has received travel expenses and accommodation from Ferring International. Ian Frayling is a member of Council and the Executive of the Royal College of Pathologists. He is also a member of the Clinical Molecular Genetics Society (CGMS) and thus also the British Society for Human Genetics (BSHG). Jenny Hewison is a member of the National Screening Committee's FAS Programme Steering Group. Gail Norbury is a member of the steering group for the NIHR-funded grant on Reliable and Accurate Non-Invasive Prenatal Diagnosis (RAPID).

P Preface

Reproduction and genetics without doubt encompass a very wide remit and, as is often the case in Study Groups, it is not possible to cover all the aspects relating to the topic that would fulfil everyone’s desires or aspirations. These constraints, however, do not prevent an attempt at addressing and discussing the relevant areas of the specialty with acknowledged experts from the UK and abroad. The progress and increased knowledge base in genetics within the past decade have impacted on this important area with interesting consequences. The ability to identify genetic defects prior to implantation, to diagnose fetal abnormalities and to introduce screening programmes means that genetic testing has a major role in preventive medicine. In parallel with these advances, there are other aspects that cannot be ignored, such as education of the public and the potential ethical dilemmas that may arise by virtue of these new methodologies. In this book, developed from the 57th RCOG Study Group, many of the above topics are discussed. It is hoped that the reader will find in a single book the diverse areas relating to reproduction and genetics, and it should be of interest to those involved in these specialist areas. As with all these expert groups, the RCOG thanks all the individuals who have selflessly given their time and expertise to ensure the publication of a unique book covering areas not available elsewhere. My thanks go to the co-editors for their patience and to all the team involved in making this book a reality. Sean Kehoe Convenor of Study Groups

1 Chapter 1

Genetic aetiology of infertility Anu Bashamboo, Celia Ravel and Ken McElreavey

Introduction In recent years there has been increasing concern about a possible decline in reproductive health.1,2 It is estimated that one in seven couples worldwide have problems conceiving and there is increasing demand for fertility treatments.1,2 These include intracytoplasmic sperm injection (ICSI) and in vitro fertilisation. In some European countries, such as Denmark, more than 6% of children are born after assisted reproduction.1,2 Sperm counts in many European countries are declining by around 2% per year.2 Although human infertility rates are high and increasing, our understanding of the genetic pathways and basic molecular mechanisms involved in gonadal development and function is limited. In this overview, we examine the various forms of infertility and the evidence that there is a genetic component, and discuss in some detail the known genetic causes of infertility.

Female infertility The main causes of female infertility are anovulation and anatomical causes such as obstruction in the genital tract (ovulatory infertility). About one-third of all cases of female infertility are due to obstruction in the genital tract.3 The obstruction can be in the fallopian tubes, uterus, cervix or vagina. Uterine abnormalities include congenitally absent (Mayer–Rokitansky–Küster–Hauser [MRKH] syndrome), bicornuate or double uterus, leiomyomas and Asherman syndrome.4 Endometriosis is an estrogen-dependent inflammatory disease that affects 5–10% of women of reproductive age in the USA.5 Its defining feature is the presence of endometrium-like tissue in sites outside the uterine cavity, primarily on the pelvic peritoneum and ovaries. Tubal and peritoneal blockades are due to tubal loss or impairment as a result of ectopic pregnancy, endometriosis or infections. Cervical factors (cervical stenosis, antisperm antibodies, non-receptive cervical mucus) and vaginal factors (vaginismus, vaginal obstruction) also contribute to ovulatory primary or secondary infertility. Primary ovarian insufficiency (POI) is characterised by primary or secondary amenorrhoea, hypergonadotrophinism and estrogen deficiency in women younger than 40 years.6,7 In the majority of cases, the aetiology remains unknown but known causes include chemotherapy, radiotherapy, surgery, associations with autoimmune © Anu Bashamboo, Celia Ravel and Ken McElreavey. Volume compilation © RCOG

2  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY

diseases and infections. POI is the preferred term for the condition that was previously referred to as premature menopause or premature ovarian failure; other terms used for this condition include primary ovarian failure, hypergonadotrophic hypogonadism and gonadal dysgenesis.6,7 The condition differs from the menopause in that there is varying and unpredictable ovarian function in approximately 50% of cases, and about 5–10% of women conceive and give birth to a child after they have received the diagnosis.8–11 Women with Turner syndrome (monosomy  X) have a normal complement of oocytes until the third month of fetal life, after which apoptosis is accelerated, generally resulting in oocyte depletion by the end of the first decade of life. As a consequence, only 10% of women with Turner syndrome achieve menarche.12–14 In contrast, women with Turner syndrome and a chromosomal mosaicism (45,X/46,XX) are more likely (40% of such women) to menstruate and can do so for several years before developing overt POI. It is possible that undetected X chromosome mosaicism may account for some cases of unexplained POI.15 Anovulation is the cause of infertility in about one-third of couples attending fertility clinics, and polycystic ovary syndrome (PCOS) accounts for 90% of such cases.16 PCOS is one of the most common endocrine disorders in women of reproductive age and the most frequent cause of hyperandrogenism and anovulation/ oligo-ovulation.17,18 The anovulatory or oligo-ovulatory infertility could also be a result of endocrine dysfunction (androgen insensitivity and follicle-stimulating hormone [FSH] and luteinising hormone [LH] imbalance, thyroid disorders, adrenal dysfunction), hypothalamic–pituitary anomalies (Kallmann syndrome, Sheehan syndrome, hyperprolactinaemia, hypopituitarism), disorders of sex development (DSD), ovarian tumours and Turner syndrome. Evidence of a genetic contribution Many aspects of female reproductive function are strongly influenced by genetic factors and consequently there have been repeated attempts to identify genetic mutations associated with disorders of female reproductive function. Endometriosis can be inherited in a polygenic manner; its incidence in relatives of affected women is up to seven times the incidence in women without such a family history.19 Several twin studies also indicate a strong heritability.20–22 This has prompted a large number of genetic association studies including, more recently, genomewide linkage analysis on affected sib pairs and on extended families.23,24 Although these studies present evidence of linkage to chromosomes 7p13–15 and 10q26, the causal gene(s) have not been described.23,24 A meta-analysis of published association studies suggests that variants in the glutathione S-transferase gene may contribute to endometriosis but further studies are required.25 The prevalence of PCOS varies by ethnicity and has been reported at between 6.5% and 8% in women younger than 40 years.26–30 Both twin and familial studies suggest that there is a genetic component to PCOS with a polygenic pattern of inheritance31,32 but genetic studies have not yet identified robust associations between gene polymorphisms and PCOS.32 There are several possible reasons for the inconsistency between association studies on PCOS. These methodological problems are potential pitfalls that arise in all association studies. These include population stratification; that is, the difference in allelic frequency between case and control populations may be due to genetic differences between the case and control populations, such as population history, that are unrelated to the phenotype. Case and control selection biases may

GENETIC AETIOLOGY OF INFERTILITY  |  3

also influence results and sample sizes are often too small to have the power to detect anything other than major causal mutations. There is a wide variation in the age at which the normal menopause begins, varying from 40 years to just over 60 years. Several studies have indicated that the variation of the age of onset of normal menopause has a very strong genetic component,33,34 with several to many genes assumed to make an additive contribution to the variation. Estimates of heritability range up to 85%. The observation from these studies that a woman with one or more first-degree relatives with a history of early menopause is likely to experience early menopause herself suggest that genes, perhaps the same genes, are also involved in early reproductive failure. Indeed, POI shows both familial inheritance and varies by ethnicity, suggesting that genetic factors play a role in some cases. The incidence of familial idiopathic POI is reported to be between 4% and 31%.35–40 The wide range in occurrence reflects the genetic heterogeneity, the wide spectrum of pathologies included and the absence of clearly defined guidelines for the diagnosis of POI. The prevalence of POI is estimated to be 0.1–1.4% in women younger than 40 years and the occurrence varies considerably with ethnicity (1.4% of African American women, 1.4% of Hispanic women, 1.0% of European women, 0.5% of Chinese women and 0.1% of Japanese women), suggesting predisposing or protective genetic factors.41 Genetic causes Androgen insensitivity syndrome is an X-linked disorder characterised by variable defects in virilisation of 46,XY individuals. This is due to the loss of function of the androgen receptor gene (Xq11–12), resulting in peripheral androgen resistance with an estimated frequency of about one in 65 000 46,XY individuals.42 Several genetic causes are known to be associated with POI. These include monosomy  X and an expansion of the CGG trinucleotide repeat in the 5′ region of the ‘fragile X mental retardation 1’ (FMR1) gene within the premutation range.43 Premutation repeats range from 55 to 200 trinucleotides and increasing repeat length is correlated with decreasing age for ovarian failure.44 Other regions on the X  chromosome may also be associated with ovarian failure. These are the POF1, POF2 and POF3 loci: although they contain several candidates, the precise gene involved has not been identified.45–47 Mutations in genes that result in ovarian insufficiency often cause other somatic anomalies. These include autosomal recessive mutations in the AIRE, EIF2B and GALT genes.48–50 POI has also been observed to be associated with the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) caused by mutations in the FOXL2 gene.51,52 In non-syndromic forms of ovarian failure, rare recessive inactivating mutations of the FSH and LH receptors have been described.53–55 Mutations in the ovary-specific homeobox transcription factor NOBOX are also a rare cause of POI.56 Some reports have suggested that mutations in X-linked oocyte-secreted factor BMP15 cause non-syndromic ovarian failure57 but others have questioned these findings.58 As discussed below in the section on male infertility, a great deal of caution needs to be exercised before it can formally be established that a candidate gene is responsible for a given phenotype. Several uterine abnormalities, including MRKH syndrome and bicornuate or double uterus, are considered to have a genetic basis on evidence from a number of familial cases with these anomalies.59–61

4  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY

Recurrent miscarriage is commonly defined as the loss of three or more consecutive pregnancies before 20–28 weeks of pregnancy and it affects up to 5% of couples.62,63 The examination of embryos from women with recurrent miscarriage indicates that most have a chromosomal anomaly.62 The majority of these chromosomal anomalies, including trisomies and structural rearrangements, are de novo but it has been suggested that as many as 10% are present in one of the parents.62

Male infertility A genetic contribution to spermatogenic failure is indicated by several families with multiple infertile or subfertile men.64–73 In some of these families an autosomal recessive mutation appears to be responsible while in others an autosomal dominant mutation with sex-limited expression is likely.64,68 In other families the genetic cause is known to involve either chromosomal anomalies or Y chromosome microdeletions.65 Familial clustering of male subfertility and impaired spermatogenesis has been well documented and considered as evidence of an important genetic contribution to the phenotype74,75 but other studies have questioned this interpretation, based on twin studies that suggest the shared environment may be more important.76 Although questions remain concerning the general genetic contribution to infertility and subfertility, several genetic causes have been documented that are responsible for a significant minority of cases. Chromosome disorders A chromosomal anomaly is carried by 5% of all infertile men.77,78 This is ten times higher than the frequency in the general population.77 In about 80% of these cases a sex chromosome is involved and in the remaining 20% the autosomes are involved.77,78 Klinefelter syndrome (47,XXY) is the most frequent cause of gonosomic anomalies, occurring in 0.1–0.2% of newborn males. The prevalence among infertile men is high, from 5% in those with severe oligozoospermia to 10% in those with azoospermia. The presence of an additional X chromosome is associated with testis hypotrophia and an increase in plasmatic gonadotrophins, thus representing the most frequent form of male hypogonadism. Before the advent of ICSI these men were regarded as being completely infertile. However, there are now more than 50 normal children born from fathers with Klinefelter syndrome, where rare spermatozoa were recovered with a testicular biopsy.79 In spite of the existence of a constitutional chromosomal anomaly, the risk for these men to transmit a chromosomal anomaly is small since fluorescence in situ hybridisation studies have shown that the rate of disomic XY or XX sperm rises only marginally in Klinefelter cases, suggesting these spermatozoa are produced from 46,XY spermatogonia.80 A 47,XYY karyotype is the second most frequent cause of gonosomic anomalies. Although the majority of these men are fertile, the frequency of this anomaly is four times higher in those who are infertile than in the general population.81 Almost 1% of men with azoospermia have a 46,XX karyotype.77,81,82 In most cases, the testis-determining gene SRY is present on the short arm of one of the X  chromosomes or on an autosome. In rare cases, SRY may be absent and the phenotype results then from a mutation in another gene involved in the formation of the testis. Although one case of a 46,XX male with a duplication of the SOX9 gene has been described,83 the genetic cause of most cases is unknown.

GENETIC AETIOLOGY OF INFERTILITY  |  5

Chromosomal translocations are found with a frequency 8–10 times higher in infertile men and may be acrocentric Robertsonian translocations (centric fusions) or reciprocal translocations.77,78,81,84 It also seems that the frequency of small supernumerary marker chromosomes, made up of the short arms of acrocentric chromosomes, is often increased in the infertile populations of men.77,78,81,84 Three Y chromosome microdeletions The male-specific portion of the Y  chromosome contains 78 genes or families of genes, many of which appear to be involved in spermatogenesis.85 These genes are located on both the short and long arms of the Y chromosome but most attention has focused on the long arm because it was deletions in this region that were first detected by conventional cytogenetic approaches in infertile men.86 Later molecular approaches defined three regions of the long arm associated with infertility: AZFa, AZFb and AZFc.87 In most studies, deletions are found at frequencies of around 10– 12% and are independent of the ethnic or geographic background.88 AZFc deletions and AZFc + AZFb deletions are the most frequent. AZFc deletions account for almost 60% of all Yq deletions, whereas AZFa and AZFb deletions are rare. Most infertile men carrying AZF deletions have either azoospermia or oligozoospermia. Men with complete AZFc deletions show a poor correlation with testicular histology, whereas complete AZFb deletions are usually associated with azoospermia and meiotic germ cell arrest on histology and men with complete AZFa deletions lack germ cells on histology.87,88 Rare partial deletions of AZFa and AZFb have been described associated with variable phenotypes.89,90 Thus, in cases of complete AZFa or AZFb deletion, a testicular biopsy is not recommended because the probability of finding viable sperm is almost zero. Apart from their infertility, men carrying AZF microdeletions do not present other known pathology. Since these deletions can be associated with some sperm production, cases of father-to-son transmission of AZF deletions has been described.91 The molecular mechanism of AZF microdeletions is due to the particular structure of the Y  chromosome consisting of complex repetitive regions.85 AZFc deletions occur as a consequence of intra-Y homologous recombination between repeat sequences of a complex palindromic structure.85,89 Partial deletions within the 3.5 Mb AZFc portion have been identified that result in the absence of some gene family members in the region.92 The deletions are structurally heterogeneous, with some of them being apparently polymorphic variants that have no obvious effect on fertility. One group of deletions, known as gr/gr deletions, are controversial since some studies have linked these deletions to infertility whereas other have not.89,92 Further studies are necessary to further characterise these deletions and define the Y chromosome background on which they occur. This is important because the class of Y chromosome (Y chromosome haplogroup or Y chromosome background) can have an effect on spermatogenesis89 and some gr/gr deletions that are fixed on Y chromosome haplogroups may in fact be acting as a surrogate marker for other Y variants that may affect spermatogenesis.89 The function of many of the Y  chromosome genes is now known or inferred from their X chromosome or autosomal homologue. Several of these genes may play a role in the epigenetic reprogramming that is known to occur in the specification and differentiation of the germline and during spermatogenesis. SMCY and UTY are known or suspected to have histone demethylase activity93,94 whereas CDY is a histone acetyltransferase.95 DAZ may be a meiotic competence factor.96 To date, only one

6  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY

point mutation has been described in any of the Y chromosome genes associated with male infertility, suggesting that these mutations are rare.97 Autosomal genes Although many genes are known to be essential for gametogenesis and murine knockout studies have identified several hundred genes specifically associated with infertility, there are surprisingly few mutations in X chromosome or autosomal genes that have been conclusively demonstrated to cause spermatogenic failure. Homozygous mutations in the SPATA16 gene that may be involved in acrosome formation are associated with globozoospermia.98 The AURKC gene (aurora kinase C) expresses a cell cycle regulatory serine/threonine kinase mainly in the testis during meiosis. A homozygous, single base pair deletion that results in a premature stop codon was found associated with large-headed multiflagellar polyploid spermatozoa.99 This is a founder mutation with a carrier frequency of one in 50 in North African populations.99 The CFTR gene (cystic fibrosis transmembrane conductance regulator) encodes a chloride channel that regulates the transport of water and salt on both sides of the plasma membrane of epithelial cells. Infertile men with obstructive azoospermia due to congenital bilateral absence of the vas deferens (CBAVD) frequently harbour mutations of the CFTR gene. Around 80% of men with isolated CBAVD carry two CFTR mutations, usually in compound heterozygosity.100 In an isolated case of unilateral absence of the vas deferens, the association with CFTR mutations is controversial. Similarly, it has been reported that CFTR may also be involved in sperm capacitation and that mutations impairing CFTR function may lead to reduced sperm fertilising capacity and male infertility other than CBAVD.101 In both these situations, additional data are required. End-organ resistance to androgens is designated as androgen insensitivity syndrome and comprises complete and partial forms.102,103 Complete androgen insensitivity in a 46,XY individual is characterised by female external genitalia, a short blind-ending vagina, the absence of Müllerian ducts, the development of gynaecomastia and the absence of pubic and axillary hair. Partial androgen insensitivity syndrome covers a wide spectrum of undervirilised phenotypes ranging from clitoromegaly at birth to infertile men.102,103 More than 600 mutations have been described in the X-linked androgen receptor gene (AR) resulting in a 46,XY DSD (androgen insensitivity).104 The first exon of the AR gene contains a polymorphic sequence of CAG triplets. Spinal and bulbar muscular atrophy (SBMA; also called Kennedy disease) is a recessive neurodegenerative adult-onset disorder in men.105 Men with SBMA show slowly progressive spinal and bulbar muscular atrophy with fasciculations and generalised muscle weakness. Partial androgen insensitivity with gynaecomastia, impotence and reduced fertility is often seen. Affected men have about twice as many CAG repeats as unaffected men (38–72 repeats compared with 10–36 repeats, respectively).105 In contrast to SBMA, a reduction in the number of CAG repeats has been reported to be associated with an increased risk of cancer of the prostate in androgen-dependent tumours.106 Expansion of the CAG triplets associated with reduced transcriptional activity and leading to reduced sperm counts has been extensively studied and remains controversial. Although some studies find an association between length of CAG triplets and male infertility, many others do not.107 These discordant results may reflect a difference in ethnicity and geography in the selection of patients and controls. The need for adequate control populations is highlighted by studies on the X-linked testis-specific USP26 gene. Three variants that form a haplotype (371insACA, 494T>C and 1423C>T) were reported to be associated with azoospermia in two

GENETIC AETIOLOGY OF INFERTILITY  |  7

independent studies.108,109 Further analyses demonstrated that two of these changes are the ancestral sequence of the gene that is present in significant frequencies in subSaharan African and South and East Asian populations, including in individuals with known fertility.110 A large number of polymorphic variants have been reported to be associated with male infertility, including polymorphisms associated with the genes DAZL, MTHFR, BOULE, POLG, FSHR, ESR1, DNAI1, DNAH5, DNAH11, KIT, KITLG, ES and PRM1.111–114 However, the contribution of mutations in these genes remains to be established, since the association studies were often based on small sample sizes, and the population substructure, which could give spurious associations, was not taken into account in most of the studies.

Genetics and epigenetics Any consideration of genetic contributions to infertility needs to take into account the recent and sharp decline in human male reproductive health that has been widely reported.115 Prospective cross-sectional studies have indicated a general birth cohort decline in sperm quantity.116 Indeed, 20% of investigated young men from Denmark and Norway had a sperm concentration of below the World Health Organization reference values for oligozoospermia.116 Testicular germ cell cancer is now the most common malignancy in males aged between 20 and 45 years and may have increased in incidence over the past 50 years.117 These phenotypes, together with undescended testes and anomalies of the male external genitalia, may have a common aetiology resulting from disruption of the gonadal environment during fetal life, and the unifying term testicular dysgenesis syndrome (TDS) has been used describe them.118 The rapid rise in the incidence of TDS suggest an environmental aetiology perhaps in genetically susceptible individuals. The influence of genetic and environmental factors is the subject of a continuing debate but reports of declining semen quality in industrialised countries, particularly in Western Europe, over the past 60  years suggests a major environmental influence.118 A considerable body of data suggests that exposure of a developing male fetus in vivo to a number of environmental factors can negatively influence sexual development and testicular function. Much of these data come from well-documented studies of wildlife and experimental laboratory animals exposed at critical periods in their life stage to synthetic chemicals that alter hormone activity in the body (endocrine disruptors). Human exposure is variable but widespread, and sources include consumer products at doses predicted to have a negative impact on reproductive health.118,119 A correlation has been demonstrated between primary metabolites of phthalates in the urine of expectant mothers and boys born with reduced anogenital distance (a sensitive measure of testosterone activity).120 A correlation with phthalate metabolites in breast milk and reduced testosterone levels in infants has also been noted.121 These suggest a link between phthalates and anomalies of the male reproductive system but, as with all association studies, other factors could be involved and the interpretation of statistical trends remains controversial. Associations can be fortuitous and other lifestyle factors could be involved. A move away from trend analyses can be seen in the increasing number of studies that directly evaluate the effects of these chemicals on the synthesis or activity of proteins known to be involved in testicular descent or spermatogenesis.122 In the longer term, data from functional studies may provide a more persuasive argument demonstrating the negative impact of these chemicals on male reproductive development and function. A number of studies have suggested that the detrimental influence of environmental agents on male germ cells may be an epigenetic phenomenon that alters DNA

8  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY

methylation. In utero exposure of fetal male rodents to fungicide at the moment of testis determination promotes heritable male germ cell defects in multiple generations.123,124 These defects are associated with an acquired and apparently permanent hypermethylation inherited through the paternal allele.123,124 In the human, several studies have examined the methylation profiles in sperm from infertile men, with differing results. Loss of hypermethylation at the paternally imprinted H19 locus was originally described in the sperm of men with unexplained reduced sperm counts.125 A further study of seven loci subjected to either maternal or paternal imprinting revealed anomalies of both types of locus in 14–20% of men with moderate or severe oligozoospermia.126 A genome-wide analysis has suggested a global hypermethylation of DNA from poor-quality sperm and improper erasure of DNA methylation during germ cell development.127 These data suggest that the epigenetic landscape may be altered in some men but, from such a limited number of studies, the relationship between these changes and infertility remains obscure.

Conclusion There is evidence for a major genetic contribution to human infertility but the genetic causes themselves remain to be identified. There is a limited basic understanding of the molecular mechanisms involved in gonad development and in gametogenesis. For example, there is considerable interest in deriving gametes from embryonic stem cells but the underlying genetic and epigenetic mechanisms are unknown. There is accumulating evidence of modifications of the epigenetic landscape in sperm from men with reduced sperm counts. These modifications may be related to infertility. Genetic association studies aimed at identifying genetic causes of infertility have also been largely unsuccessful because of limitations that are common in most genetic association studies – variable definition of patient/control phenotypes, small sample size, variable inclusion/exclusion criteria and ascertainment bias. These limitations can be overcome, at least in part, by the creation of national and international biobanks of biological material from well-characterised patient and control groups. Understanding of the genetic basis of infertility is likely to increase dramatically in the future. New technologies are available that permit high-throughput detailed genetic analysis. This includes advances in sequencing technologies such as sequencecapture methods that enable defined regions of the genome (such as the exome) to be enriched by hybridisation, then sequenced using new high-throughput technologies. A significant number of patients with infertility problems carry chromosomal rearrangements. It is likely that other chromosomal anomalies are present that are not detected by the current limits of resolution of conventional cytogenetic approaches. High-resolution comparative genome hybridisation using oligoarrays can identify changes at resolutions of up to 5–10 kb depending on the platform. Some individuals with infertility may be carrying these submicroscopic rearrangements that are causing infertility, whereas copy number variants may confer a genetic susceptibility to infertility. A recent example of such a copy number variant has been described in the ESR1 gene where an intragenic 2.2 kb deletion is present at a frequency of 10% in the Spanish population and may confer a susceptibility to infertility.128 A combination of these approaches together with strict diagnostic criteria will increase the likelihood of success in understanding the genetic basis of infertility.

GENETIC AETIOLOGY OF INFERTILITY  |  9

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Dam AH, Koscinski I, Kremer JA, Moutou C, Jaeger AS, Oudakker AR, et al. Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia. Am J Hum Genet 2007;81:813–20. Dieterich K, Soto Rifo R, Faure AK, Hennebicq S, Ben Amar B, et al. Homozygous mutation of AURKC yields large-headed polyploid spermatozoa and causes male infertility. Nat Genet 2007 39:661–5. Chillón M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, et al. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N Engl J Med 1995;332:1475–80. Xu WM, Shi QX, Chen WY, Zhou CX, Ni Y, Rowlands DK, et al. Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility. Proc Natl Acad Sci U S A 2007;104:9816–21. Brinkmann AO. Molecular basis of androgen insensitivity. Mol Cell Endocrinol 2001;179:105–9. Sultan C, Paris F, Terouanne B, Balaguer P, Georget V, Poujol N, et al. Disorders linked to insufficient androgen action in male children. Hum Reprod Update 2001;7:314–22. Gottlieb B, Beitel LK, Wu JH, Trifiro M. The androgen receptor gene mutations database (ARDB): 2004 update. Hum Mutat 2004;23:527–33. La Spada AR, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH. Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 1991;352:77–9. Giovannucci E, Stampfer MJ, Krithivas K, Brown M, Dahl D, Brufsky A, et al. The CAG repeat within the androgen receptor gene and its relationship to prostate cancer. Proc Natl Acad Sci U S A 1997;94:3320–3. Erratum in: Proc Natl Acad Sci U S A 1997;94:8272. Tüttelmann F, Rajpert-De Meyts E, Nieschlag E, Simoni M. Gene polymorphisms and male infertility – a meta-analysis and literature review. Reprod Biomed Online 2007;15:643–58. Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. Possible role of USP26 in patients with severely impaired spermatogenesis. Eur J Hum Genet 2005;13:336–40. Paduch DA, Mielnik A, Schlegel PN. Novel mutations in testis-specific ubiquitin protease 26 gene may cause male infertility and hypogonadism. Reprod Biomed Online 2005;10:747–54. Ravel C, El Houate B, Chantot S, Lourenço D, Dumaine A, Rouba H, et al. Haplotypes, mutations and male fertility: the story of the testis-specific ubiquitin protease USP26. Mol Hum Reprod 2006;12:643–6. Teng YN, Lin YM, Sun HF, Hsu PY, Chung CL, Kuo PL. Association of DAZL haplotypes with spermatogenic failure in infertile men. Fertil Steril 2006;86:129–35. Thangaraj K, Deepa SR, Pavani K, Gupta NJ, Reddy P, Reddy AG, et al. A to G transitions at 260, 386 and 437 in DAZL gene are not associated with spermatogenic failure in Indian population. Int J Androl 2006;29:510–14. Kukuvitis A, Georgiou I, Bouba I, Tsirka A, Giannouli CH, Yapijakis C, et al. Association of oestrogen receptor alpha polymorphisms and androgen receptor CAG trinucleotide repeats with male infertility: a study in 109 Greek infertile men. Int J Androl 2002;25:149–52. Oliva R. Protamines and male infertility. Hum Reprod Update 2006;12:417–35. Jørgensen N, Asklund C, Carlsen E, Skakkebaek NE. Coordinated European investigations of semen quality: results from studies of Scandinavian young men is a matter of concern. Int J Androl. 2006:29:54–61. Jørgensen N, Carlsen E, Nermoen I, Punab M, Suominen J, Andersen AG, et al. East–West gradient in semen quality in the Nordic-Baltic area: a study of men from the general population in Denmark, Norway, Estonia and Finland. Hum Reprod 2002;17:2199–208. Møller H. Clues to the aetiology of testicular germ cell tumours from descriptive epidemiology. Eur Urol 1993;23:8–13. Skakkebaek NE, Rajpert-De Meyts E, Main KM. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 2001;16:972–8. vom Saal FS, Akingbemi BT, Belcher SM, Birnbaum LS, Crain DA, Eriksen M, et al, Chapel Hill bisphenol A expert panel consensus statement: integration of mechanisms, effects in animals and potential to impact human health at current levels of exposure. Reprod Toxicol 2007;24:131–8. Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, et al. Study for Future Families Research Team. Decrease in anogenital distance among male infants with prenatal phthalate exposure. Environ Health Perspect 2005;113:1056–61.

14  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY 121. Main KM, Mortensen GK, Kaleva MM, Boisen KA, Damgaard IN, Chellakooty M, et al. Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants three months of age. Environ Health Perspect 2006;114:270–6. 122. Laguë E, Tremblay JJ. Antagonistic effects of testosterone and the endocrine disruptor mono-(2ethylhexyl) phthalate on INSL3 transcription in Leydig cells. Endocrinol 2008;149:4688–94. 123. Anway MD, Cupp AS, Uzumcu M, Skinner MK. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 2005;308:1466–9. 124. Chang HS, Anway MD, Rekow SS, Skinner MK. Transgenerational epigenetic imprinting of the male germline by endocrine disruptor exposure during gonadal sex determination. Endocrinology 2006;147:5524–41. 125. Marques CJ, Carvalho F, Sousa M, Barros A. Genomic imprinting in disruptive spermatogenesis. Lancet 2004;363:1700–2. 126. Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C, Utsunomiya T, et al. Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet 2007;16:2542–51. 127. Houshdaran S, Cortessis VK, Siegmund K, Yang A, Laird PW, Sokol RZ. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm. PLoS One 2007;2:e1289. 128. Galan JJ, Buch B, Pedrinaci S, Jimenez-Gamiz P, Gonzalez A, Serrano-Rios M, et al. Identification of a 2244 base pair interstitial deletion within the human ESR1 gene in the Spanish population J Med Genet 2008;45:420–4.

2 Chapter 2

Disorders of sex development Lin Lin and John C Achermann

Introduction Disorders of sex development (DSD) are defined as ‘congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical’.1,2 DSD therefore represent a diverse range of conditions that can present at different ages and to a range of different healthcare professionals. Typically, a baby born with ambiguous genitalia will be referred to a paediatric endocrinologist or urologist but DSD can also present in adolescence to gynaecologists because of primary amenorrhoea in a girl, or even in adulthood to fertility services because of difficulty having children. Furthermore, an increasing number of individuals with DSD are being diagnosed in fetal medicine units following a discordance between prenatal karyotyping (performed for another reason) and genital appearance on ultrasound or at birth. An awareness of this range of conditions is thus important for practitioners in many different fields. Given the diverse and complex nature of DSD, a multidisciplinary team (MDT) approach involving various healthcare professionals with experience and interest in this area is essential (for example, endocrinologist, urologist, gynaecologist, psychologist, geneticist, cytogeneticist, biochemist and ethicist).1 While this team need not necessarily be involved in every case of apparently ‘simple’ hypospadias if managed by an experienced urologist, an MDT approach is necessary for more complex cases of DSD where there are diagnostic, management and sex assignment issues. Our knowledge of the causes of DSD, especially at the molecular level, has increased significantly in the past 20 years.3 In the past, the two most frequent diagnoses made were 21-hydroxylase deficiency (congenital adrenal hyperplasia) in a 46,XX baby with ambiguous genitalia, and complete androgen insensitivity syndrome (CAIS) in a 46,XY girl with pubertal development but primary amenorrhoea. Although these two conditions remain the most frequent DSD diagnoses, it is also emerging that many other specific conditions can present as DSD and that the pathways underlying gonad development, gonad function and steroidogenesis are more complex than the simple reductionist models we tended to use. Indeed, in a study of adults with a 46,XY karyotype and DSD, the diagnosis, on review of the data available, was found to be incomplete, absent or even incorrect in a substantial proportion of cases.4 This is not entirely surprising as: n many different conditions might result in a similar phenotype (such as ambiguous genitalia) © Lin Lin and John C Achermann. Volume compilation © RCOG

16  |  LIN LIN AND JOHN C ACHERMANN n each condition probably has a spectrum of severity ranging from a complete

form to milder or partial forms n biochemical and endocrine markers have limited specificity in some cases n some of the biological events resulting in the phenotype may only be manifest during critical developmental windows. Therefore, a genetic-based approach to diagnosis may be useful in some situations. Reaching a specific diagnosis based on molecular genetics also improves our understanding of the biological basis of these conditions. It might also help in making the best decisions for sex assignment and subsequent management based on likely gender identity, urological and sexual function, likely need for endocrine replacement, fertility options, tumour risk and the possibility of associated features developing (for example, adrenal dysfunction). A genetic diagnosis can also help in appropriate counselling of individuals and their families, in stratifying long-term outcome studies based on specific diagnoses, and can in some cases be associated with a sense of resolution. However, genetic analysis in itself generates a number of scientific and ethical challenges, which are important to get right if improved knowledge in this area is to have a significant positive impact in the long term. In this chapter, we review some recent advances in our understanding of human sex development, discuss a suggested new nomenclature and classification system for DSD, provide a brief overview of some known genetic causes of DSD, and outline some challenges for research and clinical practice in this field related to the theme of ‘reproductive genetics’.

Human sex development Human sex development can be broken down into three major components: n chromosomal sex (the presence or absence of X or Y chromosomes) n gonadal sex (development of the gonad into either testis or ovary) n phenotypic (anatomical) sex (the appearance of the internal and external genitalia). Sometimes ‘brain sex’ (gender identity, sex role behaviour, sexual orientation) is also considered as a distinct entity. It is important to note that no single component dictates an individual’s sex. However, considering these separate aspects can help in focusing on a specific diagnosis, which might in some cases be confirmed by molecular analysis. Chromosomal sex The importance of the Y chromosome in testis development has been known for many years and the quest to identify a ‘testis-determining factor’ began in the 1950s. An increasingly narrow region of the Y  chromosome was identified that could contain this factor and, following a series of seminal studies published between 1989 and 1991, the gene SRY (sex-determining region, Y) was shown to be the main testis-determining factor in mice and humans.5 Even to this day, SRY seems to be most important molecular switch involved in testis development. This finding underscores the importance of an intact Y  chromosome for the testis to form, which occurs even in the presence of multiple copies of the X chromosome (for example, 47,XXY).

DISORDERS OF SEX DEVELOPMENT  |  17

Gonadal and phenotypic sex In humans, the primitive gonad develops from a condensation of mesoderm in the medioventral region of the urogenital ridge at around 4–5 weeks of gestation. At this time, primordial germ cells (PGCs) – the precursors of gametes – migrate into the gonad from their origin at the dorsal endoderm of the yolk sac. The presence of intact germ cells appears to be necessary for the ovary to develop but is not needed for testis development to occur. This primitive gonad remains ‘bipotential’ and undifferentiated until around 42 days of gestation, at which point a ‘wave’ of SRY expression occurs in the developing testis, which results in a series of downstream events and commitment to testis formation. At around 7 weeks of gestation, primitive Sertoli cells form and release Müllerian inhibiting substance (MIS) (also known as antimüllerian hormone, AMH), which results in the regression of the Müllerian structures that are destined to form the fallopian tubes, uterus and upper two-thirds of the vagina. At around 8 weeks of gestation, early fetal Leydig cells start to produce testosterone. Testosterone acts through the androgen receptor to stabilise the Wolffian structures (epididymes, vasa deferentia and seminal vesicles) (Figure  2.1). Testosterone also reaches the perineum where it is converted in target tissues by the enzyme 5α-reductase type 2 to its more active metabolite dihydrotestosterone (DHT). DHT also activates the androgen receptor resulting in elongation of the developing phallus and fusion of urethral folds (Figure 2.2).6,7 Thus, the 8- to 12-week period of fetal development is a crucial time for the phenotype to become established. During these early stages of sex development, Leydig cell steroidogenesis is largely under the control of placental human chorionic gonadotrophin (hCG) signalling through the shared luteinising hormone/hCG receptor (LHCGR). However, the hypothalamic– pituitary (gonadotrope) axis becomes active by 20 weeks of gestation, at which point pituitary-derived LH also has a role in stimulating Leydig cell steroidogenesis and testis descent (see below). Although relatively little is known about these specific processes in humans, detailed studies in mice are starting to reveal a cascade of genetic, structural and paracrine events that occur during gonad development.8 For example, following expression of SRY, and subsequent expression of SOX9, a series of discrete events occurs in the developing testis such as: n cellular proliferation n migration of cells into the testis from the neighbouring mesonephros n organisation into primitive cords n development of a coelomic vessel that runs lengthwise along the gonad n Leydig cell differentiation. During this period, testis development seems to be a much more active process morphologically than ovarian development, which in some respects has been considered a constitutive (or ‘default’) process. However, detailed studies of differential gene expression patterns are starting to show a distinct set of genes expressed in the developing ovary, so it seems that ovarian differentiation is an active process too.9,10 While some of these genes may be necessary to maintain ovarian integrity, it is emerging that certain genes expressed in the ovary (DAX1, WNT4/RSPO1) may be needed to oppose testis development – the so called ‘anti-testis’ genes.11 In contrast to the case with the testis, most data suggest that the ovary is steroidogenically quiescent during fetal life.

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Other sexually dimorphic processes: germ cell fate and gonad position Several other important differences occur between the developing testis and ovary in the fetus, such as variations in germ cell fate and gonad position. In the first few months of gestation, PGCs undergo multiple cycles of mitotic division. In the testis, a ‘self-renewable’ population of undifferentiated germ cells

Figure 2.1

Simplified overview of steroidogenesis in the testis and adrenal gland. Testis-specific pathways are shown in grey boxes, adrenal-specific pathways in white boxes, and shared testis/adrenal pathways in black boxes. During male sex development, placental human chorionic gonadotrophin (hCG) or pituitary luteinising hormone (LH) stimulates the LHCG receptor on the fetal Leydig cell to increase cholesterol update into the mitochondria by steroidogenic acute regulatory protein (StAR). Following conversion to pregnenolone by P450 side-chain cleavage (P450scc, CYP11A1), a series of enzymatic modifications occur that ultimately result in the synthesis of the androgen testosterone by 17β-hydroxysteroid dehydrogenase 3 (17β-HSD 3). Testosterone is released from the testis and converted to the more potent androgen dihydrotestosterone (DHT), where it acts on the androgen receptor to stimulate external genital development. Defects anywhere along this pathway can result in impaired androgen synthesis and action. Defects in StAR, P450ssc and 3β-hydroxysteroid dehydrogenase type 2 (3β-HSD 2) cause adrenal and gonadal failure, whereas defects on 17α-hydroxylase (which also has 17,20-lyase activity) cause hypertension due to adrenal steroid excess. In 46,XX fetuses with congenital adrenal hyperplasia, blocks in 21-hydroxylase or 11β-hydroxylase result in reduced cortisol feedback and elevated adrenocorticotrophin (ACTH) drive, shunting steroid precursors into the androgenic pathway and causing androgenisation of the female fetus. Milder effects are seen with blocks in 3β-HSD 2. All these conditions can be associated with adrenal dysfunction. In addition to the pathways shown, disruption of P450 oxidoreductase (POR) – a co-enzyme for 17α-hydroxylase and 21-hydroxylase – can cause under-androgenisation (46,XY) or over-androgenisation (46,XX) and a steroid pattern reflecting defects in both these enzyme systems; DHEA = dehydroepiandrosterone; DOC = deoxycorticosterone

DISORDERS OF SEX DEVELOPMENT  |  19

Figure 2.2 Differentiation of male external genitalia in humans between 8 and 10 weeks post conception (wpc): (A) undifferentiated human external genitalia at 8 wpc; (B) differentiation of scrotal folds and fusion of the urethral folds (asterisks mark patent regions, either side) at 10 wpc; gs = genital swelling; gt = genital tubercle; sf = scrotal folds; uf = urethral folds; scale bars = 500 µm; reproduced with permission from Goto et al.6 (© 2006 American Society for Clinical Investigation)

exists, but most PGCs commit to differentiation and, after several cycles of mitotic division, enter mitotic arrest. Subsequent testicular development can occur in the absence of this germ cell population. Meiosis only occurs during the progress of spermatogenesis after puberty. In the developing ovary, primordial ova (oogonia) undergo mitotic expansion in the first few months of gestation (5–24  weeks), followed by meiotic division (8– 36 weeks) and a process of meiotic arrest (oocytes). Retinoic acid signalling from the mesonephros may stimulate this process.12,13 The presence of the meiotic oocytes is critical for differentiation of pre-follicular cells into follicular cells and for maintaining ovarian development. More than six million oogonia and prophase oocytes exist in the developing ovary at around 16 weeks of gestation. Formation of oogonia from PGCs stops by 7 months of gestation. At this stage, some oocytes are found to be associated with somatic pregranulosa cells forming primitive or primordial follicles. However, most oogonia do not form follicles and undergo apoptosis, so that approximately one million germ cells are present in the ovary at the time of birth. These ‘resting’ primodial follicles can remain in this stage of development throughout the woman’s reproductive life, and meiosis only progresses in response to ovulation of the Graafian follicle (approximately 400 in a woman’s reproductive lifetime). Traditional doctrine has been that the population of germ cells is ‘fixed’ at birth but more recent data have raised the possibility of a potential self-renewable population of germline stem cells in the mouse ovary that are active into adult life or of potential repopulation of the ovary by germ cells from other sites.14 It is highly controversial whether such mechanisms exist in primates.15 Whereas the ovary remains in an intra-abdominal position, the developing testis undergoes a two-stage process of descent that starts at around 12 weeks of gestation and is usually complete by the middle of the third trimester. The initial transabdominal stage of testicular descent involves contraction and thickening of the gubernacular ligament. This stage is mediated by the testis itself, following secretion of factors such

20  |  LIN LIN AND JOHN C ACHERMANN

as insulin-like 3 (INSL3, relaxin-like factor) which acts through a G-protein-coupled receptor, LGR8 (GREAT). The subsequent trans-inguinal phase of testicular descent involves stimulation by androgens and LH. Thus, dysgenetic testes are usually intraabdominal whereas testes are inguinal in disorders of androgen synthesis and action. Early postnatal changes The hypothalamic–pituitary (gonadotrope)–gonadal (HPG) axis develops in midgestation and actively secretes gonadotrophins (LH and follicle-stimulating hormone [FSH]) from around 20  weeks of gestation. Around the time of birth, boys have relatively high levels of testosterone, which fall in the first week of life. After a period of postnatal quiescence, the HPG axis becomes reactivated and a so-called ‘minipuberty’ occurs from 6 weeks to 6 months with a peak of testosterone in the low adult range at around 3 months of age. Thereafter, the HPG axis remains quiescent again until puberty. The role of this HPG activation in early infancy is not known although it is associated with a small acceleration in penile growth and may occur before certain further developments in germ cell maturation.16 It is likely that postnatal HPG axis activation occurs in girls but the significance of this is not known. Two relatively new hormones are emerging as useful markers of postnatal testis function. Inhibin  B is secreted predominantly by Leydig cells. It is detectable at birth, falls during infancy and rises again at puberty, thus following the pattern of testosterone secretion to some degree. MIS/AMH is secreted predominantly by Sertoli cells and is high at birth, rises in the first few weeks of life and remains high throughout childhood before falling to adult levels during puberty. Both markers will be low if testicular dysgenesis or regression has occurred.17 Inhibin A and AMH may reflect ovarian integrity but levels are usually low in infancy and childhood. In both sexes, reactivation of the HPG axis in late childhood results in onset of puberty in adolescence. In boys, LH stimulates Leydig cells to produce androgens (testosterone) necessary for penile enlargement, hair growth (pubic, axillary and facial) and voice changes. FSH stimulates Sertoli cells to activate spermatogenesis, although intra-testicular testosterone also has a facilitative role in this regard. In girls, LH and FSH stimulate the production of estrogens by the steroidogenic pathways of the ovarian theca and granulosa cells. Estrogens promote breast development and uterine growth. In addition, gonadotrophins regulate certain aspects of follicular development so that regular ovulatory cycles develop by the end of puberty. More detailed descriptions of spermatogenesis and folliculogenesis are beyond the scope of this chapter.

Disorders of sex development The past 20 years have seen significant advances in our understanding of the underlying aetiology of many forms of DSD. Coupled with this has been the need to develop a classification system based on a more precise diagnosis wherever possible and awareness that many of the terms used to describe DSD are out of date, scientifically inaccurate and/or perceived as pejorative by many individuals with these conditions. In 2005, a consensus meeting to discuss these issues was convened in Chicago.1,2 Although this was driven by the major paediatric endocrinology societies, participants included surgeons, psychologists, geneticists and representatives of patient advocacy groups. The outcome of this meeting was published as a series of guidelines and statements in 2006. This process generated many more questions than answers, for

DISORDERS OF SEX DEVELOPMENT  |  21

example regarding the controversies of surgical intervention and lack of long-term outcome data. However, two important elements to emerge from this consensus meeting were a proposed new nomenclature system and a classification system that can be used to incorporate a specific molecular diagnosis where available. Nomenclature The need for a revised nomenclature system has been apparent for some time.18,19 Many of the traditional terms used were viewed negatively by individuals with these conditions and were not helpful diagnostically. For example, a woman with CAIS, who would typically present in late adolescence with primary amenorrhoea but who has a female gender identity and sex role behaviour, would subsequently be called ‘male pseudohermaphrodite’. The consensus meeting proposed that a more generic term – ‘disorders of sex development’ – be used instead of intersex, and that ‘46,XY DSD’ and ‘46,XX DSD’ could replace male and female pseudohermaphroditism, respectively (Table 2.1).1,2 Wherever possible, a more specific diagnosis (for example, ‘gonadal dysgenesis’ or ‘androgen insensitivity’) could be used and an exact molecular diagnosis incorporated into this if known. Classification and diagnosis Together with a revised nomenclature system, an updated classification system for DSD was proposed. This system divides the conditions into: n sex chromosome DSD n 46,XY DSD n 46,XX DSD. An overview of a potential classification system is shown in Table  2.2. It is very important to realise that the karyotype in itself is not necessarily the most important factor in determining outcome and the karyotype does not define an individual’s ‘sex’. However, rapid diagnostic tests such as fluorescence in situ hybridisation (FISH) (using probes for SRY and the X chromosome) or quantitative polymerase chain reaction (PCR) mean that a preliminary karyotype result can be available within a matter of hours or days, which can be very useful for guiding initial investigation, management and counselling, especially in a newborn child presenting with ambiguous genitalia. Thus, this form of genetic/cytogenetic analysis is very important in the initial approach to investigation of DSD. Such a wide-reaching definition and classification system can be criticised to some degree. For example, ‘sex chromosome DSD’ (sex chromosome aneuploidy) includes monosomy X (45,X; Turner syndrome) and 47,XXY (Klinefelter sydrome), which

Table 2.1

An updated nomenclature for disorders of sex development

Terminology used previously Intersex Male pseudohermaphrodite Female pseudohermaphrodite True hermaphrodite XX male

Proposed new terminology Disorders of sex development (DSD) 46,XY DSD 46,XX DSD Ovotesticular DSD Testicular DSD

C: Other 1. Syndromic associations of male genital development (≥ 50) (e.g. cloacal anomalies, Robinow syndrome, Aarskog syndrome, hand-foot-genital syndrome, popliteal pterygium syndrome) 2. Persistent Müllerian duct syndrome 3. Vanishing testis syndrome 4. Isolated hypospadias 5. Congenital hypogonadotrophic hypogonadism 6. Cryptorchidism 7. Environmental influences

B: Disorders of androgen synthesis or action 1. Disorders of androgen synthesis:  – LH receptor mutations  C: 45,X/46,XY mosaicism – Smith-Lemli-Opitz syndrome  (mixed gonadal dysgenesis) – Steroidogenic acute regulatory protein  – Cholesterol side-chain cleavage  D: 46,XX/46,XY – 3β-hydroxysteroid dehydrogenase type 2 (chimerism) – 17α-hydroxylase/17,20-lyase – P450 oxidoreductase  – 17β-hydroxysteroid dehydrogenase type 3 – 5α-reductase 2 2. Disorders of androgen action:  – androgen insensitivity syndrome  – drugs and environmental modulators

B: 45,X (Turner syndrome and variants)

46,XY DSD

A: Disorders of gonadal (testis) development 1. Complete or partial gonadal dysgenesis (e.g. SRY, SOX9, SF1, WT1, DHH) 2. Ovotesticular DSD 3. Testis regression

A: 47,XXY (Klinefelter syndrome and variants)

Syndromic associations (e.g. cloacal anomalies) Müllerian agenesis/hypoplasia (e.g. MURCS) Uterine abnormalities (e.g. MODY5) Vaginal atresia (e.g. McKusick–Kaufman syndrome) 5. Labial adhesions

1. 2. 3. 4.

C: Other

B: Androgen excess 1. Fetal:  – 3β-hydroxysteroid dehydrogenase type 2 – 21-hydroxylase  – P450 oxidoreductase  – 11β-hydroxylase – glucocorticoid receptor mutations 2. Fetoplacental:  – aromatase deficiency  – oxidoreductase deficiency 3. Maternal:  – maternal virilising tumours (e.g. luteomas)  – androgenic drugs

A: Disorders of gonadal (ovary) development 1. Gonadal dysgenesis 2. Ovotesticular DSD 3. Testicular DSD (e.g. SRY+, dup SOX9, RSPO1)

46,XX DSD

A classification system for disorders of sex development; modified with permission from Hughes et al.1 (© 2006 BMJ Publishing Group Ltd and the Royal College of Paediatrics and Child Health)

Sex chromosome DSD

Table 2.2

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DISORDERS OF SEX DEVELOPMENT  |  23

were not previously considered ‘intersex’ conditions. In addition, the androgenisation of 46,XX individuals with congenital adrenal hyperplasia (CAH) reflects an adrenal disease with reproductive consequences, rather than a true DSD. Finally, more frequently diagnosed conditions such as hypospadias or undescended testes may not represent a classic DSD and including such individuals in this global diagnosis may not be appropriate. Nevertheless, the group of conditions outlined in this classification do share some diagnostic, surgical or reproductive common ground and a subset of boys with hypospadias/undescended testes may have conditions at the milder end of the DSD spectrum. It is also worth noting that a large number (more than 100) of genetic syndromes have genital or reproductive anomalies listed as part of the phenotype. In most cases, this reflects a more frequent than expected occurrence of undescended testes, micropenis or hypospadias in boys. However, complete or partial testicular dysgenesis, or genital ambiguity can occur in some cases and several well-defined syndromic associations of DSD are described (for example, SOX9/campomelic dysplasia and ARX/X-linked lissencephaly; see Table 2.3). Furthermore, several syndromes affect female development, causing apparent clitoromegaly or anomalies in ovarian, uterine or vaginal development (Table 2.2). Using the classification system, the initial determination of karyotype together with clinical features can lead into a series of biochemical, endocrine, radiological and surgical investigations that can help determine the likely diagnostic category for the child’s or adult’s condition. Further detailed genetic analysis can then be used to reach a specific diagnosis at the molecular level. In certain well-established conditions, where the diagnosis is likely from the biochemical data (for example, disorders of steroidogenesis),20 genetic analysis can be useful to confirm the diagnosis at the molecular level and can help to counsel family members or potentially for guiding prenatal treatment. The likelihood of finding a specific genetic defect in such cases is relatively high (above 80%). In other forms of DSD, such as testicular dysgenesis without associated features or partial androgen insensitivity syndrome (PAIS), there may be few clues as to the exact cause of the condition and a more systematic approach involving analysis of several known candidate genes may be needed. Even in such cases, the current rate of identifying a specific disease-causing change may be of the order of only 20–30%. The remainder of this section briefly discusses some key issues from the three main types of DSD as shown in Table  2.2, with a focus on reproductive genetics. More detailed information of these conditions and their management can be found elsewhere.3 Sex chromosome DSD Sex chromosome DSD includes all cases of sex chromosome aneuploidy. As discussed above, monosomy  X and its variants (Turner syndrome) and 46,XXY and its variants (Klinefelter syndrome) were not classed as ‘intersex’ conditions previously, although the presence of a Y chromosome fragment in Turner syndrome can cause androgenisation and an increased gonadal tumour risk,21 and new data suggest that the prevalence of hypospadias is higher in Klinefelter syndrome than in the general population, especially when multiple copies of the X chromosome are present (for example, 48,XXXY and 49,XXXXY).22 Furthermore, fertility issues related to DSD may be relevant to some of these cases of sex chromosome aneuploidy, so the MDT can provide valuable input in some cases.

Gene

Uterus Adrenal Associated features present defect

↑ pregnenolone, progesterone, 11-deoxycorticosterone, ↓ 17-hydroxylated steroids (except in isolated 17,20-lyase deficiency) and adrenal/gonadal androgens, ↑ LH

↑ Δ5 : Δ4 ratio +/− mineralocorticoid insufficiency

Impaired production of all steroids

↑ LH, poor androgen response to hCG Impaired production of all steroids

↑ 7-dehydrocholesterol

− Androgen biosynthetic defect too − − − − − − − − −

Proteinuria

−

Diagnostic biochemical features

Some recognised single-gene or chromosomal defects found in disorders of sex development, with associated phenotypic and biochemical features; modified with permission from Achermann et al.46 (© 2002 The Endocrine Society)

45,X/46,XY mosaicism Mixed gonadal dysgenesis − +/− − +/− Turner syndrome-like features 46,XY DSD – disorders of testis development (gonadal dysgenesis) WAGR, Denys–Drash and WT1 +/− − Wilms’ tumour, renal abnormalities, gonadal tumours Frasier syndromes CBX2 CBX2 + − − Steroidogenic factor 1 NR5A1 +/− +/− +/− partial hypogonadotrophic hypogonadism SRY SRY +/− − − Campomelic dysplasia SOX9 SOX9 +/− − Desert hedgehog DHH + − +/− Minifascicular neuropathy X-linked lissencephaly ARX − − Lissencephaly, epilepsy SIDDT syndrome TSPYL1 − − Sudden infant death 9p24.3 deletion DMRT1 +/− − Developmental delay Xq13.3 deletion ATRX − − α-thalassaemia, developmental delay Xp21 duplication DAX1 +/− − − 1q35 duplication WNT4a +/− − − 46,XY DSD – disorders of androgen synthesis and action Smith–Lemli–Opitz DHCR7 − +/− Coarse facies, second-third toe syndactyly, developmental delay, syndrome cardiac and visceral abnormalities LH resistance LHGCR − − Leydig cell hypoplasia Congenital lipoid adrenal Lipid-filled adrenals, pubertal failure (46,XY), anovulation (46,XX) StAR − + hyperplasia Cholesterol side-chain CYP11A1 − + Pubertal failure cleavage deficiency + Pubertal failure 3β-hydroxysteroid dehydro- HSD3B2 − genase deficiency type II CYP17 − + Hypertension due to ↑ 11-deoxycorticosterone (except in isolated 17α-hydroxylase/17,2017,20-lyase deficiency), pubertal failure lyase deficiency

Condition

Table 2.3

24  |  LIN LIN AND JOHN C ACHERMANN

Gene

+

+

+

POR

CYP19

GRα

P450 oxidoreductase deficiency Aromatase insufficiency

Glucocorticoid resistance

Mild, partial androgenisation due to ↑ conversion of DHEA

↑ ACTH, ↑ 17-hydroxyprogesterone, +/− mineralocorticoid insufficiency Hypertension due to ↑ 11-deoxycorticosterone but often normotensive ↑ ACTH, ↑ 11-deoxycorticosterone, ↑ 11-deoxycortisol or even mild salt loss in early life, premature virilisation +/− Antley–Bixler craniosynostosis Mixed features of 21-hydroxylase deficiency, 17α-hydroxylase/17,20-lyase deficiency, salt loss rare Maternal androgenisation during pregnancy, absent breast ↑ A4, testosterone, ↓ estrogens, ↑ FSH/LH development at puberty except in partial cases, polycystic ovaries, delayed bone age Hypertension ↑ ACTH, 17-hydroxyprogesterone, cortisol, mineralocorticoids and androgens, failure of dexamethasone suppression (NB a patient with ambiguous genitalia was heterozygous for a mutation in CYP21 too)

+

+

−

−

+

+

− − Palmarplantar hyperkeratosis, squamous cell carcinoma

− − −

+ = present; − = absent; ↑ = increasing levels of; ↓ = decreasing levels of; ACTH = adrenocorticotrophin; DHEA = dehydroepiandrosterone; DHT = dihydrotestosterone; FSH = follicle-stimulating hormone; hCG = human chorionic gonadotrophin; LH = luteinising hormone; SIDDT = sudden infant death with dysgenesis of the testes a This region also contains RSPO1

CYP11B1 +

11β-hydroxylase deficiency

↑ ACTH, ↑ ∆5 : ∆4 ratio, +/− mineralocorticoid insufficiency

−

−

Premature virilisation

hCG-responsive ↑ androgens hCG-responsive ↑ androgens hCG-responsive ↑ androgens

Partial androgenisation at puberty

−

↑ testosterone : DHT ratio (often > 20, may need hCG stimulation) Variable ↑ testosterone and LH/FSH, ↑ AMH

Partial androgenisation at puberty

Mixed features of 21-hydroxylase deficiency, 17α-hydroxylase/17,20-lyase deficiency, salt loss rare ↓ testosterone : androstenedione ratio (< 0.6)

Diagnostic biochemical features

−

Uterus Adrenal Associated features present defect − + +/− Antley–Bixler craniosynostosis

POR P450 oxidoreductase deficiency HSD17B3 − 17β-hydroxysteroid dehydrogenase deficiency type III SRD5A2 − 5α-reductase deficiency type II Androgen receptor AR − 46,XX DSD – disorders of ovary development SRY translocation SRY +/− SOX9 duplication SOX9 +/− Palmarplantar hyperkeratosis RSPO1 +/− XX males (testicular DSD) 46,XX DSD – androgen excess HSD3B2 + 3β-hydroxysteroid dehydrogenase deficiency type II 21-hydroxylase deficiency CYP21A2 +

Condition

DISORDERS OF SEX DEVELOPMENT  |  25

26  |  LIN LIN AND JOHN C ACHERMANN

The two most likely forms of sex chromosome DSD to present with ambiguous genitalia are 45,X/46,XY mosaicism (‘mixed gonadal dysgenesis’) and a 46,XX/46,XY karyotype, which can result from chimerism or various rare mosaicism events. Individuals with 45,X/46,XY mosaicism often present to a paediatric endocrinologist or urologist with ambiguous genitalia, with or without Turner syndrome-like features.23 A wide spectrum of genital phenotypes, uterine structures and degrees of gonadal development and position can occur. If clitoromegaly is present and the gonads are intra-abdominal, a female sex assignment is usually recommended and gonadectomy performed because of the sizeable risk of malignancy. If hypospadias is present and the testes can be brought down into the scrotum, a male sex assignment is usually recommended, and the testes biopsied in adolescence for carcinoma in situ. Babies with very ambiguous genitalia and variable gonadal development are challenging to manage and, as with most cases of DSD, should be considered on an individual basis. Moreover, it is well established that the majority of individuals with a 45,X/46,XY karyotype develop as phenotypic boys and this chromosomal pattern is often only detected during pre- or postnatal karyotype analysis for other reasons.24 The long-term fertility, endocrine function and tumour risk of this cohort of individuals is not known. A 46,XX/46,XY karyotype is very rare and can result from several different mechanisms such as true chimerism or mosaicism (for example, following polar body fertilisation or a post-zygotic event). If ovarian tissue containing follicles and testicular tissue both develop in the same individual, the condition is termed ‘ovotesticular DSD’ (formerly ‘true hermaphroditism’). However, many cases of ovotesticular DSD have a 46,XY or 46,XX karyotype.25 46,XY DSD Underandrogenisation of the developing 46,XY fetus can result from defects in testis development (gonadal dysgenesis) or disorders of androgen synthesis and action (Table 2.2).3,26 Gonadal (testicular) dysgenesis

Testicular dysgenesis can be complete or partial. Complete gonadal dysgenesis results in a complete lack of androgenisation and a female phenotype. Müllerian structures (uterus, fallopian tubes and upper vagina) are usually present because of impaired secretion of AMH during early development. The gonads are usually located intraabdominally and are streak structures, although in some cases ovarian-like tissue may actually develop. Partial gonadal dysgenesis can be associated with varying degrees of underandrogenisation and ambiguous genitalia. Often, sufficient AMH is released during early development for Müllerian regression to occur. Residual gonadal tissue is usually detected although this is sometimes intra-abdominal or non-descended. Several single-gene disorders or chromosomal changes have been described in association with gonadal dysgenesis (Table  2.3). The most frequent causes are mutations or deletions in SRY or alterations in NR5A1 (steroidogenic factor 1).5,27 Other rare causes, such as SOX9 mutations, are usually associated with additional phenotypes such as campomelic dysplasia, and variations in WT1 can be found in Frasier syndrome or Denys–Drash syndrome together with variable renal abnormalities (Table 2.3). Tumour risk in these conditions tends to be high.21 Despite these advances, a specific genetic cause of gonadal dysgenesis is currently found in only about 20–30% of cases.

DISORDERS OF SEX DEVELOPMENT  |  27 Disorders of androgen synthesis and action

Defects in any of the receptors or enzymes involved in the pathways of androgen biosynthesis or action can result in underandrogenisation of the developing 46,XY fetus (Figure  2.1; Table  2.2; Table  2.3).3,26 Complete dysfunction of these specific receptors or enzymes results in complete underandrogenisation and a female phenotype. As this is primarily a defect in Leydig cell function or androgen action, AMH release by Sertoli cells will be unaltered and Müllerian structures will regress. Partial defects in these factors can result in a spectrum of phenotypes ranging from ambiguous genitalia through to hypospadias or even micropenis and varying degrees of testicular maldescent. High blocks in steroidogenesis affect enzymes involved in adrenal as well as Leydig cell function (for example, StAR, CYP11A1, HSD3B2) (Figure 2.1). Individuals with abnormalities in these genes are at risk of salt-losing adrenal failure and cortisol deficiency, which can be life threatening. Although these conditions are rare, it is important to realise that adrenal dysfunction due to forms of CAH can affect 46,XY individuals too. In contrast, disruption of CYP17A1 causes combined 17α-hydroxylase/17,20lyase deficiency, which results in underandrogenisation together with salt retention and hypertension. Affected individuals usually present in adolescence as phenotypic girls (46,XY) who fail to enter puberty. The associated hypokalaemia can result in lifethreatening cardiac arrhythmias if undiagnosed and untreated. P450 oxidoreductase deficiency can cause underandrogenisation owing to altered 17α-hydroxylase activity. These children also have evidence of 21-hydroxylase deficiency and often have craniosynostosis and bony abnormalities as part of Antley–Bixler syndrome. Androgen biosynthetic defects that involve enzymes not involved in adrenal steroidogenesis are deficiencies in 17β-hydroxysteroid dehydrogenase deficiency type  3 and 5α-reductase deficiency type  2 (Figure  2.1). Individuals with these conditions tend to have minimal signs of androgenisation in infancy and childhood but develop phallic enlargement and androgenisation at puberty owing largely to the peripheral conversion of increasing steroid precursors to active androgen metabolites by isoforms of these enzymes at other sites. Finally, defects in the androgen receptor result in androgen resistance and CAIS. Individuals with CAIS sometimes present in childhood owing to bilateral inguinal hernias or more often in late adolescence because of primary amenorrhoea and limited pubic hair despite normal breast development. CAIS affects approximately one in 40 000 and is an X-linked condition. PAIS results in partial androgenisation and ambiguous genitalia. A specific alteration in the androgen receptor is found in only 20–30% of cases of PAIS, suggesting that several other causes result in a similar biochemical and physical phenotype (for example, alterations in NR5A1, which can occur in sex-limited dominant fashion and which can mimic an X-linked condition).27 46,XX DSD Androgenisation of the 46,XX fetus is usually due to adrenal androgen excess (CAH), although, in rare cases, ovarian development defects or other sources of androgenic steroids may cause this (Table 2.2). Ovarian development defects

In rare situations, the ovary may develop as an ovotestis (‘ovotesticular DSD’, formerly ‘true hermaphrodite’) or as a testis (‘testicular DSD’, formerly ‘XX males’). Ovotesticular DSD with a 46,XX karyotype usually presents with ambiguous genitalia but, if mild or overlooked, can present with increasing androgenisation at

28  |  LIN LIN AND JOHN C ACHERMANN

puberty. This clinical scenario most commonly occurs in the sub-Saharan African population.25 The genetic cause of this is unknown, although mild defects in RSPO1 can also cause this phenotype. Testicular DSD results in a normal male phenotype and morphologically and endocrinologically normal testes in a 46,XX individual. Most cases result from translocation of the entire SRY gene, although complete loss of RSPO1/R-spondin1 can also cause this to occur.5,11 These individuals are infertile because genes on the Y chromosome necessary for spermatogenesis are not present. Androgen excess

The most common cause of androgen excess is CAH due to 21-hydroxylase deficiency (Figure 2.1). The most severe ‘salt-wasting’ or ‘simple virilising’ forms of this condition affect about one in 13 000 individuals, who can present with ambiguous genitalia at birth or premature hair development in childhood.28 The milder ‘non-classic’ form of 21-hydroxylase deficiency is more prevalent and usually presents with hirsuitism and polycystic ovaries in later childhood or adolescence.29 The diagnosis of classic forms of 21-hydroxylase deficiency is usually straightforward based on basal and/or stimulated levels of 17-hydroxyprogesterone (17-OHP), although 17-OHP can be mildly elevated in other steroidogenic defects too (for example, 3β-hydroxysteroid dehydrogenase deficiency, 11β-hydroxylase deficiency and P450 oxidoreductase deficiency). In many countries, analysis of 17-OHP is incorporated into the newborn screening programme to detect phenotypic boys (46,XY) with this condition before they present in a salt-losing crisis. Treatment in the newborn period includes glucocorticoid and mineralocorticoid replacement and salt supplementation. Genetic analysis often focuses on the most frequent eight to ten changes in the CYP21A2 gene, which account for 80–90% of cases of 21-hydroxylase deficiency.30 More detailed analysis is available but can be influenced by the presence of a closely related pseudogene. Other conditions that can cause excess adrenal androgen exposure in utero include 3β-hydroxysteroid dehydrogenase deficiency, 11β-hydroxylase deficiency and P450 oxidoreductase deficiency (Figure  2.1). These conditions can present with a range of phenotypes from relatively mild clitoromegaly (3β-hydroxysteroid dehydrogenase deficiency) through to a normal-appearing male with no palpable testes (11β-hydroxylase deficiency). Aromatase deficiency can also cause excessive androgen exposure owing to a lack of the breakdown of fetal adrenal androgens by placental aromatase. Mothers carrying affected fetuses tend to show virilisation in the later stages of pregnancy, which resolves when the baby is born.31

New challenges and opportunities in reproductive genetics and DSD Prenatal karyotyping/genotyping and CAH treatment In the past 20 years, much attention has focused on the potential benefits of prenatal treatment of 21-hydroxylase deficiency by administering dexamethasone to the mother to prevent or reduce androgenisation of an affected female baby’s genitalia and avoid the need for surgery.32,33 Treatment is more effective if started early (before 7–8  weeks of gestation) as this is the time that the fetus’s hypothalamic–pituitary– adrenal (HPA) axis develops and when the genitalia are sensitive to androgen exposure (Figure 2.2). Although this approach has shown good results in many cases,32,34 there have been concerns from some limited follow-up studies about the long-term effects of early steroid exposure on verbal memory and social anxiety,35 as well as data from primate studies on long-term adrenal and cardiometabolic effects.36 However, many of these psychological and behavioural effects are difficult to interpret when studies

DISORDERS OF SEX DEVELOPMENT  |  29

are performed on small numbers and child-rated concerns about social anxiety have not been reproduced when parent-rated questionnaires were used.37 It has been very important to address concerns about prenatal dexamethasone treatment as, until recently, the fetal karyotype and genotype could only be obtained following an invasive diagnostic procedure such as chorionic villus sampling so that results were not available until around 12–13 weeks of gestation. Given the fact that three out of four offspring will be non-affected and that boys do not need prenatal treatment, seven out of eight such fetuses would be exposed to dexamethasone unnecessarily. Advances in determining karyotype and genotype earlier in pregnancy could reduce the unnecessary use of dexamethasone considerably,38 although long-term and appropriately controlled outcome studies of affected girls treated with dexamethasone will still be needed. It is also possible that doses of dexamethasone could be reduced during the second trimester as the fetal HPA axis may be less active at this time, although ideally any such interventions would be part of a randomised controlled study.6 Karyotype/phenotype discordance and prenatal counselling An increasing number of cases of DSD are being diagnosed earlier owing to discordance between a karyotype performed in the first trimester (for example, for trisomy analysis) and genital appearance on prenatal ultrasound or at birth. When concerns have been raised before birth, this information can be used positively to counsel and educate the parents appropriately, so that postnatal discussions about diagnosis, management and sex assignment are easier. Conversely, when parents have been told a definitive karyotype or sex based on prenatal genital appearance, dealing with the discordance between the parents’ expectations and the appearance at birth can be very challenging. Early postnatal management The early postnatal management of a child born with ambiguous genitalia or karyotype/phenotype discordance is very important. Parent–infant interactions should not be undermined by undue medicalisation but important medical associations (such as adrenal failure, hypoglycaemia and salt loss) must not be overlooked. Normally, the local paediatric team will contact a specialist centre for advice and to plan transfer for assessment and investigations. Detailed investigations are not usually performed before day 3, during which time an urgent sample for karyotyping should be sent and the clinical situation discussed with the regional cytogenetics laboratory handling the sample. Having a rapid karyotype available is invaluable in reaching an early diagnosis and, it is hoped, allowing appropriate sex assignment. This time of uncertainty is extremely difficult for many parents; decisions should be reached as quickly as possible but without rash decisions being made. As some local hospitals may see a case of ambiguous genitalia only every 2 years it is likely that midwives, obstetricians and paediatricians will not be highly skilled in this area. Improved guidelines and educational tools are being developed (for example, through the UKDSD Taskforce and through an e-learning portal being developed by the European Society for Paediatric Endocrinology and EuroDSD). The multidisciplinary team The importance of the MDT in the management of DSD has been outlined already. The local team also have an important role to play in relaying information about

30  |  LIN LIN AND JOHN C ACHERMANN

their early interactions with the family and should be welcome to attend any MDT discussions. Improved communication between MDTs and local teams is needed and an MDT coordinator or nurse specialist could fulfil this role. It is likely that very complex cases of DSD could be best managed in a limited number of highly specialist centres with experience in the diagnosis and management of rarer forms of these conditions. Adolescent and adult MDTs that are expert in the management of DSD are becoming increasingly important and specialist gynaecological and psychological support is essential. Molecular diagnosis and high-throughput analysis Analysis of most single-gene disorders for DSD is currently only available on a research basis. Clinically approved tests for rarer conditions are not available within the UK and outsourcing to international laboratories is expensive. In many of these rarer conditions, biochemical markers do not lead to a specific diagnosis and the clinical phenotype can be variable, so several candidate genes might need to be considered, which is a slow and costly process. The development of newer highthroughput sequencing technologies or specific resequencing arrays might help with rapid diagnosis. Copy number variants (CNV) or small deletions are also well established as causes of DSD (for example, Xp21dup, 10qdel).39 Newer technologies (for example, comparative genomic hybridisation and CNV arrays) or focused approaches (for example, multiplex ligation-dependent probe amplification) should reveal how prevalent these genomic changes are as causes of DSD and whether the clinical phenotypes might differ from those associated with single-gene disorders. Epigenetic influences may also be important.40 Moving research-based analysis into a clinically approved arena will be important if patients and families are to be counselled properly. As with many aspects of high-throughput analysis, bioinformatic bottlenecks will be one of the most important immediate challenges. Long-term outcome Long-term outcome data for DSD are generally of poor quality.1,41 In many cases, no specific diagnosis has been reached. Even more common conditions such as 21-hydroxylase deficiency can be difficult to study long into adulthood as paediatric and adolescent practice changes. It is therefore not known whether nerve-sparing surgeries for clitoral reduction will produce better long-term results than older clitoral surgery techniques, or whether newer approaches to steroid replacement will improve long-term growth, bone mineralisation, weight and wellbeing.42,43 Randomised controlled trials of any new intervention would be the ideal but as these conditions are relatively rare and influenced by many variables, outcome studies would have to be long term, multicentered and suitably powered to detect subtle changes. Nevertheless, greater collaboration across centres could allow large-enough cohorts of children with certain rare conditions to be obtained to address some of these issues. Fertility Advances in reproductive medicine are starting to have an impact on broader categories of DSD. For example, cryopreservation of ovarian tissue has been proposed for some girls or women with Turner mosaicism and micro-TESE (testicular sperm extraction) followed by intracytoplasmic sperm injection has been shown in specialist centres to allow fertility in more than half of all men with non-mosaic Klinefelter

DISORDERS OF SEX DEVELOPMENT  |  31

syndrome.44,45 While these advances are exciting, fertility management in DSD has many challenges such as removal and preservation of reproductive tissue from minors, undue expectations of parents that ex vitro germ cell maturation might be possible in the future and complex bioethical considerations (for example, storage of a testis with germ cells from a 46,XY individual raised female). The Human Tissue Act 2004 has limited progress in this field in the UK. It is clear that application of newer reproductive technologies to DSD, while exciting, produce unique ethical and biological challenges. Acknowledgements John Achermann holds a Wellcome Trust Senior Research Fellowship in Clinical Science (079666). The research leading to this chapter has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement number 201444.

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Hughes IA, Houk C, Ahmed SF, Lee PA. LWPES Consensus Group; ESPE Consensus Group. Consensus statement on management of intersex disorders. Arch Dis Child 2006;91:554–63. Lee PA, Houk CP, Ahmed SF, Hughes IA. Writing Group for the International Intersex Consensus Conference Participants. Consensus statement on management of intersex disorders. International Intersex Consensus Conference. Pediatrics 2006;118:753–7. Achermann JC, Hughes IA. Disorders of sex development. In: Kronenberg HM, Melmed S, Polonsky KS, Larsen PR, editors. Williams Textbook of Endocrinology. 11th ed. Philadelphia: Elsevier; 2007. p. 783–848. Minto CL, Crouch NS, Conway GS, Creighton SM. XY females: revisiting the diagnosis. BJOG 2005;112:1407–10. Harley VR, Clarkson MJ, Argentaro A. The molecular action and regulation of the testisdeterming factors, SRY (sex-determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9]. Endocr Rev 2003;24:466–87. Goto M, Piper Hanley K, Marcos J, Wood PJ, Wright S, Postle AD, et al. In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development. J Clin Invest 2006;116:953–60. Wilhelm D, Koopman P. The making of maleness: towards an integrated view of male sexual development. Nat Rev Genet 2006;7:620–31. Brennan J, Capel B. One tissue, two fates: molecular genetic events that underlie testis versus ovary development. Nat Rev Genet 2004;5:509–21. Nef S, Schaad O, Stallings NR, Cederroth CR, Pitetti JL, Schaer G, et al. Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development. Dev Biol 2005;287:361–7. Beverdam A, Koopman P. Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes. Hum Mol Genet 2006;15:417–31. Parma P, Radi O, Vidal V, Chaboissier MC, Dellambra E, Valentini S, et al. R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat Genet 2006;38:1304–9. Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, et al. Retinoid signaling determines germ cell fate in mice. Science 2006;312:596–600. Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD, Page DC. Retinoic acid regulates sexspecific timing of meiotic initiation in mice. Proc Natl Acad Sci USA 2006;103:2474–9. Johnson J, Canning J, Kaneko T, Pru JK, Tilly JL. Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 2004;428:145–50.

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Telfer EE, Gosden RG, Byskov AG, Spears N, Albertini D, Andersen CY, et al. Editorial: on regenerating the ovary and generating controversy. Cell 2005;122:821–2. Boas M, Boisen KA, Virtanen HE, Kaleva M, Suomi AM, Schmidt IM, et al. Postnatal penile length and growth rate correlate to serum testosterone levels: a longitudinal study of 1962 normal boys. Eur J Endocrinol 2006;154:125–9. Lee MM, Misra M, Donahoe PK, MacLaughlin DT. MIS/AMH in the assessment of cryptorchidism and intersex conditions. Mol Cell Endocrinol 2003;211:91–8. Dreger AD, Chase C, Sousa A, Gruppuso PA, Frader J. Changing the nomenclature/taxonomy for intersex: a scientific and clinical rationale. J Pediatr Endocrinol Metab 2005;18:735–8. Vilain E, Achermann JC, Eugster EA, Harley VR, Morel Y, Wilson JD, et al. We used to call them hermaphrodites. Genet Med 2007;9:65–6. Payne AH, Hales DB. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev 2004;25:947–70. Cools M, Drop SL, Wolffenbuttel KP, Oosterhuis JW, Looijenga LH. Germ cell tumors in the intersex gonad: old paths, new directions, moving frontiers. Endocr Rev 2006;27:468–84. Lee YS, Cheng AW, Ahmed SF, Shaw NJ, Hughes IA. Genital anomalies in Klinefelter’s syndrome. Horm Res 2007;68:150–5. Telvi L, Lebbar A, Del Pino O, Barbet JP, Chaussain JL. 45,X/46,XY mosaicism: report of 27 cases. Pediatrics 1999;104:304–8. Chang HJ, Clark RD, Bachman H. The phenotype of 45,X/46,XY mosaicism: an analysis of 92 prenatally diagnosed cases. Am J Hum Genet 1990;46:156–67. Krob G, Braun A, Kuhnle U. True hermaphroditism: geographical distribution, clinical findings, chromosomes and gonadal histology. Eur J Pediatr 1994;153:2–10. Mendonca BB, Domenice S, Arnhold IJ, Costa EM. 46,XY disorders of sex development (DSD). Clin Endocrinol 2009;70:173–87. Lin L, Philibert P, Ferraz-de-Souza B, Kelberman D, Homfray T, Albanese A, et al. Heterozygous missense mutations in steroidogenic factor 1 (SF1/Ad4BP, NR5A1) are associated with 46,XY disorders of sex development with normal adrenal function. J Clin Endocrinol Metab 2007;92:991–9. Speiser PW, White PC. Congenital adrenal hyperplasia. N Engl J Med 2003;349:776–88. New MI. Extensive clinical experience: nonclassic 21-hydroxylase deficiency. J Clin Endocrinol Metab 2006;91:4205–14. Forest MG, Tardy V, Nicolino M, David M, Morel Y. 21-hydroxylase deficiency: an exemplary model of the contribution of molecular biology in the understanding and management of the disease. Ann Endocrinol 2005;66:225–32. Lin L, Ercan O, Raza J, Burren CP, Creighton SM, Auchus RJ, et al. Variable phenotypes associated with aromatase (CYP19) insufficiency in humans. J Clin Endocrinol Metab 2007;92:982–90. Nimkarn S, New MI. Prenatal diagnosis and treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Mol Cell Endocrinol 2009;300:192–6. New MI, Carlson A, Obeid J, Marshall I, Cabrera MS, Goseco A, et al. Prenatal diagnosis for congenital adrenal hyperplasia in 532 pregnancies. J Clin Endocrinol Metab 2001;86:5651–7. Meyer-Bahlburg HF, Dolezal C, Baker SW, Carlson AD, Obeid JS, New MI. Cognitive and motor development of children with and without congenital adrenal hyperplasia after earlyprenatal dexamethasone. J Clin Endocrinol Metab 2004;89:610–14. Hirvikoski T, Nordenström A, Lindholm T, Lindblad F, Ritzén EM, Wedell A, et al. Cognitive functions in children at risk for congenital adrenal hyperplasia treated prenatally with dexamethasone. J Clin Endocrinol Metab 2007;92:542–8. de Vries A, Holmes MC, Heijnis A, Seier JV, Heerden J, Louw J, et al. Prenatal dexamethasone exposure induces changes in nonhuman primate offspring cardiometabolic and hypothalamic– pituitary–adrenal axis function. J Clin Invest 2007;117:1058–67. Hirvikoski T, Nordenström A, Lindholm T, Lindblad F, Ritzén EM, Lajic S. Long-term follow-up of prenatally treated children at risk for congenital adrenal hyperplasia: does dexamethasone cause behavioural problems? Eur J Endocrinol 2008;159:309–16.

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Avent ND, Chitty LS. Non-invasive diagnosis of fetal sex; utilisation of free fetal DNA in maternal plasma and ultrasound. Prenat Diagn 2006;26:598–603. Barbaro M, Cicognani A, Balsamo A, Lofgren A, Baldazzi L, Wedell A, et al. Gene dosage imbalances in patients with 46,XY gonadal DSD detected by an in-house-designed synthetic probe set for multiplex ligation-dependent probe amplification analysis. Clin Genet 2008;73:453–64. Mendonca BB, Billerbeck AE, de Zegher F. Nongenetic male pseudohermaphroditism and reduced prenatal growth. N Engl J Med 2001;345:1135. Warne GL. Long-term outcome of disorders of sex development. Sex Dev 2008;2:268–77. Minto CL, Liao LM, Woodhouse CR, Ransley PG, Creighton SM. The effect of clitoral surgery on sexual outcome in individuals who have intersex conditions with ambiguous genitalia: a crosssectional study. Lancet 2003;36:1252–7. Falhammar H, Filipsson H, Holmdahl G, Janson PO, Nordenskjöld A, Hagenfeldt K, et al. Metabolic profile and body composition in adult women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 2007;92:110–16. Bondy CA, Turner Syndrome Study Group. Care of girls and women with Turner syndrome: A guideline of the Turner Syndrome Study Group. J Clin Endocrinol Metab 2007;92:10–25. Schiff JD, Palermo GD, Veeck LL, Goldstein M, Rosenwaks Z, Schlegel PN. Success of testicular sperm extraction and intracytoplasmic sperm injection in men with Klinefelter syndrome. J Clin Endocrinol Metab 2005;90:6263–7. Achermann JC, Ozisik G, Meeks JJ, Jameson JL. Genetic causes of human reproductive disease. J Clin Endocrinol Metab 2002;87:2447–54.

3 Chapter 3

Preimplantation genetic diagnosis: current practice and future possibilities Alison Lashwood and Tarek El-Toukhy

History of preimplantation genetic diagnosis Preimplantation genetic diagnosis (PGD) has developed dynamically over the past 19 years. It was first introduced for sexing embryos in the case of X-linked genetic disorders in 1990.1 In 1992, the first case of a live birth after successful PGD for the single-gene disorder cystic fibrosis was reported.2 PGD for single-gene disorders moved on a stage further in 1995 when a centre in Belgium reported the successful outcome of a pregnancy following PGD for Duchenne muscular dystrophy by detecting the dystrophin gene deletion.3 This resulted in a successful non-carrier female pregnancy. Since then, the worldwide development of PGD has expanded to offer testing for over 200 single-gene and chromosomal disorders.4,5 The technique has also diversified to include preimplantation genetic screening (PGS), which has been employed to improve in vitro fertilisation (IVF) outcome in subfertile couples and embryo testing for human leucocyte antigen (HLA) matching for sick siblings. Future developments will possibly include comparative genomic hybridisation (CGH) microarray technology that would enable testing for full chromosome aneuploidy and single-gene disorders from a single embryo biopsy.6,7 These new applications of the technology have been and remain controversial and will be discussed further below. For the purposes of this chapter, it is important to first define the various forms of treatment available and the terminology used within the context of this chapter: n preimplantation genetic diagnosis is the testing of embryonic material (polar body, blastomere or blastocyst biopsy) for a single-gene or chromosomal abnormality that is recognised as a known risk for the couple n preimplantation genetic screening involves testing embryonic material for the presence of aneuploidy where there is no underlying risk of an inherited abnormality. Internationally, the term PGD is used to describe both forms of testing.

PGD as a clinical service PGD is a complex process comprising many stages and involving a multidisciplinary team of healthcare professionals. Centres offering such treatment develop and deliver © Alison Lashwood and Tarek El-Toukhy. Volume compilation © RCOG

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a comprehensive service to patients including patient support before, during and after a treatment cycle. Practice varies widely, although it has been acknowledged that PGD should involve the medical expertise of both assisted reproduction and clinical genetics departments.8 Recommendations have been made by the European Society of Human Reproduction and Embryology (ESHRE) Consortium9 and the Preimplantation Genetic Diagnosis International Society (PGDIS)5 for all PGD services to adhere to guidelines that promote best practice. Within these guidelines, acceptable practice requires that counselling offered to couples requesting PGD is provided in a non-directive manner by an appropriately qualified professional. The combined skills of genetic counsellors and clinical geneticists from accredited genetic centres and specialists in assisted reproduction should ensure that patients receive a high-quality service in PGD. It is essential that couples who are referred for PGD have consulted a clinical geneticist in the first instance to ensure that they have the benefit of specialist advice and support relating specifically to the genetic disorder within the family, and have the knowledge to make a fully informed decision before proceeding with PGD. Clinical genetics is a rapidly progressing field and, with advances in the molecular basis of genetic conditions and improved interpretation of some chromosomal rearrangements, it is possible that past genetic information and associated risks may no longer be accurate. For example, the molecular basis of cystic fibrosis and associated congenital bilateral absence of the vas deferens is now better understood, and some men who were originally believed to be carriers are now considered compound heterozygotes and therefore clinically affected.10 As a result, the genetic risk to the couple may be higher. Likewise, some chromosomal rearrangements initially believed to carry reproductive risks in the form of miscarriage or physical or mental disability, where family members may have considered prenatal testing, may be re-evaluated and thus now represent normal variants without reproductive risk.11 A number of chromosome inversions, such as inv(10)(p11.2q21.2), are now recognised as no longer having an associated reproductive risk.12 Before proceeding with PGD, it is important that couples: n discuss their family history and reason for requesting PGD n understand the genetic risk associated with their specific diagnosis n are aware of the PGD process and the adverse effects of treatment n understand the limitations of testing n know what alternative reproductive options are available n understand the anticipated success rate of PGD per cycle started n have considered the physical, emotional and financial impact of treatment on themselves and their families n have a written summary of the consultation and accompanying patient information leaflets to help understanding and retention of clinical details given.9

Why do people request PGD? Couples requesting PGD do so for a variety of reasons. Many couples will have experienced the loss of a child or a pregnancy and possibly had prenatal diagnosis (PND) that may have ended in the termination of an affected pregnancy. Most couples cite the issue of termination as their main reason for wanting to avoid a spontaneous pregnancy.13,14 Past studies have shown that termination of pregnancy

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for fetal abnormality or ‘genetic termination’ carries with it potentially serious psychological consequences as it means the loss of a generally wanted and planned pregnancy.15 Most terminations occur in the second trimester (after chorionic villus sampling, nuchal scan or amniocentesis, which are performed towards the end of the first or in the second trimester). In one study of 84 women who had a termination in the second trimester, 20% reported psychological difficulties affecting their general wellbeing 2 years after the termination.16 Other couples may have simply reached the point where they feel they cannot contemplate trying conventional PND again and see PGD as relieving them of the burden of having PND and termination of pregnancy as well as giving them the chance of knowing from the earliest possible time that a pregnancy is unaffected. Snowden and Green17 published a study that recorded attitudes of male and female carriers of recessive disorders to the availability of PGD and other reproductive options. They concluded that the early knowledge of a pregnancy being unaffected, the biological link with both parents and the avoidance of termination of pregnancy were all deemed important advantages of PGD.

The process of PGD PGD enables a couple to conceive a pregnancy that is biologically their own and is unaffected by the genetic condition in the family. Embryos are created using assisted reproductive technology (ART) and are tested for the relevant genetic condition by polar body, blastomere or trophectoderm biopsy.4 The embryonic tissue is then analysed using fluorescence in situ hybridisation (FISH), polymerase chain reaction (PCR) or CGH to determine affected and unaffected embryos. Any embryos identified as unaffected can be used for uterine transfer with the hope that implantation will occur and any continuing pregnancy will therefore be unaffected by the relevant genetic disorder. Unaffected good-quality embryos that are excess to those used in any one transfer are stored for future use. Preparation for PGD Preparing a couple for a PGD cycle can take several months and couples need to be aware of this so that they have realistic expectations of when they may start treatment. Before starting a PGD cycle, the PGD centre will have ensured that the following preparations have been completed: n FISH or PCR analysis to ensure that a robust test with minimal associated misdiagnosis risk is available n an application for funding for treatment from a local heath authority where relevant n a full general health assessment of both partners including an early follicularphase hormonal profile, a pelvic ultrasound scan (female), semen analysis (male) and virology screening for hepatitis B and C and HIV for both. n in the UK, consent forms must be signed for treatment procedures including oocyte retrieval, fertilisation, embryo biopsy, transfer and storage and research options. Any women who have health-related problems associated with their genetic diagnosis should be referred to an obstetric or other relevant physician to discuss the impact of treatment and pregnancy. For example, women who are affected with myotonic

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dystrophy should be assessed before anaesthesia as they have an increased risk of arrhythmias, prolonged recovery from the anaesthesia and a risk of developing malignant hyperpyrexia.18 Their myotonia often deteriorates during pregnancy and they are prone to obstetric complications including prolonged labour, placenta praevia and postpartum haemorrhage. Women affected by cystic fibrosis may have their lung function compromised during pregnancy.19 Ovarian stimulation Although the method of pituitary downregulation, ovarian stimulation, oocyte collection and embryo transfer will differ from centre to centre, broadly similar protocols exist. There is a clear correlation between the success of PGD treatment and the number of oocytes available,20,21 although care should be exercised to minimise the risk of ovarian hyperstimulation syndrome.22 Controlled ovarian stimulation is performed using a standard long protocol for the majority of women.23 A short protocol can be used for ovarian stimulation if a reduced ovarian reserve is predicted based on previous history or elevated serum follicle-stimulating hormone or estradiol levels.24 Method of oocyte fertilisation Intracytoplasmic sperm injection (ICSI) is the most commonly used method for fertilisation in PGD, reaching 87% of cases in the latest ESHRE PGD Consortium report.4 ICSI is recommended for use in all cases involving PCR analysis as there is a residual risk of contamination from extraneous sperm that penetrate the zona pellucida.25–27 IVF is acceptable for use when FISH analysis is to be used for chromosome rearrangements and embryo sex determination for X-linked disorders.9 Embryo biopsy There are several techniques employed in the biopsy of embryos, with the most common (90% of cases)4 being blastomere biopsy on day 3 after fertilisation (Figure 3.1). Polar body biopsy has been supported mainly by two groups.28–32 It is used in some centres to assess maternal genotype but this is of no value where the autosomal dominant single-gene condition or chromosome rearrangement is paternally inherited. As polar bodies are considered a by-product of embryo development, biopsy of such is considered to be less invasive than blastomere biopsy. Polar body biopsy has an important role to play in countries where embryo biopsy is not permitted (Germany, Switzerland and Austria). Trophectoderm biopsy, which provides a larger tissue sample on day 5 has been attempted but to date there is no convincing evidence that embryo diagnostic rates after trophectoderm biopsy are higher than after blastomere biopsy. This technique is currently used in only a few centres around the world.33,34 Debate continues as to the value of one- compared with two-cell blastomere biopsy, with centres varying in their approach. The arguments are based on concerns about the reliability of results obtained via analysis of one blastomere and whether a single cell is representative of the embryo as a whole. Mosaicism is known to occur in up to 50% of cleavage-stage embryos.35–37 However, there is concern that if centres only use embryos where the two cells biopsied are concordant for a normal result this will result in the exclusion of a significant proportion of embryos owing to higher rates of false positive results. In addition, it is recognised that, although some embryos may initially be mosaic for aneuploid cell lines with increasing cell division, the abnormal cell line is likely to

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(b)

(c)

(d)

Figure 3.1

Cleavage-stage blastomere biopsy: (a) eight-cell cleavage-stage embryo on day 3; (b) holding pipette (left), breaching of zona pellucida using acid tyrose solution; (c) aspiration of blastomere using biopsy pipette (right); (d) single nucleus visible in free blastomere; photographs courtesy of Dr Susan Pickering, Centre for PGD, Guy’s and St Thomas’ Hospital NHS Foundation Trust, London

be selected against and the resulting embryo will be euploid. In FISH-based cases, provided there are at least two fluorescent probes that would detect viable unbalanced forms of the chromosome rearrangement, and in PCR-based cases a linked intragenic marker is available, then one-cell biopsy can be recommended.9 While the reliability of test results is of paramount importance, this must be balanced by ensuring that there are sufficient embryos with transferable results. There is evidence to suggest that two-cell biopsy might reduce the number of embryos available for transfer even though the predictive value of such results would be higher.38 Cohen et al.39 reported that the potential for embryo implantation could be reduced by twocell biopsy. Conversely, Goossens and colleagues40 found no statistically significant difference in the livebirth rate after one- or two-cell biopsies (20.2% versus 17.2%). Embryo transfer Embryo transfer is usually performed between day 4 and day 5 after fertilisation41–43 using transabdominal ultrasound guidance. If a number of unaffected embryos are available for transfer there may be value in waiting until the embryos reach the blastocyst stage as this may give a better indication of the quality of the embryo and increase the chance of successful implantation.44,45 Following oocyte retrieval, a progesterone supplementation regimen will commence for luteal support and a urinary pregnancy test is done on day 16 after oocyte retrieval. Women with a positive test continue with progesterone supplementation until 8 weeks of gestation.

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Number of embryos for transfer and cryopreservation One issue of concern related to ART is that of multiple births and its associated risks.46,47 Attempts to increase the chance of pregnancy in PGD cycles by replacing more than one embryo have led to a high multiple pregnancy rate.4,21,48 Babies born from multiple birth have a higher risk of prematurity, low birth weight, neonatal mortality and neurological disability.49 The issue of the number of embryos for transfer needs careful discussion in cases where there is a choice. A multiple pregnancy may have both clinical and social implications for a couple. Couples requesting PGD often also care for children with disabilities and special needs as a result of the genetic condition within the family and a multiple pregnancy would be a significant additional burden. In addition, as part of recommended best practice, confirmatory prenatal testing is advised following a successful PGD pregnancy9 and, although possible, prenatal testing is more complex in a multiple pregnancy. In a study by Bryan,50 and from the authors’ own experience, it appears that couples may not always fully consider the impact of multiple pregnancies. Their overriding desire for a child may affect their decision in relation to the number of embryos to transfer. The frequency of multiple pregnancies in PGD couples has reduced from 25% to 20% over two time periods, 1999–2003 and 2003–2004.51 In 2009, the ESHRE collective PGD data still reported a multiple birth rate of 27%.4 Some studies in both PGD and ART are now demonstrating improvements in livebirth outcome using single-blastocyst transfer,45,52 and that the use of single-embryo transfer, in particular in women younger than 36 years,53 is resulting in fewer multiple pregnancies without a reduction in the overall delivery rate. El-Toukhy et al.54 have demonstrated that selection of single embryos for transfer, with cryopreservation of surplus unaffected embryos, maintains a good pregnancy rate while reducing multiple births. The implantation rate using cryopreserved biopsied PGD blastocysts was comparable with that obtained after using non-biopsied frozen IVF blastocysts. This is an important step towards encouraging couples to opt, where clinically indicated, for single-embryo transfer which may have additional benefits for PGD couples.

Success of PGD and paediatric outcomes Number of cycles and pregnancy rates There is no overall worldwide data collection of the outcomes of PGD. However, ESHRE collates and publishes data on an annual basis.25–27,,40,48,51,55,56 The 2009 ninth report presents data from 57 centres from Europe, the USA and Australia.4 This series of nine data collections reports on 21 743 cycles with 3841 babies born. The ninth data collection includes a larger number of PGS cycles, which had not been included previously. Data have been collected on the number of cycles performed for singlegene disorders, chromosomal abnormalities, aneuploidy screening, HLA matching and social sex selection. In 2008, the publication of the ESHRE data included for the first time the success rates per participating centre.40 In the 2009 report, the 57 participating centres reported pregnancy rates ranging between 0% and 50% (mean 21%) per oocyte retrieval. That huge range in success rates has raised questions within the ESHRE PGD Consortium Steering Committee over whether those centres with low success rates require investigation and guidance from better-performing services.57 Outcome data for  PGD cycles (including clinical pregnancy rates) published by ESHRE are compared with outcome data from the authors’ centre in Table 3.1.

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Success rates of PGD from the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium4 compared with PGD and ART data from Guy’s and St Thomas’ Hospital NHS Foundation Trust, London Period

Type of Cycles treatment started ESHRE PGD 1999 to October PGD Not 2007 recorded GSTT PGD 1997 to October PGD 695 2008 GSTT ART 2004 to IVF/ICSI 3925 December 2008

Oocyte retrievals 8111

Embryo transfers 5850

Clinical pregnancies

606 (87%)

470 (68%)

166 (OR = 28%; ET = 35%)

3678 (94%)

3345 (85%) 1147 (OR = 31%; ET = 34%)

1542 (OR = 19%; ET =  26%)

ART = assisted reproductive technology; ESHRE = European Society of Human Reproduction and Embryology; ET = embryo transfer; OR = oocyte retrieval; PGD = preimplantation genetic diagnosis; GSTT= Guy’s and St Thomas’ Trust, London

Paediatric outcome Evidence suggests that human embryo development in vitro is not affected by biopsy at the eight-cell stage58 but the authors acknowledge that continuing pregnancies should be closely monitored by ultrasound scanning for evidence of fetal abnormality. Verlinsky et al.59 recommended establishing international collaboration for long-term follow-up of children born as a result of PGD. In the latest ESHRE PGD Consortium report,4 which amalgamates nine data sets representing outcomes of PGD up to 2007, a total of 3841 babies have been born, with outcome data available on 3393. Further in-depth analysis of these data is now being undertaken. However, in the previous 2008 report,40 cumulatively, no major or minor malformations were reported in 95% of cases within this group. The abnormalities that were reported varied in severity and ranged from significant cardiac abnormalities to mild syndactyly. These outcomes are similar to those reported in the IVF/ICSI population.60 Neonatal complications occurred in 9% of cases, with 1% of cases resulting in neonatal death. Several studies have now looked at the longer-term growth and development of children following PGD and PGS. Both Banerjee et al.61 and Desmyttere et al.62 noted that, although PGD/PGS babies were of lower birth weight, their linear growth compared well with that of ICSI and normally conceived children. Furthermore, the PGD/PGS children had the same incidence of congenital abnormality and childhood ill health as the two control groups. Psychomotor development at 2 years of age was also comparable in all groups.63 A further study of social, emotional and language development64 again found no difference in PGD children compared with matched control groups. Lashwood et al.65 reported follow-up data of 120 PGD babies born between 1999 and 2007. Data about health and development were collated at birth and at 1 and 2 years of age. At birth, seven babies had congenital abnormalities (minor and major). By 2 years of age, 12 new abnormalities were reported, leading the authors to conclude that longer-term follow up was necessary.65

Regulation of PGD The regulation of PGD varies widely within the EU member states.66 A European study67 to investigate the use of PGD practice across EU member states found that patients were travelling outside their own member states for treatment because either there were local legal restrictions, treatment was cheaper elsewhere or the PGD technology was not available in the home state. This has implications for continuing

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care of patients and the children born through PGD and there are potential difficulties in undertaking a complex process at a distance from the treatment centre and with the possibility of language barriers. In many countries where PGD is regulated, the same bodies responsible for ART will regulate PGD. ART and PGD in the UK are regulated by the Human Fertilisation and Embryology Authority (HFEA), which was established in 1991 in response to the requirements of the Human Fertilisation and Embryology Act 1990.68 The HFEA’s remit is to monitor all centres offering ART and PGD, to report outcomes of treatment and to issue PGD practice licences and disorder-specific licences for each centre offering the service. In 2001, a joint committee was set up between the HFEA and the Human Genetics Commission to look at issues relating to the clinical application of PGD. The outcome of a public consultation was published and recommendations were made which now determine good practice in centres offering PGD in the UK.69 Centres wishing to set up PGD must apply to the HFEA for a licence to practise PGD and submit details of the genetic conditions for which they are planning to offer treatment. Disorder-specific licences are required before treatment can commence. The HFEA Code of Practice70 requires that the following considerations are made when submitting an application for a treatment licence: n the seriousness of the condition being tested n the couple’s perception of their genetic risk n their previous reproductive history n the welfare of any child born following treatment and the impact of treatment on children already within the family.

Conditions for which PGD is available There are two broad groups of genetic disorders for which PGD is available: singlegene disorders (X-linked and autosomal dominant or recessive inheritance) and chromosomal abnormalities. It should be noted that while some centres offering PGD for X-linked disorders can offer direct mutation testing in the embryos, some will currently only be able to offer embryo sexing with selection of female embryos for transfer. Worldwide, PGD is now available for a wide range of genetic disorders. The PGDIS reports over 150 single-gene conditions for which PGD is available.5 Nevertheless, as PGD is a technically demanding procedure, its application is still limited to fewer conditions than is available through conventional prenatal diagnosis. Table 3.2 is not an exhaustive list of conditions for which PGD is available but it does indicate the conditions for which PGD is most widely available.

Funding In the UK, there is no overall NHS funding for PGD. Many couples are not able to proceed without funding and thus applications for funding are currently made to the couple’s local primary care trusts (PCT). Most couples are unable to self-fund the procedure and rely on funding from their PCTs. In our experience, 90% of the PGD cycles performed between 2005 and 2007 were funded by the PCTs.71 Most applications are dealt with by PCTs’ ‘exceptional treatment arrangements’.21 PGD can only commence after funding has been secured. The process of obtaining funding can be a lengthy one, with no guarantee of a favourable outcome. The mean time taken by PCTs to make a decision about funding was 4 months (range 2–14 months).

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Work is under way to develop an improved system of funding which will mean that applications are dealt with at the Specialist Commissioning Service level.

PGD for single-gene disorders and chromosomal rearrangements Single-gene disorders Those requesting PGD for single-gene disorders will usually be at risk of an autosomal dominant (50% offspring risk), autosomal recessive (25% offspring risk), X-linked dominant (50% risk to male and female offspring) or X-linked recessive (50% risk to male offspring only) disorder. The causative mutation has usually been detected within the relevant gene and thus carrier status has been confirmed. Strategies for setting up single-cell analysis for single-gene disorders employ various techniques. These will vary depending on the mode of inheritance of the disorder (autosomal or X-linked, dominant or recessive) and on the PGD centre involved (their special interest and expertise, organisational setup and funding). The accuracy of any specific test is paramount and PGD assays must minimise the risk of both contamination and allele drop-out (ADO; the failure of one or both alleles to amplify) as either would compromise the reliability of the testing. In most settings, fluorescent PCR (f-PCR) is used to amplify alleles to directly detect mutations. In addition, linked markers comprising single-nucleotide polymorphisms or simple tandem repeats near to or within the gene (intragenic) have enabled a reduction in the misdiagnosis risk by detecting ADO and contamination.72 Both ESHRE and PGDIS recommend this strategy as part of their best practice guidelines.5,9 Mutation detection strategies successfully used for PGD are summarised in Table 3.3. In 2006, a new technique was created as an alternative approach to extend PGD for multiple disorders irrespective of the causative mutation. The technique, termed preimplantation genetic haplotyping (PGH), employs the use of whole-genome amplification to amplify the single-cell DNA a million-fold. Panels of linked markers enable the disease-causing allele to be tracked through the family, avoiding the need for individual mutation detection in a PGD cycle (Figure 3.2).73 PGH is beneficial because: n it removes the need to design assays for individual pathogenic mutations with selected informative markers; one panel of markers can, for example, be used for every case of cystic fibrosis where there are over 1500 different mutations n the markers define the origin of the embryonic DNA and therefore clearly detect contamination.

Table 3.3

Mutation analyses used in PGD

Method of analysis Amplification refractory mutation system for spinal muscular atrophy Minisequencing for multiple single-gene disorders Single-strand conformation polymorphism for five singlegene disorders Temperature gradient electrophoresis for β-thalassaemia Real-time PCR for cystic fibrosis and β-thalassaemia

Reference Moutou et al. (2001)120 Fiorentino et al. (2003)121 Harper et al. (2002)122 Vrettou et al. (1999)123 Pierce et al. (2003),124 Vrettou et al. (2004)125 and Traeger-Synodinos (2006)126

X-linked (if direct testing is unavailable,   PGD by embryo sexing in UK centres) Achondroplasia Adrenoleucodystrophy α-thalassaemia Alport syndrome Adrenal hyperplasia (21β-hydroxylase deficiency) Alport syndrome Amyotrophic lateral sclerosis Androgen insensitivity syndrome (AIS), Alpha-1-antitrypsin deficiency Aniridia (PAX6) complete β-thalassaemia Beals syndrome (congenital contractural Becker muscular dystrophy Bare lymphocyte syndrome arachnodactyly) Duchenne muscular dystrophy Bloom syndrome Choroideraemia BRCA1 Canavan disease Brugada syndrome Emery–Dreifuss muscular dystrophy Congenital disorders of glycosylation type Ia Bullous congenital ichthyosiform erythroderma Congenital nephrosis, Finnish type Fragile X syndrome (BCIE) Haemophilia A and B Cystic fibrosis Charcot–Marie–Tooth disease type 1 Hunter syndrome Epidermolysis bullosa, Herlitz Crouzon syndrome Hydrocephalus, X-linked Familial dysautonomia Darier–White disease Hyper IgM syndrome Fanconi anaemia + HLA typing Ectrodactyly ectodermal dysplasia (EEC) Incontinentia pigmenti Galactosaemia Ehlers–Danlos syndrome type IV Lowe syndrome Gaucher disease Emery–Dreifuss myopathy, LMNA exostoses Menkes syndrome Glutaric aciduria type I multiple type I Mucopolysaccharidosis type II GM1 gangliosidosis Facioscapulohumeral dystrophy Nance–Horan syndrome Haemophagocytic lymphohistiocytosis Familial adenomatous polyposis Norrie disease Hyperinsulinaemic hypoglycaemia Familial amyloid polyneuropathy Ornithine transcarbamylase (OTC) Krabbe disease Glomuvenous malformations deficiency Leigh syndrome Hand-foot-genital syndrome Otopalatodigital syndrome, X-Linked Long-chain hydroxyacyl-CoA dehydrogenase Holt–Oram syndrome (OPD2) deficiency (LCHAD) Huntington disease Spinal and bulbar muscular atrophy Metachromatic leucodystrophy Hypochondroplasia Wiskott–Aldrich syndrome Netherton syndrome type II

Autosomal recessive

Deletion 22q Reciprocal translocations Robertsonian translocations Other chromosomal disorders (inversions, deletions)

Chromosome rearrangements

Conditions for which PGD is available; data from the Preimplantation Genetic Diagnosis International Society (PGDIS) 2008 guidelines5 and the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium data collection nine4

Autosomal dominant

Table 3.2

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Autosomal dominant Hypokalaemic periodic paralysis Kostmann disease Li–Fraumeni syndrome Lynch syndrome Marfan syndrome Metaphyseal chondrodysplasia Multiple endocrine neoplasia 2 (MEN2) Myotonic dystrophy Neurofibromatosis type I Neurofibromatosis type II Osteogenesis imperfecta type 1 Pfeiffer craniosynostosis Polycystic kidney disease, dominant 1 Retinitis pigmentosa Retinoblastoma, hereditary Rh D isoimmunisation Saethre–Chotzen syndrome Spastic paraplegia-4 (SPG4) Spinocerebellar ataxia 1, 2, 3, 7 Spondylometaphyseal dysplasia, Schmidt type Stickler syndrome Treacher Collins syndrome Torsion dystonia Tuberous sclerosis Von Hippel–Lindau syndrome

Autosomal recessive Non-syndromic sensorineural deafness Oculo-cutaneous albinism Osteopetrosis mucopolysaccharidosis Polycystic kidney disease, recessive Pompe disease (glycogen storage disease II) Severe combined immunodeficiency Spinal muscular atrophy Sickle cell disease HbSS and HbSC Smith–Lemli–Opitz syndrome (SLOS) Tay–Sachs disease Zellweger syndrome

X-linked

Chromosome rearrangements PREIMPLANTATION GENETIC DIAGNOSIS: CURRENT PRACTICE AND FUTURE POSSIBILITIES  |  45

(b) (c)

Figure 3.2 Phase of high- and low-risk haplotypes used in PGH cycles: (a) X-linked pedigree; (b) autosomal recessive pedigree; (c) autosomal dominant pedigree

(a)

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PGH may not be useful in situations where the family structure does not allow the determination of marker phase; for example, if relatives are unwilling to provide blood samples or in the case of a de novo autosomal dominant condition where only one family member is affected. Developments in techniques using CGH and microarrays have been described74,75 and in the future may enable concurrent testing of embryos for single-gene disorders and aneuploidy. Chromosome rearrangements and sex selection Chromosome rearrangements It is important to distinguish the difference between PGD and PGS for sporadic chromosome aneuploidy. PGS will be discussed further and is a screening technique usually used in association with treatment for the ART population to try to improve IVF success rates. PGD is offered for known inherited chromosome rearrangements that are carried by the male or female partner; two-thirds of these couples are normally fertile.4 Chromosome rearrangements, including Robertsonian and reciprocal translocations, deletions, duplications, insertions and inversions, can lead to an increased risk of recurrent miscarriage or, less commonly, liveborn children with physical and developmental disabilities.12 Accurate risk assessment associated with the specific chromosome rearrangement is essential so that couples understand the risk of abnormality before they opt for PGD. It is possible that for some fertile couples their best option for having an unaffected child would be to continue trying for a pregnancy themselves while accepting that further miscarriages might occur.76 Both chromosome paints and locus-specific probes have been used previously in PGD for chromosome rearrangements on polar body biopsy.30,31 Locus-specific probes have been used by several groups on cleavage-stage biopsied embryos. However, unless these probes span the rearrangement breakpoints, they cannot distinguish between heterozygous and normal chromosomes.77–80 Commercially available subtelomere and centromere FISH probes can be used to develop a robust and informative test to detect embryos with the unbalanced products of the chromosome rearrangement.78 The combination of probes will depend on the specific chromosome rearrangement, meiotic segregation of products and the risk of giving rise to viable offspring. In accordance with the ESHRE recommendations for best practice, a probe scheme should detect where possible all forms of the unbalanced products unless the unbalanced product has been assessed as being non-viable or of low prevalence.9 Figure 3.3 demonstrates the use of probe schemes for reciprocal and Robertsonian PGD translocation cases. Sex selection The first report of a successful PGD cycle was for X-linked disorders using PCR for Y-chromosome-specific sequences.1 Those who present for PGD are usually carriers of an X-linked recessive condition where male offspring have a 50% risk of being affected or an X-linked dominant condition where females also have a 50% risk of being affected. Sex selection for nonmedical reasons (social sexing or gender balancing) is available in some countries although prohibited in many including the UK in accordance with the Human Fertilisation and Embryology Act 2008. The ESHRE data series nine for sex selection in PGD4 reported 579 oocyte retrievals for social sexing.

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(b)

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(d)

Figure 3.3 Recommended FISH probe schemes for common Robertsonian and reciprocal translocations and deletion 22q11: (a) reciprocal 11/22 translocation: 46,Xn,t(11;22)(q23;q11.2), top – balanced/normal signal pattern, bottom – unbalanced signal pattern; 3:1 tertiary trisomy in blastomere nuclei; (b) Robertsonian 13/14 translocation: 45,Xn,t(13;14)(q10;q10), metaphase spread of balanced product in lymphocyte nucleus; (c) Robertsonian 14/21 translocation: 45,Xn,t(14;21)(q10;q10), metaphase spread of balanced product in lymphocyte nucleus; (d) 22q11 deletion: 46,Xn,del(22)(q11.2q11.2), metaphase spread of balanced product in lymphocyte nucleus; images courtesy of Dr Paul Scriven, Centre for PGD, Guy’s and St Thomas’ Hospital NHS Foundation Trust

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Sex selection alone is inefficient because it excludes the 50% of male embryos that will be unaffected and it cannot detect carrier females in X-linked recessive conditions, which owing to non-random X chromosome inactivation may develop some clinical features. The use of PCR-based assays to detect specific mutations on the X chromosome alongside linked multiplex markers has been reported.30,40 In the absence of a direct test, or where the specific mutation is not known, FISH provides a robust technique81,82 using commercially available centromeric probes for X and Y with an additional control probe on an autosome to determine ploidy. PGH has now improved the diagnosis for such families as the linked markers can distinguish affected from unaffected alleles within the X chromosomes, which means that unaffected male embryos can also be diagnosed and considered for transferral. This means that, on average in X-linked recessive conditions, 75% of the embryos can be considered for embryo transfer.

Preimplantation genetic screening PGS is distinct from PGD and it is mainly used within the ART arena as an adjunct to fertility treatment with the aim of improving IVF outcome. It is recognised that success rates of ART are affected by female age owing to the increased risk of oocyte chromosome aneuploidy. Screening embryos by testing polar bodies, blastomeres from cleavage-stage embryos or blastocysts for common aneuploidies before transfer was introduced to improve the likelihood of successful implantation and a continuing pregnancy by excluding aneuploid embryos that would fail to implant or result in early miscarriage.53,83–85 PGS is controversial and questions remain as to whether there is any clinical benefit.86,87 There is no evidence from randomised trials88–93 of any improvement in the chance of live birth per cycle started. Indeed, in one study there was evidence of harm with a significant reduction in pregnancy rates.89 Meta-analysis of the randomised trials showed it would be unethical to conduct any further cleavage-stage PGS trials for advanced maternal age.94 As a result, a task force convened by ESHRE to reach a consensus about how to move forward with PGS agreed that no further trials should be undertaken using cleavage-stage biopsied embryos. Instead, it is proposed that a randomised controlled trial be started using polar body biopsy and 24 chromosome analysis.95 In the UK, the concerns about PGS and a review of the available evidence led the British Fertility Society to recommend that PGS should only be offered as part of a randomised trial in a recognised experienced centre and that patients should be told that there is no evidence that it works for any of the indications for which it is currently used.96 The American Association for Reproductive Medicine has also issued a practice opinion, which concluded that there was no evidence that PGS worked using current techniques.97

Difficult issues PGD for HLA matching The first case of using PGD to create an HLA-matched embryo to act as a haematopoietic stem cell donor for a sick sibling was reported in 2001.98 This application of PGD is now used for families where there is a sick child affected with one of many immunodeficiencies such as Blackfan–Diamond anaemia.99 Initially introduced for couples with an underlying risk of a genetic disorder such

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as thalassaemia major and requiring stem cell donation for another sick child, it rapidly developed to offer treatment to those requiring stem cell donation with no underlying genetic risk as in the case of childhood leukaemia. The term ‘saviour sibling’ was quickly coined. Critics of this indication for PGD argue that it is unethical to create a child as a means to an end, rather than for the end itself. Indeed, ethical concerns have been raised over the subsequent expectations of that child and the impact should the transplant fail. On the other hand, several authors have argued that the ethical and psychological concerns do not justify prohibition of PGD-HLA.100–103 Whatever the moral or ethical stance taken on this issue, the fact remains that the chance of success for any couple is low, especially when there is also an underlying genetic risk. In such cases, the chance of creating an embryo that is both unaffected and an HLA match is approximately 1 in 6. Given that the average number of embryos created and biopsied per PGD cycle is 6.1,4 the overall chance of success remains low, despite a 62–72% embryo transfer rate reported in two case series of 26 and 18 PGD-HLA cycles.99,104 Late-onset disorders Many centres offering PGD are now able to do so for genetic disorders that may not affect an individual until their adulthood, such as Huntington disease.105 Those who request PGD for Huntington disease have either had a presymptomatic test that indicates that they are a gene carrier106 or are at 50% risk of the disease having decided against gene testing. The latter is a choice often made by those who are uncertain about their ability to cope with knowing they carry the gene.107 Offering PGD for late-onset disorders presents the clinician and the couple with some additional specific considerations where the implications of the welfare of any child born following PGD may be compromised by the health of the parent when they become symptomatic at a future stage. An open discussion with the couple to address issues around the future care of the affected partner and that of any children born as a result of PGD is critical. On the whole, it is generally accepted that as long as one parent is able to offer care to a child then there is no justification for withholding PGD.108 Best practice guidelines5,9 recommend that confirmatory prenatal or cord blood testing be offered in successful PGD cycles. One of the difficulties of such testing at birth in late-onset disorders is that it could result in mutation detection in a child if a misdiagnosis has occurred. Testing children for late-onset disorders, where no intervention prevents or ameliorates the symptoms, is not morally advocated.109–111 While the risk of misdiagnosis is low, this issue requires discussion before the start of treatment. Questions arise as to whether PGD should be used for couples who have not confirmed their genetic status, such as those who are at 50% risk of Huntington disease, and may undertake PGD with its inherent risks when it may not be clinically indicated.72 Non fully penetrant disorders PGD has been used for other late-onset disorders such as breast cancer and Alzheimer’s disease112 and where the pathogenic mutation is not fully penetrant as in the case of a BRCA1 or BRCA2 mutations where carrier status confers a lifetime risk of breast cancer of 60–80% or ovarian cancer of 5–60%.113,114 Once again, this issue has courted

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controversy owing to the late onset of the disease, the fact that the carrier may never be affected or at least will experience several decades of healthy life before developing symptoms and the possibilities for future successful treatment exist. In the UK, the HFEA has granted licences for PGD for BRCA1 on a case-by-case basis.115 Selection for disability Requests for PGD treatment have been made by couples that are deaf or have achondroplasia116 who have asked for the transfer of a similarly affected embryo. For many deaf couples, deafness is seen as the cultural norm and the concern is that hearing children born into such a community will be disadvantaged. While this has been discussed in several forums, the Human Fertilisation and Embryology Act 2008 prohibits positive selection for a genetic disability.117

The future of PGD PGD is advancing rapidly, with future improvements expected in both availability and accuracy. The list of genetic disorders for which PGD is applicable will expand beyond the 200 or so conditions currently amenable to PGD. New indications for PGD such as inherited cancer predisposition, susceptibility to late-onset conditions (such as presenile Alzheimer’s disease) and rhesus incompatibility will no doubt contribute to the wider availability of PGD. The potential use of PGD to select embryos with desirable nonmedical traits such as intelligence, beauty or longevity perhaps represents a more distant future for PGD. Much of the future growth in PGD will also depend on advances in genetic knowledge and analysis techniques. It is hoped that the science of functional genomics will provide sufficient information to improve our understanding of human genetics in health and disease. PGD test accuracy of 100% is possible using newer analysis techniques.118 In addition, microarray or ‘genetic-chip’ technology promises to allow efficient whole-genome analysis using DNA from a single blastomere, thus opening up new possibilities in screening for and diagnosing a vast number of disease alleles and trait polymorphisms in a rapid, highly accurate and cost-effective manner.72,119 Equally important, the scope of future application of PGD will depend on ethical and social acceptability of the new indications for PGD. Ethical and moral controversy surrounding the creation, screening and selection of embryos for new indications of PGD, particularly the nonmedical indications, is likely to intensify. Indeed, the future challenge for PGD will be to regulate its use for an expanding list of medical conditions while preventing or limiting its use in eugenic selection. Acknowledgements Alison Lashwood has drawn upon material used for her chapter ‘Genetic counselling’ in the recently published book Preimplantation Genetic Diagnosis, edited by Joyce Harper and published by Cambridge University Press. The authors would like to thank Dr Paul Scriven and Dr Pamela Renwick, both from The Centre for Preimplantation Genetic Diagnosis, Guy’s and St Thomas’ Hospital NHS Foundation Trust, for their valuable advice and contribution.

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in couples with advanced maternal age: a prospective randomized controlled trial. Hum Reprod 2004;19:2849–58. Mastenbroek S, Twisk M, van Echten-Arends J, Sikkema-Raddatz B, Korevaar JC, Verhoeve HR, et al. In vitro fertiization with preimplantation genetic screening. N Engl J Med 2007;357:9–17. Schoolcraft WB, Katz-Jaffe M, Stevens J, Rawlins M, Munne S. Preimplantation aneuploidy testing for infertile patients of advanced maternal age: a randomized prospective trial. Fertil Steril 2009;92:157–62. Hardarson T, Hanson C, Lundin K, Hillensjö T, Nilsson L, Stevic J, et al. Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial. Hum Reprod 2008;23:2806–12. Jansen RP, Bowman MC, de Boer KA, Leigh DA, Lieberman DB, McArthur SJ. What next for preimplantation genetic screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy. Hum Reprod 2008;23:1476–8. Staessen C, Verpoest W, Donoso P, Haentjens P, van der Elst J, Liebaers I, et al. Preimplantation genetic screening does not improve delivery rate in women under the age of 36 following singleembryo transfer. Hum Reprod 2008;23:2818–25. Mastenbroek S, Scriven P, Twisk M, Viville S, Van der Veen F, Repping S. What next for preimplantation genetic screening? More randomized controlled trials needed? Hum Reprod 2008;23:2626–8. Gerhaedts J. Preimplantation genetic screening:Task Force opts for polar body biopsy and 24-chromosome analysis. Focus on Reproduction January 2009:15 [www.eshre.com/binarydata. aspx?type=doc/FOR_JANUARY2009.pdf]. Anderson RA, Pickering S. The current status of preimplantation genetic screening: British fertility Society Policy and Practice Guidelines. Hum Fertil (Camb) 2008;11:71–5. Practice Committee of the Society for Assisted Reproductive Technology and the Practice Committee of the American Society for Reproductive Medicine. Preimplantation genetic testing: a practice committee opinion. Fertil Steril 2007;88:1497–504. Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A. Preimplantation genetic diagnosis for Fanconi anemia combined with HLA matching. JAMA 2001;285:3130–3. Verlinsky Y, Rechitsky S, Sharapova T, Laziuk K, Barsky I, Verlinsky O, et al. Preimplantation diagnosis for immunodeficiencies. Reprod Biomed Online 2007;14:214–23. Devolder K. Preimplantation HLA typing: having children to save our loved ones. J Med Ethics 2005;31:582–6. Shenfield F, Pennings G, Cohen J, Devroey P, Tarlatzis B; ESHRE Task Force on Ethics and Law. Taskforce 9: the application of preimplantation genetic diagnosis for human leukocyte antigen typing of embryos. Hum Reprod. 2005;20:845–7. Pennings G, Schots R, Liebaers I. Ethical considerations on preimplantation genetic diagnosis for HLA typing to match a future child as a donor of haematopoietic stem cells to a sibling. Hum Reprod 2002;17:534–8. Baetens P, Van de Velde H, Camus M, Pennings G, van Steirteghem A, Devroey P, et al. HLAmatched embryos selected for siblings requiring haematopoietic stem cell transplantation: a psychological perspective. Reprod Biomed Online 2005;10:154–63. Kahraman S, Karlikaya G, Sertyel S, Karadayi H, Findikli N. Clinical aspects of preimplantation genetic diagnosis for single gene disorders combined with HLA typing. Reprod Biomed Online 2004;9:529–32. Online Mendelian Inheritance in Man (OMIM). Huntington Disease; HD. +143100. Baltimore: Johns Hopkins University [www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=143100]. Tyler A, Ball A, Craufurd D. Presymptomatic testing for Huntington’s disease in the UK. BMJ 1992;304:1593–6. Tibben A, Frets PG, van de Kamp JJ, Niermeijer MF, Vegtervan der Vlis M, Roos RA, et al. On attitudes and appreciation 6 months after predictive DNA testing for Huntington’s disease in the Dutch program. Am J Med Genet 1993;43:103–11. Robertson JA. Extending preimplantation genetic diagnosis: the ethical debate. Ethical issues in the new uses of preimplantation genetic diagnosis. Hum Reprod 2003;18:465–71.

58  |  ALISON LASHWOOD AND TAREK EL-TOUKHY 109. Bloch M, Fahy M, Fox S, Hayden MR. Predictive testing for Huntington’s disease. Demographic characteristics, lifestyle patterns, attitudes and psychological assessments of the first fifty one test candidates. Am J Med Genet 1989;32:217–24. 110. Clinical Genetics Society. The Genetic Testing of Children. Report of a Working Party, Dr Angus Clarke (Chairman). March 1994 [www.bshg.org.uk/documents/official_docs/testchil.htm].. 111. British Medical Association. Human Genetics: Choice and Responsibility. Oxford; Oxford University Press; 1998. 112. Verlinsky Y, Rechitsky S, Verlinsky O, Masciangelo C, Lederer K, Kuliev A. Preimplantation diagnosis for early-onset Alzheimer disease caused by V717 L mutation. JAMA 2002;287:1018–21. 113. Ford D, Easton DF, Stratton M, Narod S, Goldgar D, Devilee P, et al. Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium. Am J Hum Genet 1998;62:676–89. 114. Antoniou A, Pharoah PD, Narod S, Risch HA, Eyfjord JE, Hopper JL, et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet 2003;72:1117–30. 115. Human Fertilisation and Embryology Authority. Choices and Boundaries. London: HFEA; 2006 [www.hfea.gov.uk/docs/Choices_and_Boundaries.pdf]. 116. Baruch S, Kaufman D, Hudson KL. Genetic testing of embryos: practices and perspectives of U.S. IVF clinics. Fertil Steril 2008;89:1053–8. 117. Human Fertilisation and Embryology Act 2008. London: HMSO; 2008 [www.opsi.gov.uk/acts/ acts2008/pdf/ukpga_20080022_en.pdf]. 118. Kuliev A, Verlinsky Y. Preimplantation genetic diagnosis: technological advances to improve accuracy and range of applications. Reprod Biomed Online 2008;16:532–8. 119. Salvado CS, Trounson AO, Cram DS. Towards preimplantation diagnosis of cystic fibrosis using microarrays. Reprod Biomed Online 2004; 8:107–14. 120. Moutou C, Gardes N, Rongières C, Ohl J, Bettahar-Lebugle K, Wittemer C, et al. Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy. Prenat Diagn 2001;21:498–503. 121. Fiorentino F, Magli MC, Podini D, Ferraretti AP, Nuccitelli A, Vitale N, et al. The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders. Mol Hum Reprod 2003;9:399–410. 122. Harper JC, Wells D, Piyamongkol W, Abou-Sleiman P, Apessos A, Ioulianos A, et al. Preimplantation genetic diagnosis for single gene disorders: experience with five single gene disorders. Prenat Diagn 2002;22:525–33. 123. Vrettou C, Palmer G, Kanavakis E, Tzetis M, Antoniadi T, Mastrominas M, et al. A widely applicable strategy for single cell genotyping of beta-thalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis. Prenat Diagn 1999;19:1209–16. 124. Pierce KE, Rice JE, Sanchez JA, Wangh LJ. Detection of cystic fibrosis alleles from single cells using molecular beacons and a novel method of asymmetric real-time PCR. Mol Hum Reprod 2003;9:815–20. 125. Vrettou C, Traeger-Synodinos J, Tzetis M, Palmer G, Sofocleous C, Kanavakis E. Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis. Hum Mutat 2004;23:513–21. 126. Traeger-Synodinos J. Real-time PCR for prenatal and preimplantation genetic diagnosis of monogenic diseases. Mol Aspects Med 2006;27:176–91.

4 Chapter 4

Ethical aspects of saviour siblings: procreative reasons and the treatment of children Mark Sheehan

‘Saviour siblings’ are children who are born through in vitro fertilisation (IVF) and are the product of the use of preimplantation genetic diagnosis (PGD) to select embryos that are a tissue match for a sibling who is in need of tissue or organ donation.* Two cases that received a good deal of attention in the UK were the Hashmi and the Whitaker cases. In the former, Zain Hashmi had β-thalassaemia and could be cured using the cord blood of a tissue-matched sibling. In the latter, Charlie Whitaker had Blackfan– Diamond anaemia, which similarly could have been cured by a cord blood donation from a tissue-matched sibling. The relevant difference for the Human Fertilisation and Embryology Authority (HFEA) was that in the Hashmi case the tissue-matched sibling would also be selected so as to avoid having β-thalassaemia. In the Whitaker case, the embryo would have been selected solely as a match for Charlie.1,2 In 2004, after conducting a review of the policy that led to these decisions, the HFEA modified its view. The current position is that preimplantation tissue typing will continue to be considered on a case-by-case basis for serious or life-threatening conditions and as a last-chance treatment. Preimplantation tissue typing ‘may be acceptable in cases in which the embryo to be tested is not itself at risk from the condition affecting the existing child’ and it may be used ‘with a view to using bone marrow from the resulting child.’3,4 Given the regulatory history and current situation in the UK, I assume that there are ethically ‘easy’ cases and ‘hard’ cases of saviour siblings. Cases where the ‘saving’ involves donating cord blood, stem cells or bone marrow are counted as easy, whereas hard cases are those where the required donation is more onerous, say a kidney, part of a liver or part of an intestine. I take it that the severity of these interventions influences the acceptability of the choice to use PGD to select for a saviour sibling.† A great deal has been written on the ethical aspects of saviour siblings, with views largely converging on a similar basic position: easy case saviour siblings are permissible * The term ‘saviour sibling’ might be taken to have something of a sensationalist element to it. In this paper, I will ignore this element and where I do use the term it will be simply as shorthand for the process described above. † This distinction is consistent with there being borderline cases that depend on both the severity of the intervention and other factors.

© Mark Sheehan. Volume compilation © RCOG

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and hard cases are not.1,2,5–12 This chapter will attempt to capture the main thrust of this convergence. In what follows, I consider two questions: n What makes the easy cases justified?’ n What, in justificatory terms, makes the hard cases different from the easy cases? The challenge is to avoid the justification for permitting the easy cases also applying to the hard cases.

Two distinctions Reasons and actual treatment The important distinction to draw here is between the reasons for having a child and the treatment of the resulting child. This distinction is important because we generally seem prepared to countenance more when it comes to the reasons for having a child than we are when it comes to the actual treatment of children. The distinction between the reasons for procreation and the actual treatment of a child is not new in this context.5,7–9 In particular, Pennings7 uses ‘the postnatal test’: ‘It is ethically acceptable to conceive a child for a certain reason if it is acceptable to use an existing child for the same reason.’* Although this test looks circular at first glance, we should find it plausible because it seems to follow from the more general idea that it is wrong to intend to do X when doing X would be wrong. As Pennings7 puts the point, ‘if it is permitted to use an existing child as a bone marrow donor, then how can it be wrong to intend to use it as a bone marrow donor at the time of its conception?’ This distinction provides a useful way to address the differences between easy and hard saviour sibling cases. There is an important asymmetry between the justifying considerations that are relevant to permitting the easy cases and those that are relevant to disallowing the hard cases. This asymmetry is made more salient by separating the reasons for procreation from the treatment of the actual child early in the discussion. Third-party intervention A second distinction to draw at the outset concerns the use of PGD and IVF technologies as opposed to non-technologically assisted reproduction. Importantly, the fact that these technologies involve the assistance of others unlike that required in non-assisted reproduction (or conception, at least), is morally significant. Although people conceive children, privately, in dubious circumstances and for dubious reasons, the costs associated with their doing so are not outweighed by the costs involved in the breach of privacy required to prevent them from behaving irresponsibly.13 The privacy and liberty infringements associated with ensuring that couples reproduce for the right reasons and in the right circumstances are severe enough to outweigh the moral costs of conceiving children for the wrong reasons. In the context of assisted reproductive technology, the same privacy and liberty infringements are not present. As a result, the reasons and context of reproduction are often dominant moral considerations. It may be objected that holding those in need of reproductive assistance to higher moral standards than those who can reproduce privately is to discriminate against those who happen to be unable to reproduce ‘naturally.’ But this is to miss the argument. * ‘Use’ is an unfortunate expression here because it can imply a particular (unintended) relationship to the child. The important point is the treatment of the child in accordance to (or in line with) the reason.

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Broadly, considerations of justice or discrimination do not apply when the behaviour in question is unethical – it cannot be unfair that one is caught doing the wrong thing.*

Reasons and saviour siblings Our focus here is on prospective ‘second’ parents whose explicit reason for having a second child importantly involves reference to their first child. Their relevant reason might be: ‘We want to have a second child who will be able to “save” our first child by having a matched tissue type.’ Normally, prospective parents have many different reasons for wanting children. When reproductive technologies are involved, these reasons are likely to come under more scrutiny and so will be subject to greater sanctions than in the unassisted case. This is partly to do with the issues of ‘third-party involvement’ discussed above but also, given the added hardship, complexity and cost, seems likely to be a result of more reflection on the part of the parents. Prospective parents who do not need reproductive assistance do not need to consider their reasons for becoming a parent, let alone in a reflective manner. As others have suggested, it is important to consider the range of reasons that prospective parents may have for having a child or for having a second child.5,6,10 High on the list here is having a second child to be company for the first child or to satisfy the parents’ conception of a balanced family. The first of these is a milder form of the ‘saviour sibling’ reason while the second is a version of the much discussed ‘family balancing’ reason usually raised in the context of sex selection.14 Neither case requires PGD and nor are they likely, often, to be exclusive or dominant reasons for procreation. More problematic reasons for procreation include having a child ‘who will be the professional athlete that I always wanted to be’ or having children to increase social benefits – Wasserman10 cites Charlie Whitaker’s father as remarking: ‘Teenagers have babies in this country to get a subsidised flat.’ How do we deal with bad reasons? There are two fundamental principles that increasingly govern the bioethical debate in this area and that may well represent the orthodoxy among bioethicists. These two principles, the right to reproductive autonomy and the harm principle, provide a framework for dealing with the reasons that people have for procreating and the kinds of intervention by third parties that are justified. The right to reproductive autonomy can be thought of as related both to the general right to found a family and the right to privacy. In terms of broad moral principles, it is connected to the principle of respect for autonomy. The central idea here is that individuals are understood to have the right to determine when, where and with whom to reproduce.13,15–17 There are a number of ways of understanding this principle. We can point to the distinction between negative and positive rights. The right to reproductive autonomy looks like a negative right where the obligation imposed on others is not to limit the freedom to choose entailed by the right; that is, third parties (society, the state or the * An important qualification on the discussion that follows is that my interest is with the ethical questions rather than the legal or regulatory options. Conclusions about the acceptability (or unacceptability) of the use of PGD for saviour siblings might not imply that such uses should be permitted (or not) in law or through regulation. Despite the issues raised by the involvement of a third party in assisted reproduction cases, there is some distance between there being a moral case for an individual refusing to perform a procedure (and being entitled to do so) and there being regulation to that effect.

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medical profession) should refrain from interfering in the reproductive practices of individuals. This is distinct from the claim that reproductive autonomy is a positive right, where there is an obligation on the part of some particular third party to enable or provide resources for those who are unable to have children. It might be further argued that, while the state should provide facilities for infertile couples to have children, there is no obligation to facilitate couples choosing the kind of child they have. It is unclear that the plausible justification of the negative conception of the right leads to the positive conception and to the provision of a full range of ‘reproductive choice’ services. Being free to reproduce is a long way from an obligation on others to provide the desired kind of child. We need to be very clear about the scope of the right to reproductive freedom. If this right is a negative one or is limited to the provision of fertility treatment, there will be scope for judgement by third parties as to whether to proceed. By itself, the right to reproductive autonomy, narrowly construed, does not allow us to decide between reasons. The generally accepted constraint, captured in the harm principle, is given by Mill18 in On Liberty as: ‘the only purpose for which power can be rightfully exercised over any member of a civilized community, against his will, is to prevent harm to others.’

In the reproductive context, the question becomes whether particular reproductive choices will result in harm to others.* If so, then the right to reproductive autonomy can be overruled. Substantial attention is given to the extent to which parents’ reasons for having a child affect the way in which the child’s life goes. Clearly, if procreative reasons matter for child welfare then we want parents to have good reasons rather than bad ones.2,5–9 The key then is the connection between procreative reasons and harm: the way in which acting on particular reasons will affect the resulting child’s welfare. Here, we need to be careful when comparing harms and ‘harmed lives’ of the resulting children.19–21 We should remember that the particular child is a product of particular choices without which that child would probably not have existed. Instead, we should understand these harms to be risks of harm – where doing something that is more risky is more problematic. What matters are the grounds that we have for thinking that certain reasons are more risky than others for the child’s welfare.2,5,6 The first observation to make in this regard is that prospective parents usually have a large range of variously important reasons for having a child or indeed a second child. These reasons may be unstated (or non-conscious) and only become apparent retrospectively. This complexity makes the task of determining the connection between any particular procreative reason and child welfare very difficult and uncertain – it is clearly hard to determine which reason has what kind of consequence independently of the other variously weighted reasons, and independently of intervening contextual circumstances. A common connection made between procreative reasons and the treatment of the child involves the importance of valuing children for their own sake. In the saviour sibling case, the worry is that the created child is being treated as a mere means or is being exploited or commodified – the child is simply a source of spare parts. It seems reasonably clear that the saviour sibling reason is one that ‘instrumentalises’ the resulting child but it is unclear whether this amounts to viewing the child as a mere means or that there is anything wrong with this degree of instrumentalisation. Because parents’ * Of course, the ‘others’ here can be specific others or society more generally; that is, if society is harmed by allowing particular reproductive choices then this may constitute grounds for limiting those choices. In the saviour sibling context, given the likely numbers involved, it is difficult to see how these sorts of choices will undermine or cause harm to society more generally.

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reasons for having children and ways of valuing them are complex and varied, we cannot conclude from the saviour sibling reason that the child is less likely to be valued for its own sake. Moreover, we only need to look at the range of procreative reasons described earlier to see perfectly usual and acceptable examples of instrumentalisation. Related concerns have been raised about psychological harms that follow from the ‘spare parts’ idea. These include harms related to the child being adversely affected by learning of their saviour sibling status. Furthermore, the child may feel responsible if the transplantation is unsuccessful. In both of these cases, the difficulty lies in attributing the harm to a particular procreative reason rather than another or to other independent circumstances. Even with this link, there is a further question about why these are different to other harms inflicted on children by their families and not condemned by society.* Let us return to the more problematic reasons mentioned above. When Mr Whitaker complained about teenagers having children to collect more social welfare benefits, the implication is that his reason ‘is not as bad as one that society allows’. In response, we might point out that such parents may still care about their ‘social welfare’ children. Although we may think that having children for this reason is problematic, it does not follow that the child will suffer as a result of the parent acting on this reason. The parents may come to know better, they may have a change of heart or they may have always had a range of reasons and attitudes to procreation, only one of which involves exploiting the social welfare system. In any of these cases, other reasons and circumstances mean that the child’s life unfolds in an unproblematic way. Recall that, on the orthodox view, we have grounds for interference only when we have concerns about the welfare of the child; that is, when there is a significant relationship between the procreative reason in question and the likelihood of the harms associated with that reason occurring. Two further thoughts There are various ways of challenging this standard view. Most of these involve making the prospective parents more accountable for their choices. Since the outcome of parental choices is mostly either uncertain or incalculable, this strategy must then be to consider ways in which these choices might be wrong rather than bad. The important shift here is from the chances of the bad outcomes to the responsibility for decisions that have particular risks associated with them. Here I briefly consider two strategies for challenging the standard view: 1. qualifying conditions for the right to reproductive autonomy 2. an account of the right choice or choices for prospective parents through the character of the chooser or the nature and demands of parenting. The first challenge is an account of the qualifying criteria needed for the possession of the right to reproductive autonomy.13,20,21 In the context of Mill’s original statement of the harm principle, he certainly excluded children and ‘those who are still in a state to require being taken care of by others’ from possessing the liberty in question.† Only * There are clearly physical harms associated with the process of PGD and IVF but the HFEA report found that these were not significant enough to warrant distinguishing between selecting away from a particular condition and selecting for a tissue match with an existing sibling. We will return to this issue later in the chapter. † ‘It is, perhaps, hardly necessary to say that this doctrine is meant to apply only to human beings in the maturity of their faculties. We are not speaking of children, or of young persons below the age that the law may fix as that of manhood or womanhood. Those who are still in a state to require being taken care of by others must be protected against their own actions as well as against external injury.’16

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those satisfying certain criteria were bearers of the right. Similarly, we might develop criteria for the right to reproductive autonomy based on fitness to make decisions about reproduction. Onora O’Neill22 suggests that: ‘the right to beget or rear is not unrestricted, but contingent upon begetters or bearers having or making some reasonable plan for their child to be adequately reared by themselves or by willing others. Persons who beget or bear without making any such plans cannot claim that they are exercising a right.’

The work to be done here is to develop a defensible account of the relevant ‘fitness to parent’ conditions and then to flesh out the consequences of these conditions for particular kinds of choices. It seems likely that an account of this sort might justify an unfavourable judgement of the ‘social services’ reason but it is less obvious that it would yield a similarly unfavourable account of the saviour sibling reason. A second challenge to the more orthodox way of handling procreative reasons involves developing an account of the kinds of decisions that responsible prospective parents might make. This approach develops the thought that those who wish to procreate for bad reasons somehow fail to understand what it means to be a parent. Here we might develop an account of parental character and parental virtue or we could focus on what it is to be a good parent. In the former case, good procreative reasons demonstrate parental virtue.23 In the latter, bad reasons undermine the point of the activity.24,25 Both options face similar challenges: it is not clear that there is sufficient agreement about what counts as being a good or virtuous parent for such an approach to generate specific criticisms in the saviour sibling case. It seems more likely, if this strategy is to be effective at all, for it to apply to more extreme cases. Conclusions: judging reasons As things stand, it is not clear that there is a compelling argument based on prospective parental reasons for denying PGD for saviour siblings. This is based largely on: n the current lack of force (or completeness) of the attempts to resist the orthodoxy that is represented in the harm principle/reproductive autonomy combination n the uncertainty associated with the supposedly harmful outcomes of particular procreative decisions. In the latter case, there is an important gap between the kinds of reasons that prospective parents have for having children and the way in which the resulting child is treated.26 This conclusion about our ability to judge parental reasons is pending the development of each of the ‘further thoughts’ as well as the investigation of the strength and extent of the right to reproductive autonomy. My suspicion is that neither of these lines of investigation will cause trouble for the intuitive acceptability of the easy saviour sibling cases. First, any account of good parenting or qualifying criteria for reproductive rights should allow for the possibility that parents will need to prioritise between children in certain circumstances. It is inevitable that parents will be required to make choices that will benefit one child and put another at risk of harm. The good parent will be able to make such decisions well. It is difficult to see how such an account will be able to make room for such decisions and yet fail to allow the easy saviour siblings cases. Second, there is relatively little argument about the strength of the right to reproductive autonomy. When faced with a decision about whether to permit a

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reproductive decision, how much weight should be given to the right to reproductive autonomy? The default position, consistent with the harm principle, is that the right to reproductive autonomy should trump all other considerations with the exception of those that involve harm to others. Even here, though, there is a question about how much harm is required in order for the exception to be triggered. Overall then, it is difficult to arrive at any clear answers to our initial questions based on an examination of parental reasons. The default view is one that privileges parental freedom in the absence of clear evidence of harm connected with the reasons in question. It is unclear, however, whether this gives us enough to separate the easy cases from the hard cases in the saviour sibling context. To do this, we need to consider the questions from the perspective of the actual treatment of children.

The treatment of saviour siblings By definition, saviour siblings are children whose tissue or organs will be donated so that a brother or sister will be saved. The treatment of existing saviour siblings immediately involves ethical questions about live organ donation by children. In what follows, I maintain that once the focus has shifted away from procreative reasons, we should use the permissibility of live organ donation by children as a guide in decisions about the permissibility of using PGD to select a saviour sibling. In short, if the ‘saving’ act is not a permissible live organ donation, then PGD and selection is not warranted. Before turning to the permissibility of live organ donation, we need to be clear about the distinction between reasons and treatment. Pennings7,8 characterises this distinction via the ‘postnatal test’: the ethical acceptability of particular reasons is implied by the acceptability of treating an actual child in the manner specified in the reason. Specifically, ‘If the parents can decide that an existing child should have an operation in order to give bone marrow to a sibling, then it is difficult to argue that they should not desire, as part of their set of motives for having a child, a child that can give bone marrow to a sibling.’*7

The difficulty here is that the kinds of decisions that the parents make about an actual child are arguably very different from decisions made about potential children. In the former case, the parents can reflect on what they know of the developing character of the child, the child’s actual relationships and interactions with others, and, importantly, the child’s relationship with the potential recipient of the organ. The parents in such a situation will have a real sense of the dynamics of the family as well as the kinds of support available both within and external to the family unit. In some cases, particularly those where there are already a number of children, some of this sort of information and experience will be available to prospective parents. However, even in these cases, the decision to allow this particular child to become a donor is different from the decision to have a child intending that the child become a donor. The difference between the two kinds of decision matters in this context: given the differences, nothing significant follows about the permissibility of saviour sibling reasons from the fact that the parents are permitted to decide about organ donation for an existing child. So although we might generally think that the connection between intending to do something wrong and actually doing it holds, here, perhaps because * Of course we are actually comparing expressed reasons rather than desires – we are interested in the justification of having and acting on certain reasons rather than whether or not people should have particular desires.

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of the distance between the intending and the actual treatment as well as the different nature of the decision itself, the connection is not so obvious. The difference matters most in the context of dealing with hard saviour sibling cases. Pennings8 seems to assume that in these hard cases the reasons that the prospective parents have are wrong or unjustified because of the intending/acting relation – it is the harm associated with the fulfilment of the intention that is wrong. However, given the separation between reasons for parenting and what takes place in actual parenting, together with the qualitative difference between the decisions, it seems hard then to base the problem on this connection. Parental freedom The first point to consider about the treatment of children is the basis of parents’ freedom to determine the course of their child’s life. Of course, the obvious basis for parental freedom lies in the central liberal values surrounding the plurality of conceptions of the good life. The value of the individual freedom to determine how one’s life goes extends to one’s children and the family is generally seen to be the locus of individual ‘experiments in living’. Consequently, we think it is appropriate that parents shape their children through their choices. This extends to schooling, values, religion, diet and discipline. Increasingly, where there is broad consensus and evidence of harmful effects, there is some suggestion that society is taking a stand about some aspects of these areas of parental choice. The recent focus on diet and exercise in school is a good example. In medicine, the situation looks slightly different. There is much greater debate between healthcare professionals and parents about the treatment of children. This seems to be partly a result of the immediacy and magnitude of the harms involved as compared with those involved with more general social decisions, but it is also connected to the idea of third-party intervention mentioned earlier – healthcare professionals have more of a say in the medical treatment of children because they are tasked with looking after the child and have specialist expertise in the area. Overall, in medicine also, parents have a good deal of authority to decide for their child. Live organ donation by children The major issues surrounding live organ donation by children are fairly clear:27–31 n there are questions about consent, both the ability of children of various ages to do so and the possibility of genuinely free choice even when they have the ability n there are worries about the welfare of the donor child that involve questions about whether children can be asked or made to do things that are detrimental to their wellbeing n perhaps pulling in the other direction, there are questions about the role of family obligations and commitments alluded to above. The consent issue does a good deal of the ethical work in these cases. As with other cases of self-sacrifice, there is a residual worry about the voluntariness of the decision. This worry is greater in the case of children, where there may be general capacity issues or serious power imbalances that are clearly open to exploitation. Most significant, however, is the lack of the ability to consent for most children – without consent there is no clear way of getting ‘proper’ permission for the donation. There are limits

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to the amount of harm or risk of harm that can be inflicted on a child, particularly if the child is unable to consent. There may also be similar limits that apply even if the child is able to consent. For suitably minor risks, there is a good case that we should allow live organ donation by children. Again, what counts as minor and the degree of risk that is acceptable will partly depend on the ability of the child to give consent. In some cases, we can resort to assent or to the failure to dissent in an effort to ease concerns about the lack of consent. One strand of the argument about children donating organs that follows immediately from the difficulties with consent is a concern for the welfare of the donor child. As a result, one condition that is often placed on child organ donation is that it is in the best interests of the donor child. Of course, unless we understand best interests very broadly, this looks to be difficult condition to meet. What is required in these cases is that the benefits to the donor of the recipient being saved outweigh the harms associated with the donation. The more permissive argument in favour of allowing live organ donation by children, and as decided by the parents, stems from the importance that we give to parental freedoms and to the value of familial ties. Included within this is the recognition that parents may in some cases require a certain level of sacrifice on the part of an individual member of the family either for the family as a whole or for another member. The family unit – here understood simply as parent(s) and children – and its independence is of value and so decisions within it should be respected. Overall, the standards set for minors providing live organ donations largely match the distinction between easy cases and hard cases and incorporate, to varying degrees, each of the considerations above. There is some variation across countries, with the most significant difference being in the USA, where the trend is to allow children to be live kidney donors. In August 2008, the American Academy of Pediatrics published guidance on minors as living solid-organ donors and which consisted of the following five conditions:32 1. ‘Both the potential donor and recipient are to be highly likely to benefit.’ Because the benefit to the donor is likely to be psychological, this condition is taken to limit transplants to within families. 2. The surgical risk to the donor should be ‘extremely low’. This means that minors are limited to live kidney donation. 3. All alternative opportunities for donation have been exhausted. 4. ‘The minor is to agree to the donation freely and without coercion.’ This is to be established by an independent advocate. 5. ‘The emotional and psychological risks to child donors should be minimized.’ This involves both preparation of the child as well as support after the donation for the child and the family.

Conclusion There are two final questions to be mentioned here. First, there may be some contexts in which the tissue type being selected for (the target tissue type) brings with it an associated, undesirable condition. That is, by instructing the clinicians to select for a particular tissue type, the parents are also running the risk that the resulting child will be born with another associated, undesirable condition. Where this risk is significant, the harm involved tells against the permissibility of the reason. So a high chance of the saviour sibling having a debilitating condition tells against acting on the saviour

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sibling reason – this is a situation where acting on the reason directly has an associated risk of harm. In this case, the harm is proximal to the reason. Second, both Pennings7,8 and Devolder9 argue that the creation of tissue-matched children to be donors should not be limited to siblings and that tissue typing should be permitted both for parents and non-family members. Nothing I have argued for here rules out this possibility since this will entirely depend on the permissibility of such an arrangement in terms of live organ donation. The argument against allowing this type of donation turns on the reliability of the judgements about consent, assent, best interests or relationship. In practical terms, a major danger in these cases is some form of skewed authority and power relationship. So, while siblings might be usually more equal, this is less likely for parents. In the case of non-family members, it seems more difficult to judge the proper model of the relationship and so to be confident that something was not awry. In this chapter, I have not argued for the standards described for live organ donation by children. Instead, I have suggested that the appropriate standards for live organ donation by children should be adopted to decide the acceptability of the use of PGD for saviour siblings. This amounts not to an ethical judgement about the procreative reasons of prospective parents; instead, it is a practical judgement based on the permissibility of particular ways of treating children.

References 1. 2. 3.

4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Dickens B. Preimplantation genetic diagnosis and ‘savior siblings.’ Int J Gynaecol Obstet 2005;88:91–6. Ram NR. Britain’s new preimplantation tissue typing policy: an ethical defence. J Med Ethics 2006;32:278. Baldwin T (Deputy Chair, Human Fertilisation and Embryology Authority). Announcement of the Report of the Preimplantation Tissue Typing Policy Review. 4 August 2004 [www.hfea.gov. uk/2677.html]. Human Fertilisation and Embryology Authority. Report of the Preimplantation Tissue Typing Policy Review 2004 [www.hfea.gov.uk/docs/PolicyReview_PreimplantationTissueReport.pdf]. Sheldon S, Wilkinson S. Should selecting saviour siblings be banned? J Med Ethics 2004;30:533–7. Sheldon S, Wilkinson S. Hashmi and Whitaker: an unjustifiable and misguided distinction? Med Law Rev 2004;12:137. Pennings G, Schots R, Liebaers I. Ethical considerations on preimplantation genetic diagnosis for HLA typing to match a future child as a donor of haematopoietic stem cells to a sibling. Hum Reprod 2002;17:534–8. Pennings G. Saviour siblings: using preimplantation genetic diagnosis for tissue typing. Int Congr Ser 2004;1266:311–17. Devolder K. Preimplantation HLA typing: having children to save our loved ones. J Med Ethics 2005;31:582–6. Wasserman D. Having one child to save another: a tale of two families. Philos Public Policy Q 2003;23:21–7. Aulisio MP, May T, Block JT. Procreation for donation: the moral and political permissibility of ‘having a child to save a child’. Camb Q Healthc Ethics 2001;10:408–19. Spriggs M, Savulescu J. Saviour siblings. J Med Ethics 2002;28:289. O’Neill O. Autonomy and Trust in Bioethics. Cambridge: Cambridge University Press; 2002. Wilkinson S. Sexism, sex selection and ‘family balancing.’ Med Law Rev 2008;16:369–89. Robertson J. Procreative liberty in the era of genomics. Am J Law Med 2003;29:439. Robertson J. Children of Choice – Freedom and the New Reproductive Technologies. Princeton: Princeton University Press; 1994. Steinbock B. A philosopher looks at assisted reproduction. J Assist Reprod Genet 1995;12:543–51. Mill JS. On Liberty (1859). London: Penguin Classics; 2006.

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Parfit D. Reasons and Persons. Oxford: Clarendon Press; 1984. Archard D. Wrongful life. Philosophy 2004;79:403–20. Heyd D. Genethics: Moral Issues in the Creation of People. San Francisco: University of California Press; 1992. O’Neill O. Begetting, bearing and rearing. In: O’Neill O, Ruddock W, editors. Having Children: Philosophical and Legal Reflections on Parenthood. Oxford: Oxford University Press; 1979. p. 25–38. McDougall R. Parental virtue: a new way of thinking about the morality of reproductive actions. Bioethics 2007;21:181–90. Norman R. Interfering with nature. J Appl Philos 1996;13:1–11. Sheehan, M. Making sense of the immorality of unnaturalness. Camb Q Healthc Ethics 2009;18:177–88. Lotz M. Procreative reasons–relevance: on the moral significance of why we have children. Bioethics 2008;23:291–9. Webb N, Fortune P. Should children ever be living kidney donors? Pediatr Transplant 2006:10;851–5. Fleck LM. Children and organ donation: some cautionary remarks. Camb Q Healthc Ethics 2004;13:161–6. Jansen LA. Child organ donation, family autonomy, and intimate attachments. Camb Q Healthc Ethics 2004;13:133–42. Holm S. The child as organ and tissue donor: discussions in the Danish Council of Ethics. Camb Q Healthc Ethics 2004;13:156–60. Sheldon M. Children as organ donors: a persistent ethical issue. Camb Q Healthc Ethics 2004;13:119–22. Ross LF, Thistlethwaite JR Jr, the Committee on Bioethics. Minors as living solid-organ donors. Pediatrics 2008;122:454–61.

5 Chapter 5

Epigenetics, assisted reproductive technologies and growth restriction Jennifer M Frost, Sayeda Abu-Amero, Caroline Daelemans and Gudrun E Moore

Epigenetics: overview Epigenetics is both heritable and a reversible interaction on the DNA, resulting in a change in expression or phenotype without altering the DNA sequence. Epigenetic effectors include the modification of DNA by methylation, the configuration and modification of nucleosomal histone proteins and the involvement of antisense RNA molecules. Epigenetics, in association with the transcriptional apparatus, regulates transcriptional profiles which dictate changes in cellular phenotype, controlling cell differentiation, division and death. During mammalian development, a specific mechanism of epigenetic regulation is in place that acts on a small number of genes controlling some aspects of fetal growth and development and postnatal behaviour. This mechanism, known to involve approximately 100 genes in the mouse genome and 50 genes in the human genome, is characterised by parent-of-origin-specific monoallelic expression, and is known as genomic imprinting (see www.otago.ac.nz/IGC). Genomic imprinting Genomic imprinting was discovered in 1984 by pronuclear transfer experiments. Bipaternal and bimaternal embryos were created in parallel to reveal opposite lethal phenotypes.1–3 The genomes inherited from the mother and the father during sexual reproduction were found to be asymmetrical, and each indispensible in normal development (Figure 5.1). Naturally occurring examples of these phenotypes can be found in humans and they are also incompatible with life. Ovarian teratomas are equivalent to gynogenotes and are characterised by varying stages of developing embryonic tissues, which are grossly disorganised. Hydatidiform moles are equivalent to androgenotes and take the form of overgrown and cystic trophoblast tissue, which can become highly invasive.4,5 The creation of uniparental disomic mice then permitted the precise regions of parental genome non-equivalence to be narrowed down. Uniparental disomy (UPD) is where the normal biparental chromosomes are all present except for one pair, or part © Jennifer M Frost, Sayeda Abu-Amero, Caroline Daelemans and Gudrun E Moore. Volume compilation © RCOG

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Figure 5.1 The creation of uniparental mice. Murine gestation at day 10 of normal development is compared with that of embryos created during pronuclear transfer experiments. Diploid gynogenote embryos contain only maternal genetically inherited material and androgenotes contain only paternally inherited material. Gynogenotes develop embryonic tissue but no placenta, whereas androgenotes develop placental tissue but no embryo. The diagram is a representation of mouse phenotypes based on figures in Barton et al.,1 McGrath and Solter2 and Surani et al.3

of one pair, which is derived from the same parent. For example, where two copies of maternal chromosome 7 are present, this is known as maternal uniparental disomy 7, or mUPD7; or as pUPD7 if both copies are paternal. Using known Robertsonian translocations, mice were created harbouring UPD of each chromosome, or part of each chromosome, and were found to each have different phenotypes. Some of the mice appeared to be normal, indicating that in these mice there were no oppositely imprinted genes present on that chromosome or chromsome segment. One of the main observations of these experiments was that UPD mice for a particular chromosome exhibited an opposing phenotype to mice with UPD for that same chromosome inherited from the opposite parent. Mice with pUPD11, for example, were 30% larger than wild type, whereas mUPD11 mice were 30% smaller.6 The regions of non-equivalence have since been narrowed down to individual imprinted genes, including isolated genes, but most frequently arranged in clusters.7 DNA methylation controls genomic imprinting Monoallelic expression of imprinted genes is brought about through allelic DNA methylation at key loci, which acts to control expression of surrounding genes in cis. Such differentially methylated regions (DMRs) of DNA are established during

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gametogenesis of the previous generation.8 Targeted mutation of individual DMRs in mouse models has established several of the known DMR loci as definitive imprint control regions (ICRs) necessary for the appropriate expression of the imprinted genes under their control.9 Evidence of the conservation of such ICRs in the human may also be gathered experimentally using cell lines or introducing human DNA into mouse models. The natural occurrence of mutations in putative ICRs in the human, leading to various congenital disorders, has also enabled verification of ICRs.10–14 In the germ cells of the new fetus, imprints are erased as primordial germ cells enter the genital ridge and are then reset to reflect fetal sex (Figure  5.2). Allelic methylation at ICRs then follows during gametogenesis, catalysed specifically by DNMT3a in conjunction with its non-enzymatic cofactor DNMT3L.17,18 In the male germline, all imprint methylation is set by mouse embryonic day  17.5, equivalent to the prospermatogonia stage of spermiogenesis.19,20 In the female, however, the acquisition of imprints occurs in a stepwise manner during folliculogenesis, a process starting at puberty and lasting several months for each ovulatory cycle (Figure 5.3).

Assisted reproductive technologies The development of in vitro fertilisation (IVF), celebrated by the birth of Louise Brown in 1978, led to a rapidly and currently still expanding field of assisted reproductive technologies (ART) available to people with subfertility. In addition to superovulatory therapy and cryopreservation, many different treatment combinations are now available to an individual or couple. Intracytoplasmic sperm injection (ICSI)

Figure 5.2 The genomic imprinting cycle. Upon fertilisation, the haploid genomes of the sperm and egg are demethylated. Imprint control region (ICR) methylation is maintained during this time. The paternal genome is demethylated by an active mechanism, occurring more rapidly than maternal pronuclei demethylation.15 During developmental progression, differential methylation of ICRs is maintained in the soma. At around mouse embryonic stay 11.5 (E11.5), the primordial germ cells (PGCs) enter the genital ridge and begin to colonise it, during which time ICR methylation is erased16

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Figure 5.3 Imprint initiation in the female germline. Gene names in lower case correspond to data from mouse studies, upper case to human. Several stages of folliculogenesis, in concert with oocyte growth, are shown alongside the timing of imprint initiation of various imprint control regions (ICRs) in the female germline. The images are not to scale but the diameter of the oocyte at each stage, shown to be instrumental in the timing of ICR methylation, is indicated for the mouse and the human;20–25 DMR = differentially methylated region; LH = luteinising hormone

is commonly used and has been attempted with immature sperm.26 In vitro maturation of oocytes is an important new technique and may avoid the use of superovulation, which is invasive for women. Each manipulation bypasses several steps of natural selection, making it difficult to distinguish the actual effects of ART from pathologies inherently associated with infertility. Embryo culture ART involving embryo culture, such as IVF and ICSI, are being examined to see how safe the technologies are with regard to a healthy outcome. Studies in animal models have demonstrated the sensitivity of the early embryo to culture conditions.27–29 The observations of low birth weight and an increase in certain imprinting syndromes following IVF and ICSI have led to fears that the culture medium does not provide ideal nutrition at this sensitive developmental stage (Figure 5.4). Fetal bovine serum is a routine and important component of tissue culture media but its use has been linked to large offspring syndrome (LOS), a common disorder in cattle and sheep that have undergone embryo culture. LOS is characterised by overgrowth in utero, organ defects, breathing difficulties and an increased risk of perinatal death.30 Alteration to the specific concentration of serum has been shown to have an impact on imprinted gene expression in the mouse blastocyst and to affect growth and development pre- and postnatally.31 Another concern regarding culture medium is the provision of a methyl donor concentration to mirror the in vivo environment.32 The preimplantation embryo

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Figure 5.4 Preimplantation development in vitro. Following fertilisation, the genome of the zygote undergoes two waves of genome-wide epigenetic reprogramming. Embryos resulting from in vitro fertilisation treatments are remodelled while in an in vitro environment. ICM =  inner cell mass; TE = trophectoderm

undergoes two waves of de- and remethylation to reprogramme the terminally differentiated nuclei of the sperm and oocyte into that of a pluripotent cell (Figure 5.4).33 An apparent lack of active demethylation of the paternal pronucleus has been observed in human pre-cleavage embryos in vitro.34 Aberrant pronuclear demethylation has been shown to have adverse effects on development and viability.35 Studies pertaining specifically to errors in epigenetic regulation are most informative in species other than human. The monoallelic expression of the imprinted IGF2R gene can be disrupted in sheep following single-cell nuclear transfer or embryo culture owing to loss of maternal DMR methylation.36 Loss of imprinting of this gene has also been implicated in LOS following nuclear transfer or in vitro culture.31,36–38 Epigenetic defects in mouse embryos are particularly widespread, involving changes in DNA methylation and the deregulation of several imprinted gene clusters.28,39–42 Superovulation One of the earliest steps in ART treatment is usually the stimulation of the woman’s ovaries in an attempt to increase the number of potential embryos and therefore the chance of success. As well as being cultured in vitro, ART embryos are thus normally produced following fertilisation of superovulatory oocytes. Primordial follicles are arrested in dictyate during female development and from puberty onwards begin a continual recruitment into maturation. Oocyte development, concurrent with the stepwise methylation of ICRs, thus occurs throughout a woman’s life, as outlined in Figure 5.3.21 Drugs specifically designed to speed up folliculogenesis and oocyte development may well have a negative impact on the integrity of ICR methylation, and an increased incidence of imprinting disorders following ART has been suggested to be a consequence of superovulation rather than embryo culture.43,44 Superovulation has been shown to cause defects in genome methylation levels and specifically in the differential methylation at ICRs, notably at the ICRs of H19 and MEST/Mest in human and mouse oocytes.23,35 Biallelic expression of H19 and Snrpn were observed in the mouse placenta and upregulation of Igf2 expression in superovulated mouse embryos both with and without embryo transfer, distinguishing

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the effects of superovulation and in vitro culture.45 The authors also suggested that, as both maternally and paternally set imprints were affected, superovulatory drug regimens may adversely affect the oocyte cytoplasm, reducing its ability to maintain imprints during preimplantation development.45 ART children’s growth and development Observations of human singleton births following ART embryo culture report an increased incidence of preterm birth, low birth weight, neonatal mortality and admissions to intensive care when compared with naturally conceived singletons.46,47 The incidence of congenital malformations, however, does not seem to be increased following ART but rather correlates positively with parental infertility.48,49 Interestingly, a small study carried out in New Zealand indicated that children born through IVF actually grew to be prepubertally taller, perhaps owing to an increase in serum insulinlike growth factor I (IGF-I) and II (IGF-II) and a better lipid profile compared with their non-IVF peers.50

Genomic imprinting and growth The relationship between ART and a deregulation of genomic imprinting is thought to have particular relevance to fetal growth because fetal growth is suggested to be the main drive for the evolution of imprinting or the conflict between the parental genomes. This has become known as the conflict theory, the premise of which is that maternally expressed imprinted genes act to curb the growth of the fetus, preserving the mother’s ability to give birth to a live baby and also her future fecundity. Paternally expressed genes need to promote the growth of the fetus, possibly to the cost of other fetuses, which may not be his, both in the current and in future pregnancies.51 The theory is supported, and elegantly demonstrated, by the example of the first imprinted gene to be discovered in the mouse, insulin-like growth factor II (Igf2), and its receptor Igf2r.52,53 The Igf2 protein is a potent enhancer of growth and Igf2r, which acts as an Igf2 sink, is a growth suppressor. In marsupials and in the mouse, Igf2 is paternally expressed and Igf2r is maternally expressed, although IGF2R is not imprinted in humans.54,55 Disruption of the Igf2 imprinting system leads to opposing phenotypes in the mouse (Figure 5.5).56 Other genes behave in a manner consistent with the conflict theory: mouse knockouts of paternally expressed Mest, Peg3 and Ins result in fetal growth restriction, and ablation of maternally expressed H19 results in fetal overgrowth.57,58 In the large imprinting cluster on human chromosome 11, maternally expressed growth-suppressing PHLDA2 has been shown to have a negative correlation with birth weight.59 Genomic imprinting and the placenta The placenta is directly responsible for bringing the maternal and fetal blood supplies into contact, facilitating nutrient exchange and determining resource allocation.60 The size, development and efficiency of the placenta thus have a primary influence on fetal growth. In addition, the placenta, particularly the invasive trophoblast lineages, is an important focus for potential parental conflict. Almost all imprinted genes known to date are expressed in the placenta and many have crucial roles in its development and function.61 The deregulation of imprinted gene expression following ART can therefore be expected to have a significant impact

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Figure 5.5 Theoretical model for reciprocal phenotypes through opposite imprinting mutations in the mouse; M = maternal allele; P = paternal allele; biallelic Igf2, or a lack of expression of its receptor, would in theory lead to excess available Igf2 protein and increased growth; biallelic Igf2r or a lack of Igf2 would lead to a deficiency in Igf2 protein and decreased growth56

on the function and growth of the placenta, subsequently affecting the growth of the fetus. Mest, Peg3 and Igf2 have been shown to be promoters of placental growth.62–64 Murine Igf2 is expressed from several different promoters, one of which, P0, is placental specific. Deletion of P0 reduces placental size close to that of complete Igf2 knockout; that is, around 40% smaller than normal.64 Interestingly, IGF2 P0 in the human is not placental specific and is most highly expressed in fetal muscle.65 Placental invasion is also controlled by the decidua, which expresses a wide variety of IGF binding proteins, balancing invasion and fetal provision. Decidual control is accentuated by maternal-specific imprinted gene expression in the placenta and in the fetus. Paternally expressed MEST is thought to play a role in angiogenesis in human trophoblast and decidua.66 In the mouse, deletion of maternally expressed H19, Igf2r, Cdkn1c, Mash2 or Phlda2 results in placental hyperplasia, indicative of the regulatory influence of these transcripts in invasion.67–69 The importance of transport systems in nutrient acquisition is highlighted by the imprinting of several soluble nutrient carriers, including murine placental-specific Slc22A2 and Slc22A3, and Slc22a18/SLC22A18 and Slc22a1ls/SLC22A1LS in both mouse and human.70 Maternally expressed growth suppressors such as PHLDA2 may reduce nutrient supply through action in the placenta, or reduce nutrient demand, through the fetus.59,71 ART and genomic imprinting syndromes The link between embryo culture and phenotype is thought to be, at least in part, epigenetic. Evidence for this includes an increase in the incidence of certain rare imprinting disorders following ART, often linked to changes in methylation at control loci.72–80 Abnormalities in Igf2 and H19 imprinting in the mouse following ART were shown to be due to a deregulation of both DNA methylation and histone modification.81 The disruption of monoallelic, parent-of-origin-specific expression of imprinted genes can result in severe developmental abnormalities in humans. There are several, although rare, well-characterised syndromes, each associated with a specific imprinted gene or imprinted gene cluster. There are two that have been linked in several studies with the use of ART: Beckwith–Wiedemann syndrome and Angelman syndrome.

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Beckwith–Wiedemann syndrome is a fetal overgrowth disorder characterised by macroglossia, pre- and postnatal growth greater than the 90th centile and abdominal wall defects. Angelman syndrome is associated with mental restriction, problems with movement and balance and severe limitations in speech and language. The incidence of both of these syndromes in the normally conceived population is low, both currently affecting one in 15 000 live births. Beckwith–Wiedemann syndrome may result from several epimutations but the majority, and almost all cases following from ART, are caused by hypomethylation of the KvDMR.74,75,82 It has been suggested that procedures such as IVF and ICSI may increase the risk of the syndrome in these children to as much as 3–6%.73 Angelman syndrome is caused by hypomethylation at the SNRPN promoter and has been particularly linked with the ICSI procedure.44,72,76 As a subgroup of these rare populations, the small number of patients conceived through ART make statistical comparison difficult and it is still not clear whether the perceived increase in imprinting disorders following ART is significant The significance of postnatal genomic imprinting There are a number of imprinting syndrome phenotypes that cannot easily be reconciled with the conflict theory, notably Prader–Willi syndrome and the previously discussed Angelman syndrome. After weaning, the father has a vastly increased role in provision of food for the offspring. This has led to the recent suggestion that some imprinted genes exist to control appetite, functioning specifically after the mother’s primary role in gestation and lactation has ended; that is, at weaning. Ubeda83 suggests that maternally expressed genes that act before weaning are resource inhibiting (RI) and that a second set of maternally expressed genes exist that are resource enhancing (RE) and act after weaning. This is combated by RE genes expressed paternally before weaning, and RI genes expressed paternally after weaning. Prader–Willi syndrome, caused by paternal inheritance of deletions on 15q11–13, or by mUPD, is characterised by poor suckling, hypotonia and low weight after birth, but upon weaning by an insatiable appetite, leading to obesity. Angelman syndrome is caused by maternal inheritance of deletions on 15q11–13, or by pUPD, and children actually delay weaning, having an extended suckling period. Both syndromes are associated with mental restriction. Consistent with Ubeda’s theory, loss of paternal RE expressed before weaning, and RI after weaning, would result in reduced demand for resources by the offspring before weaning and an increase in it after weaning, as seen in Prader–Willi syndrome. In Angelman syndrome, maternally expressed RI are lost, manifesting in extended suckling.83

Summary Genomic imprinting is an important epigenetic mechanism through which some aspects of fetal growth and development are regulated. Imprinting is controlled by allelic methylation, and the setting up of imprints in the female germline is a process that occurs throughout reproductive life. Inappropriate methylation or expression of imprinted gene clusters cause a range of pathologies, often involving aberrant growth and development. Similarly, the use of ART has been shown to cause disorders of growth in humans and other mammals. Suggested links between aberrant imprinting and ART are supported by a potential increase in the incidence of rare imprinting syndromes following ART. Whether

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embryo culture or the use of superovulated oocytes is responsible for these problems is not clear at present. The continued influence of imprinted gene expression after birth, as demonstrated by Prader–Willi and Angelman syndromes, stresses the importance of follow-up of ART children in the investigation of whether ART is truly in part a cause of developmental disorders.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

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Barton SC, Surani MA, Norris ML. Role of paternal and maternal genomes in mouse development. Nature 1984;311:374–6. McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell 1984;37:179–83. Surani MA, Barton SC, Norris ML. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 1984;308:548–50. Kajii T, Ohama K. Androgenetic origin of hydatidiform mole. Nature 1977;268:633–4. de Grouchy J. Human parthenogenesis: a fascinating single event. Biomedicine 1980;32:51–3. Cattanach BM, Kirk M. Differential activity of maternally and paternally derived chromosome regions in mice. Nature 1985;315:496–8. Morison IM, Ramsay JP, Spencer HG. A census of mammalian imprinting. Trends Genet 2005;21:457–65. Wilkins JF. Genomic imprinting and methylation: epigenetic canalization and conflict. Trends Genet 2005;21:356–65. Thorvaldsen JL, Bartolomei MS. SnapShot: imprinted gene clusters. Cell 2007;130:958. Buiting K, Lich C, Cottrell S, Barnicoat A, Horsthemke B. A 5-kb imprinting center deletion in a family with Angelman syndrome reduces the shortest region of deletion overlap to 880 bp. Hum Genet 1999;105:665–6. Ohta T, Buiting K, Kokkonen H, McCandless S, Heeger S, Leisti H, et al. Molecular mechanism of Angelman syndrome in two large families involves an imprinting mutation. Am J Hum Genet 1999;64:385–96. Horike S, Mitsuya K, Meguro M, Kotobuki N, Kashiwagi A, Notsu T, et al. Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith–Wiedemann syndrome. Hum Mol Genet 2000;9:2075–83. Paulsen M, El-Maarri O, Engemann S, Strodicke M, Franck O, Davies K, et al. Sequence conservation and variability of imprinting in the Beckwith–Wiedemann syndrome gene cluster in human and mouse. Hum Mol Genet 2000;9:1829–41. Arima T, Yamasaki K, John RM, Kato K, Sakumi K, Nakabeppu Y, et al. The human HYMAI/ PLAGL1 differentially methylated region acts as an imprint control region in mice. Genomics 2006;88:650–8. Mayer W, Niveleau A, Walter J , Fundele R, Haaf T. Demethylation of the zygotic paternal genome. Nature 2000;403:501–2. Lee J, Inoue K, Ono R, Ogonuki N, Kohda T. Kaneko-Ishino T, et al. Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells. Development 2002;129:1807–17. Hata K , Okano M, Lei H, Li E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development 2002;129:1983–93. Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, et al. Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 2004;429:900–3. Li JY, Lees-Murdock DJ, Xu GL, Walsh CP. Timing of establishment of paternal methylation imprints in the mouse. Genomics 2004;84:952–60. Hiura H, Obata Y, Komiyama J, Shirai M, Kono T. Oocyte growth-dependent progression of maternal imprinting in mice. Genes Cells 2006;11:353–61. Obata Y, Kono T. Maternal primary imprinting is established at a specific time for each gene throughout oocyte growth. J Biol Chem 2002;277:5285–9. Lucifero D, Mann MR, Bartolomei MS, Trasler JM. Gene-specific timing and epigenetic memory in oocyte imprinting. Hum Mol Genet 2004;13:839–49.

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Sato A, Otsu E, Negishi H, Utsunomiya T, Arima T. Aberrant DNA methylation of imprinted loci in superovulated oocytes. Hum Reprod 2007;22:26–35. Khoueiry R, Ibala-Rhomdane S, Mery L, Blachere T, Guerin JF, Lornage J, et al. Dynamic CpG methylation of the KCNQ1OT1 gene during maturation of human oocytes. J Med Genet 2008;45:583–8. Chotalia M, Smallwood SA, Ruf N, Dawson C, Lucifero D, Frontera M, et al. Transcription is required for establishment of germline methylation marks at imprinted genes. Genes Dev 2009;23:105–17. Georgiou I, Pardalidis N, Giannakis D, Saito M, Watanabe T, Tsounapi P, et al. In vitro spermatogenesis as a method to bypass pre-meiotic or post-meiotic barriers blocking the spermatogenetic process: genetic and epigenetic implications in assisted reproductive technology. Andrologia 2007;39:159–76. Bowman P, McLaren A. Viability and growth of mouse embryos after in vitro culture and fusion. J Embryol Exp Morphol 1970;23:693–704. Khosla S, Dean W, Brown D, Reik W, Feil R. Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol Reprod 2001;64:918–26. Lane M, Gardner DK. Ammonium induces aberrant blastocyst differentiation, metabolism, pH regulation, gene expression and subsequently alters fetal development in the mouse. Biol Reprod 2003;69:1109–17. Young LE, Sinclair KD, Wilmut I. Large offspring syndrome in cattle and sheep. Rev Reprod 1998;3:155–63. Fernandez-Gonzalez R. Moreira P, Bilbao A, Jimenez A, Perez-Crespo MA, Rodriguez De FF, et al. Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. Proc Natl Acad Sci U S A 2004;101:5880–5. Steele W, Allegrucci C, Singh R, Lucas E, Priddle H, Denning C, et al. Human embryonic stem cell methyl cycle enzyme expression: modelling epigenetic programming in assisted reproduction? Reprod Biomed Online 2005;10:755–66. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science 2001;293:1089–93. Fulka H, Mrazek M, Tepla O, Fulka J Jr. DNA methylation pattern in human zygotes and developing embryos. Reproduction 2004;128:703–8. Shi W, Haaf T. Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure. Mol Reprod Dev 2002;63:329–34. Young LE, Schnieke AE, McCreath KJ, Wieckowski S, Konofortova G, Fernandes K, et al. Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer. Mech Dev 2003;120:1433–42. Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M, Rideout WM, et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci U S A 2001;98:6209–14. Young LE, Fernandes K, McEvoy TG, Butterwith SC, Gutierrez CG, Carolan,C, et al. Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture. Nat Genet 2001;27:153–4. Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E, et al. Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc Natl Acad Sci U S A 2001;98:13734–8. Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout WM III, Biniszkiewicz D, et al. Epigenetic instability in ES cells and cloned mice. Science 2001;293:95–7. Mann MR, Lee SS, Doherty AS, Verona RI, Nolen LD, Schultz RM, et al. Selective loss of imprinting in the placenta following preimplantation development in culture. Development 2004;131:3727–35. Rivera RM, Stein P, Weaver JR, Mager J, Schultz RM, Bartolomei MS. Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development. Hum Mol Genet 2008;17:1–14.

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Chang AS, Moley KH, Wangler M, Feinberg AP, Debaun MR. Association between Beckwith– Wiedemann syndrome and assisted reproductive technology: a case series of 19 patients. Fertil Steril 2005;83:349–54. Ludwig M, Katalinic A, Gross S, Sutcliffe A, Varon R, Horsthemke B. Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Genet 2005;42:289–91. Fortier AL, Lopes FL, Darricarrere N, Martel J, Trasler JM. Superovulation alters the expression of imprinted genes in the midgestation mouse placenta. Hum Mol Genet 2008;17:1653–65. Jackson RA, Gibson KA, Wu YW, Croughan MS. Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004;103:551–63. Schieve LA, Cohen B, Nannini A, Ferre C, Reynolds MA, Zhang Z, et al. A population-based study of maternal and perinatal outcomes associated with assisted reproductive technology in Massachusetts. Matern Child Health J 2007;11:517–25. Sutcliffe AG, Ludwig M. Outcome of assisted reproduction. Lancet 2007;370: 351–9. Zhu JL, Basso O, Obel C, Bille C, Olsen J. Infertility, infertility treatment, and congenital malformations: Danish national birth cohort. BMJ 2006;333:679. Miles HL, Hofman PL, Peek J, Harris M, Wilson D, Robinson EM, et al. In vitro fertilization improves childhood growth and metabolism. J Clin Endocrinol Metab 2007;92:3441–5. Moore T, Haig D. Genomic imprinting in mammalian development: a parental tug-of-war. Trends Genet 1991;7:45–9. Barlow DP, Stoger R, Herrmann BG, Saito K, Schweifer N. The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus. Nature 1991;349:84–7. DeChiara TM, Robertson EJ, Efstratiadis A. Parental imprinting of the mouse insulin-like growth factor II gene. Cell 1991;64:849–59. Monk D, Arnaud P, Apostolidou S, Hills FA, Kelsey G, Stanier P, et al. Limited evolutionary conservation of imrpinting in the human placenta Proc Natl Acad Sci U S A 2006;103:6623–8. Killian JK, Nolan CM, Wylie AA, Li T, Vu TH, Hoffman AR, et al. Divergent evolution in M6P/IGF2R imprinting from the Jurassic to the Quaternary. Hum Mol Genet 2001;10:1721–8. Fowden AL. The insulin-like growth factors and feto-placental growth. Placenta 2003;24:803–12. Tycko B, Morison IM. Physiological functions of imprinted genes. J Cell Physiol 2002;192:245–58. Reik W, Davies K, Dean W, Kelsey G, Constancia M. Imprinted genes and the coordination of fetal and postnatal growth in mammals. Novartis Found Symp 2001;237:19–31. Apostolidou S, Abu-Amero S, O’Donoghue K, Frost J, Olafsdottir O, Chavele KM, et al. Elevated placental expression of the imprinted PHLDA2 gene is associated with low birth weight. J Mol Med 2007;85:379–87. Reik W, Constancia M, Fowden A, Anderson N, Dean W, Ferguson-Smith AC, et al. Regulation of supply and demand for maternal nutrients in mammals by imprinted genes. J Physiol 2003;547:35–44. Abu-Amero S, Moore GE. Imprinting. In: Pijnenborg R, Brosens I, Romero R. Human Placental Bed Vascular Failure: Basic Science and Clinical Management. Cambridge: Cambridge University Press (in press). Lefebvre L, Viville S, Barton SC, Ishino F, Keverne EB, Surani MA. Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest. Nat Genet 1998;20:163–9. Li L, Keverne EB, Aparicio SA, Ishino F, Barton SC, Surani MA. Regulation of maternal behavior and offspring growth by paternally expressed Peg3. Science 1999;284:330–3. Constancia M, Hemberger M, Hughes J, Dean W, Ferguson-Smith A, Fundele R, et al. Placentalspecific IGF-II is a major modulator of placental and fetal growth. Nature 2002;417:945–8. Monk D, Sanches R, Arnaud P, Apostolidou S, Hills FA, Abu-Amero S, et al. Imprinting of the IGF2 P0 transcript and novel alternatively spliced INS-IGF2 isoforms show differences between mouse and human. Hum Mol Genet 2006;15:1259–69. Mayer W, Hemberger M, Frank HG, Grummer R, Winterhager E, Kaufmann P, et al. Expression of the imprinted genes MEST/Mest in human and murine placenta suggests a role in angiogenesis. Dev Dyn 2000;217:1–10.

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Eggenschwiler J, Ludwig T, Fisher P, Leighton PA, Tilghman SM, Efstratiadis A. Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith–Wiedemann and Simpson–Golabi–Behmel syndromes. Genes Dev 1997;11:3128–42. Takahashi K, Kobayashi T, Kanayama N. p57(Kip2) regulates the proper development of labyrinthine and spongiotrophoblasts. Mol Hum Reprod 2000;6:1019–25. Frank D, Fortino W, Clark L, Musalo R, Wang W, Saxena A, et al. Placental overgrowth in mice lacking the imprinted gene Ipl. Proc Natl Acad Sci U S A 2002;99:7490–5. Zwart R, Sleutels F, Wutz A, Schinkel AH, Barlow DP. Bidirectional action of the Igf2r imprint control element on upstream and downstream imprinted genes. Genes Dev 2001;15:2361–6. Constancia M, Kelsey G, Reik W. Resourceful imprinting. Nature 2004;432:53–7. Cox GF, Burger J, Lip V, Mau UA, Sperling K, Wu BL, et al. Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet 2002;71:162–4. Debaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith– Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 2003;72:156–60. Gicquel C, Gaston V, Mandelbaum J, Siffroi JP, Flahault A, Le Bouc Y. In vitro fertilization may increase the risk of Beckwith–Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene. Am J Hum Genet 2003;72:1338–41. Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, et al. Beckwith– Wiedemann syndrome and assisted reproduction technology (ART). J Med Genet 2003;40:62–4. Orstavik KH, Eiklid K, van der Hagen CB, Spetalen S, Kierulf K, Skjeldal O, et al. Another case of imprinting defect in a girl with Angelman syndrome who was conceived by intracytoplasmic semen injection. Am J Hum Genet 2003;72:218–19. Lucifero D, Chaillet JR, Trasler JM. Potential significance of genomic imprinting defects for reproduction and assisted reproductive technology. Hum Reprod Update 2004;10:3–18. Arnaud P, Feil R. Epigenetic deregulation of genomic imprinting in human disorders and following assisted reproduction. Birth Defects Res C Embryo Today 2005;75:81–97. Maher ER. Imprinting and assisted reproductive technology. Hum Mol Genet 2005;14 Spec No 1:R133–8. Amor DJ, Halliday J. A review of known imprinting syndromes and their association with assisted reproductive techniques. Hum Reprod 2008;23:2826–34. Li T, Vu TH, Ulaner GA, Littman E, Ling JQ, Chen HL, et al. IVF results in de novo DNA methylation and histone methylation at an Igf2-H19 imprinting epigenetic switch. Mol Hum Reprod 2005;11:631–40. Lim D, Bowdin SC, Tee L, Kirby GA, Blair E, Fryer A, et al. Clinical and molecular genetic features of Beckwith–Wiedemann syndrome associated with assisted reproductive technologies. Hum Reprod 2009;24:741–7. Ubeda F. Evolution of genomic imprinting with biparental care: implications for Prader–Willi and Angelman syndromes. PLoS Biol 2008;6:e208.

6 Chapter 6

Fetal stem cell therapy Jennifer Ryan, Michael Ting and Nicholas Fisk

Introduction Recent advances in molecular diagnostics and imaging technology now provide an unprecedented capacity for prenatal identification of a wide range of serious genetic and chromosomal disorders. Early diagnosis has far exceeded our ability to correct debilitating disorders, especially those for which there is no satisfactory postnatal treatment, and this has been the impetus for research into prenatal therapy. The rationale is that early detection allows early treatment, thus potentially curing a uniformly fatal disorder or preventing irreversible postnatal sequelae, especially those affecting the central nervous system. Stem cells hold far-reaching possibilities for the treatment of both acquired and congenital diseases. They can be used therapeutically to replace dysfunctional cells and tissues,1 or via ex vivo genetic manipulation to reconstitute a missing gene product (stem cell-based gene therapy).2 Stem cell transplantation in utero offers the exciting prospect of effectively treating inherited haematological, metabolic and other earlyonset genetic diseases. This chapter addresses the current status of fetal stem cell therapy, its limitations and its future development.

Sources of stem cells Stem cells are rare primitive cells that share two distinct properties regardless of their source: 1. the capacity for self-renewal 2. multi-lineage potential. Embryonic stem (ES) cells from the inner cell mass have the advantage of pluripotency or even totipotency but their clinical use is hindered by the real possibility of teratoma formation in vivo, which largely limits clinical application to tissue engineering rather than cell therapy. There are also ethical concerns in some sectors, although these are obviated by the increasing ability to reprogramme somatic cells into inducible pluripotent stem (iPS) cells, with properties otherwise similar to ES cells.3 Adult stem cells have been found in a diverse range of tissues and organs (neural stem cells in the brain, myogenic stem cells from muscle and stem cells can also be derived from adipose tissue) but are traditionally isolated from the bone marrow. The best© Jennifer Ryan, Michael Ting and Nicholas Fisk. Volume compilation © RCOG

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characterised populations in bone marrow are haematopoietic stem cells (HSCs) that can reconstitute haematopoietic lineages in vivo and which are widely used in bone marrow transplantation (BMT).4 Bone marrow also contains a population of mesenchymal stem cells (MSCs).5 These are multipotent stromal-like cells capable of differentiating into several lineages, chiefly fat, cartilage and bone.6,7 Adult MSCs, unlike ES cells, do not form teratomas and are free of any ethical concern but grow more slowly and have limited differentiation potential. Haematopoietic stem cells Cord blood is a rich source of haematopoietic progenitors8 and is now banked at birth for this purpose in families at risk of requiring a human leucocyte antigen (HLA)matched HSC transplant.8,9 Since the first use of HSCs from umbilical cord blood to treat children with Fanconi anaemia,10 more than 10 000 cord blood transplants have been performed, with more than 300 000 units of cord blood banked and ready for clinical use.11 Cord blood HSCs are now an accepted alternative to BMT for a wide range of childhood and even adult indications. They have also been used as vehicles for ex vivo gene therapy, largely successfully, to treat children with X-linked severe combined immunodeficiency (SCID).12 HSCs from cord blood have greater proliferative capacity than their adult counterparts but are limited in number.13 First-trimester fetal blood,14 bone marrow, liver15 and the placenta16 are also rich sources of HSCs. These peak in fetal blood in the early mid trimester, most likely owing to HSC migration as part of the shift from liver- to marrow-based myelopoiesis.17 Fetal HSCs have a number of functional advantages over cord blood and adult bone marrow HSCs, including a greater proliferative rate and a ten-fold competitive engraftment advantage (fetal liver HSC) when given to SCID mice in utero, with better reconstitution of all haematopoietic lineages.18 Translating this clinically, however, has been largely unsuccessful19 and has only been achieved in fetuses with X-linked SCID. This was first demonstrated by Flake et al.20 and Wengler et al.,21 both studies using paternal HSC to provide robust evidence of immune cell reconstitution after serial transplants of haematopoietic cells in utero. A single injection of fetal liver cells in utero was then given to a male fetus with X-linked SCID at 14 weeks of gestation. Donor cell engraftment reached 50% at 33 weeks of gestation with complete T- and NK-cell reconstitution at birth.22 However, it is now generally recognised that, because of the defective immune system in fetuses, transplantation should instead be delayed until after birth. Accordingly, this review will focus on tissue or mesenchymal stem cells. Fetal mesenchymal stem cells Fetal MSCs (fMSCs) have properties intermediate between ES and adult stem cells and can be derived from a range of pregnancy-related tissues, including first-trimester fetal blood, liver and bone marrow,23 second-trimester liver, lung, pancreas, bone marrow24 and amnotic fluid,25,26 and term placenta, cord and membranes.27 Although these cells express embryonic pluripotency markers as do ES and iPS, fMSCs do not form teratomas in vivo.28,29 Bone marrow, liver and blood The fetal haematopoietic organs are a ready source of MSCs but the frequency of circulating MSCs decreases after the first trimester of human pregnancy, when

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stromal/mesenchymal progenitors make up 0.4% of nucleated cells.23 Previously, these cells were collected by cardiocentesis at termination of pregnancy. However, advances in embryofetoscopy and ultrasound-guided cord sampling now allow fetal blood MSCs to be collected with sufficient safety during continuing pregnancies to harvest fMSC for ex vivo manipulation and autologous gene therapy (Figure 6.1).30 fMSCs share several characteristics with adult MSCs when grown in vitro, including fibroblastic morphology and surface marker expression. Early fMSCs are arguably the best characterised and have several advantages over their adult counterparts: n fMSCs have greater expansion capacity and a faster doubling time than adult MSCs, achieving 50 population doublings without differentiating.23 When compared with adult adipose tissue (AT) MSCs and cord blood MSCs, fMSCs had the faster doubling time (32 versus 54–111 hours) and the greatest colonyforming unit-fibroblast (CFU-F) capacity (1.6–2.0×), demonstrating that the gestational age and anatomical origins of MSCs have profound influences on their proliferative capacity.31 n fMSCs express telomerase and hTERT, and have longer telomeres, thus rendering them relatively resistant to senescence.28 n fMSCs have a greater differentiation capacity than adult MSCs and more readily differentiate into lineages such as skeletal muscle,32 neurons, oligodendrocytes,33 hepatocytes, endothelial cells and even blood in vivo.34 This more primitive status is consistent with fetal but not adult MSCs expressing the pluripotent stem cell markers Oct-4, Nanog, Rex-1, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81.28,35 Further advantages of fMSCs include their lack of intracellular HLA class  II and relative resistance to induction with interferon-gamma (IFN-γ),36,37 making them

Figure 6.1 Ultrasound-guided fetal blood sampling at 10 weeks of gestation to harvest circulating mesenchymal stem cells; adapted with permission from Chan et al. (2008)30

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less immunogenic.36 Additionally, fMSCs express the lowest HLA-I (55%) compared with adult AT-MSCs and cord blood MSCs (95–99%).31 Like adult MSCs, fMSCs are immunoprivileged and do not elicit an allogeneic immune response, favouring transplantation across immune barriers without the need for tissue typing.36,38 The ontological and anatomical origins of MSCs also influence their lineage predisposition. fMSCs show preferential bone differentiation in vitro and in vivo, suggesting utility in both congenital and acquired disease.39 In vivo, fMSCs compared with adult and term sources resulted in the most robust mineralisation after implantation onto cellular scaffolds in immunodeficient mice.31 There seems to be a hierarchy within fetal sources as well, as first-trimester fetal bone marrow-derived fMSCs had higher osteogenic gene expression compared with fetal blood and fetal liver fMSCs.39 Furthermore, Stro-1, a marker associated with the osteogenic progenitor fraction found within MSC cultures, was more frequently expressed by fMSCs compared with AT-MSCs and cord blood MSCs (51% versus 10–27%), which may in part explain their osteogenic superiority over later MSC types.31 Placenta and membranes fMSCs can also be isolated from human placental tissues at any stage of gestation.27,34,40,41 At or near term, placental tissues are otherwise discarded at birth, while earlygestation placental tissue can be obtained from surplus diagnostic yield at chorionic villus sampling (CVS) or in greater quantity from tissue discarded after termination of pregnancy. Placental-like fMSCs have increased differentiation potential compared with adult MSCs and give rise to endothelial and neurogenic lineages, as confirmed by several groups.41–43 Studies have shown that fMSCs can also be isolated from the amnion and the chorionic membranes of the placenta.44–46 Ontological stage is likely to influence the biological properties of placental fMSCs as well, and fMSCs isolated from first-trimester chorionic villi should be a more primitive source compared with later gestational placental fMSCs.43,47 Notwithstanding this, a study comparing early with late adnexal MSCs suggested that cell source rather than gestational age was the more important influence on differentiation capacity. Osteogenic, chondrogenic and myogenic differentiation was higher in chorion-derived fMSCs, whereas adipogenic and neuronal cell differentiation was higher in amnion-derived fMSCs.43 As expected, fetal membrane-derived MSCs express very low major histocompatibility complex (MHC) class I and almost no MHC class II on their surface and have immunosuppressive ability.48 They also express the primitive markers SSEA-4, Oct-4 and Nanog.49 Fetal placenta represents a heterogeneous population of stem cells, including human amniotic epithelial cells, human amniotic mesenchymal stromal cells, human chorionic mesenchymal stromal cells and human chorionic trophoblastic cells.50 Cells from each layer demonstrate variable plasticity and make it difficult to determine true stemness. It is of note that it is readily possible to grow maternal decidual MSCs from term placenta51 and in this regard an international consensus statement requires that placental MSCs be shown to be fetal in origin.50 Amniotic fluid Amniotic fluid (AF) is another attractive source of MSCs for transplantation as, like CVS, it can be obtained from routine diagnostic procedures, including as an autologous source in continuing pregnancies. MSCs have now been isolated, cultured and enriched from second-trimester AF by several groups.25,26,52 The prevalence of AF-MSCs in second-trimester samples ranges from 0.9% to 1.5%,

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evaluated by gating a CD45-negative and CD90, CD73, CD105 and CD44 population.53 AF-MSCs were successfully isolated from 80 of 118 liquor samples collected at between 15 and 18 weeks of gestation.53 In contrast, MSCs were found in only two of ten term AF samples.54 Consistent with the primitiveness of fetal and placental-derived MSCs, these cells also express the pluripotency markers Oct-4, Nanog and SSEA-4.53,55 In contrast to early work on fMSCs, AF-MSCs and amnion-derived MSCs have been shown to be immunosuppressive in mixed lymphocyte reactions and to inhibit T-cell proliferation.56 Interestingly, these differences cannot be explained by gestational age, since first-trimester fetal liver38 and second-trimester AF-MSCs and amnion-derived MSCs were the sources for these two respective studies.56 Thus, AF and amnion might be the better source of immunosuppressive MSCs compared with fetal liver, although this has yet to be tested in vivo and no alloreactivity against donor cells could be discerned after in utero transplantation of a first-trimester liver MSC during third-trimester pregnancy.57 These findings may have clinical relevance, given the increasing recognition that allogeneic fMSCs may be used for in utero and possibly postnatal transplantation without the risk of rejection. Umbilical cord In contrast to HSCs, MSCs are rare in cord blood and are typically only found in around one-third of cord blood samples.9,58 This belies the heavy marketing of commercial directed cord blood banks as a long-term source of autologous MSCs.11 On the other hand, Wharton’s jelly (WJ), the connective tissue of the human umbilical cord, is a rich and reliable source of MSCs.59 The major advantage of WJMSCs compared with cord blood is that they can be isolated from close to 100% of samples, even from umbilical cords processed as late as 48 hours after delivery.60 Cord blood MSCs and WJ-MSCs share several properties, such as poor ability to differentiate into adipocytes,58,61,62 shorter doubling times than adult bone marrow MSCs and karyotypic stability over many passages before reaching senescence.61,63 Similar to fMSCs, cord MSCs express the primitive stem cell markers Nanog, Oct-4, Sox-2, Rex-1, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81.31,64,65 As well as mesenchymal tri-lineage potential, these cells can differentiate into muscle and neural cells.66 Perhaps the richest postnatal source of MSCs is human umbilical cord perivascular cells,, which occur as frequently as one in 300 nucleated cells.67 They exhibit a short doubling time of 20 hours at passage 2 and can produce over 1010 cells in 1 month of culture.67,68 Table 6.1 summarises the various mesenchymal stem cell sources.

Development of in utero transplantation A wide range of metabolic and haematological disorders in children can now be successfully treated by BMT. These require both irradiation and chemoablation before haematopoietic reconstitution to rid the body of recipient immune cells that might otherwise reject the transplant. The considerable morbidity associated with BMT could be avoided if stem cells were transplanted in utero. Attempts at HSC transplantation indicate that the window of immunological naïvety in the human may be over by the end of the first trimester. However, this barrier may not be so problematic with MSC transplantation in utero, given their immunomodulatory and immunotolerant properties.

88  |  JENNIFER RYAN, MICHAEL TING AND NICHOLAS FISK Table 6.1

Summary of mesenchymal stem cell sources

Source Adult Bone marrow Adipose tissue Fetal Umbilical cord blood Umbilical cord Placenta Amniotic fluid

Stem cell Bone marrow haematopoietic stem cells Bone marrow mesenchymal stem cells Adipose tissue mesenchymal stem cells Haematopoietic stem cells Mesenchymal stem cells Wharton’s jelly mesenchymal stem cells Human umbilical cord perivascular cells Amniotic mesenchymal stromal cells Chorionic mesenchymal stromal cells Amniotic fluid mesenchymal stem cells

Rationale In utero trasplantation (IUT) of stem cells has several potential advantages over postnatal transplantation, as listed in Box 6.1. These include a clear stoichiometric advantage in that the fetus weighs only 30 g at the end of the first trimester, which is less than 1% of the weight at term let alone that of a child, thus allowing transplantation of a much larger number of cells as a proportion of total body mass. The relative immaturity of the fetal immune system should facilitate graft acceptance and long-term take. Moreover, the ability of the first-trimester immune system to become tolerant of foreign antigens opens up the possibility of postnatal top-up allogeneic cell transplantation without risk of immunological rejection.69 The fetal circulation largely bypasses the lungs, where MSCs preferentially sequester after intravenous delivery in adults.70 The fetal environment is highly conducive to expansion of stem cell compartments; indeed, large-scale migration of stem cells occurs naturally only in fetal life. Consistent with this, there is evidence that fMSCs cross the placenta to engraft in pregnant women, and contribute endogenously to postreproductive tissue repair71,72 Although some of these postulated benefits of cell delivery in utero have been challenged, the scientific basis for IUT is supported by animal studies, as discussed below. Preclinical development in wild types Proof of concept for IUT has been demonstrated using HSCs in a variety of xenogeneic models, including human–mouse,73 human–sheep,74 human–baboon,75,76 goat–sheep77 Box 6.1 Rationale and theoretical advantages of in utero transplantation • Early treatment of disease in utero prior to development of irreversible tissue damage • Immune naïvety of fetus • Induction of immune tolerance to alloantigens • Avoidance of myeloablation and its complications • Rapid growth of fetus provides for engraftment and expansion of donor cells • Small size of fetus yields a stoichiometric advantage • Fetal circulation bypasses lungs, avoiding mesenchymal stem cell entrapment

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and rat–mouse78 combinations. Furthermore, murine HSCs transplanted in utero have been shown to prevent fatal defects in autosomal recessive osteopetrosis.79 However, successful outcomes in humans have been limited to immunodeficient fetuses, for which transplantation can thus be more safely deferred postnatally.20–22 In contrast, MSCs have unique immunological characteristics that allow persistence in a xenogeneic environment, seemingly regardless of gestational age or immunological competence.80 Long-term engraftment and site-specific differentiation of human adult and fetal MSCs has been demonstrated after IUT into fetal lamb recipients.80,81 First-trimester human fetal blood MSCs transplanted in utero into immunocompetent Mf1 wild type fetal mice engraft and persist postnatally.82 In a more recent study, human fetal placental MSCs showed long-term persistence in immunocompetent rats after IUT.34 Engrafted cells were found in multiple fetal tissues (brain, heart, lung, liver and spleen) and survived postnatally for at least 12 weeks, with evidence of hepatocyte differentiation in the liver and haematocyte differentiation in the rat circulation.34 A feature of all these studies is the low level of donor chimerism achieved, with increased engraftment likely to require tissue injury at the time of transplantation to facilitate homing. Preclinical development in disease models A number of animal models of human diseases suitable for fetal stem cell therapy have been investigated, mainly in the field of muscle and bone repair. Muscular dystrophy Duchenne muscular dystrophy is an X-linked recessive disease characterised by the loss of dystrophin.83 This disease affects one in 3500 males, making it the most prevalent of muscular dystrophies. People with severe forms often succumb from respiratory or cardiac failure, with average life expectancy varying from the early teens to the mid-30s.84,85 fMSCs have been shown to differentiate down the myogenic lineage and can contribute to muscle regeneration in both injured and dystrophic muscle, which underpins their potential use in fetal transplantation for muscle regeneration.6,86 Supporting this, Gang et al.87 showed that MSCs isolated from human cord blood have the capacity to differentiate into skeletal myogenic cells. Early fetal but not adult MSCs differentiate readily into myotubes when cultured with galectin-1, achieving the highest myoconversion rate of any untransformed human stem cell type.86 A further rationale for an IUT approach in Duchenne muscular dystrophy is that systemic delivery to all muscle groups is required, in particular cardiac and respiratory muscles, whereas postnatal approaches seem limited to local delivery because MSCs get trapped in the lungs by virtue of their size. IUT may circumvent these obstacles by taking advantage of the fetal circulation to access global muscle groups with systemic therapy for dystrophic fetuses. We evaluated this in an in utero xenotransplantation paradigm of human fMSCs into fully immunocompetent dystrophic fetal mice, which resulted in widespread, albeit low-level, long-term engraftment over 19  weeks in multiple organ compartments with evidence of myogenic differentiation in skeletal and myocardial muscle.82 Although there was a five-fold predilection for muscle compared with non-muscle tissues, the low-level chimerism achieved (less than 1%) was insufficient for phenotypic improvement. We attributed this to the minimal muscle pathology seen in utero in mdx mice, and next moved to models with more substantial tissue injury in utero, such as skeletal dysplasia.

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Osteogenesis imperfecta Osteogenesis imperfecta (OI) is a rare genetic disorder, occurring in one in 10 000 births, where systemic osteopenia results in skeletal deformities, bone fragility, fractures and short stature. It ranges from the milder type I through the moderately severe types III and IV to the perinatally lethal type II.88,89 OI results from mutations predominantly affecting one of two collagen genes, COL1A1 or COL1A2, which in turn produce type I collagen with abnormal helical folding, leading to an unstable and more hydrophilic protein structure. Currently, bisphosphonate treatment is used empirically to increase bone density and possibly reduce fractures but it does not address the underlying collagen defect.90 Our group previously showed that IUT of early fMSCs into a mouse model of type  III OI can reduce bone fractures by over 60% (Figure  6.2), thus supporting their potential to treat bone defects in human fetuses.91 A fetal source for this fetalto-fetal approach was chosen based on the superior osteogenic differentiation of fMSCs compared with adult or human umbilical cord MSCs.31,39 Transplantation was associated with increased bone strength, thickness and length and a reduction in abnormal growth plate height in IUT-treated mice.91 fMSCs preferentially engrafted in bone, where there was evidence they were recruited to sites of active bone formation, differentiated into osteoblasts, and produced the missing α2 chain of type  I collagen. The observed improvement in skeletal phenotype from fMSC transplantation was associated with engraftment levels in bone of only around 5%. However, this was comparable to engraftment levels seen in a paediatric bone marrow/MSC transplantation trial associated with phenotypic improvement, as well

Figure 6.2 Reduction in incidence of fractured bones in humerus, ulna, femur and tibia of 4-weekold (n = 15), 8-week-old (n = 15) and 12-week-old (n = 6) osteogenesis imperfecta mice (OIM) treated with in utero transplantation (IUT) compared with untreated OIM; n = 20 for each group; * P < 0.01; adapted with permission from Guillot et al.91

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as a single clinical case of fMSC IUT in a human fetus with OI resulting in long-term chimerism and lack of alloreactivity to donor MSCs.57,92

Clinical development of in utero transplantation The range of candidate diseases includes not only the classic haemoglobinopathies, immunological disorders and in-born errors of metabolism (such as lysosomal storage disorders and leucodystrophies) attempted so far with HSC IUT, but also genetic diseases more recently suggested as amenable to MSC therapy and that feature chronic tissue injury beginning in utero such as skeletal dysplasias and muscular dystrophies. In utero transplantation with haematopoietic stem cells Over 40 IUTs have been attempted to date with HSCs.19 Currently only IUT for human immunodeficiency disorders such as SCID has proved successful. However, because these transplants occurred after the window of fetal immunological naïvety, HSC transplantation in this disease is now deferred until postnatally, when it does not expose the pregnancy to any procedure-related risk. Notwithstanding this, there is some evidence in a mouse model of X-SCID to support greater efficacy of engraftment and superior B- and T-cell reconstitution with IUT compared with neonatal and adolescent transplantation.93 IUT with HSCs has also been attempted for a number of other haematological and non-haematological diseases, including sickle cell anaemia,94 thalassaemia95 and the lysosomal storage disorders Hurler syndrome96 and Niemann–Pick disease,97 but resulted in only low-level chimerism without significant clinical improvement. Mesenchymal stem cells for osteogenesis imperfecta Paediatric experience Postnatal stem cell therapy, such as BMT in children with OI, has also been attempted to ameliorate peripheral tissue damage. Horwitz et al.92,98 first reported that allogeneic BMT in three of five children with type III OI in which osteoblasts engrafted was associated with a 44–77% increased bone mineral content, improved linear growth and reduced fracture frequency. However, bone marrow contains not only precursors for the haematopoietic system but also MSCs that can give rise to mesenchymal lineages, including bone and cartilage. Although BMT postnatally may lead to clinical improvement, there are a number of major potential complications, including transplant-related mortality of 15–35%, morbidity related to conditioning therapy and the development of graft-versus-host disease. The same authors then infused same-donor MSCs (two doses 8–21  days apart, of 1 and then 3–5 × 106 per kg body weight) in an expanded cohort of previously transplanted children with type  III OI.99 Growth velocity increased from 20% of predicted before to 70% afterwards in five of six children with osteoblast engraftment, while the child with no engraftment, attributed to anti fetal bovine serum antibodies, showed no improvement.98 This makes OI the first genetic condition in humans in which MSC has been used therapeutically. Fetal experience Following the paediatric trial, an affected human fetus was administered rescue therapy by MSC transplantation in utero.57 Type  III OI was suspected based on

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ultrasound detection at 24  weeks of gestation and an IUT performed with fetal liver-derived fMSCs (6.5 × 106 cells) injected via the umbilical vein at 32  weeks (approximately 4.3 × 106 per kg body weight). The baby was delivered at 35  weeks of gestation in good health. Since then, she suffered two spontaneous fractures and had reasonable bone mineralisation of 76% of age-matched controls at 22  months, which may be attributed in part to parallel treatment with bisphosphonates. Bone biopsy at 9 months of age demonstrated a chimerism rate of 7.4% (range 6.8–16.6%), detected by Y chromosome fluorescence in situ hybridisation, with evidence of sitespecific differentiation into osteocytes detected as donor cells. Moreover, there was no alloreactivity found between the donor cells and the patient’s peripheral blood lymphocytes.57 Thus, the clinical effects were difficult to discern over the first 2 years, both in the presence of confounding biphosphonate therapy and in the absence of any control. However, the case is of interest in that it demonstrates the ability of allogeneic fetal liver-derived fMSCs to be transplanted across full MHC barriers and undergo site-specific differentiation. Anecdotally, a further four in utero transplants have been performed, mainly with adult MSCs, but few details are yet available.19

Route to clinical translation The future success of other IUT-like stem cell therapies will require in-depth knowledge of the barriers that impede stem cell engraftment together with development of novel methods to enhance the competiveness of donor stem cells within the local environment, here the fetus. The safe and efficient ex vivo expansion of clinical-grade MSCs is also a key issue that needs to be addressed before introduction of stem cell therapy into clinical practice. Notwithstanding this, MSC transplantation has been attempted clinically in a handful of cases on a rescue basis. Barriers to engraftment Postnatal BMT typically requires myeloablation by irradiation or administration of cytotoxic drugs before infusion of HSCs so as to achieve host immunosuppression and free up niches for stem cell engraftment. Although advantageous for postnatal transplantation, it is not feasible for transplantation in utero owing to toxicity concerns, and may not be necessary given the immune naïve and ontological fetal environment. One alternative to promote engraftment is to administer a larger dose of donor cells to give a numerical advantage to donor over endogenous stem cells. Indeed, this is the stoichiometric basis of the rationale for IUT. The concept of spatial niches represents a dynamic equilibrium between stem cell proliferation and affinity for cellular niches. In terms of further development of MSC therapy for OI, Horwitz et al.100 demonstrated that engraftment of transplantable marrow osteoprogenitors is saturable with a maximal engraftment of about 15% of all bone cells in the epiphysis and metaphysis of the femur 3 weeks after transplantation. This suggests that the capacity for initial osteopoietic engraftment after BMT is limited and ‘megadose’ stem cell transplantation is unlikely to enhance engraftment. Thus, novel strategies to foster osteopoietic chimerism are required, such as manipulating chemokine and adhesion signalling in MSCs, for instance integrin subunit CD49d, or sialofucosylating CD44 to augment adhesion of stem cells and enhance homing to bone, as demonstrated experimentally in non-IUT paradigms.101–103 Transplantation of MSCs in animal models of tissue injury have demonstrated that they can migrate specifically to the site of damage and undergo tissue-specific differentiation.104,105 Indeed, significant injury appears to be responsible for the

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enhanced homing and engraftment of MSC seen in injured animals compared with wild type.82 This raises an important issue in regard to the use of fetal stem cell therapy as, on the one hand, significant tissue injury is required to facilitate uptake of MSCs but, on the other, the extent of tissue injury must not be so severe that it is refractory to therapy. This requirement for moderate injury in utero limits the number of diseases that may be treated by IUT. Type III OI is a disease that features sufficient early tissue damage in utero to be potentially amenable to IUT. However, there are difficulties associated with OI as a model for IUT therapies, including a recurrence risk of only 7%.106 Thus, most will be new diagnoses without prior family history, and will be unclear phenotypic diagnoses given the difficulty of making a precise molecular diagnosis with a new presentation on ultrasound in the absence of family history against the background of over 250 causative mutations. Production of clinical-grade mesenchymal stem cells Clinical trials using MSCs will require ready access to safe and functionally intact cells in sufficient numbers for transplantation into human patients. Despite the need for vast numbers of expanded human MSCs for therapeutic purposes, there is only limited information on the optimisation of culture conditions required for such production. Specifically, knowledge regarding selection, including optimal tissue sources50,107 and growth factors for proliferation enhancement,108 is needed, as is the effect of donor age and cryopreservation on stem cell function.109 Currently, there is considerable variation between laboratories concerning media, the starting and passaging cell-plating density, culture surfaces and supplementary factors for isolation and expansion of MSCs. Preparation of MSCs without the need for human or animal products is now essential for cell therapy applications. The avoidance of prion, viral or zoonose contamination is a significant advantage of serum-free conditions for MSCs. Indeed, it is hoped that production of chemically defined media will lead to less variability in growth characteristics than currently achieved with ill-defined fetal calf serum characterised by considerable inter-batch variability. Also, serum contains a variety of proteins that may attach to cells and act as antigenic substrates for immunological reactions once transplanted. In a number of clinical studies in which patients received infusions of MSCs cultured in fetal calf serum, newly formed antibodies against fetal calf serum could be detected, thus indicating that adverse immune complex formation, capable of affecting engraftment, can complicate cell transplantation.99,110 Until a chemically defined serum-free medium becomes widely available, autologous serum supplementation or the use of platelet lysate may offer safe alternatives to animal serum. Their use is particularly useful in the clinical setting on a single-patient basis. Autologous serum as well as platelet lysate has been shown to be indistinguishable from fetal calf serum with regard to both isolation and expansion of human MSCs.111–113 As the use of MSCs moves closer to clinical translation, there is a need for serum-free culture conditions that meet GMP (good manufacturing practice) standards. This will ensure that MSCs can be safely expanded ex vivo and maintain their important functional characteristics, such as homing, engraftment and differentiation. Rescue treatment Considerable preclinical development is required before clinical translation, as above. Even for OI, more work in mice models is needed on maximising cell delivery, on optimising engraftment, on comparing or packaging with bisphosphonate therapy and

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on the longevity of effect. Notwithstanding the above, the clinician may encounter a woman who wishes to continue a pregnancy with a fetus with a presumptive diagnosis of intermediate-severity OI. Given the limited but promising human and animal experience to date, experimental ‘rescue’ therapy may be able to be justified after informed consent on a one-off case-by-case basis, subject to stringent ethical, safety and oversight arrangements.

Conclusion The ability to deliver primitive cells to damaged tissues in utero offers a new therapeutic paradigm with the potential to cure a broad range of conditions not achieved by contemporary approaches. fMSCs have properties intermediate between ES and adult stem cells, making them attractive stem cells for tissue repair. fMSCs can be readily derived from a range of pregnancy-related tissues, including fetal blood, liver, bone marrow and amniotic fluid, and placenta, cord and membranes. Studies showing enhanced fetal stem cell engraftment in bone and muscle provide an early scientific basis for downstream translation to treat acquired and inherited diseases. Stem cell transplantation in utero offers the prospect of delivering ontologically appropriate stem cells across immune boundaries without the need for postnatal myeloablation, and so effectively treating a range of inherited haematological, metabolic and other early-onset genetic diseases. However, clinical experience with over 40 cases of HSC transplantation is that it only works in immunodeficiency conditions, which can instead be treated postnatally. More recent attention has turned to fMSCs, which owing to their immunosuppressive properties can be transplanted across MHC barriers. Preclinical and early clinical experience in OI suggests MSCs preferentially engraft in bone, undergo osteoblastic differentiation and ameliorate the phenotype. Fetal stem cell therapy requires considerable preclinical development before clinical translation but rescue treatment has been attempted in a few continuing pregnancies with intermediate-severity fetal OI. From a research perspective, therapeutic interventions using stem cell technology are still in their infancy and the challenge is now to optimise cell engraftment and tissue repair in in utero recipients.

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Parolini O, Alviano F, Bagnara GP, Bilic G, Buhring HJ, Evangelista M, et al. Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells. Stem Cells 2008;26:300–11. Barlow S, Brooke G, Chatterjee K, Price G, Pelekanos R, Rossetti T, et al. Comparison of human placenta- and bone marrow-derived multipotent mesenchymal stem cells. Stem Cells Dev 2008;17:1095–107. In ‘t Anker PS, Scherjon SA, Kleijburg-van der Keur C, Noort WA, Claas FH, Willemze R, et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003;102:1548–9. Roubelakis MG, Pappa KI, Bitsika V, Zagoura D, Vlahou A, Papadaki HA, et al. Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells. Stem Cells Dev 2007;16:931–52. In ‘t Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, Fibbe WE, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004;22:1338–45. Prusa AR, Marton E, Rosner M, Bernaschek G, Hengstschlager M. Oct-4-expressing cells in human amniotic fluid: a new source for stem cell research? Hum Reprod 2003;18:1489–93. Roelen DL, van der Mast BJ, in’t Anker PS, Kleijburg C, Eikmans M, van Beelen E, et al. Differential immunomodulatory effects of fetal versus maternal multipotent stromal cells. Hum Immunol 2009;70:16–23. Le Blanc K, Gotherstrom C, Ringden O, Hassan M, McMahon R, Horwitz E, et al. Fetal mesenchymal stem-cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta. Transplantation 2005;79:1607–14. Bieback K, Kern S, Kluter H, Eichler H. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 2004;22:625–34. Wang HS, Hung SC, Peng ST, Huang CC, Wei HM, Guo YJ, et al. Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem Cells 2004;22:1330–7. Weiss ML, Medicetty S, Bledsoe AR, Rachakatla RS, Choi M, Merchav S, et al. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson’s disease. Stem Cells 2006;24:781–92. Karahuseyinoglu S, Cinar O, Kilic E, Kara F, Akay GG, Demiralp DO, et al. Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys. Stem Cells 2007;25:319–31. Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24:1294–301. Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells 2007;25:1384–92. Hoynowski SM, Fry MM, Gardner BM, Leming MT, Tucker JR, Black L, et al. Characterization and differentiation of equine umbilical cord-derived matrix cells. Biochem Biophys Res Commun 2007;362:347–53. Jo CH, Kim OS, Park EY, Kim BJ, Lee JH, Kang SB, et al. Fetal mesenchymal stem cells derived from human umbilical cord sustain primitive characteristics during extensive expansion. Cell Tissue Res 2008;334:423–33. Troyer DL, Weiss ML. Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells 2008;26:591–9. Sarugaser R, Ennis J, Stanford WL, Davies JE. Isolation, propagation, and characterization of human umbilical cord perivascular cells (HUCPVCs). Methods Mol Biol 2009;482:269–79. Sarugaser R, Lickorish D, Baksh D, Hosseini MM, Davies JE. Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells 2005;23:220–9. Lee PW, Cina RA, Randolph MA, Goodrich J, Rowland H, Arellano R, et al. Stable multilineage chimerism across full MHC barriers without graft-versus-host disease following in utero bone marrow transplantation in pigs. Exp Hematol 2005;33:371–9. Schrepfer S, Deuse T, Reichenspurner H, Fischbein MP, Robbins RC, Pelletier MP. Stem cell transplantation: the lung barrier. Transplant Proc 2007;39:573–6. O’Donoghue K, Chan J, de la Fuente J, Kennea N, Sandison A, Anderson JR, et al. Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy. Lancet 2004;364:179–82.

98  |  JENNIFER RYAN, MICHAEL TING AND NICHOLAS FISK 72. 73.

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Santos MA, O’Donoghue K, Wyatt-Ashmead J, Fisk NM. Fetal cells in the maternal appendix: a marker of inflammation or fetal tissue repair? Hum Reprod 2008;23:2319–25. Pallavicini MG, Flake AW, Madden D, Bethel C, Duncan B, Gonzalgo ML, et al. Hemopoietic chimerism in rodents transplanted in utero with fetal human hemopoietic cells. Transplant Proc 1992;24:542–3. Zanjani ED, Pallavicini MG, Ascensao JL, Flake AW, Langlois RG, Reitsma M, et al. Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero. J Clin Invest 1992;89:1178–88. Muirhead DY, Kuehl TJ, Vandeberg JL, Menchaca EM, Downs MP, Roodman GD. Mixed lymphocyte culture reactivity of fetal baboons: application for in utero bone marrow transplantation. Bone Marrow Transplant 1990;6:263–7. Shields LE, Bryant EM, Easterling TR, Andrews RG. Fetal liver cell transplantation for the creation of lymphohematopoietic chimerism in fetal baboons. Am J Obstet Gynecol 1995;173:1157–60. Colas G, Hollands P, Locatelli A, Le Vern Y, Cotinot C, Canepa S, et al. The xenotransplantation of goat and human hematopoietic cells to sheep fetuses. Transplantation 1999;67:984–90. Rice HE, Hedrick MH, Flake AW. In utero transplantation of rat hematopoietic stem cells induces xenogeneic chimerism in mice. Transplant Proc 1994;26:126–8. Frattini A, Blair HC, Sacco MG, Cerisoli F, Faggioli F, Cato EM, et al. Rescue of ATPa3deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero. Proc Natl Acad Sci U S A 2005;102:14629–34. Liechty KW, MacKenzie TC, Shaaban AF, Radu A, Moseley AM, Deans R, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000;6:1282–6. MacKenzie T, Campagnoli C, Almeida-Porada G, Radu A, Fisk NM, Flake AW. Circulating human fetal stromal cells engraft and differentiate in multiple tissues following transplantation into pre-immune fetal lambs. Blood 2001;98:798a. Chan J, Waddington SN, O’Donoghue K, Kurata H, Guillot PV, Gotherstrom C, et al. Widespread distribution and muscle differentiation of human fetal mesenchymal stem cells after intrauterine transplantation in dystrophic mdx mouse. Stem Cells 2007;25:875–84. Hoffman EP, Brown RH Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:919–28. Melacini P, Vianello A, Villanova C, Fanin M, Miorin M, Angelini C, et al. Cardiac and respiratory involvement in advanced stage Duchenne muscular dystrophy. Neuromuscul Disord 1996;6:367–76. Carpenter S, Karpati G. Duchenne muscular dystrophy: plasma membrane loss initiates muscle cell necrosis unless it is repaired. Brain 1979;102:147–61. Chan J, O’Donoghue K, Gavina M, Torrente Y, Kennea N, Mehmet H, et al. Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration. Stem Cells 2006;24:1879–91. Gang EJ, Jeong JA, Hong SH, Hwang SH, Kim SW, Yang IH, et al. Skeletal myogenic differentiation of mesenchymal stem cells isolated from human umbilical cord blood. Stem Cells 2004;22:617–24. Forlino A, Marini JC. Osteogenesis imperfecta: prospects for molecular therapeutics. Mol Genet Metab 2000;71:225–32. Sillence DO, Senn A, Danks DM. Genetic heterogeneity in osteogenesis imperfecta. J Med Genet 1979;16:101–16. Glorieux FH. Experience with bisphosphonates in osteogenesis imperfecta. Pediatrics 2007;119 Suppl 2:S163–5. Guillot PV, Abass O, Bassett JH, Shefelbine SJ, Bou-Gharios G, Chan J, et al. Intrauterine transplantation of human fetal mesenchymal stem cells from first-trimester blood repairs bone and reduces fractures in osteogenesis imperfecta mice. Blood 2008;111:1717–25. Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WW, Gordon PL, Neel M, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–13.

FETAL STEM CELL THERAPY  |  99 93. 94.

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Liuba K, Pronk CJ, Stott SR, Jacobsen SE. Polyclonal T cell reconstitution of X-SCID recipients following in utero transplantation of lymphoid-primed multipotent progenitors. Blood 2009;113:4790–8. Westgren M, Ringden O, Eik-Nes S, Ek S, Anvret M, Brubakk AM, et al. Lack of evidence of permanent engraftment after in utero fetal stem cell transplantation in congenital hemoglobinopathies. Transplantation 1996;61:1176–9. Touraine JL, Raudrant D, Royo C, Rebaud A, Barbier F, Roncarolo MG, et al. In utero transplantation of hemopoietic stem cells in humans. Transplant Proc 1991;23:1706–8. Flake AW, Zanjani ED. In utero hematopoietic stem cell transplantation. A status report. JAMA 1997;278:932–7. Touraine JL, Raudrant D, Golfier F, Rebaud A, Sembeil R, Roncarolo MG, et al. Reappraisal of in utero stem cell transplantation based on long-term results. Fetal Diagn Ther 2004;19:305–12. Horwitz EM, Prockop DJ, Gordon PL, Koo WW, Fitzpatrick LA, Neel MD, et al. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 2001;97:1227–31. Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci U S A 2002;99:8932–7. Marino R, Martinez C, Boyd K, Dominici M, Hofmann TJ, Horwitz EM. Transplantable marrow osteoprogenitors engraft in discrete saturable sites in the marrow microenvironment. Exp Hematol 2008;36:360–8. Kumar S, Ponnazhagan S. Bone homing of mesenchymal stem cells by ectopic alpha 4 integrin expression. FASEB J 2007;21:3917–27. Chavakis E, Urbich C, Dimmeler S. Homing and engraftment of progenitor cells: a prerequisite for cell therapy. J Mol Cell Cardiol 2008;45:514–22. Sackstein R, Merzaban JS, Cain DW, Dagia NM, Spencer JA, Lin CP, et al. Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone. Nat Med 2008;14:181–7. Chapel A, Bertho JM, Bensidhoum M, Fouillard L, Young RG, Frick J, et al. Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiationinduced multi-organ failure syndrome. J Gene Med 2003;5:1028–38. Herrera MB, Bussolati B, Bruno S, Morando L, Mauriello-Romanazzi G, Sanavio F, et al. Exogenous mesenchymal stem cells localize to the kidney by means of CD44 following acute tubular injury. Kidney Int 2007;72:430–41. Thompson EM, Young ID, Hall CM, Pembrey ME. Recurrence risks and prognosis in severe sporadic osteogenesis imperfecta. J Med Genet 1987;24:390–405. Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001;98:2615–25. van den Bos C, Mosca JD, Winkles J, Kerrigan L, Burgess WH, Marshak DR. Human mesenchymal stem cells respond to fibroblast growth factors. Hum Cell 1997;10:45–50. Kotobuki N, Hirose M, Takakura Y, Ohgushi H. Cultured autologous human cells for hard tissue regeneration: preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs 2004;28:33–9. Sundin M, Ringden O, Sundberg B, Nava S, Gotherstrom C, Le Blanc K. No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients. Haematologica 2007;92:1208–15. Capelli C, Domenghini M, Borleri G, Bellavita P, Poma R, Carobbio A, et al. Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts. Bone Marrow Transplant 2007;40:785–91. Kobayashi T, Watanabe H, Yanagawa T, Tsutsumi S, Kayakabe M, Shinozaki T, et al. Motility and growth of human bone-marrow mesenchymal stem cells during ex vivo expansion in autologous serum. J Bone Joint Surg Br 2005;87:1426–33. Stute N, Holtz K, Bubenheim M, Lange C, Blake F, Zander AR. Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use. Exp Hematol 2004;32:1212–25.

7 Chapter 7

Prenatal gene therapy Khalil Abi-Nader and Anna David

Summary Prenatal gene therapy aims to deliver genes to cells and tissues early in prenatal life, allowing correction of a genetic defect before long-term tissue damage has occurred. In contrast to postnatal gene therapy, prenatal application has a number of advantages, including targeting genes to a large population of dividing stem cells, and the smaller fetal size, which allows a higher vector to target cell ratio to be achieved. Early-gestation delivery may result in the fetus developing immune tolerance to the transgenic protein, which would allow postnatal repeat vector administration if needed. Further treatment options include using transduced autologous or allogeneic stem cells to treat congenital fetal disease, or to treat obstetric conditions such as fetal growth restriction by delivering gene therapy to the mother. Recent advances in vector design and stem cell research have benefited this potential treatment. Although still in the preclinical stage, proof-of-principle studies in animal models of congenital disease, such as the haemophilia mouse, have shown the potential of prenatal gene therapy to cure disease. Investigators have devised delivery strategies in large animals that could be used clinically to apply gene therapy to the human fetus, and prenatal gene therapy may become available for the treatment of certain life-threatening congenital disorders in the near future. In this way, pregnant women and their partners would have a third choice when faced with an affected fetus where currently the only options are either terminating the pregnancy or continuing with an affected fetus with a poor prognosis. A number of issues need to be resolved before translation of therapy into clinical practice can be achieved. These include: n how best to target and regulate therapeutic gene expression for particular diseases n the long-term safety and efficacy of the therapy n the fetal and maternal immune response n the effect of transgene expression on fetal development n the potential for germline gene transfer n gaining informed consent from patients n regulatory and ethical questions.

© Khalil Abi-Nader and Anna David. Volume compilation © RCOG

102  |  KHALIL ABI-NADER AND ANNA DAVID

Introduction Gene therapy aims to correct an existing cellular abnormality by delivering genetic material to cells via a vector. Recent breakthroughs in the field of gene therapy for cancer1,2 and congenital blindness3 have shown the potential for this kind of therapy. Prenatal gene therapy, as the name implies, involves the delivery of genetic material to either the fetus, in what is referred to as fetal gene therapy, or the mother in an attempt to treat a pregnancy-related complication such as placental insufficiency. When the vector delivers the therapeutic gene to the fetal somatic cells in vivo, the process is termed fetal somatic gene therapy. On the other hand, stem cells of fetal origin, such as the fetus’s own amniotic fluid stem cells, could be gene-treated ex vivo and then transplanted into the fetus; this is called fetal autologous stem cell gene therapy. When healthy allogeneic tissue such as haematopoietic stem cells from a sibling, parent or other fetus is being transplanted, the process is termed fetal allogeneic stem cell therapy or in utero allogeneic stem cell transplantation and does not involve gene therapy per se. The latter is discussed in Chapter 6. Progress in postnatal gene therapy has been relatively slow since the first trials of gene therapy were conducted in the 1980s, for a number of reasons that will be highlighted below. Prenatal gene therapy aims to overcome these difficulties. The field is still in development at the preclinical stage. Experiments in small-animal models are being carried out by limited groups around the world and by only a few groups in large-animal models. It is hoped that this technology can be carried into the clinic in the years to come for specific disease indications.

The advantage Prenatal gene therapy has the ability to target rapidly dividing cells, thus providing a large population of transduced cells that should provide a better therapeutic effect. Organs that are difficult to target for gene transfer after birth, such as the lung in cystic fibrosis and the skin in epidermolysis bullosa, might be better reached prenatally. Prenatal gene therapy could also deliver a therapeutic gene before the onset of organ damage, an important issue for metabolic diseases such as the mucopolysaccharidoses where brain damage occurs before birth. Furthermore, the fetus has a size advantage, allowing a higher vector to target cell ratio, especially in cases where vector titres are limited. The most important barrier facing postnatal or adult gene therapy remains the development of immunity against the transgenic protein or the vector itself, which limits long-term therapeutic transgene expression and prevents repeated administration. For example, after application of adeno-associated virus serotype 2 (AAV2) vectors containing human factor IX, significant coagulation factor levels could be sustained for years in a dog haemophilia model but expression only lasted a few weeks in human adult gene therapy trials.4 In this case, the limiting factor seems to be preexisting T-cell immunity to the AAV2 vector in humans. Even without pre-existing immunity, immune responses are commonly detected after adult gene therapy. For example, antibodies to transgenic dystrophin protein develop in mice with muscular dystrophy after treatment with an adenovirus containing the dystrophin gene.5 Although neonatal gene transfer has shown some promise in small-animal studies of haemophilia treatment, concurrent immunosuppression may be required.6 In contrast, a number of studies on prenatal gene therapy application have shown long-term expression of proteins at therapeutic levels and induction of immune

PRENATAL GENE THERAPY  |  103

tolerance7 in both small8 and large animals.9 Immune tolerance can occur when a foreign protein is maintained in an individual during the development of its immune system. Evidence of long-term tolerance to a new chimeric state can be found in human fetuses with X-linked severe combined immunodeficiency (SCID) after transplantation with allogeneic haematopoetic stem cells during the early second trimester.10 So, in human fetuses there exists a potential therapeutic window of opportunity during the late first trimester and early second trimester during which exposure to a foreign protein might result in induction of tolerance and a real opportunity of long-term expression with the potential for cure. A 2008 study11 has suggested that early as compared with late intrauterine gene transfer in the fetal sheep (at 54–65 days of gestation rather than 72 days or more, where term is 145 days) takes advantage of multiple tolerogenic mechanisms promoting both central and peripheral tolerance to the transgene products. Induction of immune tolerance after gene therapy in the immune-competent human fetus has yet to be clearly demonstrated.

Candidate diseases As with any potential therapeutic modality, the risks are not well characterised and the efficacy is still undetermined. In this regard, the report of the US National Institutes of Health Recombinant DNA Advisory Committee12 proposed that initial application of prenatal gene therapy should be limited only to those diseases where all of the following are present: n there are serious morbidity and mortality risks for the fetus either in utero or postnatally n there is no effective postnatal therapy n associated serious abnormalities can be corrected by the transferred gene n prenatal diagnosis is possible and there is a well-defined genotype/phenotype relationship n an animal model of the disease is available. For example α-thalassaemia major causes fetal hydrops, which is by definition lethal to the fetus, while β-thalassaemia major presents during infancy. Fetal gene therapy, if successful, is thus an obvious option for α-thalassaemia major but needs to be proven to be safe and more effective than postnatal therapies to be considered clinically applicable in β-thalassaemia. The same applies to disorders presenting during early childhood. However, for some conditions such as the haemophilias and in particular factor VII deficiency, there is a high risk of perinatal haemorrhagic complications,13 which gives a good reason for prenatal application. It is likely that, with clinical experience, the indications and the risk–benefit balance for prenatal gene therapy will become more defined. The list of candidate diseases includes not only many single-gene disorders but also some pregnancy-specific conditions and structural abnormalities of the fetus (Table  7.1). Severe placental insufficiency and fetal growth restriction affects one in 100 pregnancies and is a major cause of neonatal morbidity and mortality but there is no effective treatment. The underlying abnormality is a lack of sufficient uteroplacental circulation. We have recently demonstrated that local overexpression of vascular endothelial growth factor (VEGF) mediated via adenovirus vector delivery to the uterine arteries increases uterine artery blood flow and reduces vessel constriction in pregnant sheep.14 Current work is studying the long-term effects with the aim

Airway and intestinal In utero epithelial cells Haematopoietic precursor cells

Dystrophin

CF transmembrane conductance regulator

γc cytokine receptor (X-linked SCID)

Neurotrophic factors

Vascular endothelial growth factor (VEGF), placental growth factor

Lung growth factors

Cystic fibrosis

Severe combined immunodeficiency (SCID)

Hypoxic ischaemic encephalopathy

Severe fetal growth restriction

Congenital diaphragmatic hernia

Alveoli

Uterine arteries, trophoblast

Cortical neurons

Myocytes

In utero

In utero

Birth

Birth

2 years

Birth

Muscular dystrophy, e.g. Duchenne

Keratinocytes

Type VII collagen

Epidermolysis bullosa

In utero (type 0), 6 months (type 1)

Survival motor neuron protein

Spinal muscular atrophy

Motor neurons

Uridine diphosphate (UDP) glucuronosyl­ transferase in Crigler–Najjar type 1 syndrome

9–11 years

In utero

Age at onset

Metabolic disorders

Hepatocytes

Lysosomal storage diseases Glucocerebrosidase in Gaucher disease

Hepatocytes Erythrocyte precursors

Human factor VII

Factor VII deficiency

Target cells/organ

Homozygous α-thalassaemia α-globin

Therapeutic gene product

Candidate diseases for prenatal gene therapy

Disease

Table 7.1

Mid-30s

25 years

Adulthood

2 years

< 2 years

Lethal

Life expectancy

1 : 2200

1 : 100

1 : 500

Adulthood if individual survives neonatal surgery

Adulthood if individual survives the neonatal period

Adulthood

1 : 1 000 000 < 6 months if no bone marrow transplant or neonatal gene therapy

1 : 4000

1 : 4500

1 : 40 000

1 : 10 000

1 : 9000 overall

1 :‌ 2700

Incidence

104  |  KHALIL ABI-NADER AND ANNA DAVID

PRENATAL GENE THERAPY  |  105

of reversing severe fetal growth restriction in animal models.15 Another potentially important application of prenatal gene therapy is in fetuses affected with congenital diaphragmatic hernia, a condition where lung hypoplasia causes significant morbidity and mortality. Prenatal CFTR expression mediated by adenovirus vector appears to enhance saccular density and air space in the lungs of a rat model of congenital diaphragmatic hernia.16 It is possible that short-term expression of growth factors at a critical stage of lung growth in combination with other therapies, such as balloon occlusion, may be useful for this serious condition.

Vectors for prenatal gene therapy Vectors are used to deliver the therapeutic gene to the target cell population. The ideal vector for fetal somatic gene therapy is one that can produce long-term regulated and therapeutic expression of the transferred gene through the use of a single and efficient gene delivery method. The ideal vector should also be safe to the mother and fetus, allowing incorporation into clinical practice. Other characteristics include a vector with good transduction efficiency, a specific tropism to the target organ, a carrying capacity large enough to incorporate the therapeutic gene and regulatory elements, a low immunogenicity and a low teratogenic and mutagenic potential. Vectors used in gene therapy have been classically divided into replication-deficient viral vectors and non-viral vectors. Comprehensive reviews of the vector systems are available elsewhere17,18 and we will discuss here mainly the issues that relate to their use prenatally. Table 7.2 summarises the main characteristics of the various vector systems. Adenoviruses are highly efficient at gene transfer and as such have been important tools in proof-of-principle studies.19 The vector does not integrate in the host genome and it remains episomal, becoming rapidly diluted by cellular proliferation resulting in transient gene expression, which, for most prenatal indications, is not desirable. For some conditions, however, transient gene expression may be an advantage, particularly those specifically related to pregnancy, such as fetal growth restriction, or those related to fetal structural development, such as congenital diaphragmatic hernia. Most of the investigations in prenatal gene therapy using adenoviral vectors have applied first- and second-generation adenoviral vectors that are highly immunogenic. With elimination of the viral coding sequences, less immunogenic types have been developed, including the ‘gutless vector’ where all the adenoviral coding sequences are eliminated.20 These helper-dependent adenoviral vectors have a higher packaging capacity and a lower immunogenicity because of elimination of the viral antigens. This allows for longterm expression in quiescent cells by avoiding problems associated with the cellular immune response against viral gene products.21 As an alternative to adenovirus vectors in proof-of-principle studies, Sendai virus gives short-term high levels of gene transfer to the mouse fetus and placenta.22 Adeno-associated viruses (AAVs) are less immunogenic than adenoviruses and are non-pathogenic although their definitive host is still the human. AAV vector-mediated gene transfer of coagulation factor IX to the skeletal muscle or to liver has resulted in sustained correction of haemophilia  B in mice and dogs. However, only short-term therapeutic expression has been observed in human trials, probably because of immune responses to vector capsid.4,23 The wild type AAV and AAV vector genomes mostly remain episomal. While wild type AAV integrates at very low frequencies but predictably at a functionally unimportant site on chromosome 19, AAV vectors integrate randomly24,25 and may preferentially integrate into active genes.26 This poses some concerns prenatally where cell populations have a more rapid turnover such as in the fetus.

++

+

Adeno-associated 4 kb virus (AAV)

10 kb

10 kb

7.5 kb ++

30 kb

Retrovirus

Lentivirus

Adeno–retro hybrids

Herpes simplex

+ = low level; ++ = average; +++ = high level

++

++

+++

7.5 kb +++

Adenovirus

Helper-dependent 35 kb adenovirus

No limit +

Non-viral DNA complexes

Long-term expression; low immunogenicity

Miscarriage risk with some subtypes; integrates into active genes so mutagenesis risk

May be associated with fetal abnormalities

Prenatal considerations

Latent infection

Highly efficient gene transfer with long- Insertional mutagenesis Insertional mutagenesis risk may be increased term expression

Broad: central Retro-axonal transduction; infects nonnervous system dividing cells

Broad

Depends on Long-term gene transfer; infects dividing Insertional mutagenesis Potential for germline transmission; insertional pseudotyping of and non-dividing cells mutagenesis risk may be increased viral envelope

Insertional mutagenesis; Potential for germline transmission; virus infects dividing cells only inactivated by amniotic fluid; insertional mutagenesis risk may be increased

Liver toxicity

Inefficient production

Short-term expression; immunogenic

Can grow to high titre; highly efficient gene transfer Low immunogenicity; high capacity; long-term expression in quiescent cells

Low transduction efficiency

Disadvantages

Low toxicity; low immunogenicity

Advantages

Depends on Long-term gene transfer pseudotyping of viral envelope

Depends on subtype

Broad

Broad

Limited

Efficiency Tropism

DNA

Characteristics of the various vector systems for gene therapy

Vector

Table 7.2

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PRENATAL GENE THERAPY  |  107

Retroviruses and lentiviruses are integrative viruses that provide long-term transgene expression because they are not diluted by cell division. They do, however, have the potential to cause mutagenesis and retroviruses based on the murine leukaemia virus (MLV) were implicated as a cause of T-cell leukaemia when used in human trials of neonatal stem cell gene therapy for SCID.27 Lentiviruses have been associated with insertional mutagenesis when applied fetally. Mice that were prenatally treated with the equine infectious anaemia virus (EIAV) lentivirus vector had a high prevalence of postnatal liver tumours.28 Insertion sites in these instances were preferentially associated with genes involved in development and cell growth, suggesting that the fetus and neonate may be particularly sensitive. Integration-deficient lentiviral vectors have thus been developed to avoid this problem. These vectors can sustain expression in postmitotic tissues such as the brain while virtually eliminating the risk of insertional mutagenesis.29 Although there is no reported experience with fetal application, foamy virus vectors may be a useful alternative to retrovirus or lentivirus vectors because of their high efficiency in targeting haematopoietic stem cells, their favourable integration profile and the non-pathogenic nature of the parent virus.30 Non-viral vectors are considered to have a better safety profile than their viral counterparts, with an ability to transfer very large fragments of genetic material. These vectors include the cationic liposomes, the cationic polymers, the yeast artificial chromosomes, and the Epstein–Barr virus plasmids and semi-viral systems as in the case of the sleeping beauty (SB) transposon.31,32 Although many non-viral vectors are limited by their low transduction capacity and short expression period, manipulating the formulation improved the expression of a marker gene in fetal mouse liver.33 Gene transfer levels were 40-fold higher in the fetus than in adults,33 suggesting that fetal tissues may be very amenable to the uptake and expression of naked DNA. The integrating ability of SB transposons combines the advantages of plasmid-based vectors with targeted integration into the genome that can provide long-term expression of the therapeutic protein, which would be advantageous for a prenatal approach.34 The ability to custom-design a non-viral vector creates an exciting opportunity to enhance the vector properties at each level, including stability, transduction efficiency and tropism. This is evident in the field of nano-carriers35 where DNA is combined with a carrier molecule(s) that, for example, can shield the DNA, enhance endocytosis and escape from endosomes, and improve nuclear entry to enhance vector tropism.

The evidence: preclinical proof-of-principle studies Over the past few years, proof-of-principle studies in small-animal models have shown that prenatal gene therapy can result in the permanent phenotypic correction of early single-gene disorders. We present some of the landmark evidence in support of such an approach. Waddington et al.36 demonstrated a permanent phenotypic correction of fetal immune-competent haemophilic mice using intravascular injection of a lentivirus vector encoding the human factor  IX protein at 16  days of gestation (term is 22 days). Plasma factor IX levels remained at around 10–15% of normal in the treated haemophilia B mice until the last measurement at 14 months postnatally; the usual life-time of a mouse is around 2–3 years. The therapeutic goal in the treatment of haemophilia is modest, as an increase in the circulating levels of clotting factors to just 1% of normal is sufficient to ameliorate the bleeding diathesis.37,38 Substantial improvement in blood coagulability was seen in the treated mice, which rapidly

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stopped bleeding after venipuncture. No humoral or cellular immunity against the protein, elevation of serum liver enzymes or vector spread to the germline or maternal circulation were detected. Sabatino et al.39 subsequently documented the induction of tolerance after AAV1-hFIX administration in Factor IX-deficient fetal mice. Using a self-complementary AAV8-hFIX vector that has been described to have a high transduction capacity in mice and macaques,40 we showed long-term hFIX expression after intraperitoneal injection of fetal sheep in early and late gestation.41 No functional antibodies could be detected initially against the vector or transgene product and there was no liver toxicity observed. This is the first report of long-term hFIX expression after in utero application in a large animal. Tolerance could not be induced, however, and antibodies to the therapeutic gene were detectable when challenged postnatally with the hFIX recombinant protein. A notable success in small-animal models is in the long-term correction of bilirubin uridine diphosphate (UDP) glucuronosyltransferase deficiency in fetal rats using a lentivirus vector injected on day 19 (term is 22 days).42 The human condition is Crigler–Najjar type 1 syndrome and individuals suffer severe brain damage early in childhood owing to the inability to conjugate and excrete bilirubin. A rat model of Criggler–Najjar was injected with a lentivirus vector carrying the gene for bilirubin UDP glucuronosyltransferase. The treated rats sustained a 45% decrease in serum bilirubin levels for more than a year, a level that would be considered therapeutic in the human.42 Despite the long-term expression, these rats developed antibodies against bilirubin UDP glucuronosyltransferase,43 which may be related to the timing of fetal injection that occurred late in fetal life. Leber congenital amaurosis (LCA) describes a group of congenital recessively inherited diseases causing early-onset severe retinal dystrophy leading to poor vision at birth and complete loss of vision in early adulthood. The genetic mutations involve the gene encoding retinal pigment epithelium-specific 65 kD protein (RPE65) among others. In adults, gene therapy using AAV2-RPE65 injected subretinally was safe and resulted in modest improvement in measures of retinal function, creating some optimism.44 Human studies of LCA indicate that earlier intervention may be more likely to restore vision.45 A window of therapeutic opportunity exists as there is a period of photoreceptor cell dysfunction that precedes retinal degeneration.46 In utero subretinal gene therapy in a murine model of LCA using AAV2/1-RPE65 at day 14 of gestation resulted in efficient transduction of retinal pigment epithelium and restoration of visual function.45 Similar findings were achieved in an avian model using a lentivirus vector carrying the retinal guanylate cyclase-1 gene, a gene whose mutation is also associated with the development of LCA.46 In this study, the vector was injected into the neural tube of the embryonic chick to determine whether photoreceptor function and sight could be restored. Of seven treated animals, six exhibited sight as assessed by optokinetic and volitional visual behaviours. Electroretinographic responses were also partially restored in treated animals and morphological analyses indicated there was slowing of the retinal degeneration. The lysosomal storage diseases are inherited disorders where lysosomal enzymes are deficient and this leads to intracellular substrate accumulation. In mucopolysaccharidosis (MPS) type  VII, for example, a deficiency of β-glucuronidase activity leads to accumulation of glycosaminoglycans in lysosomes,47 resulting in hepatosplenomegaly, skeletal deformities, developmental delay and death from cardiac failure. Because of the difficulties in attaining widespread gene expression in the postnatal brain and spinal cord, prenatal gene delivery is an attractive strategy. Injection of adenovirus into the cerebral ventricles of fetal mice led to widespread and long-term transgene

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expression throughout the brain and the spinal cord.48 In the same study, delivery of a therapeutic gene to the cerebral ventricles of fetal MPS type VII mice prevented damage in most of the brain cells before and until 4  months after birth. A study using an AAV vector had even more impressive results. A single AAV serotype  1 vector injection into the ventricle at 15.5  days of gestation resulted in widespread distribution and lifelong expression of the normal gene in the brain and spinal cord. This prevented the development of storage lesions throughout the central nervous system and the survival in the treated animals was significantly increased after 1 year.49 Our studies in the fetal sheep showed that ultrasound-guided injection of adenovirus vectors into the cerebral ventricles could be achieved in early gestation (day 60 out of 145 days; unpublished data), with widespread transduction of the choroid plexus and ependymal cells when sampled 3 days later. However, the long-term safety of such an approach requires validation. Prenatal gene transfer has also been applied with some success in glycogen storage disease type II, which is caused by a deficiency in acid α-glucosidase (GAA). This leads to lysosomal accumulation of glycogen in all cell types and abnormal myofibrillogenesis in striated muscle with death from respiratory failure. Delivery of the AAV2/1-GAA vector by intraperitoneal injection to the mouse embryo in knockout models gave high-level transduction of the diaphragm and restoration of its normal contractile function.50 Replication of these successes in a large-animal model, or at least demonstration of safety and feasibility where a large-animal model is not available, will be a further step towards translation into the human fetus.

Issues facing prenatal gene therapy The main issues facing translation of prenatal gene therapy into clinical practice are discussed below. Some problems have been already observed in preclinical studies whereas others remain a potential threat. Targeting therapy to the correct organ Targeting therapy to the correct organ(s) is a desired strategy. It enhances the efficacy of gene therapy by delivering the vector load to where it is most needed, for example lung and gut in cystic fibrosis. Furthermore, it reduces the chances of transgene or vector-mediated side effects in other organ systems, such as the possibility of retinal neovascularisation after VEGF gene delivery. The few strategies that could help specific-organ targeting are briefly discussed below. Manipulating the vector The vector properties can be manipulated to enhance gene transfer generally. Expression from AAV vectors can be improved by packaging complementary dimers of a mini-hFIX liver-specific expression cassette within a single AAV virion to produce an scAAV vector. This overcomes the need for conversion of the single-stranded genome into transcriptionally active double-stranded forms, which significantly improves gene transfer.40 The vector can also be manipulated to direct expression to specific organs or cell types, through pseudotyping for example, as discussed above, or by altering the vector promoter, for example to a liver-specific promoter to increase hepatic transgene expression.

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Methods and timing of delivery to the fetus If fetal gene therapy is to be clinically applicable, developments in vector technology will have to be accompanied by improvements in minimally invasive methods of delivering vectors to the fetus. Traditionally, invasive surgical techniques such as maternal laparotomy or hysterotomy have been performed to access the fetus in small- and even large-animal models. However, in clinical practice, minimally invasive techniques such as ultrasound-guided injection, or even fetoscopy, could be used to deliver gene therapy to the fetus with less morbidity and mortality. It is likely that non-human primates will be the ultimate animal model that will be used for safety studies in the immediate preparation for a clinical trial of fetal gene therapy. However, the high maintenance costs and breeding conditions prohibit their use in the routine development of novel injection techniques. Sheep are much easier to breed and maintain and are a well-established animal model of human fetal physiology. Sheep have a consistent gestation period of 145 days; the development of the fetus and of the immune system is very similar to humans. Using the pregnant sheep, we have adapted ultrasound-guided injection techniques from fetal medicine practice and developed new methods to deliver gene therapy to the fetal sheep, including ultrasound-guided intratracheal injection to target the distal respiratory epithelium51,52 and ultrasoundguided intragastric injection to target the intestinal mucosa.53 Table 7.3 describes the various injection methods that have been tested in fetal sheep and the fetal organs that can be targeted using them. Maternal mortality in the pregnant sheep was negligible and fetal mortality was between 3% and 15%, depending on the route of injection. Over 90% of the fetal mortality was due to iatrogenic infection, usually with known fleece commensals. Invasive procedures such as tracheal injection had a complication rate of 6%, which was related to blood vessel damage within the thorax.54 Intracardiac and umbilical vein injection had an unacceptably high procedure-related fetal Table 7.3

Clinically applicable routes for gene delivery that have been evaluated in the fetal sheep using ultrasound-guided injection

Route of application Gestational age at application Target organ(s) Sheep fetus Equivalent gestational age in the human fetus Intra-amniotic From day 33 From week 10 Skin, fetal membranes, airways Intraperitoneal From day 50 From week 14 Peritoneum, liver, diaphragm, haematopoietic system Intrahepatic From day 50 From week 14 Liver, haematopoietic system Intramuscular From day 50 From week 14 Muscle Cerebral ventricles Days 55–65 Weeks 15–17 Choroid plexus, lateral ventricle and neurocortex From day 60 From week 16 Stomach, small and large bowel, liver Intragastric Intrapleural From day 60 From week 16 Intercostal and diaphragm muscles Umbilical vein From day 70 From week 20 Systemic delivery (predominantly liver, adrenal gland) Intratracheal Days 80–115 Weeks 22–32 Airways From day 100 From week 20 Systemic delivery (predominantly liver, Intracardiac adrenal gland)

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mortality in first-trimester fetal sheep55 and umbilical vein injection was only reliably achieved from 70 days of gestation, which is equivalent to 20 weeks of gestation in humans. The relevant time windows for the various application routes in humans still need to be established with respect to technical feasibility, fetal physiology and the development of the fetal immune system. Effect of fetal exposure on vectors The effect of fetal exposure on vectors is also important to consider, since many routes of fetal application require delivery into fluid compartments such as the serum, airways or amniotic fluid. Human serum can inactivate retroviruses56 and amniotic fluid inhibits retrovirus infection.57 Altering vector production can make them more robust58 and lentiviruses are relatively immune to damage. Germline transmission Germline transmission to the fetus or the mother might cause transgenerational mutations or even induce secondary infertility if a severe immune reaction were to be mounted against the foreign vector and transgene. Compartmentalisation of fetal primordial germ cells in the gonads is complete by 7 weeks of gestation and there is a blood–follicle barrier present in the ovary, thus in practice this is unlikely. After intraperitoneal injection of a retroviral vector to first-trimester fetal sheep, the born rams were estimated to have a testicular germ cell transduction frequency of one in 6250 germ cells.59 This is well below the calculated frequency of naturally occurring endogenous insertions of one in 50 to one in 10060 and below the recommended upper limit of one in 6000 for exogenous insertions in human gene therapy trials. There was evidence of fetal oocyte transduction after intraperitoneal injection of vesicular stomatitis virus glycoprotein (VSVG) pseudotyped HIV vectors in late firsttrimester but not in second-trimester non-human primates.61 The risk of germline transmission is probably confined to vectors that preferentially integrate. Choice of disease The first human application will most probably parallel successes in large-animal models but, unfortunately, few exist. Neuronal ceroid lipofuscinosis (Batten disease) is an autosomal recessive condition in which accumulation of ceroid lipofuscin leads to degeneration of neuronal cells. Mouse, sheep and dog animal models exist and have been used to demonstrate efficacy in postnatal gene therapy. Application of AAV containing human ceroid lipofuscinosis neuronal  2 cDNA to the brain of affected neonates suggested a slowing of progression of the condition in the treated children.62 Earlier treatment during fetal life, therefore, may prevent the damage already sustained by neonates. As the short-term efficacy of prenatal gene therapy is more predictable and longterm outcomes remain uncertain, translation into the human fetus may be more achievable where only short-term correction is required or particularly beneficial. An example is factor  VII deficiency, which carries a very high risk of perinatal cerebral haemorrhage.63 Gene therapy with a non-integrating vector such as AAV a few weeks before term may ensure that the fetus has sufficient factor VII to prevent the occurrence of bleeding. Another condition where only short-term expression is required is severe fetal growth restriction. Gene therapy with adenovirus-VEGF delivered locally into the maternal uterine arteries increases uteroplacental blood

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flow for at least a month after vector delivery.15 This may lead to a prolongation of pregnancy for 3–4 weeks, which could improve the chances of delivering a viable and more mature neonate since small increases in fetal size and gestational age at delivery translate into large reductions in the neonatal morbidity and mortality rates. Length of expression Growth of the vector recipient The fetal and neonatal periods manifest one of the most accelerated growth patterns. A certain number of transduced cells that are enough to provide therapeutic gene expression at a specific fetal weight might not be enough to maintain high-level long-term gene expression as the individual increases in size. This is especially true in the case of non-integrating vectors targeted to organs with a high cell turnover rate. The genetic material gets diluted as the organ grows and cells are lost, limiting the duration and level of expression. This situation is evident when, for example, the respiratory epithelium is targeted with adenoviral vector,16 or even in organs with a much lower turnover rate such as the liver. We have noted a high level of factor IX expression 3  weeks after in utero gene transfer of AAV8-hFIX to the pre-immune fetal sheep. Despite the absence of neutralising antibodies, and long-term expression that persisted for over 1 year, hFIX levels dropped rapidly as the fetal liver and lamb weight increased.41 Vector silencing Silencing of vectors is widely acknowledged to be a drawback that can limit their gene therapy applications. Silencing is particularly well documented in MLV vectors.64 The phenomenon includes complete transcriptional silencing observed shortly after infection which is thought to occur via methylaton, and variegation in which genetically identical sister cells that inherit the same provirus may either express or be silenced. Extinction occurs where there is progressive silencing of an initially expressed provirus during long-term culture or differentiation, which may be a particular problem in the fetus where cells may be relatively immature.65 Removal of silencing elements in the MLV long terminal repeats, such as occurs in the production of self-inactivating gammaretrovirus (SIN) vectors, or introduction of insulator elements can be used to counteract silencing. Lentivirus vectors are similarly affected by silencing but, because they can infect noncycling cells and express efficiently because of multiple copy integrations, they provide more efficient gene transfer. Integrating vectors and insertional mutagenesis Insertional mutagenesis is a concern when using integrating vectors. Retroviruses used in the ex  vivo transduction of patients with X-linked SCID were implicated in the development of a form of T-cell leukaemia due to insertion near the site of a protooncogene.66 This event may be disease specific or related to the strong promoter activity of early-generation retroviral vectors. Another form of mutagenesis was evident in mice prenatally treated with the EIAV lentivirus vector where these mice developed a high incidence of postnatal liver tumours.28 Insertion sites were preferentially associated with genes involved in development and cell growth, and this observation suggests that the developing mouse fetus and neonate may be particularly sensitive to the effects of

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integrating vectors. This drawback in fetal gene transfer may provide a good method to test for insertional mutagenesis and to identify new cancer genes. Pre-existing maternal immunity Maternal immunoglobulin  G antibodies can cross the placental barrier and theoretically prevent long-term expression. This is especially a consideration in AAVmediated gene transfer where the limiting factor seems to be pre-existing memory T-cell immunity to AAV2 in humans, who are the only natural hosts to this virus.4 This can be circumvented by applying AAV serotypes that humans are not naturally exposed to, such as AAV8. Regulating transgene expression Regulation of gene expression represents a long-sought goal of gene therapy. Inducible systems such as Tet-dependent expression67 have been used in adult animal studies to alter levels of expressed transgenic protein such as hFIX.68 The Tet system combines elements of viral and bacterial origin to generate chimeric transcription factors that control the activity of a chimeric promoter (Ptet) driving transgene expression. Although not yet studied in prenatal gene therapy studies, regulated transgene expression may be particularly important in the fetus to prevent adverse developmental effects of the transgenic protein. Fetal and maternal immune response to vector and transgene Despite the notable evidence described in the previous section, the fetal immune response is still a relative barrier to long-term expression. Jerebtsova et al.69 evaluated multiple routes and viral dose combinations of adenoviral and adeno-associated vectors carrying the marker β-galactosidase gene in the fetal mouse at 13–15 days.69 In utero injection of either viral vector at any route and dose combination resulted in the production of low titres of neutralising antivirus and antitransgene antibodies. This primary immune response only partially blocked transgene expression after the readministration of viral vectors postnatally. However, delivery of the virus postnatally triggered an immune response that completely blocked transgene expression after a third viral injection. Another example has been mentioned earlier: despite the long-term expression obtained in the rat Criggler–Najjar model that was gene-treated prenatally, the rats developed antibodies against bilirubin UDP glucuronosyltransferase,43 which may be related to the fact that the fetal injection was done late in fetal life. These fetal and potentially maternal immune responses to the vector and transgene are a reminder that prenatal gene transfer is still subject to immunological barriers that relate to differences in biodistribution, timing of expression and the levels of expression. Designing less immunogenic vectors and considering immune conditioning before gene delivery, a less desirable option, could be ways to partially overcome the problems. There is a need to elucidate the pathways of fetal immune development and regulation further. Reversion to wild type vector Many replication-deficient lentiviruses are based on the immunodeficiency virus and there is the theoretical possibility of reversion to the wild type. With the advent of third- and fourth-generation lentivirus vectors, the risk of in  vivo generation of

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replication-competent viruses has been reduced by removal of the Tat gene and usage of the self-inactivating strategy.70 Clinical-grade production of vectors will need to adhere closely to guidelines regarding testing for replication-competent viruses. Safety of fetus, mother and her future progeny Teratogenicity is a concern. AAV infection has been proposed as a possible cause of first-trimester miscarriage71 although the AAV genome could not be amplified in aborted tissues in another study.72 Adenoviruses have also been implicated as fetal pathogens associated with the presence of echogenic liver lesions and neural tube defects.73 Since antenatal infections with adenoviruses and AAVs are common, it is possible that concerns in this area have been over-emphasised. The more immunogenic vectors can often produce a local inflammatory reaction, thus promoting vector-related pneumonia or hepatotoxicity depending on the target organ. A severe reaction was observed in a human trial of adult gene therapy for ornithine transcarbamylase deficiency,74 where in one case a systemic inflammatory response ended with the death of a patient.75,76 An inflammatory response in the fetal sheep lung has also been detected after intratracheal in utero adenoviral vector delivery in the third trimester.77 The immature immune system of the early-gestation fetus would have a protective effect against such reactions and achieving tolerance would certainly be regarded as a major advantage. However, one would have to make sure that vector tolerance does not compromise the immune defence against the wild type virus. Even if the vector is safe, the expression of a therapeutic gene at a particular stage in fetal development may be damaging to the fetus. This has been explored using CFTR where adenovirus-mediated expression of CFTR in fetal rats and mice resulted in altered lung development and morphology.78,79 The long-term effect of any transgenic protein applied to a fetus at a specific time will require long-term monitoring into neonatal and adult life. There are particular risks associated with the use of prenatal gene therapy for the mother and the fetus. Any invasive procedure used to deliver the vector could potentially affect the health of the fetus and the mother. Intrauterine procedure such as those performed under ultrasound guidance carry a finite but definitive risk of miscarriage, infection and preterm labour.80,81 Earlier gene transfer may be beneficial as there are profound increases in the numbers of circulating T-cells observed between 12 and 14  weeks of fetal life.82 However, gene therapy before this time limits the routes that can be safely used. In addition, fetal gene treatment during a certain pregnancy could pose a risk to future pregnancies by affecting the mother’s health through the effects of gene therapy or the delivery procedure itself. For example, significant bleeding complications requiring hysterectomy and even maternal deaths have been reported with the use of fetoscopic procedures.83 Post-amniocentesis chorioamnionitis may occur in 0.1% of cases and can evolve into a systemic infection with marked maternal morbidity if appropriate treatment is not instituted.84 On the other hand, although low-level transplacental gene transfer to the mother is possible, it seems that it is unlikely to reach significant levels, as evidenced by studies in the non-human primate.61,85–87 Ethical concerns The consideration to apply gene therapy in the human fetus raises multiple ethical questions and research into this aspect is largely lacking. It is unclear whether society

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will accept prenatal gene therapy as a useful technology for certain conditions. The public remains largely unaware of gene therapy and public education such as meetings organised by the Gene Therapy Advisory Council are important. Issues regarding short- and long-term safety, balancing the best interests of the fetus and the mother, risk–benefit analysis and the availability of other options such as termination of pregnancy or preimplantation genetic diagnosis should be considered. Ethical discussions of the issues relating to the field of reproductive genetics and genetic therapy are presented in Chapters 4 and 8. Despite these concerns, fetal gene therapy offers a third option to parents where currently the choice is limited to either terminating the pregnancy or continuing with an affected pregnancy and an uncertain prognosis at best. Application in humans Obviously, prenatal gene therapy should not be performed except in cases where long-term safety and efficacy have been established in pertinent animal models. Regulatory procedures should be satisfied before human application could be attempted. This will involve lengthy toxicological animal studies, including transgenerational experiments. Phase I human trials might also face hurdles because of difficulties in testing pregnant women, where toxicological studies are usually contraindicated. Thus, when human application becomes possible, extensive unbiased parental counselling and informed consent will be paramount because of the uncertainties about the efficacy and long-term safety of prenatal gene therapy,88 which may not become evident until much later in the individual’s life. This can be difficult because the decision to participate in a fetal gene therapy trial will occur close to the time of prenatal diagnosis of the condition. Because the risks involve the mother, fetus and possibly future progeny, parents will also be required to consent their offspring and themselves to lifelong follow-up.

Fetal somatic gene therapy in practice Assuming that a safe and effective fetal gene therapy approach were possible, how might it work in practice for treatment of a fetal congenital condition? Figure  7.1 shows a possible scheme for a hypothetical syndrome  X, an autosomal recessive condition that results in severe morbidity. Without an effective screening strategy with accurate prenatal diagnosis for syndrome  X, many families would not know that they were at risk until an affected child were born. For the next pregnancy, the parents could choose to have prenatal diagnosis of syndrome X in the fetus before the gestational age for optimum gene therapy treatment, by non-invasive prenatal diagnosis using cell-free fetal DNA if available, or by chorionic villus sampling. The mother would undergo the invasive procedure to treat the fetus at the best time to target the affected organ. The option of further invasive testing to confirm expression of the curative gene product later in the pregnancy could be available. An alternative strategy is preimplantation genetic diagnosis, which is often proposed as the most sensible option for parents at risk of having an affected fetus. The main limitations of in vitro fertilisation and preimplantation genetic diagnosis are the ovulation induction and invasive procedures that the woman is required to have, that only 20–30% of couples achieve a pregnancy per cycle89 and that some embryos will be disposed of, which for some individuals is of concern.90

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Figure 7.1 Fetal gene therapy offers a potential third option to parents faced with a pregnancy complicated by a fetal genetic disease; CVS = chorionic villus sampling

The future of prenatal gene therapy Research into prenatal gene therapy will need to be directed to a number of areas before its clinical applicability will be accepted. It will be important to determine the diseases that are most amenable to prenatal therapy. Neonatal gene transfer may prove to be more suitable for some candidate diseases, and for other conditions preimplantation genetic diagnosis and replacement of a fertilised embryo that is free from disease may be most acceptable. It is likely, however, that a prenatal approach may be the best for certain early-onset and severe genetic conditions. Prenatal gene therapy in humans will depend critically on our ability to demonstrate its safety and efficiency in preventing or treating severe genetic disease. Improvements in vector design and safety and in delivery techniques to the fetus are key. Vectors must be able to provide long-term regulated gene expression, preferably for the lifetime

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of the individual, that does not interfere with normal fetal development in other organ systems. The ability to custom-design nano-carriers offers a huge opportunity to create the ideal non-viral vector.35 Risks due to the procedures involved in vector application can be well defined since these techniques are used routinely in fetal medicine. However, the optimum gestational age at which to target gene therapy for specific diseases will define which is the best route of administration since direct access to the fetal circulation cannot safely be achieved before mid-gestation. With the enhanced resolution of the new-generation ultrasound systems, clinical application is expected to prove more accurate and safe in the late first and early second trimester. A better understanding of the development of the fetal immune response to vector and gene products is also vital. Animal models of severe genetic diseases in the mouse and generation of large-animal transgenic models will be useful to demonstrate proof of principle for in utero treatment. Transgenerational aspects may need to be studied in small-animal models. Ultimately, however, it is likely that some safety studies will need to be performed in non-human primates. Regulating the expression of transgenic protein must be explored to prevent any adverse effects by overexpression. This should allow specific gene control with very limited interference with the normal physiology of the cell, making use of systems that either permit transcriptional regulation of a particular gene in a reversible fashion such as the tet, lac and Gal4/UAS, or manipulate gene expression based on site-specific recombination reactions such as the Cre/lox and FLP/FRT systems.91 One criticism levelled at fetal gene therapy is a belief that couples pregnant with an affected child would be unlikely to proceed with prenatal gene therapy and would opt for a termination instead. The general public remains concerned that ethical discussion about issues such as gene therapy, cloning and the Human Genome Project are falling behind the technology.92 There is almost no research in this area and the views of the general public and patient groups need to be solicited as this technology comes closer to the clinic. Research is also needed into how adequate information on the risks and benefits of these novel techniques can best be provided for the general public. This will enable couples to have an educated involvement in the decision-making process alongside healthcare professionals. Finally, there needs to be an acknowledgement that prenatal gene transfer may be a useful approach for certain genetic conditions and is worthy of study.93

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Philpott NJ, Thrasher AJ. Use of nonintegrating lentiviral vectors for gene therapy. Hum Gene Ther 2007;18:483–9. Rethwilm A. Foamy virus vectors: an awaited alternative to gammaretro- and lentiviral vectors. Curr Gene Ther 2007;7:261–71. Hackett PB, Ekker SC, Largaespada DA, McIvor RS. Sleeping beauty transposon-mediated gene therapy for prolonged expression. Adv Genet 2008;54:189–232. Hollis RP, Nightingale SJ, Wang X, Pepper KA, Yu XJ, Barsky L, et al. Stable gene transfer to human CD34(+) hematopoietic progenitor cells using the Sleeping Beauty transposon. Exp Hematol 2006;34:1333–43. Gharwan H, Wightman L, Kircheis R, Wagner E, Zatloukal K. Nonviral gene transfer into fetal mouse livers (a comparison between the cationic polymer PEI and naked DNA). Gene Ther 2003;10:810–17. Aronovich EL, Bell JB, Belur LR, Gunther R, Koniar B, Erickson DC, et al. Prolonged expression of a lysosomal enzyme in mouse liver after Sleeping Beauty transposon-mediated gene delivery: implications for non-viral gene therapy of mucopolysaccharidoses. J Gene Med 2007;9:403–15. Li SD, Huang L. Gene therapy progress and prospects: non-viral gene therapy by systemic delivery. Gene Ther 2006;13:1313–19. Waddington SN, Mitrophanous KA, Ellard FM, Buckley SM, Nivsarkar M, Lawrence L, et al. Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice. Gene Ther 2003;10:1234–40. McVey JH, Boswell E, Mumford AD, Kemball-Cook G, Tuddenham EG. Factor VII deficiency and the FVII mutation database. Hum Mutat 2001;17:3–17. Furie B, Limentani SA, Rosenfield CG. A practical guide to the evaluation and treatment of hemophilia. Blood 1994;84:3–9. Sabatino DE, Mackenzie TC, Peranteau W, Edmonson S, Campagnoli C, Liu YL, et al. Persistent expression of hF.IX after tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice. Mol Ther 2007;15:1677–85. Nathwani AC. Self-complementary adeno-associated virus vectors containing a novel liverspecific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood 2006;107:2653–61. David AL, McIntosh J, Weisz B, Boyd M, Cook T, Wigley V. Long-term perinatal gene transfer after clinically applicable delivery of prenatal gene therapy in the sheep. Hum Gene Ther 2008. Seppen J, van der Rijt R, Looije N, van Til NP, Lamers WH, Oude Elferink RP. Long-term correction of bilirubin UDPglucuronyltransferase deficiency in rats by in utero lentiviral gene transfer. Mol Ther 2003;8:593–9. Seppen J. Immune response to lentiviral bilirubin UDP-glucuronosyltransferase gene transfer in fetal and neonatal rats. Gene Ther 2006;13:672–7. Maguire AM, Simonelli F, Pierce EA, Pugh EN Jr, Mingozzi F, Bennicelli J, et al. Safety and efficacy of gene transfer for Leber’s congenital amaurosis. New Engl J Med 2008;358:2240–8. Dejneka NS, Surace EM, Aleman TS, Cideciyan AV, Lyubarsky A, Savchenko A, et al. In utero gene therapy rescues vision in a murine model of congenital blindness. Mol Ther 2004;9:182–8. Williams ML, Coleman JE, Haire SE, Aleman TS, Cideciyan AV, Sokal I, et al. Lentiviral expression of retinal guanylate cyclase-1 (RetGC1) restores vision in an avian model of childhood blindness. PLoS Med 2006;3:e201. Sly WS, Quinton BA, McAlister WH, Rimoin DL. Beta glucuronidase deficiency: report of clinical, radiologic, and biochemical features of a new mucopolysaccharidosis. J Pediatr 1973;82:249–57. Shen JS, Meng XL, Yokoo T, Sakurai K, Watabe K, Ohashi T, et al. Widespread and highly persistent gene transfer to the CNS by retrovirus vector in utero: implication for gene therapy to Krabbe disease. J Gene Med 2005;7:540–51. Karolewski BA, Wolfe JH. Genetic correction of the fetal brain increases the lifespan of mice with the severe multisystemic disease mucopolysaccharidosis type VII. Mol Ther 2006;14:14–24. Rucker M, Fraites TJ Jr, Porvasnik SL, Lewis MA, Zolotukhin I, Cloutier DA, et al. Rescue of enzyme deficiency in embryonic diaphragm in a mouse model of metabolic myopathy: Pompe disease. Development 2004;131:3007–19.

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Matovina M, Husnjak K, Milutin N, Ciglar S, Grce M. Possible role of bacterial and viral infections in miscarriages. Fertil Steril 2004;81:662–9. Baschat AA, Towbin J, Bowles NE, Harman CR, Weiner CP. Is adenovirus a fetal pathogen? Am J Obstet Gynecol 2003;189:758–63. Harvey BG, Maroni J, O’Donoghue KA, Chu KW, Muscat JC, Pippo AL, et al. Safety of local delivery of low- and intermediate- dose adenovirus gene transfer vectors to individuals with a spectrum of morbid conditions. Hum Gene Ther 2002;13:15–63. Raper SE, Yudkoff M, Chirmule N, Gao GP, Nunes F, Haskal ZJ, et al. A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency. Hum Gene Ther 2002;13:163–75. Raper SE, Chirmule N, Lee FS, Wivel NA, Bagg A, Gao GP, et al. Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 2003;80:148–58. Vincent MC, Trapnell BC, Baughman RP, Wert SE, Whitsett JA, Iwamoto HS. Adenovirusmediated gene transfer to the respiratory tract of fetal sheep in utero. Hum Gene Ther 1995;6:1019–28. Morrow SL, Larson JE, Nelson S, Sekhon HS, Ren T, Cohen JC. Modfication of development by the CFTR gene in utero. Mol Genet Metab 1998;65:203–12. Larson JE, Delcarpio JB, Farberman MM, Morrow SL, Cohen JC. CFTR modulates lung secretory cell proliferation and differentiation. Am J Physiol 2000;279:L333–41. Ghidini A, Sepulveda W, Lockwood CJ, Romero R. Complications of fetal blood sampling. Am J Obstet Gynecol 1993;168:1339–44. CEMAT Group. Randomised trial to assess safety and fetal outcome of early and midtrimester amniocentesis. The Canadian Early and Mid-trimester Amniocentesis Trial (CEMAT) Group. Lancet 1998;351:242–7. Pahal GS, Jauniaux E, Kinnon C, Thrasher AJ, Rodeck C. Normal development of human fetal hematopoiesis between eight and seventeen weeks’ gestation. Am J Obstet Gynecol 2000;183:1029–34. Morales WJ. Outcomes and complications of the surgical treatment of twin–twin transfusion syndrome. In: Quintero R, ed. Twin–Twin Transfusion Syndrome. London: Informa Healthcare; 2007. p. 148. Hamoda H, Chamberlain PF. Clostridium welchii infection following amniocentesis: a case report and review of the literature. Prenat Diagn 2002;22:783–5. Lai L, Davison BB, Veazey RS, Fisher KJ, Baskin GB. A preliminary evaluation of recombinant adeno-associated virus biodistribution in rhesus monkeys after intrahepatic inoculation in utero. Hum Gene Ther 2002;13:2027–39. Tarantal AF. Fetal gene transfer using lentiviral vectors: in vivo detection of gene expression by microPET and optical imaging in fetal and infant monkeys. Hum Gene Ther 2006;17:1254–61. Tarantal AF, O’Rourke JP, Case SS, Newbound GC, Li J, Lee CI, et al. Rhesus monkey model for fetal gene transfer: studies with retroviral-based vector systems. Mol Ther 2001;3:128–38. Noble R, Rodeck CH. Ethical considerations of fetal therapy. Best Pract Res Clin Obstet Gynaecol 2008;22:219–31. Wells D, Delhanty JD. Preimplantation genetic diagnosis: applications for molecular medicine. Trends Mol Med 2001;7:23–30. Snowdon C, Green JM. Preimplantation diagnosis and other reproductive options: attitudes of male and female carriers of recessive disorders. Hum Reprod 1997;12:341–50. Mallo M. Controlled gene activation and inactivation in the mouse. Front Biosci 2006;11:313–27. Brown P. Regulations not keeping up with developments in genetics, says poll. BMJ 2000;321:1369. Coutelle C. Why bother? Is in utero gene therapy worth the effort? Mol Ther 2008;16:219–20.

8 Chapter 8

Ethical aspects of stem cell therapy and gene therapy Søren Holm

Introduction A therapy can raise ethical issues because of: n its nature – what kind of therapy it is n its complexity n the risks it entails n the uncertainty surrounding estimates of risk and benefits in general or in the specific clinical situation n its cost and/or cost-effectiveness n the use to which it is being put. Most stem cell and gene therapies that are likely to be developed will actualise some of these elements, for example initially most are likely to be costly. However, most will actualise them in ways that are not significantly different from complex surgical procedures or chemotherapy (at least in the foreseeable future). This could therefore be a very short chapter leading to the conclusion that stem cell and gene therapy raise no ethical issues that have not already been discussed extensively in the literature. This conclusion might initially surprise some readers because it is so obvious that stem cell and gene therapy research has caused very significant and sustained ethical controversy.1 However, many of the intractable issues raised in these debates are of considerably less relevance to therapy than to basic research and they will mostly have been resolved at the policy level before the therapies move from research to therapy. Before clinical use becomes possible it will be decided, or in some cases has already been decided, whether there is something so problematic in deliberately making lasting genetic alterations or deriving stem cells from embryos that no therapy should be developed. On the other hand, it can be argued that there are therapy-related ethical issues that do need to be discussed because, although stem cell and gene therapy are similar to other treatments, there are still morally relevant and potentially significant differences. It is important to note that the analysis here is primarily an analysis of the ethical issues raised by ‘therapies’; that is, well-researched interventions that have reached a © Søren Holm. Volume compilation © RCOG

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stage of development where they have been deemed sufficiently safe and effective by the regulatory authorities to be introduced into clinical practice outside of the context of clinical trials. As therapies they may still be highly complex and technical and restricted to use by highly skilled specialists but they have been deemed ready for clinical practice and are therefore not completely untested or purely experimental. Stem cell and gene therapies may involve either permanent or temporary insertion of stem cells or genetic material. The analysis here will be focused on cases where stem cells that are not the patient’s own are intended to be permanently integrated in the body or where gene therapy is intended to permanently alter the patient’s nuclear or mitochondrial DNA.

Implication in evil The first of the potentially morally significant differences between these therapies and others is related almost exclusively to stem cell therapy. In so far as stem cell therapy will involve cells derived from embryonic or fetal stem cells, the therapy will be seen by some as unethical because it involves implication in an evil act.* If the act of deriving stem cells from embryos and/or fetuses and destroying them in the process is morally problematic then the moral badness may be transferred to any downstream uses of the problematically derived cells. This is clearly not a problem for anyone who does not believe that the use of embryos or fetuses for stem cell derivation is morally problematic. However, whether it is a problem for those who, for example, believe that the destruction of embryos is akin to murder is a more complicated issue. It is important to note that many of us benefit from historical evil to some degree, for instance from the evils of colonialism or the evils of (poorly regulated) animal experimentation. What is at issue is thus not the mere fact of benefiting from morally problematic acts but the way this benefit comes about and the link between the act and the person who is now benefiting. In relation to stem cell therapy, it will very rarely be the case that a person benefits from his or her own evil acts and we therefore only need to consider circumstances where the benefit flows from the evil acts of others. In a 2002 paper, Ronald Green2 helpfully distinguished between three different ways of how benefiting from the evil acts of another can be problematic: 1. if that person is my agent 2. if acceptance of the benefit encourages repetition of the act 3. if acceptance of benefit legitimises the act. For most ordinary people with ordinary incomes, situation 1 will not arise in relation to therapies derived from stem cell lines. We do not own or otherwise control the firms and institutions where stem cell research takes place and our relation to, for example, state-funded universities is too remote and inconsequential to make the stem cell deriver ‘my agent’ in any meaningful sense. Even if I pay for therapy myself and not, for example, through healthcare insurance or taxation, I do not pay directly for the derivation of the stem cells if the therapy is an ‘off the shelf ’ therapy. There are, however, circumstances where an agency relation might be established. If therapies * In this section, ‘evil act’ and cognates are simply used as technical terms equivalent to ‘morally very problematic act’ and not with any implication of evil intention or motivation. This is the classical use in the, mainly Catholic, implication in evil debate.

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are developed that rely on personalised stem cells and the derivation of these uses somatic cell nuclear replacement techniques it would be much more natural to see the stem cell deriver (and by implication the destroyer of embryos) as the agent of the patient for whom these cells are being prepared, even if the patient is not funding the treatment. If personalised stem cell therapies are developed using induced pluripotent stem cells,3 and thus not involving the destruction of embryos, this problem will not occur.4 (There is a discussion about whether induced totipotent stem cells would be morally equivalent to embryos but as long as the induced stem cells are merely pluripotent this question does not arise.) In relation to encouragement of repetition, the issue is to some extent one of counterfactual prediction. Will my using/buying this therapy lead to evil acts (that is, embryo or fetus destruction) in the future? In a situation where most potential patients are not worried about the moral status of the embryo or have other reasons for believing that the destruction of embryos or fetuses for stem cell derivation is acceptable, it becomes unlikely that the withdrawal from the market of those with moral scruples will have any appreciable effect on that market. Given that most of the market for these therapies will be in economically developed and in general rather secular societies, we can therefore conclude that use of the therapies is unlikely to encourage repetition (for recent data on secularisation, see Crabtree and Pelham5). This leaves the third category of problematic benefit. Will my use of a stem cell therapy in some way legitimise the destruction of embryos or fetuses? Will it, for instance, validate the decision to destroy them? It seems to be perfectly possible and consistent to hold both that it is good that a cure for a disease exists and that some step on the way to developing that cure is morally problematic.* Wanting something to be the case or being happy that something is the case is not necessarily wanting every possible means of making it the case. I may want to have your job but not want it to become vacant owing to your death. There is also no inconsistency in, for example, strongly desiring that a cure for Parkinson’s disease should be developed while at the same time holding that some of the ways such a cure can be developed are morally problematic and should not be pursued. If I, for example, think that great apes (chimpanzees, bonobos, gorillas and orangutans) have the same moral status as human beings, I can consistently hold that no research involving great apes should be pursued, even if such research is likely to lead to a cure for Parkinson’s disease.7 But maybe there is hypocrisy hidden in the fact that, while being against human embryonic stem cell research, I nevertheless (secretly) hope that it is successful. Is that not hypocritical? Let us consider the situation where I know that you are going to perform some immoral action but where I am in no position to stop you. Would it be wrong or hypocritical of me to hope that your action succeeds, in the sense of hoping that if you do the action it will have good consequences? Clearly not! I may be required to think that you should not benefit from your immoral act but there is no requirement to hope that no good consequences should follow or to hope that no other person benefits. If you decide to kill someone to get an organ that your child needs to survive, I must as a moral person hope that your killing does not succeed and that if it succeeds you are caught and appropriately punished, but at the same time I must hope that if your killing succeeds the transplant succeeds as well and your child’s life is saved. A person’s use of stem cell therapies does not, therefore, in and of itself validate or legitimise the destruction of embryos. * The rest of this section is based on Holm.6

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Reproductive effects Both stem cell therapies and gene therapies can have reproductive effects in the sense that the genetic changes introduced are transmitted in the germline or the stem cells give rise to germ cells. Such effects will mainly be unintended and unwanted and part of the research before the introduction of these therapies will be aimed at estimating the risks of any reproductive effects. These risks are, for biological reasons, likely to be significantly higher for fetal and early childhood therapies.8 Let us consider the scenario where research before the introduction of a given treatment has shown that there is a quantifiable risk of germline alteration. Does this give rise to any ethical problems? One problem that arises is that, whereas having the treatment and running the risk may well be in the best interests of the person in question (see more below in the section on proxy consent), society may have an interest in preventing any risk of unwanted changes to the germline. There may thus be a conflict between the societal interest or ‘the common good’ and the interests of the individual patient. This conflict of interest could be resolved if the reproduction of the person at risk were controlled but that would potentially require significant intrusions in personal liberty in general and in reproductive freedoms more specifically. In discussions about xenotransplantation and the risk of introducing new viral diseases, it has been suggested that patients should sign up to lifelong follow-up as a precondition for getting the transplant, so there is some support for liberty restrictions in a similar context. Welin,9 for instance, argues that: ‘In order to safeguard the public, the opt-out clause in the Helsinki declaration should not be fully applied. Legally binding rules on obligatory monitoring and restrictions should be imposed – before clinical trials start.’

It is important to note that many current treatments for cancer and for a number of other conditions create a similar problem. These treatments, which comprise a range of chemo- and radiotherapies, may induce mutations in germ cells and will, when used on patients of reproductive age, increase their risk of transmitting a mutation to their progeny, unless the treatment renders them completely infertile. We have, as a society, accepted this increase in risk as an acceptable adverse effect of effective treatment for serious conditions and there is no obvious reason for taking a different view in the context of gene therapies. Accepting the risk does, however, not entail that patients or their proxies should not be informed about it. There are possible future scenarios where the reproductive effects become intended and wanted. If it were possible to perform gene therapy in a way that either restored the faulty gene to normal or in some other way established completely normal gene function and regulation, then germline genetic therapy would become attractive. Not only would the genetic defect be treated in the patient, it would also be eradicated from his or her germline. In such ideal conditions, it is difficult to find any ethical arguments against germline genetic engineering for serious genetic disorders.10 It does, however, seem unlikely that such ideal conditions will eventuate in the near future.

Blurring of the line between research and treatment This chapter is, as stated in the introduction, primarily concerned with ‘therapies’ but the line between research and treatment is not always easy to draw and when a given intervention is initially introduced there may still be a large research element.11

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This is well recognised in relation to surgical innovations but also occurs in cases where regulatory agencies allow some kind of early access outside of clinical trials to promising therapies for life-threatening or otherwise severe conditions.12 Another situation where the research–treatment distinction becomes blurred is when treatments are used ‘off label’ for conditions outside the set of conditions for which they are registered and where there is (presumably) research-based evidence of effectiveness. Not all off-label use is experimental; there may. for example, be strong non-research-based evidence for effectiveness from a long tradition of offlabel use or there may be research evidence that the producer has not used for registration purposes. In relation to stem cell and gene therapies, it is likely that both kinds of blurring of the research–treatment distinction are going to take place to some extent. As many of the proposed uses for these therapies are for the treatment of very serious conditions, it is likely that they will be introduced in general use earlier than other treatments. The information gathered through post-market surveillance and other follow-up studies will therefore be a more important part of the evidence base for the therapy than is usually the case. It is also likely that when they are introduced there will be some clearly experimental off-label use of some of these therapies. This means that the context of consent will neither be a clear treatment context nor a clear research context. The estimation of benefits and risks will be less certain than in the standard treatment context and the patient will in some circumstances be asked to allow considerable data collection and processing for non-treatmentrelated purposes. In our regulatory systems we are not particularly good at handling cases that do not neatly fall into our predefined categories but it is important to come to grips with these betwixt and between cases. This is complicated in situations where doctors themselves may not be clear about their role and where they may have both an interest in pursuing experimental treatments and an interest in not making the experimental aspects of the treatment and the attending uncertainties too obvious. Off-label use is both legitimate and important and requiring full research ethics approval for each instance would be extremely onerous but there need to be reporting mechanisms to deter planned research from evading scrutiny by masquerading as offlabel use.

Blurring of the line between treatment and enhancement Another issue that may arise is that therapies developed as a treatment for disease may be found also to be able to enhance normal function in the healthy. Some stem cell or gene therapies may therefore actualise the continuing treatment–enhancement debate; that is, whether there is an ethical difference between treating disease and enhancing normal function and whether such a difference makes some or all enhancement morally problematic. This is a debate far beyond the scope of this chapter and it is sufficient to note that it is not specific for these kinds of therapies and that they do not make qualitatively new forms of enhancement possible, except as part of much larger and highly speculative anti-ageing and transhumanist research programmes.

Consent for fetuses and children Many stem cell and gene therapies will have to be given in the fetal period or during early childhood to be effective or to have their full effect. This raises the issue of

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proxy consent for very young children.*13,14 This is very well-trodden ground and the only specific issues that arise in relation to stem cell and gene therapies is how a proxy decision maker ought to deal with risks of reproductive effects. This is not straightforward and is especially difficult when these treatments are first introduced because it will be difficult to quantify the risk or estimate the consequences for the person if the risk eventuates. The mere theoretical risk of reproductive effects will not be sufficient to make a treatment that is otherwise in the child’s best interests suddenly change its valence. Whatever the merits of precaution, sacrificing a likely therapeutic benefit for a theoretical reproductive problem is neither sensible nor rational in the context of a serious condition.† At some point on the risk continuum, the balance changes but for serious conditions this may well be at a fairly high degree of risk. If the child is not going to live to reproductive age or if his or her welfare is going to be severely compromised if not treated, then it may be in the best interests of the child to have the treatment even at the cost of later reproductive problems.

Cost-effectiveness and resource allocation A final set of issues arises in relation to resource allocation within the healthcare system. All healthcare systems operate under resource constraints and no system can deliver clinically optimal treatment and care to everyone (in the same way that no education system provides optimal education to everyone and no policing system provides optimal security). As stem cell and gene therapies are likely to be expensive when they are introduced, they will be the subject of explicit analysis and decision making in relation to whether a given healthcare system can and will fund their use. This decision will not only be based on whether a therapy is clinically effective but will also involve cost-effectiveness or cost–utility calculations. Within the confines of this chapter it is not possible to provide an in-depth analysis of the ethics of resource allocation but it is nevertheless important to note that both the benefits and the costs of treatment are undeniably important factors in resource allocation under resource constraint. There may be good reasons to give added priority to treatments for severe conditions, to treatments for conditions where there is currently no effective treatment, or to the first treatments to come to market in completely new classes of treatment in order to promote innovation. All of this may add up to good reasons to give added priority to early stem cell and gene therapies. However, added priority is not the same as being oblivious to cost and it is legitimate for a healthcare system not to fund clinically effective but very costly treatments even if they are novel and exciting. This conclusion is further supported by the observation that the price of commercially produced and marketed therapies bears little direct relation to production or development costs.16 Drugs are often priced according to what the producer thinks the market is willing to bear and not according to some notion of a fair return on investment.

* Legally, no issue of proxy consent arises for fetuses in most jurisdictions but in so far as we treat them with the hope that the treatment is effective and that they will therefore become children and adults, very similar ethical issues arise in regard to the considerations the proxy decision maker ought to take into account. † For a critique of the so-called ‘precautionary principle’, see Harris and Holm.15

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Conclusion This chapter has argued that, although stem cell and gene therapies raise a range of ethical issues that require significant analysis and reflection, none of the issues is truly novel or uniquely specific to these therapies. We may not be able to transfer our way of handling, for example, consent for other complex and risky treatments completely unmodified but there is also no reason to believe that we need radically new regulatory frameworks for these kinds of therapies.* Acknowledgements The research for this chapter has been supported by CESAGEN, the ESRC Centre for the Social and Economic Aspects of Genomics.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17.

Holm S. Going to the roots of the stem cell controversy. Bioethics 2002;16:493–507. Green RM. Benefiting from ‘evil’: an incipient moral problem in human stem cell research. Bioethics 2002;16:544–56. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861–72. Holm S. Time to reconsider stem cell ethics – the importance of induced pluripotent cells. J Med Ethics 2008;34:63–4. Crabtree S, Pelham B. What Alabamians and Iranians Have in Common [www.gallup.com/ poll/114211/Alabamians-Iranians-Common.aspx]. Holm S. Are countries that ban human embryonic stem cell research hypocritical? Regen Med 2006;1:357–9. Great Ape Project [www.greatapeproject.org]. Porada CD, Park PJ, Tellez J, Ozturk F, Glimp HA, Almeida-Porada G, et al. Male germ-line cells are at risk following direct-injection retroviral-mediated gene transfer in utero. Mol Ther 2005;12:754–62. Welin S. Starting clinical trials of xenotransplantation – reflections on the ethics of the early phase. J Med Ethics 2000;26:231–6. Harris J. Wonderwoman & Superman: The Ethics of Human Biotechnology. Oxford: Oxford University Press; 1992. Bortolotti L, Heinrichs B. Delimiting the concept of research: an ethical perspective. Theor Med Bioeth 2007;28:157–79. U.S. Food and Drug Administration. Expanded Access and Expedited Approval of New Therapies Related to HIV/AIDS [www.fda.gov/ForConsumers/ByAudience/ForPatientAdvocates/ HIVandAIDSActivities/ucm134331.htm]. Buchanan AE, Brock DW. Deciding for Others: The Ethics of Surrogate Decision Making. Cambridge: Cambridge University Press; 1989. Ross LF. Children, Families and Health Care Decision Making. Oxford: Oxford University Press; 1998. Harris J, Holm S. Extending human lifespan and the precautionary paradox. J Med Philos 2002;27:339–54. Frank RG. Prescription drug prices: why do some pay more than others do? Health Aff (Millwood) 2001;20:115–28. Expert Scientific Group on Phase One Clinical Trials. Final Report. London: TSO (The Stationery Office); 2006 [www.dh.gov.uk/en/Publicationsandstatistics/Publications/ PublicationsPolicyAndGuidance/DH_063117].

* This does, again, not imply anything about whether we need new, or perhaps just tighter, regulation in relation to research with these therapies in humans. Given that they are technically novel and very complex and may give rise to new types of risk, we may well decide that they should be regulated separately until we know enough about the risks they entail. With hindsight, it would, for example, have been a good thing if research with immune-modulating monoclonal antibodies had been regulated and monitored more tightly.17

9 Chapter 9

Fetal dysmorphology: the role of the geneticist in the fetal medicine unit in targeting diagnostic tests Tessa Homfray

Introduction A clinical geneticist can have several roles in a fetal medicine unit: assisting with management of the family at high risk of a genetic condition, aiding the interpretation of complex chromosome rearrangements and facilitating the diagnosis of a fetus with structural anomalies but apparently normal chromosomes. With increasing routine use of prenatal sonography for the diagnosis of fetal anomalies and the technological improvements in ultrasound machines, more fetal abnormalities are being detected. Defining the prognosis for a fetus with structural abnormalities if the karotype is abnormal can be reasonably straightforward but cases with apparently normal chromosomes are more challenging. Accurate prenatal diagnosis of rare syndromes is becoming an increasing reality with the advances in ultrasonography and molecular technology. Such accuracy is not always possible prenatally, and postnatal confirmation is thus essential. For the past two decades, increasing numbers of gene mutations have been identified that allow invasive prenatal diagnosis in subsequent pregnancies in early gestation, giving parents in many countries around the world choices in the management of affected pregnancies. This does, however, require a couple to have a family history of a disorder, with accurate confirmation of the diagnosis, usually before pregnancy. In the absence of a family history, an abnormality will only be identified by a screening test. Screening tests during pregnancy fall into two groups: n those identified on blood tests, such as sickle cell trait, and first- and secondtrimester biochemical screening for Down syndrome n those identified on ultrasound. Biochemical screening tests can identify an unexpected result not in keeping with a karyotypic abnormality, such as low unconjugated estriol,1 which might suggest a diagnosis of steroid sulphatase deficiency that causes severe ichthyosis, or very high alpha-fetoprotein that suggests Finnish nephropathy,2,3 but it is ultrasound that more frequently gives unexpected results. © Tessa Homfray. Volume compilation © RCOG

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Confirmation of the underlying diagnosis for ultrasound abnormalities is important both for the prognosis of the abnormalities identified and for the associated potential abnormalities that cannot be seen on ultrasound. For example, tetralogy of Fallot identified antenatally may have a very good prognosis for later cardiac function but if associated with a chromosome 22q11.2 microdeletion will have other abnormalities such as learning difficulties that may have a much more significant impact on the child and the family than the congenital heart disease itself. Major advances in genetics have identified the genes involved with many syndromes, which has been made possible by clinical syndrome recognition and also by rapid advances in molecular technology. Mutational analysis can now be undertaken much faster than previously, allowing a specific diagnosis within the time frame of the pregnancy. The clinical utility of molecular cytogenetics has yet to be realised but this is another major advance that will change our ability to confirm the underlying cause of many multiple malformation syndromes identified prenatally. The management of a pregnancy at known high risk for fetal abnormality because of an affected family member can be more straightforward but there are still many cases where a joint consultation between the geneticist and fetal medicine unit could be advantageous. If a parent is affected with an autosomal dominant disease, the offspring risk is 50% but these diseases are often very variable within a family. There are a number of possibilities that may concern the parents: they may be only concerned about severe manifestations of the disease; they may not want to take any risks of passing on the disease; or they may not be concerned either way. If the mother is the affected parent, there may be concerns about her own health during the pregnancy. For example, a parent with achondroplasia is rarely concerned about having an affected child but if the partner also has a skeletal dysplasia, the combination of the two disorders might be lethal and they would wish for a prenatal diagnosis to exclude this possibility. A woman with Ehlers–Danlos syndrome type  IV may have high risk of arterial rupture during pregnancy and will therefore need intensive surveillance throughout the pregnancy, and her baby will be at high risk of carrying the disorder.4 Treacher Collins syndrome is an autosomal dominant disorder with extremely variable penetrance. In its mildest form, a gene carrier may have an underdeveloped zygomatic arch and in its severest form there may be severe disfigurement with deafness, micrognathia and a cleft palate, and the baby may require a tracheostomy and occasionally may die from recurrent aspirational pneumonia. Ultrasound would miss a mildly affected baby, which in any case would not be associated with major complications, but examination for specific features on the 18–20  week scan may identify a severely affected fetus, possibly with the help of 3-D ultrasound.5 A specialist in fetal medicine would not be expected to know the variability of a condition, the likely gestation at which the abnormality could be identified or the prognosis of a rare disorder in the absence of the geneticist. There are a number of conditions whereby the specific anxiety for a couple can be discussed in advance and then the presence of the geneticist may be unnecessary but in these circumstances, if there are unexpected findings, if the geneticist is present these can be discussed at the time of diagnosis. Branchio-oto-renal syndrome is an autosomal dominant disorder associated with branchial pits, hearing loss and variable renal abnormalities ranging from a dysplastic kidney to bilateral renal agenesis.6 More detailed counselling may be required if the renal function is predicted to be compromised but there is not renal agenesis.

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Increased nuchal translucency with a normal karyotype A markedly raised nuchal translucency (NT) with a normal karyotype may be associated with a cardiac abnormality and cardiac scanning can identify abnormalities in expert hands from 13 weeks of gestation,7–9 but it is also known to be associated with other abnormalities including diaphragmatic hernia and severe skeletal dysplasias such as the short-rib polydactyly syndromes. Many of these syndromes are so rare that it is difficult to know whether an increased NT is a consistent finding in these conditions.10,11 However, the finding of an enlarged NT in the presence of multiple anomalies and dysmorphic features but a normal karyotype should alert the clinician to the possibility of an underlying genetic syndrome such as Noonan syndrome.12 Many of the dysmorphic features of Noonan syndrome, with low-set posterior rotated ears and a low posterior hairline, are probably secondary to an increased NT. Pulmonary stenosis may be identified later on in the pregnancy but it may be progressive and therefore may not be identified until after birth. Parental examination by a clinical geneticist may identify some of the subtle dysmorphic features associated with Noonan syndrome, thereby aiding prenatal diagnosis. The development of a cardiomyopathy and polyhydramnios suggests a much more sinister prognosis such as Costello syndrome.13 PTPN11 mutations can be identified in 50% of children with Noonan syndrome and HRAS mutations in more than 90% of babies with Costello syndrome.14 The latter may be particularly helpful as children with Costello may have a severe cardiomyopathy as well as have a significant degree of developmental delay. If the detailed 20 week scan shows no obvious abnormality following detection of increased NT, the parents should be advised that the risk of undetected long-term problems is decreased, but a high NT and the persistence of a nuchal fold later on in the pregnancy continue to raise the risk of undiagnosed fetal abnormalities.15

Identification of fetal abnormalities at the 11–13 week scan The 11–13 week scan is primarily a screening scan for Down syndrome but the increasing resolution of ultrasound machines and the expertise of sonographers are resulting in the identification of an increasing number of abnormalities. Parents are often disappointed when an abnormality is picked up at the 20 week scan that had been missed at the 13 week scan and it is important that expectations are managed appropriately. Abnormalities that may be identified at the early scan that warrant input from a clinical geneticist include holoprosencephaly and omphalocele. Holoprosencephaly A failure of division of the forebrain results in holoprosencephaly, for which three types can be recognised: alobar (Figure 9.1), semilobar and lobar. Alobar holoprosencephaly can be recognised on the 12 week scan and the prognosis, whatever the underlying aetiology, is appalling. The majority of couples choose to terminate the pregnancy on the basis of this abnormality alone. It is essential to establish the underlying diagnosis for future pregnancies. Trisomy 13 is the most common cause of holoprosencephaly but other abnormalities of chromosome 13 may also cause this malformation.16 These abnormalities may arise from a balanced translocation in the parent and therefore there would be a significant chance of a recurrence. Other causes of holoprosencephaly include mutations in a number of genes, many of which are autosomal dominantly inherited with very variable penetrance.17 All cases with holoprosencephaly but normal

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Figure 9.1 Alobar holoprosencephaly, in which no division of the brain can be identified

chromosomes should be referred for further genetic evaluation. A family history may reveal previous affected cases, and fetal tissue and parental bloods should be screened for mutations in genes known to cause holoprosencephaly. Expert examination of the parents may reveal subtle dysmorphic features not obvious to the obstetrician. In the presence of a family history or familial mutation, recurrence risks may be as high as 50%.18 As the condition can be extremely variable, in these cases prenatal diagnosis based on DNA analysis may be the only reliable method, but prediction of outcome can be very difficult in the absence of ultrasound or magnetic resonance imaging (MRI) anomalies. Following the diagnosis of a severe abnormality, parents are always very upset and may not wish to contemplate invasive testing. However, chromosomes frequently fail to grow after termination and hence the diagnostic opportunity has passed. DNA can be stored even from a surgical termination and molecular techniques can now be employed to confirm a diagnosis, so it is very important that this is arranged with the surgical team before the termination. Omphalocele Thirty percent of omphaloceles will have an associated chromosome abnormality, with trisomy  18 being the most common cause. If the karyotype is normal then other genetic conditions should be considered, such as Beckwith–Wiedemann syndrome.19 Babies with Beckwith–Wiedemann syndrome may develop macroglossia and nephromegaly. The omphalocele is most likely to be identified antenatally at the routine scan, with the other common finding being nephromegaly with increased liquor. The genetics of Beckwith–Wiedemann syndrome are complicated but the prognosis is good. Those cases with associated learning difficulties are said to be

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secondary to unrecognised hypoglycaemia in the neonatal period unless there is a paternal duplication of 11p15. There is an association with the development of Wilms’ tumours in early childhood. Wilms’ tumour only develops in a subgroup of those with Beckwith–Wiedemann syndrome and this testing can now be undertaken antenatally, which may go some way to reassuring the parents of an optimistic prognosis for their child.20 It is not known what proportion of patients with an omphalocele and normal chromosomes have Beckwith–Wiedemann syndrome as the underlying cause but in a prospective study of omphaloceles identified prenatally the proportion that had a methylation abnormality at 11p15 was six of 30.21

Anomalies detected at the 18–20 week scan Fetal anomalies are most frequently recognised at the 18–20  week scan but there are a few notable exceptions of what might be missed. Some syndromes evolve with time and therefore no abnormalities will be present at 20 weeks of gestation. The heart is fully formed by 20  weeks and therefore a major cardiac abnormality is likely to be identified. Hypoplastic left or right heart syndromes can become more or less significant during the pregnancy depending on flow of blood through rudimentary heart valves but in the majority of cases an accurate prognosis can be given by the fetal cardiologist by this gestation. On the other hand, development of the brain continues throughout the pregnancy and many abnormalities will not yet be detectable at the 20 week scan, for example many cases of hydrocephalus and neuronal migrational abnormalities. Achondroplasia cannot be recognised on ultrasound at 20 weeks, as the long bones are not unusually short at this gestation. The rest of this chapter will discuss the differential diagnosis and further investigations of fetal abnormalities identified on the anomaly scan. Specific brain malformations will not be discussed further as they are beyond the scope of this chapter. Accurate diagnosis of fetal brain malformations remains challenging when isolated, although fetal MRI is proving to be a useful adjunct to ultrasound in antenatal diagnosis.22 Skeletal dysplasias A large number of skeletal dysplasias resulting in varying degrees of short stature and other orthopaedic problems have been described. Many are not detectable prenatally as significant limb shortening does not occur until postnatal life. For those that do present prenatally, precise definition of the diagnosis may be difficult as most of the genes involved are large and are not amenable to analysis during the time frame of a pregnancy. Many of the X-ray abnormalities diagnosed postnatally cannot be identified by ultrasound antenatally, partly because ultrasound is not as good at looking at the skeleton as X-rays but also because the development of the epiphyses is late and these may help identify the correct diagnosis. However, this is an area where an expert in fetal dysmorphology may be helpful as knowledge of the associated features and patterns of affected limb shortening may aid accurate prenatal diagnosis.23 It must also be remembered that, while a short femur is often associated with an underlying skeletal dysplasia, it can also be a feature of aneuploidy or a sign of early fetal growth restriction.24 Other features of skeletal dysplasias include a narrow chest and hypomineralisation of boney structures. A narrow chest at the 20  week scan is often a sign of a very poor prognosis and can suggest that the pregnancy will result in neonatal death or stillbirth secondary to pulmonary hypoplasia, but some conditions present prenatally with a small chest and may have a better prognosis. Ellis–van Creveld syndrome can

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present early with very short long bones, a narrow chest and polydactyly, all of which are features suggestive of the short-rib polydactyly syndromes that are all lethal, but this syndrome has a much better prognosis and must always be considered in the differential diagnosis.25 Jeune’s asphyxiating dystrophy should also be considered and, although frequently lethal, prolonged survival is well recognised.26 Many of the short-rib polydactyly syndromes will have a raised NT followed by the development of hydrops; the latter confirms that the outcome will be lethal. Ellis–van Creveld syndrome may have associated congenital heart disease although this may be an atrial septal defect that will not be recognised antenatally. Other features of the short-rib polydactyly syndromes include cleft lip and palate, sex reversal and omphalocele.23,27 All of these syndromes are autosomal recessive with a one in four recurrence risk. Confirmation by DNA analysis is not possible in the first affected pregnancy. In a couple of Amish origin, the ‘Amish mutations’ could be looked for in the Ellis–van Creveld gene. The first short-rib polydactyly syndrome gene has recently been identified and joins the large number of diseases caused by mutations in cilial proteins.28 Diastrophic dysplasia is another autosomal recessive skeletal dysplasia that is allelic to atelosteogenesis type II and achondrogenesis type 1B, and these all have mutations in the DTD sulphate transporter gene of varying severity. Careful ultrasound examination may suggest the diagnosis by the presence of talipes: if talipes is identified in the presence of short limbs, the thumb should be examined as it is usually fully abducted in the typical hitchhiker posture. Three-dimensional ultrasound may show a growth in the ear. Bent bones may be symmetrical or asymmetrical; the differential between bent and fractured bone is not possible to distinguish on a still image but only by real-time ultrasound. An isolated bent short femur has an excellent prognosis and seems to be secondary to an antenatal insult in the first trimester; it may be part of the spectrum of the femur fibula ulna (FFU) syndrome. Fractures are most likely to be due to osteogenesis imperfecta. The severity can be variable but the mildest type I rarely has an antenatal presentation. Type II is lethal and type  III (Figure  9.2) is non-lethal but can be extremely severe, with multiple fractures occurring on normal handling. Independent mobility is usually not possible owing to recurrent fractures,29 although postnatal treatment with bisphosphonates may improve outcome. The division between the different types is an X-ray diagnosis but genetically the majority have mutations in the COL1A1 and COL1A2 genes, with the different severity partially being explained by the affect of the mutation on protein

Figure 9.2 Osteogenesis imperfecta type III, with shortened and bent long bones with fractures visible in this severe case

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function. The recurrence risk is low for all types of osteogenesis imperfecta and is normally secondary to germinal mosaicism.30 Mutations in LEPRE1 and CRTAP have been described and cause autosomal recessive osteogenesis imperfecta.31 The differential for osteogenesis imperfecta is hypophosphatasia (Figure 9.3) caused by mutations in the tissue non-specific alkaline phosphatase gene (ALPL). Measurement of parental serum alkaline phophatase may be low in cases of hypophosphatasia, which will help to distinguish it from osteogenesis imperfecta. Ultrasound features that may suggest hypophosphatasia are clear identification of the brain secondary to a very poorly calcified skull, and very poor ossification of the bones.32,33 Thanatophoric dysplasia is one of the easiest skeletal dysplasias to recognise as the long bones are very short; the femurs are bent and described as telephone handle femurs.34,35 Mutation analysis of the FGFR3 gene for the common mutations for thanatophoric dysplasia is possible antenatally but confirmation is rarely required. Two types of thanatophoric dysplasia can be identified: type 1 with bent femur and a normal-shaped skull and type 2 with straight femur and a cloverleaf skull. Type  II collagenopathies are increasingly recognised as having an antenatal phenotype of macrocephaly and short long bones at 20 weeks of gestation but this may be only just below the fifth centile. There may be talipes unilateral or bilateral. The most serious of the type II collagenopathy syndromes are achondrogenesis type 2, Kniest dysplasia and spondyloepiphyseal dysplasia congenita, which are epiphyseal disorders that can result in severe arthritis and kyphoscoliosis. It is not yet possible to have any assistance from DNA testing in a case arising de novo in pregnancy, although molecular testing can be offered to families where the mutation has been identified in an affected parent before pregnancy.36Achondroplasia occurring de novo in low-risk pregnancies will not be identified at the 18–20 week scan as the limbs are not short until the third trimester. Cardiac abnormalities Fetal echocardiography is a highly specialised prenatal sonographic investigation usually undertaken following detection of a raised NT or suspicion of a cardiac anomaly at the time of the routine anomaly scan. The geneticist will not be useful at this examination but once the abnormality has been identified further investigations may be indicated. Atrioventricular septal defects have a particularly high risk of having associated abnormalities.37

(a)

(b)

Figure 9.3 Hypophophatasia, with (a) severe undermineralisation of the whole skeleton and severe shortening of the long bones and (b) very poor mineralisation

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Chromosome  22q11.2 microdeletions are now well recognised as causing conotruncal abnormalities. A geneticist will not help with this diagnosis but will be essential when discussing the diagnosis once confirmed as the condition can be very variable. Occasionally, a chromosome abnormality might be identified before the cardiac scan, for instance cat eye syndrome secondary to a marker chromosome 22. This may be associated with total anomalous pulmonary venous drainage, which is a difficult cardiac diagnosis and is important to identify as it can otherwise be missed postnatally and present with sudden death. Cardiac rhabdomyomas can be identified on routine examination of the fourchamber view of the heart and confirmation is by fetal cardiological assessment. These are benign tumours that rarely cause problems themselves and disappear over the first 2 years of life. Occasionally, they cause obstruction of the ventricular outflow tract and dysrhythmias but their significance lies in their association with tuberous sclerosis. Tuberous sclerosis is a multisystem disease with very variable penetrance: it can range from being almost asymptomatic to severe developmental delay associated with intractable epilepsy and autism. Many cases arise as de novo mutations and only a cardiac rhabdomyoma gives an indication of the diagnosis antenatally. A fetus with a single rhabdomyoma is said to have a 50% chance of having tuberous sclerosis but multiple rhabdomyomas are invariable associated with tuberous sclerosis.38,39 A single rhabdomyoma may be associated with tuberous sclerosis in a much higher percentage of cases than previously quoted: as the accuracy of fetal echocardiography diagnosis improves the number that appear to be multiple increase and the definitive diagnosis becomes more certain. Careful examination of the parents by a clinical geneticist may reveal subtle skin changes associated with carrier status. Fetal brain MRI may help with confirmation of the diagnosis but, if a couple would consider a termination of pregnancy, invasive prenatal diagnosis should be undertaken at diagnosis to try to identify a mutation in one of the two tuberous sclerosis genes. Cleft lip and palate Clefting of the lip and alveoli are frequently identified antenatally. Isolated cleft palate is rarely identified routinely but can occasionally be seen later on in the pregnancy when the baby opens its mouth to swallow. Isolated cleft lip and palate have an excellent prognosis but many syndromes are known that have a cleft lip and palate as a feature. Detailed scanning including cardiac scanning should thus always be undertaken when it has been seen. Chromosomal abnormalities such as trisomy  13 and 4p may be present and karyotyping should be discussed, particularly if other anomalies or risk factors are present.40,41 Arthrogryposis Arthrogryposis may present at the 12 week scan with multiple symmetrical contractures and often with an increased NT. The prognosis for this group is usually extremely poor and the underlying diagnosis is often not obvious. A further scan should be undertaken after 2  weeks and if the contractures appear fixed then termination of pregnancy should be discussed. A post-mortem is essential to try to ascertain whether this a primary muscular or primary neurological disorder. Talipes may be the first sign of a progressive joint disorder. The causes that need to be considered are fetal restriction, fetal joint dislocation and neuromuscular disorders. The majority of isolated talipes have an extremely good prognosis but a further scan

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needs to be performed 4 weeks later to confirm that there has been no progression of the fetal contractures.42,43 Careful examination of the parents is recommended as very rarely evidence of contractures in one may indicate the diagnosis of a distal dominant arthrogryposis that can present prenatally with contractures and increased liquor. In these rare cases the prognosis may be better. Congenital muscular dystrophies may present with arthrogryposis or later with polyhydramnios and hydrops. The development of polyhydramnios in the presence of fetal contractures is a very poor sign and suggests severe muscular dysfunction. It is not currently possible in the presence of a normal brain ultrasound and fetal MRI to distinguish primary neurological causes from muscular causes of arthrogryposis. The recurrence risks and prenatal diagnosis in future pregnancies depend on accurate postnatal diagnosis and hence the collection of relevant specimens is essential. This may require muscle biopsy taken soon after birth and then rapid freezing to allow full analysis including mitochondrial enzyme analysis and electron microscopy. Fetal movements as felt by the mother appear to be a very poor indication of underlying severity. Development of unexplained acute polyhydramnios at about 30  weeks of gestation may suggest a diagnosis of myotubular myopathy. The most common type is X linked and there may be a positive family history of a neonatal death.44 As amniodrainage may need to be undertaken, it is possible that other tests can be undertaken on the fluid. The MTM gene is a small gene and hence rapid analysis is possible. Maternal myasthenia gravis may rarely present with recurrent arthrogryposis even in an asymptomatic mother.44–46 Antibodies to the fetal acetylcholinesterase receptor can be identified. Mutations in the acetylcholine receptor genes may also cause arthrogryposis, mutations in these genes can be identified and the recurrence risk in subsequent pregnancies is one in four.44,47,48 Myotonic dystrophy may have its first presentation in a family with a severely affected fetus with arthrogryposis and polyhydramnios, which may be secondary to the mother carrying the expansion in the myotonin gene. Maternal examination for signs of myotonic dystrophy should be undertaken as well as a family history of cataracts and fetal loss requested. Maternal mutational analysis of the myotonin gene should be undertaken.49 Fetal constriction may occur secondary to premature rupture of membranes or other causes of oligohydramnios, and the prognosis will depend on the underlying cause of the oligohydramnios. Uterine abnormalities can cause major fetal constriction and therefore should be examined for on ultrasound. These can be difficult to identify in pregnancy and if after birth this is suggested then further examination of the uterus is recommended. Fetal joint dislocations can occur in Larsen syndrome. Nail patella syndrome may show multiple joint dislocations but may be very variable within a family: examination of the parental patellae and nails and urine analysis for proteinuria may suggest this diagnosis. Hydrops Fetal hydrops is a frequent conundrum for the fetal medicine team. By the time they have summoned the geneticist, they will have excluded most of the common causes of the condition such as aneuploidy, fetal anaemia, viral infections and chorioangioma. Cardiac rhythm disturbance may lead to fetal hydrops and in the presence of a fetal bradycardia it is important to measure anti-rho antibodies in the mother.50 Fetal bradycardias can also be caused by inherited or de novo cardiac rhythm disorders such

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as long QT syndrome. A parental history for faints or epilepsy should be taken and electrocardiograms performed. Absence of the ductus venosus can lead to hydrops more commonly if there is direct drainage of the umbilical vein into the right atrium.51 Noonan syndrome and Turner syndrome have both been associated with this anomaly but in the absence of a raised NT these diagnoses are unlikely. Pleural effusions can lead to hydrops and if these are isolated drainage is indicated but the underlying cause will determine the outcome.52 Many genetic syndromes and metabolic conditions have been associated with fetal hydrops53–55 but the underlying abnormality is rarely identified during the pregnancy. It is essential that specimens are collected, either at the time of termination of pregnancy or at birth, as it may be too late at post-mortem to undertake enzyme analysis. Carbohydrate-deficient glycoprotein syndrome may be suggested by cerebellar lobar hypoplasia. Hepatosplenomegaly increases the chances of a metabolic syndrome. If a feticide is performed, amniotic fluid should be frozen as well as a fibroblast culture established for possible future metabolic tests. Blood should be taken for a full blood count including vacuolated lymphocytes. DNA should be stored and a karyotype performed if this has not already been undertaken earlier in the pregnancy.56 Sex reversal Micropenis or ambiguous genitalia may be detected antenatally but in the absence of any other abnormalities are unlikely to be identified unless a karyotype shows discordance of the fetal sex with the ultrasound. Many parents do not wish to know the sex of the baby so this information will need to be sought before examination if there are concerns. A family history may be suggestive of androgen insensitivity syndrome, which can be partial or complete.57 Other investigations of isolated genital anomalies include measurement of amniotic fluid steroid metabolites to exclude congenital adrenal hyperplasia.58 Early diagnosis may prevent an adrenal crisis occurring after birth. Total sex reversal of a 46,XY fetus may occur with congenital lipoid adrenal hyperplasia secondary to mutations in the StAR gene but this is very rare.59 21-hydroxylase deficiency resulting in clitoromegaly of 46,XX fetuses is the cause of congenital adrenal hyperplasia in over 90% of cases. Identification antenatally will allow correct sexual identification postnatally, which will be very helpful to the parents and allow rapid treatment of steroidal insufficency. In the presence of genital ambiguity and other sonographic abnormalities, the clinical geneticist may be very helpful in defining the precise diagnosis as many syndromes are associated with genital anomalies.60 If the long bones are short and bent then campomelic dysplasia needs to be considered. Other clues to this diagnosis would be small scapula, cleft palate and talipes.61 Smith–Lemli–Opitz syndrome is an autosomal recessive disorder with 46,XY sex reversal associated with dysmorphic features, mild fetal growth restriction, occasionally major brain malformations and moderate to severe developmental delay. It is secondary to an enzyme deficiency of 7-dehydrocholesterol reductase. Measurement of maternal urinary sterols may indicate this diagnosis and 7-dehydrocholesterol levels can be examined in the amniotic fluid.62,63 CHARGE syndrome secondary to mutations in the CDH7 gene is a difficult antenatal diagnosis. Features are frequently identified antenatally and with de novo mutations now being able to be identified within a 1 month time frame this may be of great value. These babies may have ambiguous genitalia, congenital heart

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Figure 9.4 CHARGE ear, with (a) a malformed and thickened right ear and (b) the left ear set low and posteriorly rotated but without the classical morpholosty seen postnatally in a CHARGE ear

disease, fetal growth restriction, oesophageal atresia and cleft lip and palate that may be identified antenatally.64 The cardinal features of the syndrome of choanal atresia and coloboma will be missed. Identification of a CHARGE ear on 3-D ultrasound (Figure 9.4) is possible and may help to direct investigations appropriately. Children with CHARGE syndrome can have severe mental handicap and it is an important diagnosis not to miss. Isolated limb abnormalities Isolated limb abnormalities frequently have a very good prognosis but there are a few conditions that need to be considered. Ectrodactyly (lobster claw hand) is frequently genetic but is normally isolated and counselling is reassuring. Cornelia de Lange syndrome65 is a serious disorder that is associated with severe developmental delay and is caused by a new dominant mutation in the majority of cases. Fetal growth restriction occurs late in gestation and is unlikely to be identified at the 20 week scan. Ectrodactyly is frequently the only manifestation on ultrasound (Figure 9.5). If a limb abnormality is identified before 20 weeks, pregnancy-associated plasma protein A (PAPP-A) measured in the second trimester is consistently low.66 Three-dimensional ultrasound can help to identify the dysmorphic features of this syndrome. Radial ray defects should always be referred for a second opinion as they are frequently associated with a syndromic diagnosis such as VACTERL association, Holt–Oram syndrome and Fanconi anaemia. Trisomy 18 can also cause radial aplasia and this should be looked for before other diagnoses are considered. A genetic opinion may be of value in some situations such as Holt–Oram syndrome, where subtle anomalies in a parent may aid prenatal diagnosis. Gastrointestinal disorders Bowel obstructions are the most frequently diagnosed gastrointestinal disorder. Duodenal atresia with the familiar double bubble is characteristically associated with Down syndrome. Cystic fibrosis can cause bowel perforation and obstruction and hyperechogenic bowel is the only ultrasound feature that will be identified.67

142  |  TESSA HOMFRAY (a)

(b)

(c)

(d)

Figure 9.5 Facial features and limb abnormalities of Cornelia de Lange syndrome: the limb shows ectrodactyly (a,b) and the face shows arched eyebrows (c) with a long smooth philtrum (d)

Oesphageal atresia has associations with a number of genetic syndromes.68 It can be suspected at the time of the second trimester anomaly scan if the stomach bubble is persistently absent, but it may only be suspected late in the second trimester if a tracheo-oesphageal fistula is present as a stomach may still be identified and the only sonographic sign will be polyhydramnios. CHARGE syndrome should be considered and the karyotypic sex should be checked against the genital sex on ultrasound. There is rarely full sex reversal but a micropenis should raise concerns. Ear malformations could be identified on 3-D ultrasound. Other common conditions that should be considered include the VACTERL association, which can be associated with radial, renal, cardiac and spinal anomalies.19 Renal disorders Renal anomalies are among those most frequently diagnosed prenatally and, while the majority are isolated, they also have many genetic associations69 and can have a significant recurrence risk. Renal agenesis is usually isolated but can carry a significant recurrence risk if one of the parents has unilateral renal ageneis or if it is associated with other anomlies. In one study, de novo RET mutations were identified in 37% of stillborn infants with bilateral renal agenesis70 when the recurrence risk is low. Other syndromes such as

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branchio-oto-renal syndrome and renal urodysplasia may present with bilateral renal agenesis and the parents should both be examined and renal ultrasound undertaken.71 Identification of large echogenic fetal kidneys72 requires renal ultrasound of both parents to be undertaken as autosomal dominant kidney disease can present prenatally73 and an affected parent may be asymptomatic at the time of the fetal diagnosis. Large echogenic fetal kidneys, with or without cystic change, are associated with a wide range of conditions69 and some such as autosomal recessive polcystic kidney disease, Meckel–Gruber syndrome and Bardet–Beidel syndrome have high recurrence risks. The latter both have associated malformations that can be detected prenatally, including polydactyly and central nervous system malformations, and it can be difficult to distinguish between the two conditions.74 The genes causing these syndromes code for a variety of cilial proteins. Mutation testing is not possible at the time of an ultrasound diagnosis but may be helpful to facilitate early molecular prenatal diagnosis in future pregnancies, although frequently ultrasound diagnosis remains the only option.

Summary Advances in prenatal diagnosis have been very rapid and the number of syndromes that can accurately be diagnosed prenatally continue to increase. Confirmation of the diagnosis requires the doctor to recognise features suggestive of the disease as testing is very specific for each disease. New genes are being identified daily and new technology expands the availability of specific tests all the time. In view of the rarity of the diseases, geneticists are the most likely to be able to recognise the syndromes from their experience in dysmorphology. Examination of parents and the family history can give helpful clues to the underlying diagnosis of a dysmorphic fetus and this is best done by the clinical geneticist. The pattern of malformations can suggest targeted investigations but, as still images can be difficult to interpret, ideally the geneticist should be present in the fetal medicine unit to aid scan interpretation. Prompt referral for detailed ultrasound with a genetics opinion allows appropriate investigations to be undertaken in a timely manor. Many syndromes remain undiagnosed antenatally and the post-mortem examination is essential. The expanding number of disease confirmatory tests means that the correct collection of relevant samples at the time of feticide or soon after birth needs to be carefully coordinated.

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Glass IA, Lam RC, Chang T, Roitman E, Shapiro LJ, Shackleton CH. Steroid sulphatase deficiency is the major cause of extremely low oestriol production at mid-pregnancy: a urinary steroid assay for the discrimination of steroid sulphatase deficiency from other causes. Prenat Diagn 1998;18:789–800. Heinonen S, Ryynänen M, Kirkinen P, Saarikoski S. Endometrial and fetoplacental markers in pregnancies with fetal congenital nephrosis. Acta Obstet Gynecol Scand 1996;75:526–30. Patrakka J, Martin P, Salonen R, Kestilä M, Ruotsalainen V, Männikkö M, et al. Proteinuria and prenatal diagnosis of congenital nephrosis in fetal carriers of nephrin gene mutations. Lancet 2002;359:1575–7. Rudd NL, Nimrod C, Holbrook KA, Byers PH. Pregnancy complications in type IV Ehlers– Danlos syndrome. Lancet 1983;1:50–3. Tanaka Y, Kanenishi K, Tanaka H, Yanagihara T, Hata T. Antenatal three-dimensional sonographic features of Treacher Collins syndrome. Ultrasound Obstet Gynecol 2002;19:414–15.

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Weber S, Moriniere V, Knüppel T, Charbit M, Dusek J, Ghiggeri GM, et al. Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study. J Am Soc Nephrol 2006;17:2864–70. Hyett J, Sonek J, Nicolaides K; FASTER Consortium. Nuchal translucency and the risk of congenital heart disease. Obstet Gynecol 2007;109:1455–6. Simpson LL, Malone FD, Bianchi DW, Ball RH, Nyberg DA, Comstock CH, et al. Nuchal translucency and the risk of congenital heart disease. Obstet Gynecol 2007;109:376–83. Carvalho JS. Early prenatal diagnosis of major congenital heart defects. Curr Opin Obstet Gynecol 2001;13:155–9. Souka AP, Von Kaisenberg CS, Hyett JA, Sonek JD, Nicolaides KH. Increased nuchal translucency with normal karyotype. Am J Obstet Gynecol 2005;192:1005–21. Erratum in: Am J Obstet Gynecol 2005;192:2096. Brady AF, Pandya PP, Yuksel B, Greenough A, Patton MA, Nicolaides KH. Outcome of chromosomally normal livebirths with increased fetal nuchal translucency at 10–14 weeks’ gestation. J Med Genet 1998;35:222–4. Lee KA, Williams B, Roza K, Ferguson H, David K, Eddleman K, et al. PTPN11 analysis for the prenatal diagnosis of Noonan syndrome in fetuses with abnormal ultrasound findings. Clin Genet 2009;75:190–4. Smith LP, Podraza J, Proud VK. Polyhydramnios, fetal overgrowth, and macrocephaly: prenatal ultrasound findings of Costello syndrome. Am J Med Genet A 2009;149A:779–84. Schulz AL, Albrecht B, Arici C, van der Burgt I, Buske A, Gillessen-Kaesbach G, et al. Mutation and phenotypic spectrum in patients with cardio-facio-cutaneous and Costello syndrome. Clin Genet 2008;73:62–70. Bilardo CM, Muller MA, Pajkrt E, Clur SA, van Zalen MM, Bijlsma EK. Increased nuchal translucency thickness and normal karyotype: time for parental reassurance. Ultrasound Obstet Gynecol 2007;30:11–18. Quélin C, Bendavid C, Dubourg C, de la Rochebrochard C, Lucas J, Henry C, et al. Twelve new patients with 13q deletion syndrome: genotype-phenotype analyses in progress. Eur J Med Genet 2009;52:41–6. Dubourg C, Bendavid C, Pasquier L, Henry C, Odent S, David V. Holoprosencephaly. Orphanet J Rare Dis 2007;2:8. David AL, Gowda V, Turnbull C, Chitty LS. The risk of recurrence of holoprosencephaly in euploid fetuses. Obstet Gynecol 2007;110:658–62. Hurst J, Firth HV, Chitty LS. Syndromic associations with congenital anomalies of the fetal thorax and abdomen. Prenat Diagn 2008;28:676–84. Scott RH, Douglas J, Baskcomb L, Huxter N, Barker K, Hanks S, et al. Constitutional 11p15 abnormalities, including heritable imprinting center mutations, cause nonsyndromic Wilms tumor. Nat Genet 2008;40:1329–34. Scott RH, Walker L, Olsen ØE, Levitt G, Kenney I, Maher E, et al. Surveillance for Wilms tumour in at-risk children: pragmatic recommendations for best practice. Arch Dis Child 2006;91:995–9. Guibaud L. Contribution of fetal cerebral MRI for diagnosis of structural anomalies. Prenat Diagn 2009;29:420–33. Chitty LS, Griffin DR. Prenatal diagnosis of fetal skeletal anomalies. Fetal Matern Med Rev 2008;19:135–64. Papageorghiou AT, Fratelli N, Leslie K, Bhide A, Thilaganathan B. Outcome of fetuses with antenatally diagnosed short femur. Ultrasound Obstet Gynecol 2008;31:507–11. Guschmann M, Horn D, Gasiorek-Wiens A, Urban M, Kunze J, Vogel M. Ellis–van Creveld syndrome: examination at 15 weeks’ gestation. Prenat Diagn 1999;19:879–83. den Hollander NS, Robben SG, Hoogeboom AJ, Niermeijer MF, Wladimiroff JW. Early prenatal sonographic diagnosis and follow-up of Jeune syndrome. Ultrasound Obstet Gynecol 2001;18:378–83. Hill LM, Leary J. Transvaginal sonographic diagnosis of short-rib polydactyly dysplasia at 13 weeks’ gestation. Prenat Diagn 1998;18:1198–201. Merrill AE, Merriman B, Farrington-Rock C, Camacho N, Sebald ET, Funari VA, et al. Ciliary abnormalities due to defects in the retrograde transport protein DYNC2H1 in short-rib polydactyly syndrome. Am J Hum Genet 2009;84:542–9.

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Engelbert RH, Uiterwaal CS, Gulmans VA, Pruijs H, Helders PJ. Osteogenesis imperfecta in childhood: prognosis for walking. J Pediatr 2000;137:397–402. Bodian DL, Madhan B, Brodsky B, Klein TE. Predicting the clinical lethality of osteogenesis imperfecta from collagen glycine mutations. Biochemistry 2008;47:5424–32. Willaert A, Malfait F, Symoens S, Gevaert K, Kayserili H, Megarbane A, et al. Recessive osteogenesis imperfecta caused by LEPRE1 mutations: clinical documentation and identification of the splice form responsible for prolyl 3-hydroxylation. J Med Genet 2009;46:233–41. Simon-Bouy B, Taillandier A, Fauvert D, Brun-Heath I, Serre JL, Armengod CG, et al. Hypophosphatasia: molecular testing of 19 prenatal cases and discussion about genetic counseling. Prenat Diagn 2008;28:993–8. Zankl A, Mornet E, Wong S. Specific ultrasonographic features of perinatal lethal hypophosphatasia. Am J Med Genet A 2008;146A:1200–4. Li D, Liao C, Ma X. Prenatal diagnosis and molecular analysis of type 1 thanatophoric dysplasia. Int J Gynaecol Obstet 2005;91:268–70. Sahinoglu Z, Uludogan M, Gurbuz A, Karateke A. Prenatal diagnosis of thanatophoric dysplasia in the second trimester: ultrasonography and other diagnostic modalities. Arch Gynecol Obstet 2003;269:57–61. Chitty LS, Tan AW, Nesbit DL, Hall CM, Rodeck CH. Sonographic diagnosis of SEDC and double heterozygote of SEDC and achondroplasia – a report of six pregnancies. Prenat Diagn 2006;26:861–5. Berg C, Kaiser C, Bender F, Geipel A, Kohl T, Axt-Fliedner R, et al. Atrioventricular septal defect in the fetus – associated conditions and outcome in 246 cases.Ultraschall Med 2009;30:25–32. Chao AS, Chao A, Wang TH, Chang YC, Chang YL, Hsieh CC, et al. Outcome of antenatally diagnosed cardiac rhabdomyoma: case series and a meta-analysis. Ultrasound Obstet Gynecol 2008;31:289–95. Jóźwiak S, Kotulska K. Are all prenatally diagnosed multiple cardiac rhabdomyomas a sign of tuberous sclerosis? Prenat Diagn 2006;26:867–9. Liu Y, Freitas Rda S, Magriples U, Persing JA, Shin JH. Fetal diagnosis of cleft lip: natural history and outcomes. J Craniofac Surg 2008;19:1195–8. Bergé SJ, Plath H, von Lindern JJ, Appel T, Niederhagen B, Van de Vondel PT, et al. Natural history of 70 fetuses with a prenatally diagnosed orofacial cleft. Fetal Diagn Ther 2002;17:247–51. Swamy R, Reichert B, Lincoln K, Lal M. Foetal and congenital talipes: interventions and outcome. Acta Paediatr 2009;98:804–6. Bakalis S, Sairam S, Homfray T, Harrington K, Nicolaides K, Thilaganathan B. Outcome of antenatally diagnosed talipes equinovarus in an unselected obstetric population. Ultrasound Obstet Gynecol 2002;20:226–9. Jungbluth H, Wallgren-Pettersson C, Laporte J. Centronuclear (myotubular) myopathy. Orphanet J Rare Dis 2008;3:26. Hoff JM, Daltveit AK, Gilhus NE. Artrogryposis multiplex congenita – a rare fetal condition caused by maternal myasthenia gravis. Acta Neurol Scand Suppl 2006;183:26–7. Oskoui M, Jacobson L, Chung WK, Haddad J, Vincent A, Kaufmann P, et al. Fetal acetylcholine receptor inactivation syndrome and maternal myasthenia gravis. Neurology 2008;71:2010–12. Steinlein OK. Genetic disorders caused by mutated acetylcholine receptors. Life Sci 2007;80:2186–90. Hoffmann K, Muller JS, Stricker S, Megarbane A, Rajab A, Lindner TH, et al. Escobar syndrome is a prenatal myasthenia caused by disruption of the acetylcholine receptor fetal gamma subunit. Am J Hum Genet 2006;79:303–12. Zaki M, Boyd PA, Impey L, Roberts A, Chamberlain P. Congenital myotonic dystrophy: prenatal ultrasound findings and pregnancy outcome. Ultrasound Obstet Gynecol 2007;29:284–8. Clancy RM, Buyon JP, Ikeda K, Nozawa K, Argyle DA, Friedman DM, et al. Maternal antibody responses to the 52-kd SSA/RO p200 peptide and the development of fetal conduction defects. Arthritis Rheum 2005;52:3079–86. Berg C, Kamil D, Geipel A, Kohl T, Knöpfle G, Hansmann M, et al. Absence of ductus venosusimportance of umbilical venous drainage site. Ultrasound Obstet Gynecol 2006;28:275–81.

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Sohan K, Carroll SG, De La Fuente S, Soothill P, Kyle P. Analysis of outcome in hydrops fetalis in relation to gestational age at diagnosis, cause and treatment. Acta Obstet Gynecol Scand 2001;80:726–30. Ismail KM, Martin WL, Ghosh S, Whittle MJ, Kilby MD. Etiology and outcome of hydrops fetalis. J Matern Fetal Med 2001;10:175–81. Van Maldergem L, Jauniaux E, Fourneau C, Gillerot Y. Genetic causes of hydrops fetalis. Pediatrics 1992;89:81–6. Burin MG, Scholz AP, Gus R, Sanseverino MT, Fritsh A, Magalhães JA, et al. Investigation of lysosomal storage diseases in nonimmune hydrops fetalis. Prenat Diagn 2004;24:653–7. Beke A, Joó JG, Csaba A, Lázár L, Bán Z, Papp C, et al. Incidence of chromosomal abnormalities in the presence of fetal subcutaneous oedema, such as nuchal oedema, cystic hygroma and nonimmune hydrops. Fetal Diagn Ther 2009;25:83–92. Riskin A, Koren I, Bader D, Grün M, Dar H, Leibovitz Z, et al. The approach to a neonate with a possible prenatal diagnosis of androgen insensitivity syndrome. J Pediatr Endocrinol Metab 2006;19:1437–43. Forest MG, Morel Y, David M. Prenatal treatment of congenital adrenal hyperplasia. Trends Endocrinol Metab 1998;9:284–9. Jean A, Mansukhani M, Oberfield SE, Fennoy I, Nakamoto J, Atwan M, et al. Prenatal diagnosis of congenital lipoid adrenal hyperplasia (CLAH) by estriol amniotic fluid analysis and molecular genetic testing. Prenat Diagn 2008;28:11–14. Pajkrt E, Petersen OB, Chitty LS. Fetal genital anomalies: an aid to diagnosis. Prenat Diagn 2008;28:389–98. Natasha G, Ghai R, Shah D, Kiran PS. Camptomelic dysplasia: prenatal diagnosis by ultrasound. Skeletal Radiol 2006;35:699–701. Griffiths WJ, Wang Y, Karu K, Samuel E, McDonnell S, Hornshaw M, et al. Potential of sterol analysis by liquid chromatography-tandem mass spectrometry for the prenatal diagnosis of Smith– Lemli–Opitz syndrome. Clin Chem 2008;54:1317–24. Porter FD. Smith–Lemli–Opitz syndrome: pathogenesis, diagnosis and management. Eur J Hum Genet 2008;16:535–41. Sanlaville D, Verloes A. CHARGE syndrome: an update. Eur J Hum Genet 2007;15:389–99. Chong K, Keating S, Hurst S, Summers A, Berger H, Seaward G, et al. Cornelia de Lange syndrome (CdLS): prenatal and autopsy findings. Prenat Diagn 2009;29:489–94. Aitken DA, Ireland M, Berry E, Crossley JA, Macri JN, Burn J, et al. Second-trimester pregnancy associated plasma protein-A levels are reduced in Cornelia de Lange syndrome pregnancies. Prenat Diagn 1999;19:706–10. Simon-Bouy B, Satre V, Ferec C, Malinge MC, Girodon E, Denamur E, et al.; French Collaborative Group. Hyperechogenic fetal bowel: a large French collaborative study of 682 cases. Am J Med Genet A 2003;121A:209–13. Shaw-Smith C. Oesophageal atresia, tracheo-oesophageal fistula, and the VACTERL association: review of genetics and epidemiology. J Med Genet 2006;43:545–54. Wellesley D, Howe DT. Fetal renal anomalies and genetic syndromes. Prenat Diagn 2001:21:992–1003. Skinner MA, Safford SD, Reeves JG, Jackson ME, Freemerman AJ. Renal aplasia in humans is associated with RET mutations. Am J Hum Genet 2008;82:344–51. Roume J, Ville Y. Prenatal diagnosis of genetic renal diseases: breaking the code. Ultrasound Obstet Gynecol 2004;24:10–18. Avni FE, Garel L, Cassart M, Massez A, Eurin D, Didier F, et al. Perinatal assessment of hereditary cystic renal diseases: the contribution of sonography. Pediatr Radiol 2006;36:405–14. Erratum in: Pediatr Radiol 2006;36:731. Brun M, Maugey-Laulom B, Eurin D, Didier F, Avni EF. Prenatal sonographic patterns in autosomal dominant polycystic kidney disease: a multicenter study. Ultrasound Obstet Gynecol 2004;24:55–61. Karmous-Benailly H, Martinovic J, Gubler MC, Sirot Y, Clech L, Ozilou C, et al. Antenatal presentation of Bardet–Biedl syndrome may mimic Meckel syndrome. Am J Hum Genet 2005;76:493–504.

10 Chapter 10

Fetal karyotyping: what should we be offering and how? John Crolla

Background Prenatal diagnosis (PND) for fetal chromosome abnormalities (karyotyping) began in the UK in the late 1970s with the introduction of amniocentesis followed by chorionic villus sampling (CVS). Amniotic fluid (AF) and CVS cell culture technologies were adopted by the 23 regional cytogenetic laboratories and the patient ascertainment group was initially restricted exclusively to women with age-related risk of Down syndrome pregnancies. Since then, a PND service has become available in all developed countries. Within the UK, approximately 30 000 chromosomal PNDs are performed each year, the vast majority of which are following risk screening protocols and tests defined by the UK National Screening Committee (NSC) policy on Down syndrome screening in pregnancy1 or, more rarely, because of maternal age alone or maternal anxiety. Following screening, women defined as ‘at risk’ for Down syndrome pregnancies or other trisomies are offered invasive PND and, for the vast majority of women, their AF or CVS samples are sent for karyotyping at the local regional cytogenetic centre. Although prenatal karyotyping has historically been considered the ‘gold standard’ test following invasive PND, the karyotyping technology has developed very little from that first introduced in the 1970s and, despite some improvements in the recent past, turnaround times in the UK for a prenatal karyotype still remains around 2 weeks for most women and clinicians (Figure  10.1), and these turnaround times show significant interlaboratory variation (Figure 10.2). Cytogenetic PND has always used full karyotype analysis to produce the diagnosis and is therefore capable of detecting visible chromosomal changes such as aneuploidy, numerical and structural mosaicism and balanced and unbalanced structural and numerical chromosome changes but detection rates are restricted by the resolution of the light microscope and the human eye to relatively ‘large’ chromosomal imbalances. In genomic terms, conventional prenatal karyotyping is only capable of detecting imbalances representing 8–10 Mb (million base pairs) or more of DNA. This becomes an important consideration in later discussions (see below). More recently, service delivery models for chromosomal PND were improved and speeded up by the introduction and widespread adoption of so called ‘rapid aneuploidy testing’ using either fluorescence in situ hybridisation (FISH) or © John Crolla. Volume compilation © RCOG

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Figure 10.1 National reporting times for prenatal karyotyping in the UK for the period 1995–2005; the variation in reporting times is shown by arrows; although the trend is towards faster conventional reporting times, the national average for the 2006–2007 audit period was still approximately 12 days; data from the Professional Standards Committee of the Association for Clinical Cytogenetics15

Figure 10.2 National reporting times by laboratory for prenatal karyotyping in the UK for the period 1995–2005; each bar represents an individual laboratory, with the variation ranging from 7 to 16 days; data from the Professional Standards Committee of the Association for Clinical Cytogenetics15

quantitative fluorescence polymerase chain reaction (QF-PCR). These tests are designed to detected trisomies 13, 18 and 21 and the sex chromosome aneuploidies using either uncultured AF or CVS cells (FISH) or extracted DNA (QF-PCR).2 The NSC recommended that prenatal FISH or PCR tests should only include trisomies 13, 18 and 21 and in some parts of the UK full karyotyping was replaced by QF-PCR

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as the standalone test in cases identified as being at high risk after Down syndrome screening, a decision that was considered controversial at the time.3 The steer towards more rapid prenatal testing results was also driven in part by the Government’s 2003 white paper Our Inheritance, Our Future,4 which set laboratory reporting times as ‘within three days where the result is needed urgently (e.g. for prenatal diagnosis)’. Consequently, QF-PCR and FISH are now widely used as a supplementary prenatal test targeted to pregnancies at high risk following antenatal screening (Figure 10.3).

Current PND service delivery models in the UK The antenatal subgroup of the NSC has been proactive in the development and implementation of a series of screening initiatives designed primarily to identify subpopulations of women at the highest risk of carrying a Down syndrome fetus.5 The standards and policies for Down syndrome screening are regularly revised and their principal aim is to improve the sensitivity of the screening tests used (serum markers, first- and second-trimester ultrasound, or combinations of both) and to reduce loss of normal pregnancies and reduce health service costs.6 The current defined programme outcomes are:1 n a detection rate for Down syndrome of greater than 75% of affected pregnancies with a screen positive rate of less than 3% (benchmark time frame: April 2007 to April 2010) n a detection rate of greater than 90% of affected pregnancies with a screen positive rate of less than 2% (benchmark time frame: by April 2010).

Figure 10.3 Current service delivery models for chromosomal prenatal diagnosis in the UK; the model on the left (no karyotyping in the absence of ultrasound abnormalities) is now restricted to London, the model on the right is now virtually non-existent, and the majority of laboratories now provide the central model of targeted rapid testing with full karyotyping; CVS = chorionic villus sampling

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It is beyond the scope of this chapter to discuss the merits or otherwise of this screening approach except to note that, particularly in the past 5 years, the UK-wide application of antenatal screening has led to a significant reduction in the overall number of invasive procedures and the 2010 benchmark, if successfully implemented, will see further reductions in the invasive procedure rates. The current UK PND service delivery models are outlined diagramatically in Figure  10.3 and almost universally follow a screening → rapid aneuploidy → full karyotyping pathway.

New technologies and PND In the context of developing PND service delivery models, there is no doubt that the most significant innovation will come from the application of a technology known as array comparative genomic hybridisation (aCGH), which in some postnatal clinical scenarios will soon replace conventional karyotyping as the front-line ‘whole-genome test’. In simple terms, aCGH is a way of identifying copy number changes between a ‘test’ (that is, the patient DNA sample) and a ‘control’ (that is, a normal DNA sample) using the principles of CGH.7 However, instead of targeting metaphase chromosomes, the differentially labelled patient DNA is competitively hybridised with control DNA and placed onto a glass slide on which thousands or millions of genomic probes (that is, with defined genomic sequence identities) have been spotted using modifications of high-resolution inkjet printing technology. Thus, an array can be composed of a variety of genomic ‘targets’, all with defined sequence identity, so that either specific regions of the genome or the whole genome (or a combination of a whole-genome ‘backbone’ and targeted regions) can be designed and covered on the array (Figure 10.4). Each target sequence on the array can be thought of as an individual FISH probe, so, in effect, an array result represents the sum of thousands or millions of individual FISH tests. The original research applications of arrays in postnatal genetics used relatively low-density arrays (approximately 1000 targets) and large insert clones (so-called bacterial artificial chromosomes, BACs) as the target sequences. These original arrays conclusively demonstrated the clinical utility of aCGH when applied to selected cohorts of children with idiopathic developmental delay8 by more than doubling the detection of pathogenic de novo chromosomal changes, thereby significantly improving the clinical diagnosis and management of patients with developmental delay, dysmorphism and/or congenital abnormalities.9 The postnatal application of aCGH in the UK has to date largely been targeted to retrospective cohorts of patients previously reported with normal chromosomes together with a long list of other negative investigations. It is important to stress that the aCGH results to date in this cohort have also conclusively shown that the arrays not only detect all the chromosome imbalances that would have been visible microscopically (that is, deletions or duplications of 6–10  Mb or more) but, when applied to some classes of visible structural and numerical chromosome abnormalities, also reveal cryptic pathogenic changes not resolvable by the light microscope.10,11 These early reports and subsequent larger population studies also showed that copy number changes occur with relatively high frequency to the extent that all genomes carry one or more deletions or amplifications of defined regions without any apparent deleterious phenotypic effects.12 These apparently benign copy number changes are frequently called copy number variants (CNVs) and their frequency and distribution in normal populations are summarised at the Database of Genomic Variants.13 An ability to distinguish between potentially pathogenic and ‘normal’ copy number variation is one of the cardinal analytical and interpretational issues surrounding the design and application of aCGH in postnatal diagnosis but particularly in PND (see below).

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Figure 10.4 Simple schematic of array comparative genomic hybridisation (aCGH); the patient DNA (e.g. extracted from amniocytes or chorionic villi) is labelled with a red fluorochrome and the control (‘normal’) DNA is differentially labelled with a green fluorochrome; the labelled DNA is mixed with a DNA enriched with repetitive sequences (Cot 1) and then hybridised to the array; following stringent washing, the ratio of red to green fluorescence is quantitatively analysed using a high-resolution scanner with extraction software that converts the red to green fluorescence at each target point on the array into a log2 ratio, which is then analysed using an analytical programme (see Figure 10.6); the arrays can be printed in many formats and 4 and 8 plex formats are shown on the left of the diagram

Low-resolution BAC arrays have largely been replaced by high-density and specifically designed arrays that use either oligonucleotides (oligos) or singlenucleotide polymorphisms (SNPs), or a combination of both, as array targets. It is beyond the remit of this chapter to discuss the relative merits of all these aCGH formats except to say that oligoarrays have specific advantages in a prenatal setting because they can be designed to focus on specific regions of the genome and to reduce the problems of ‘normal’ copy number changes (CNVs) in an antenatal setting. In this context, the International Standard Cytogenomic Array (ISCA) Consortium14 has been established and has agreed on a consensus postnatal design of an oligoarray platform that simultaneously provides defined targets for approximately 500 known microdeletion/duplication regions and high-resolution ‘backbone’ genomic coverage at an average spacing of approximately 25 kb. Over 70 laboratories worldwide now subscribe to the ISCA and the first iteration of the consensus array design is being formally evaluated for postnatal applications in various formats (4 × 44K; 2 × 105K; 4 × 180K) with a view to widespread adoption later in 2009. This point is being made here because the ISCA consensus design provides a model for the design of antenatal diagnosis-specific arrays that this author believes will be an important prerequisite for

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the adoption of this technology in an invasive PND setting. The ISCA is not platform specific in that the design principles can be applied to other aCGH providers using SNP- and oligo-/SNP-based approaches. In summary, with aCGH we have now moved from the analysis of the chromosomal band to the DNA base pair (Figure 10.5).

Figure 10.5 A conventional karyotype is shown on the left while an array slide with the scanned image is shown on the right; prenatal karyotyping can detected imbalances of 6–10 Mb or more while an array’s resolution is only limited by the design and density of probes used; aCGH = array comparative genomic hybridisation

aCGH and PND The proven utility of aCGH in the postnatal setting is leading to an investigation into the possibility of the use of specifically designed constitutional arrays for PND. However, before this can be achieved, consideration will need to be given to how the current cytogenetic and molecular genetic laboratory infrastructures will have to be adapted to provide reconfigured service delivery models. There are a number of key components related to the interpretation of aCGH results that will apply particularly to a prenatal setting: 1. The primary interpretation of the aCGH result will be critically dependent on (a) the quality of the AF or CVS DNA used on the array and (b) the confidence with which copy number changes can be identified using the analytical software. 2. Virtually all large copy number changes (5 Mb or more), particularly amplifications, will need to be further characterised using metaphase chromosomes and/or FISH. aCGH will not, therefore, completely replace karyotyping but will significantly supplant it in this cohort. Smaller copy number changes will need follow-up of parental samples (see point 4 below).

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3. A number of recently described recurrent pathogenic copy number changes (for example, 1q21.1, 15q11.2q13, 17q12, 17q21.1 and 22q11.23) are associated with incomplete or variable penetrance and thus careful and systematic genetic counselling will have to be a feature of result interpretation involving aCGH. This is, in effect, no different from the current situation sometimes encountered with the 22q11.2 DiGeorge/velocardiofacial microdeletion that can be carried by an apparently asymptomatic parent but is ascertained in a child with the full abnormal clinical spectrum. 4. Copy number changes found in the proband may be carried by a phenotypically normal parent and thus analysis of the trio (fetus, mother and father) by aCGH, FISH, quantitative PCR (qPCR) or other dosage-sensitive molecular tests will need to be carried out quickly and systematically. 5. There will be a number of copy number changes that are not reported in the Database of Genomic Variants13 and may therefore be novel CNVs and not potentially pathogenic changes. Access to and the availability of large databases of copy number changes will be required to facilitate genotype/phenotype correlations, particularly in an antenatal setting. 6. Start-up capital costs for arrays are relatively high but the costs of the associated consumables are being driven down by widespread uptake of the technology and open competition between a number of innovative commercial array providers. 7. The cost and training for UK cytogenetic service provision is currently focused on the provision of conventional karyotyping and transfer of parts of the service to aCGH will require significant workforce retraining and financial restructuring. Despite the need for implementation of all of the above components (and possibly others not documented here), the rapidly accumulating data from postnatal applications of aCGH provide compelling evidence that aCGH will not only detect all currently diagnosed unbalanced chromosome abnormalities but will do so with greater accuracy, precision and speed. Furthermore, appropriately designed prenatal aCGH will also detect all the clinically significant recurring genomic disorders (the numbers of which are growing steadily), most of which are associated with some degree of postnatal developmental delay and/or congenital malformations (Figures 10.6 and 10.7). The hardware, software and analytical platforms for implementation of prenatal aCGH are all in place but what is now needed is a significant shift in infrastructure development, training and funding so that prenatal aCGH can be delivered. As far as this author is aware, there are no systematic continuing prenatal studies in the UK or Europe designed to compare aCGH with conventional karyotyping. There is, however, a US National Institute of Child Health and Human Developmentfunded aCGH study that will prospectively compare results from approximately 4000 prenatal diagnoses (AF and CVS samples combined) achieved contemporaneously by both karyotyping and aCGH. The karyotyping is being carried out by a single laboratory, with a number of participating laboratories performing the DNA extraction from the same sample and then running customised prenatal arrays. All results are being centrally collated and coordinated by a project coordinator (Figure 10.8). The preliminary results of this trial are due to be published in 2010 and will provide a framework for local (UK) implementation of aCGH technology.

154  |  JOHN CROLLA

Figure 10.6 An example of an array comparative genomic hybridisation (aCGH)-detected deletion at 17q21.31 that is not present in the parents and is therefore a de novo chromosome abnormality; on the left of the figure is an ideogram of chromosome 17 (International System for Human Cytogenetic Nomenclature [ISCN]) with the summary of the array result for the whole of chromosome 17; the detail on the right shows the ~633 kb deleted region; each black dot on the vertical axis represents a log2 ratio of 0 and therefore no copy number change; the green dots show an average log2 ratio shift of −1, indicating a 2 : 1 ratio of normal : patient DNA, i.e. a deletion (see also Figure 10.7)

Potential PND service reconfiguration driven by aCGH There are a number of scenarios that may provide drivers for the adoption of aCGH as the first-line whole-genome test following invasive PND, of which the following two appear the most probable: n cases that, following invasive PND, are reported to be normal following QF-PCR and/or karyotyping but have abnormal ultrasound findings (first trimester or at the 18-20 week anomaly scan). n cases that are normal following non-invasive PND (NIPD) for the autosomal aneuploidies but have abnormal ultrasound findings (first trimester or at the 18–20 week anomaly scan). The possible time frames associated with the implementation of NIPD, particularly for trisomy 21, is the focus of Chapter 11. There is no doubt, however, that if NIPD for trisomy 21 is successfully introduced, either as a highly sensitive screening tool or as a standalone diagnostic test, the effects on current cytogenetic PND service delivery models will be profound. If we make an assumption that NIPD for trisomy 21 is successfully introduced within the next 5  years, this will reduce the number of

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Figure 10.7 The deletion coordinates for the case shown in Figure 10.6 have been entered into the Ensembl Genome Browser (www.ensembl.org/Homo_sapiens/Info/Index); this shows that MAPT (microtubule-associated protein; OMIM +157140) is deleted and that this region is part of a recently described recurring microdeletion syndrome (see DECIPHER; decipher. sanger.ac.uk/application/); this deletion is therefore fully cryptic, can only be detected by molecular technology (e.g. multiplex ligation-dependent probe amplification [MLPA], fluorescence in situ hybridisation [FISH] or quantitative polymerase chain reaction [qPCR]) and is associated with a variable but recognisable clinical phenotype that includes low birthweight (0.4th to 0.9th centile), neonatal hypotonia, poor feeding in infancy and oromotor dyspraxia together with moderate developmental delay/learning disability but friendly/amiable behaviour; other clinically important features include epilepsy, heart defects (atrial septal defect and ventricular septal defect) and kidney/urological anomalies

invasive procedures requiring karyotyping by more than 80%, although confirmatory QF-PCR may be required before clinical decisions are made on the basis of a positive NIPD trisomy 21 test (Figure 10.9). The UK’s regional cytogenetic laboratories currently provide prenatal cytogenetic diagnoses based on ascertainments arising from the current NSC screening protocols (see above). In the first of the two scenarios outlined above, the 20% or so of ‘chromosomally normal’ cases remaining following successful NIPD still requiring

156  |  JOHN CROLLA

Figure 10.8 The continuing US National Institute of Child Health and Human Developmentfunded array comparative genomic hybridisation (aCGH) study; the aim is to compare approximately 4000 prenatal diagnoses achieved contemporaneously both by karyotyping and aCGH

Figure 10.9 The possible effects on chromosomal prenatal diagnosis when non-invasive prenatal diagnosis (NIPD) for the common aneuploidies is introduced into the UK healthcare system; in this scenario, NIPD will detect approximately 80% of the abnormalities currently being detected by either quantitative fluorescence polymerase chain reaction (QF-PCR) and/or karyotyping; the remaining cases for ‘chromosomal diagnoses’ will therefore be restricted largely to fetuses with ultrasound abnormalities; these are the cases for which array comparative genomic hybridisation (aCGH) would produce the greatest benefit

FETAL KARYOTYPING: WHAT SHOULD WE BE OFFERING AND HOW?  |  157

invasive PND will be those presenting with an abnormal ultrasound scan, either nuchal thickening in the first trimester or a congenital abnormality detected at the 18–20 week anomaly scan; these cases will be the primary focus of PND using aCGH. The second scenario does not require successful NIPD but asks the question whether in the genomic era conventional karyotyping is sufficient to detect the full range of potentially pathogenic chromosomal changes in this highly selected cohort of fetuses with abnormal scans. It should be stated here that some level of conventional karyotyping will need to be maintained so that aCGH-detected abnormalities can be fully characterised (see above) and to provide a service to phenotypically normal carriers of balanced structural chromosome rearrangements to determine the chromosomal status of the fetus, which may potentially be carrying an unbalanced form of the balanced parental rearrangement. Laboratories that wish to introduce prenatal aCGH technology will need to meet several stringent technical and scientific criteria, including: n full infrastructure, including hardware, software and bioinformatic resources to deliver prenatal aCGH n rapid turnaround times for DNA extraction, purification and quality assurance n rapid turnaround times for multiple aCGH hybridisations, scanning and analyses n the ability to quickly produce FISH probes and/or specific primers to determine the structural complexity (if any) of any copy number changes as well as the parental origins of copy number changes detected by aCGH n established lines of communications between molecular cytogeneticists, clinical geneticists, fetal medicine units and bioinformatic experts to facilitate the interpretation of prenatally detected aCGH abnormalities. All of the above are achievable and in some centres are being addressed now in the context of the delivery of a postnatal application of aCGH to both retrospective and, increasingly, prospective cases. The two scenarios given above differ in one very important respect, which is that the successful introduction of NIPD for trisomy 21 will very substantially reduce the workload of clinical cytogenetic prenatal laboratories to the extent that consideration should be given to whether prenatal aCGH technology and its stringent requirement for rapid follow-up studies should be centralised in several larger centres with the capacity to deliver all aspects of a prenatal aCGH service. Even if NIPD is not introduced in the next 5 years, the increasing success of the NSC’s prenatal screening programme is already resulting in a significant reduction in invasive PND and this author believes that for aneuploidy-negative, ultrasound-positive prenatally diagnosed fetuses, prenatal aCGH will soon become the front-line test in several UK laboratories. Acknowledgements The author is very grateful to Professor Laird Jackson for discussions concerning the continuing US National Institute of Child Health and Human Development-funded prenatal array study.

References 1.

NHS Fetal Anomaly Screening Programme. Screening for Down’s Syndrome: UK NSC Policy Recommendations 2007–2010: Model of Best Practice [fetalanomaly.screening.nhs.uk/getdata. php?id=10848].

158  |  JOHN CROLLA 2.

3.

4.

5. 6. 7.

8.

9.

10.

11.

12. 13. 14. 15.

Mann K, Fox SP, Abbs SJ, Yau SC, Scriven PN, Docherty Z, et al. Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis. Lancet 2001;358:1057–61. Caine A, Maltby AE, Parkin CA, Waters JJ, Crolla JA; UK Association of Clinical Cytogeneticists (ACC). Prenatal detection of Down’s syndrome by rapid aneuploidy testing for chromosomes 13, 18, and 21 by FISH or PCR without a full karyotype: a cytogenetic risk assessment. Lancet 2005;366:123–8. Department of Health. Our Inheritance, Our Future: Realising The Potential of Genetics in the NHS. London: Department of Health; 2003 [www.dh.gov.uk/en/Publicationsandstatistics/Publications/ PublicationsPolicyAndGuidance/DH_4006538]. UK National Screening Committee. NHS Fetal Anomaly Screening Programme [www.screening.nhs. uk/fetalanomaly-england]. NHS Fetal Anomaly Screening Programme. Standards and Policies [fetalanomaly.screening.nhs.uk/ standardsandpolicies]. Kallioniemi OP, Kallioniemi A, Sudar D, Rutovitz D, Gray JW, Waldman F, et al. Comparative genomic hybridization: a rapid new method for detecting and mapping DNA amplification in tumors. Semin Cancer Biol 2009;4:41–6. Shaw-Smith C, Redon R, Rickman L, Rio M, Willatt L, Fiegler H, et al. Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features. J Med Genet 2004;41:241–8. Hochstenbach R, van Binsbergen E, Engelen J, Nieuwint A, Polstra A, Poddighe P, et al. Array analysis and karyotyping: workflow consequences based on a retrospective study of 36,325 patients with idiopathic developmental delay in the Netherlands. Eur J Med Genet 2009;52:161–9. Baptista J, Mercer C, Prigmore E, Gribble SM, Carter NP, Maloney V, et al. Breakpoint mapping and array CGH in translocations: comparison of a phenotypically normal and an abnormal cohort. Am J Hum Genet 2008;82:927–36. De Gregori M, Ciccone R, Magini P, Pramparo T, Gimelli S, Messa J, et al. Cryptic deletions are a common finding in “balanced” reciprocal and complex chromosome rearrangements: a study of 59 patients. J Med Genet 2007;44:750–62. Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, Andrews TD, et al. Global variation in copy number in the human genome. Nature 2006;444:444–54. Centre for Applied Genomics. Database of Genomic Variants [projects.tcag.ca/variation/]. International Standard Cytogenomic Array (ISCA) Consortium [https://isca.genetics.emory.edu/ iscaBrowser/]. Association for Clinical Cytogenetics [www.cytogenetics.org.uk].

11 Chapter 11

Non-invasive prenatal diagnosis: the future of ptrenatal genetic diagnosis? Lyn Chitty, Gail Norbury and Helen White

Introduction Current prenatal diagnosis of fetal genetic status or aneuploidy depends on the use of the invasive diagnostic tests amniocentesis and chorionic villus sampling (CVS), which carry a small but significant risk of miscarriage.1 For many years, researchers have sought alternative sources of fetal material on which to base genetic prenatal diagnosis. It was initially thought that fetal cells in the maternal circulation would fulfil this role but it is now recognised that this approach is unlikely to be clinically useful because of the scarcity of these cells and the inability to analyse them reliably.2 The identification of cell-free fetal DNA (cffDNA) in both maternal plasma and serum offered potential as an alternative source of fetal genetic material for prenatal diagnosis when molecular determination of fetal sex was achieved using the Y-chromosome-derived gene TSPY, found in the DYS14 marker sequence using a nested polymerase chain reaction (PCR).3 Lo’s group then went on to show that cffDNA can be detected in maternal plasma from 4 weeks of gestation4 and is cleared rapidly from the maternal circulation after delivery,5 making it a potentially ideal source of fetal material for prenatal diagnosis. It is now thought that cffDNA emanates from the placenta as normal levels of cffDNA have been reported in anembryonic pregnancies.6 However, the vast majority of cellfree DNA (cfDNA) in the circulation is maternal in origin. It was initially estimated that cffDNA constitutes around 3% of total cfDNA in maternal plasma, rising to 6% towards term,4 but recent studies using deep sequencing and digital PCR methods suggest that the concentration may be as high as 19%.7,8 Existing methodologies do not allow complete separation of fetal from maternal cell-free DNA and thus current clinical applications focus on the detection or exclusion of genes not present in the mother; that is, those inherited from the father such as Y-chromosome sequences or rhesus D (RHD) in D-negative women, or arising de novo at the time of conception. There have been rapid advances in this field secondary to developments in genetic technology and, although much work is required to validate methods based on noninvasive prenatal diagnosis (NIPD) before widespread implementation, there seems little doubt that it will significantly reduce the need for invasive diagnostic procedures in the not too distant future. In this chapter, we will describe the current clinical applications of NIPD for genetic conditions and the potential use for aneuploidy screening or diagnosis. We will also highlight ethical, educational and laboratory aspects that require evaluation before introduction into routine clinical practice. © Lyn Chitty, Gail Norbury and Helen White. Volume compilation © RCOG

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Current clinical applications Fetal sex determination The main clinical indications for fetal sex determination are risk of X-linked genetic disorders and a family history of congenital adrenal hyperplasia (CAH), with occasional indications including some fetal ultrasound findings and discrepancy between genetic sex and the appearances of the external genitalia on fetal ultrasound. Since the original report by Lo et al.3 there have been many publications reporting high degrees of accuracy (Table 11.1). Most reports describe the use of the Y-chromosome gene SRY or the DYS14 marker sequence of the TSPY gene. SRY is a single-copy gene whereas the DYS14 sequence is present in multiple copies and is, therefore, easier to detect in maternal plasma when a male fetus is present, having a ten-fold lower detection limit than SRY.9 It is, however, less specific.10 Early reports used conventional PCR followed by nested PCR in an attempt to increase the sensitivity of the assay (Table 11.1) but this introduced risk of contamination of the assay from the millions of amplicons created. Real-time fluorescence PCR has now become the method of choice in most laboratories as this eliminates the contamination risk by using a closed analytical system. In addition to the reagents used in conventional PCR, probes with fluorescently labelled reporter and quencher dyes are used in the PCR mix, which increases specificity. When the target gene is present, exponential DNA amplification causes a proportional increase in the reporter dye fluorescence during each PCR cycle, shown after analysis of the reaction as an amplification plot (Figure 11.1). The cycle threshold is the cycle of PCR at which the reporter dye reaches a specific level of fluorescence (usually 10 standard deviations above the background level). A further advantage of real-time PCR is its sensitivity, enabling detection of very low copy numbers of DNA, which is essential when analysing samples taken early in pregnancy. One major disadvantage of this method is that a female fetus is inferred following failure of amplification; that is, when fetal DNA sequences are not detected in maternal plasma. This could, of course, also happen in the event of failure to amplify fetal DNA, for example if the amount of cffDNA present were very low. Ideally, the presence of cffDNA should be confirmed by detecting another gene, not on the Y chromosome, inherited from the father, but not present in the maternal genome. The use of a panel of ten bi-allelic polymorphic markers to demonstrate the presence of fetal DNA has been described.11 The main drawback to this technique is that a large number of alleles need to be tested to increase the likelihood that parents will be informative (that is, parents will carry a different allele and therefore be distinguishable) and it is time-consuming because maternal (and ideally paternal) DNA must be typed first so that only those markers absent from the maternal genome are targeted in maternal plasma. Our experience in the North East Thames Regional Molecular Genetics Laboratory showed that these markers are of limited use as they were informative in only 40% of cases. The limitations of this approach have led to the search for a fetal-specific marker that could be used universally in NIPD. Such candidates include loci that are subject to differential epigenetic modification in the mother and fetus. Sequences within the tumour suppressor genes MASPIN and RASSF1A are differently methylated in blood and trophoblast. MASPIN is preferentially methylated in the maternal DNA and hypomethylated in fetal (placental) DNA.12 However, the preferential detection of an unmethylated fetal sequence is more tedious and less sensitive than the detection of methylated material. Fortunately, in the case of the RASSF1A promoter, it is the fetal DNA that is preferentially hypermethylated and maternal DNA unmethylated. By digestion with methylation-sensitive enzymes,

NON-INVASIVE PRENATAL DIAGNOSIS: THE FUTURE OF PRENATAL GENETIC DIAGNOSIS?  |  161 Table 11.1. Studies reporting the use of cell-free fetal DNA in the maternal circulation for determination of genetic sex in pregnancy; adapted from Finning and Chitty17 Study Lo et al. (1997)3 Lo et al. (1998)4 Smid et al. (1999)34

n

Gestation (weeks) 43 12–40 50 11–17; 37–43 16 7–32

Zhong et al. (2000)35 9 Honda et al. (2001)36 61 Nelson et al. (2001)37 18 Al Yatama et al. (2001)38 80 Costa et al. (2001)39 121 Rijnders et al. (2001)40 45 Sekizawa et al. (2001)41 302 Zhong et al. (2001)42 34 Hromadnikova et al. (2002)43 37 Farina et al. (2002)44 63 Honda et al. (2002)45 81 Mazza et al. (2002)14 18 Rijnders et al. (2003)46 13 Guibert et al. (2003)47 22 Hromadnikova et al. (2003)48 44 Rijnders et al. (2004)49 72 Hwa et al. (2004)50 56 Hyett et al. (2005)15 35 Boon et al.c (2007)51 58 54 54 Bustamante-Aragones et al. 196 (2008)52 Picchiassi et al. (2008)10 145 145 Vecchione et al. (2008)53 26

PCR technique Gene

16 10–17 9–34 7–40 8–14 8–17 7–16 13–17 15–22 10–12 5–10 12 5–10 4–9 10–18 11–19 6–16 7–14 11 ± 3.3 11 ± 3.3 11 ± 3.3 5–12

Accuracy (males) Conventional 80% Real time SRY 100% Conventional 93% Nested 100% [Not given] Nested 100% Conventional DYS14, DYZ3 87% Conventional 100% Nested [Not given] 96% Real timeb SRY 100% Real time SRY 96% Real time DYS14 97% Real time 100% Real time SRY 92% Real time SRY 95.2% Real timeb DYS14 100% Nested AMELOGENIN 50% Real time SRY 100% Real time SRY 100% Real time SRY 100% Real time SRY 97.2% Real time SRY 87% Real time SRY 100% Real time SRY 100% SRY 100% PAP 100% Real time + PAP SRY Real time SRY 100%

11–12 11–12 18

Real time Real time QF-PCR

Specificity 100% 100% 45% 91% 100% 100% no dataa 88% 100% 100% 100% 100% 100% no dataa 100% 100% 100% 100% 100% 100% 100% 97.7% 98.1% 100% 100%

DYS 97.6% 96.8% SRY 98.2% 98.4% STRs and 100% 100% AMELOGENIN

PAP = pyrophosphorolysis-activated polymerisation; PCR = polymerase chain reaction; QF-PCR = quantitative fluorescence PCR; STR = short tandem repeat only male cases reported

a

freeDNA extracted from serum rather than plasma

b

high test failure rate

c

the maternal unmethylated sequence can be removed and the methylated fetal material assayed by real-time PCR analysis.13 This technique is currently being used by some laboratories as an independent fetal marker. However, as this is an independent assay, it does not provide an internal control for the presence of fetal material. In the UK, cffDNA is being used to determine fetal sex in women at risk of X-linked disorders, where early identification of a male fetus indicates a need for an

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Figure 11.1. Real-time polymerase chain reaction (PCR) analysis showing the log of the change in fluorescence (delta Rn) versus cycle number: (a) amplification of control DNA and maternal sample with SRY signal, indicating that there is a male fetus; (b) Amplification of control DNA and a maternal sample from a pregnancy with a female fetus, as demonstrated by the lack of amplification of SRY

invasive diagnostic test to determine whether the affected X chromosome has been inherited, but no invasive test is required if the fetus is female. It is also widely used in pregnancies at risk of CAH, where early treatment of pregnancies with an affected female fetus has been shown to reduce the degree of virilisation of the external genitalia. In these situations, NIPD has been shown to reduce the uptake of invasive diagnostic testing by nearly 50% and allow for early cessation of dexamethasone treatment in pregnancies at risk of CAH where the fetus is found to be male.14,15 A national audit of all cases done in the two UK laboratories offering this test has demonstrated concordance of 97.8% between sex reported using cffDNA performed at 7 weeks or later and sex confirmed at invasive testing or birth.16,17 Testing before 7 weeks was less accurate, with two of the five cases giving discordant results. The aetiology of discordant results requires further investigation but causes include low

NON-INVASIVE PRENATAL DIAGNOSIS: THE FUTURE OF PRENATAL GENETIC DIAGNOSIS?  |  163

levels of cffDNA in early pregnancy and the persistence of trophoblastic material in a multiple pregnancy, discordant for fetal sex, where there has been demise of one fetus. Single-gene disorders The techniques described above have been applied to the diagnosis of single-gene disorders and there are a few reports in the literature describing the detection18,19 or exclusion of the paternal allele inherited from an affected father with an autosomal dominant condition such as Huntington’s disease20 (Table 11.2), but currently available techniques are unsuitable for the diagnosis of X-linked and most recessive disorders as the fetal genes inherited from the mother are swamped by the excess of the mother’s own cfDNA. In recessively inherited conditions such as thalassaemia or cystic fibrosis, if the parents carry different mutations, then exclusion of the paternal allele from the maternal plasma indicates that the fetus would be unaffected, but if present an invasive test is required to determine whether the fetus has inherited the abnormal maternal allele and is thus affected. The possibility for increasing the scope for NIPD of singlegene disorders to include recessive disorders or maternally inherited conditions was demonstrated by Lun and colleagues in 20088 using digital PCR. By exploiting the size difference in fetal and maternal DNA, these authors were able to enrich for fetal DNA. They then developed a process that they called digital relative mutation dosage (RMD) which is based on the ability to detect very small imbalances in levels of the mutant and wild type alleles of a disease-causing gene in maternal plasma. By determining whether the ratio of alleles was balanced or unbalanced, they correctly predicted the fetus to be unaffected or affected in five of ten fetuses at 18–20 weeks with an increased risk of thalassaemia.8 In one there was an incorrect classification and in the remaining four the concentration of cffDNA was too low. The authors went on to propose measures that might be taken to improve the reliability of this technique.

Future possibilities Diagnosis of aneuploidy NIPD of Down syndrome poses different challenges. It is not feasible to use the PCR methods described above as these are not sufficiently sensitive to detect the relatively small increase of fetal chromosome 21. If the figure of 6% is used for the proportion of fetal DNA in maternal blood and if the ratio of chromosome 21 relative to another autosome, say 9, is calculated then if the fetus is euploid (that is, has two copies of each autosome) the ratio will be 1 (Box 11.1). If, however, the fetus has trisomy 21

Box 11.1

Increase in the proportion of cffDNA in a pregnancy complicated by Down syndrome, assuming that the percentage of fetal DNA in a maternal plasma sample is 6%

Euploid situation: Total amount of Chr 21 ( 0.94 maternal + 0.06 fetal) =1 Total amount of Chr 9 (0.94 maternal + 0.06 fetal) Trisomy 21: Total amount of Chr 21 (0.94 maternal + 0.09 fetal) = 1.03 Total amount of Chr 9 (0.94 maternal + 0.06 fetal)

α0-thalassaemia (Hb Bart’s)

Haemoglobin E

β-thalassaemia

Condition Achondroplasia

Table 11.2.

β-globin

β-globin (β41/42)

Real-time nested QF-PCR63

Digital NASS-RMD8

Nested PCR and restriction digestion61 β-globin (βE, β41/42, β17) Semi-nested and nested realtime PCR62

β-globin (βE)

β-globin SNPs

8–20 weeks

18–20 weeks

7–23 weeks

8–18 weeks

10–12 weeks

APEX60

β-globin 1 case positive 1 case negative 4 carry paternal SNP 3 negative for paternal SNP 3 cases positive 2 cases negative 11 affected βE 15 carrier βE 3 carry paternal β41/42 1 β17 3 β17 β41/42 6 negative 5 correct 1 incorrect 4 unclassified 8 carriers 1 Hb H 2 Hb Bart’s 2 normal

2 cases positive, one negative

32–35 weeks

Real-time PCR19

Result Positive

2 cases positive

Gestation 30 weeks

Size separation and restriction digest55 MALDI-TOF MS56 34 weeks

Method and reference Restriction digest54

Real-time PCR57 Size separation and PNA-clamp PCR58 AS-PCR for SNP59 11 weeks

Gene and mutation FGFR3 p.Gly3880Arg FRFR3 p.Gly3880Arg FGFR3 p.Gly3880Arg FGFR3 p.Gly3880Arg

Non-invasive prenatal diagnosis of single-gene disorders reported in the literature

CVS, amniocentesis or cordocentesis

Invasive testing

CVS and cord blood

CVS (1 paternal SNP not detected, i.e. 1 FN)

CVS

2 confirmed at birth by X-ray 1 intrauterine growth restriction

Confirmed on amniotic fluid

Confirmation Confirmed on amniotic fluid

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RP2 (c.400C>T) CRB1 p.Cys896ter PCCB c.1218del14ins12 12 weeks

Melt curve and primer extension74

Positive for paternal mutation

Positive for paternal mutation

12 weeks

dHPLC73

29 and 32 weeks 1 case positive

Restriction digestion19 10 and 19 weeks Positive

2 cases positive

7–9 weeks

Real-time PCR19

Sequencing72

Negative

Positive for paternal allele 2 cases positive for paternal allele 1 case negative for paternal allele

Negative for high-risk paternal allele 2 show paternal expansion 1 negative for paternal expansion 1 no paternal allele detected

Result Negative for paternal allele

Allele-specific standard PCR71 Real-time PCR19 12 weeks

Restriction digestion69 SNaPshot®70

Fluorescent SNPs68 13 weeks 12 weeks and 15 weeks

12 weeks (6) 12 weeks

STR65 Fragment and STRs66

Nested PCR67

Gestation 13 weeks

Method and reference (semi) QF-PCR20,64

Confirmed at CVS

Confirmed at CVS

Carrier status confirmed at CVS

Confirmed postnatally

Confirmed postnatally

Normal live birth

Carrier status confirmed at CVS Status confirmed at CVS

Confirmed at CVS Confirmed at CVS 3 affected and 1 normal fetus, i.e. 1 nondiagnosis (misdiagnosis)

Confirmation Confirmed at CVS

APEX = arrayed primer extension; CVS = chorionic villus sampling; dHPLC = denaturing high-performance liquid chromatography; FN = false negative; Hb = haemoglobin; MALDI-TOF MS = matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry; NASS-RMD = nucleic acid size selection, relative mutation dosage; PCR = polymerase chain reaction; PNA = peptide nucleic acid; QF-PCR = quantitative fluorescence PCR; SNP = single-nucleotide polymorphism; STR = short tandem repeat

Retinitis pigmentosa (X-linked) Leber congenital amaurosis Propionic acidaemia

Myotonic dystrophy DMPK (CTG)n Congenital adrenal hyperplasia Cystic fibrosis CFTR CFTR p.Arg668Cys, p.Lys710ter, p.Tyr1092ter Hb Lepore Crouzon syndrome FGFR2 c.1040C>G Torsion dystonia DYT1 c.946delGAG Apert syndrome FGFR2 c.755C>G

Condition Gene and mutation Huntington disease IT15 (CAG)n

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then the relative proportion of chromosome  21 will be increased, but only very slightly (approximately 3%), as most of the cfDNA is maternal in origin. Detection and quantification of this small difference requires either the analysis of targets that are free from maternal background interference and/or the use of technologies that enable extremely accurate copy number ‘counting’. An approach that enables the analysis of a fetal-specific target has been described by Lo and colleagues21,22 who analysed mRNA in maternal plasma rather than cffDNA. They tested cell-free mRNA from a gene located on chromosome 21 (PLAC4) that was found to be expressed in the placenta but not in maternal blood (that is, it is fetal specific). By extracting cell-free RNA (rather than cfDNA) from maternal plasma and testing a single-nucleotide polymorphism (SNP, a common sequence variation found in the normal population) located in the PLAC4 fetal mRNA sequence, the chromosome  21 allelic ratio can be determined either using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry21 (Figure 11.2a) or digital PCR22 (Figure 11.2b). For both methods, an SNP allele ratio of 1 : 1 indicates a normal fetus (one copy of A and one copy of G = two copies of chromosome 21) and a ratio of 2 : 1 is indicative of a trisomy 21 fetus (one copy of A and two copies of G = three copies of chromosome 21). If a fetus is homozygous for the SNP, no quantitative information is obtained and the result will be noninformative. In some cases (approximately 60% of pregnancies in Lo’s paper), this test will not be informative as the fetus will be homozygous for the SNP; that is, the sequence of the inherited maternal and paternal allele will be identical and no quantitative information can be obtained. In Lo’s original report, 67 cffRNA samples were from cases where the fetus was known to be informative for the PLAC4 SNP. In this small cohort, 90% of Down syndrome fetuses and 55 of 57 normal fetuses were correctly identified (sensitivity 90%; specificity 96.5%). Data presented more recently at scientific meetings indicate that, by increasing the number of SNPs analysed, the test could be used successfully in approximately 95% of pregnancies in the certain populations, making it more widely applicable.23 Two small series were reported in 2008 where deep, or massively parallel, genomic sequencing was used to identify cases with aneuploidy. In the first report,7 12 cases of aneuploidy were successfully detected (nine trisomy 21, two trisomy 18 and one trisomy 13) in a total of 18 cases, and in the second study24 of 28 cases, all 14 Down syndrome cases were correctly identified, with no false positive results. The method used is time-consuming and expensive, with estimates of around US$700 per case and requiring more than 3 days per sample.7 The highly fragmented cfDNA is extracted from maternal plasma and sequenced to generate millions of short DNA sequences from random genomic locations. By comparing these sequences with the known human genome sequence, it is possible to work out how many sequences have been derived from each chromosome. By comparing the total number of chromosome 21 sequences obtained from a cfDNA sample with the number obtained from a normal genomic DNA sample, very small increases (less than 3%) in the amount of chromosome 21 can be detected in the cfDNA sample if the fetus carries an additional chromosome 21. This methodology clearly has potential and would be more widely applicable as it does not depend on there being differences between the maternal and paternal alleles. However, currently both cost and time preclude its routine clinical application. Other proposed methods for NIPD of Down syndrome include those based on the epigenetic differences between maternal and fetal DNA, and also the use of digital PCR. The possibility for the diagnosis of both trisomy 1825 and trisomy 2126,27 has been demonstrated. However, the application of these methods in clinical practice

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may be limited by the fact that the laboratory methods used to detect methylation often degrade DNA, thereby reducing the already small amounts available for analysis and making quantification problematic. Regardless of which approach is taken, results from relatively small numbers have been reported to date and therefore considerable effort is now needed, including formal validation, standardisation, education and other work, before NIPD for aneuploidy can be considered ready for implementation in routine clinical practice. Whether these new methods will be used primarily to replace current invasive prenatal diagnosis or whether they will be used as a screening test must await further evaluation, including more accurate information on sensitivity and specificity from large-scale studies.

(a)

(b)

Figure 11.2. Analysis of the PLAC4 RNA single-nucleotide polymorphism (SNP) allele ratio on chromosome 21.(a) Matrix-assisted laser desorption/ionisation time-of-flight (MALDITOF) mass spectrometry. The relative quantity of the two SNP alleles in a heterozygous fetus can be determined by primer extension and MALDI-TOF mass spectrometry. The intensity of the resulting peak height is proportional to the amount of allele present. (b) Digital polymerase chain reaction (PCR). Cell-free RNA is reverse transcribed and the cDNA is diluted to one copy per well of the 96-well analysis plate and analysed for the presence of the A allele (red wells) and G allele (green wells) using real-time PCR. The number of red (A) and green (G) wells are counted to determine the ratio of red (A) to green (G) wells, that is, the SNP allele ratio. Yellow wells indicate the presence of both alleles (more than one copy present) and are excluded from the analysis. Light blue wells indicate no amplification

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Other laboratory issues There seems little doubt that, with time, NIPD using fetal nucleic acids will become a reality for an increasing number of indications. However, with the exception of fetal sex determination, most studies so far reported have described small numbers of cases and large-scale evaluations must be undertaken before implementation in clinical practice. One issue that will need to be addressed is that of confined placental mosaicism (CPM). In CVS samples taken in the late first trimester, CPM is reported to occur in 1–2% of cases. As cffDNA appears to be derived from the trophoblast, it is likely that CPM will occur at similar rates. Furthermore, cffDNA as a predictor of fetal genetic status will not be valid in the case of multiple pregnancies where the fetuses may be discordant for a given genotype. Demise of a twin discordant for an allele may result in discordant NIPD results being obtained, since trophoblastic material may continue to shed fetal DNA into the maternal circulation after fetal death. On occasion, evidence of an empty gestational sac on ultrasound raise suspicion of this possibility, but in many cases the ‘vanishing twin’ may go unrecognised. The circulating fragments of cffDNA are short (fewer than 300 bp). This means that it will not be possible to use this technology to detect disease-causing mutations involving large nucleotide repeat expansions, such as in fragile X syndrome, where a mutation associated with the disease can involve more than 600 bp. Furthermore, this technique may not be applicable to mutations involving rearrangements such as inversions and duplications. In these situations, it may be possible to adopt an indirect linkage approach that would be limited to familial disease where there is an appropriate family structure and sample availability to assign phase. Alternatively, the direct sequencing methods may be more applicable if the degree of enrichment of cffDNA can be increased sufficiently.

Ethics, education and counselling It seems clear that NIPD based on cell-free fetal nucleic acids circulating in the maternal plasma will gradually move into clinical practice. Indeed, they are already being used for some indications and molecular prenatal diagnosis based on cffDNA constituted around 25% of all molecular prenatal diagnostic tests done in the UK in 2007–8.28 There is some evidence that the potential change in practice that removes the risk of miscarriage associated with invasive testing may influence pre-test counselling. In a small pilot study, van den Heuvel et al.29 showed that obstetricians and midwives in the UK considered NIPD akin to Down syndrome screening and were less likely to consider it necessary to obtain written consent for NIPD for Down syndrome than when offering invasive testing. More professionals also thought it reasonable to counsel women and perform the test on the same day when offering NIPD and Down syndrome screening than when offering invasive testing. Concerns have also been raised that removing the barrier of the risks associated with invasive procedures may mean that women will not consider the possible outcomes of testing as fully and not appreciate that the result of NIPD, that is, the potential diagnosis of an affected fetus and the choices they may then face, are the same as when undergoing invasive testing.30,31 Other wider issues, such as commercialisation31 and use of NIPD for non-clinical indications such as fetal sex determination32 or paternity testing,33 are also areas for concern but are beyond the scope of this chapter.

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The way forward This technology heralds a more accessible and safer future for prenatal diagnosis but for its potential to be fully realised much work is required, not just in developing the laboratory standards, determining the sensitivity and specificity and reviewing service delivery, but also in public and healthcare professional education and further investigation of the ethical and social issues. We have an opportunity now to develop the standards and infrastructure required for safe, equitable implementation if we take a broad approach and resist the technological ‘creep’ that so often accompanies exciting developments in medical technology. Acknowledgements Lyn Chitty is partially supported by UK Biomedical Research Centre funding. Her research in this area has been supported by the European Commission through the Special Non-invasive Advances in Fetal and Neonatal Evaluation (SAFE) Network of Excellence (LSH-CT-2004–503241) and currently by the National Institute for Health Research.

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Zhong XY, Holzgreve W, Hahn S. Detection of fetal Rhesus D and sex using fetal DNA from maternal plasma by multiplex polymerase chain reaction. BJOG 2000;107:766–9. Honda H, Miharu N, Ohashi Y, Ohama K. Successful diagnosis of fetal gender using conventional PCR analysis of maternal serum. Clin Chem 2001;47:41–6. Nelson M, Eagle C, Langshaw M, Popp H, Kronenberg H. Genotyping fetal DNA by noninvasive means: extraction from maternal plasma. Vox Sang 2001;80:112–16. Al-Yatama MK, Mustafa AS, Ali S, Abraham S, Khan Z, Khaja N. Detection of Y chromosomespecific DNA in the plasma and urine of pregnant women using nested polymerase chain reaction. Prenat Diagn 2001;21:399–402. Costa JM, Benachi A, Gautier E, Jouannic JM, Ernault P, Dumez Y. First trimester fetal sex determination in maternal serum using real-time PCR. Prenat Diagn 2001;21:1070–4. Rijnders RJ, van der Schoot CE, Bossers B, de Vroede MA, Christiaens GC. Fetal sex determination from maternal plasma in pregnancies at risk for congenital adrenal hyperplasia. Obstet Gynecol 2001;98:374–8. Sekizawa A, Kondo T, Iwasaki M, Watanabe A, Jimbo M, Saito H, et al. Accuracy of fetal gender determination by analysis of DNA in maternal plasma. Clin Chem 2001;47:1856–8. Zhong XY, Holzgreve W, Hahn S. Risk free simultaneous prenatal identification of fetal Rhesus D status and sex by multiplex real-time PCR using cell free fetal DNA in maternal plasma. Swiss Med Wkly 2001;13:70–4. Hromadnikova I, Houbova B, Hridelova D, Voslarova S, Calda P, Nekolarova K, et al. Quantitative analysis of DNA levels in maternal plasma in normal and Down syndrome pregnancies. BMC Pregnancy Childbirth 2002;2:4. Farina A, Caramelli E, Concu M, Sekizawa A, Ruggeri R, Bovicelli L, et al. Testing normality of fetal DNA concentration in maternal plasma at 10–12 completed weeks’ gestation: a preliminary approach to a new marker for genetic screening. Prenat Diagn 2002;22:148–52. Honda H, Miharu N, Ohashi Y, Samure O, Kinutani M, Hara T, et al. Human gender determination in early pregnancy through qualitative and quantitative analysis of fetal DNA in maternal serum. Hum Genet 2002;110:75–9. Rijnders RJ, Van Der Luijt RB, Peters ED, Goeree JK, Van Der Schoot CE, Ploos Van Amstel JK, et al. Earliest gestational age for fetal sexing in cell-free maternal plasma. Prenat Diagn 2003;23:1042–4. Guibert J, Benachi A, Grebille AG, Ernault P, Zorn JR, Costa JM. Kinetics of SRY gene appearance in maternal serum: detection by real time PCR in early pregnancy after assisted reproductive technique. Hum Reprod 2003;18:1733–6. Hromadnikova I, Houbova B, Hridelova D, Voslarova S, Kofer J, Komrska V, et al. Replicate realtime PCR testing of DNA in maternal plasma increases the sensitivity of non-invasive fetal sex determination. Prenat Diagn 2003;23:235–8. Rijnders RJ, Christiaens GC, Soussan AA, van der Schoot CE. Cell-free fetal DNA is not present in plasma of nonpregnant mothers. Clin Chem 2004;50:679–81. Hwa HL, Ko TM, Yen ML, Chiang YL. Fetal gender determination using real-time quantitative polymerase chain reaction analysis of maternal plasma. J Formos Med Assoc 2004;103:364–8. Boon EM, Schlecht HB, Martin P, Daniels G, Vossen RH, den Dunnen JT, et al. Y chromosome detection by real time PCR and pyrophosphorolysis-activated polymerisation using free fetal DNA isolated from maternal plasma. Prenat Diagn 2007;27:932–7. Bustamante-Aragones A, Rodriguez de Alba M, Gonzalez-Gonzalez C, Trujillo-Tiebas MJ, Diego-Alvarez D, Vallespin E, et al. Foetal sex determination in maternal blood from the seventh week of gestation and its role in diagnosing haemophilia in the foetuses of female carriers. Haemophilia 2008;14:593–8. Vecchione G, Tomaiuolo M, Sarno M, Colaizzo D, Petraroli R, Matteo M, et al. Fetal sex identification in maternal plasma by means of short tandem repeats on chromosome X. Ann N Y Acad Sci 2008;1137:148–56. Saito H, Sekizawa A, Morimoto T, Suzuki M, Yanaihara T. Prenatal DNA diagnosis of a singlegene disorder from maternal plasma. Lancet 2000;356:1170. Li Y, Holzgreve W, Page-Christiaens GC, Gille JJ, Hahn S. Improved prenatal detection of a fetal point mutation for achondroplasia by the use of size-fractionated circulatory DNA in maternal plasma – case report. Prenat Diagn 2004;24:896–8.

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12 Chapter 12

Non-invasive prenatal diagnosis for fetal blood group status Geoff Daniels, Kirstin Finning, Peter Martin and Edwin Massey

Introduction When Denis Lo and his colleagues1 in Oxford identified the presence of free fetal DNA in the blood of pregnant women, the implications for prenatal diagnostics without the requirement for invasive procedures were obvious. The main complication is that a very low concentration of fetal DNA is present in the maternal plasma, representing between 3% and 6% of the total free DNA.2 Although enrichment of fetal DNA can be achieved by exploiting the differences in fragment size between fetal and maternal DNA,3 complete separation has not proved possible. The only diagnostic tests using free fetal DNA in maternal plasma that are used routinely are those where the target gene or allele is not present in the mother. These are fetal sexing by detection of a Y-borne gene and fetal blood grouping in women whose red cells lack the corresponding antigen. Alloimmunisation against the D (RH1) red cell surface antigen of the Rh blood group system is the most common cause of haemolytic disease of the fetus and newborn (HDFN), which, before the introduction of post-delivery anti-D prophylaxis in the 1960s, accounted for the death of one baby in 2200. In the following 40 years, the effect of the anti-D prophylaxis programme and improved neonatal care reduced the prevalence to one death in 21 000. In England and Wales, about 500 fetuses develop HDFN each year, of which 25–30 babies die, and at least 20 pregnancies per year are lost to miscarriage before 24 weeks of gestation.4,5 The reason for testing for fetal D phenotype of pregnant women with anti-D is to assist in the management of pregnancy. If the fetus is D-positive, appropriate management of a pregnancy at risk from HDFN can be arranged; if it is D-negative, then it is not at risk and no invasive procedures are required. Obtaining fetal red cells is a difficult and risky procedure, so the only practical method available is to predict D phenotype from the genotype obtained from fetal DNA. This can now be done with a high degree of accuracy. Fetal typing from DNA has been provided as a service in England since 1994. Initially, the source of this fetal DNA was amniocytes or chorionic villi. The procedures for obtaining these materials, however, are expensive and invasive, and present a risk to the fetus: amniocentesis is associated © Geoff Daniels, Kirstin Finning, Peter Martin and Edwin Massey. Volume compilation © RCOG

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with a 0.5–1% risk of miscarriage.6 In addition, amniocentesis is associated with a 17% risk of transplacental haemorrhage,7 which, if the fetus were D-positive, could boost the maternal anti-D, increasing the risk of severe HDFN. Chorionic villus sampling (CVS) is associated with a similar risk of miscarriage but a reduced risk of transplacental haemorrhage. Free fetal DNA in maternal blood is now the preferred source of fetal DNA for D testing, at least in Europe.

Molecular basis for D polymorphism The antigens of the Rh system are encoded by a pair of homologous genes on chromosome 1, RHD and RHCE. These genes each have 10 exons, share 94% sequence identity, and are in opposite orientation (5′RHD3′-3′RHCE5′) on chromosome  1 (Figure 12.1).8 RHD is flanked by 9 kb regions of 98.6% identity, the Rh boxes. The Rh genes produce homologous proteins of 417 amino acids that are palmitoylated but not glycosylated. They traverse the red cell membrane 12 times, with both termini in the cytosol and six extracellular loops, the potential sites for antigen activity. In whites, almost all D-negative individuals are homozygous for a deletion of RHD, the deletion encompassing the whole of RHD (Figure 12.1).8 D-positive individuals may have one or two copies of RHD. The D-negative phenotype, therefore, almost always results from an absence of the RhD protein from the red cell membrane, although the RhCcEe protein is almost universally present. Consequently, anti-D produced as a result of alloimmunisation by D-positive red cells introduced by blood transfusion or pregnancy comprises antibody molecules recognising a variety of epitopes on the external loops of the RhD protein. In black Africans, the situation is different: only 18% of D-negative black Africans are homozygous for an RHD deletion. Sixty-six percent of D-negative black Africans have an inactive RHD gene, called RHDΨ, that has a 37 bp duplication in exon 4 and a nonsense mutation in exon 6.9 In addition, 15% of D-negative black Africans have a hybrid gene, RHD-CE-Ds, that contains exons 1, 2, part of 3, 9 and 10 from RHD but part of exon 3 and exons 4–8 from RHCE (Figure 12.1).10,11 Neither RHDΨ nor RHD-CE-Ds produces any epitopes of D. There are numerous rare variants of the D antigen, recognised by absence of some or many D epitopes and/or weakness of expression of D.12–14 In some cases, individuals

Exon 1

RHD

37 bp insert

10

1

RHCE

10

D+ D− RHD deletion D+ RHDVI D− RHDΨ

stop

D− RHD-CE-DS Figure 12.1 The Rh genes RHD and RHCE in five haplotypes, two producing D (D+) and three producing no D (D−); black boxes represent RHD exons, white boxes represent RHCE exons and grey boxes represent mutated exons

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with variant D antigens, if alloimmunised by normal D-positive red cells, can make antibodies to the D epitopes they lack from their own red cells. D variant antigens result either from single-nucleotide changes in RHD encoding amino acid substitutions or from hybrid Rh genes comprising sequences derived from both RHD and RHCE. One example of a hybrid Rh gene is that encoding DVI, the most common D variant associated with production of anti-D, an RHD-CE-D gene consisting of exons 1–3 and 7–10 of RHD but exons 4–6 of RHCE (Figure 12.1).15

Fetal D testing in alloimmunised (high-risk) pregnant women DNA is usually isolated from maternal plasma by centrifugation of EDTAanticoagulated blood to remove the plasma, followed either by a second centrifugation at high speed or by filtration through a 0.2 µm filter, to remove all cellular material, which could originate from a previous pregnancy.16 These procedures should take place as soon as possible after phlebotomy to prevent breakdown of leucocytes and an increase in the proportion of maternal DNA. Most laboratories carrying out fetal D typing on fetal DNA in maternal plasma employ real-time quantitative polymerase chain reaction (RQPCR) technology with TaqMan® chemistry.17–19 The main advantages of RQPCR over conventional PCR are that it is quantitative, making it easy to distinguish fetal and maternal contributions, and the amplification and analysis takes place in closed tubes, reducing the risks of contamination. Some amplification plots for RQPCR testing are shown in Figure 12.2. Although amplification of a single region of RHD will provide a correct phenotype prediction in the majority of cases, most protocols involve amplification of two or three exons to avoid obtaining false results with the more common variants of RHD. It is important that false positives do not result from the presence of the inactive African genes RHDΨ and RHD-CE-Ds. RHDΨ contains several single-nucleotide polymorphisms (SNPs) in exons 4 and 5 and a negative result can be achieved by the use of primers or probes specific for the common sequence.9,20 Amplification of any RHD sequence within exons 4–7 will give a correct negative result with RHD-CE-Ds. Where possible, RHD genes producing variant D  antigens should give a positive result and this can be achieved by including an amplification of exon 7 or 10. Many laboratories prefer to include an amplification of exon 7, which appears to provide a higher affinity reaction than exon 10.21.22 Methods that only employ amplification of exons 7 and 10 are not suitable for testing any population containing people of African origin, as they will give false positive results when the fetus has RHDΨ. In addition, when the mother has RHDΨ, the very strongly positive reaction given by the maternal DNA will prevent identification of an active fetal RHD. Institut de Biotechnologies Jacques Boy in Reims, France,23 the patent holder for non-invasive fetal D typing by RQPCR in Europe, has developed a kit for fetal RHD genotyping (Free DNA Fetal Kit RhD®) but this kit is limited by currently targeting only RHD exons 7 and 10. Sequenom in the USA, the worldwide licence holder for non-invasive prenatal testing, is developing a technology for fetal D typing on DNA in the maternal plasma based on their MassARRAY® system, a process involving mass spectrometry. This will permit a large number of loci to be analysed simultaneously, enhancing detection of fetal variant RHD genes. This platform will also permit the incorporation of a panel of fetal markers to control for the presence of fetal DNA in D-negative female fetuses.24 Sequenom is currently applying this technology in multicentre trials of fetal D testing on plasma from 550 D-negative pregnant women.25

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Figure 12.2 Real-time quantitative polymerase chain reaction (RQPCR) amplification plots obtained from DNA in maternal plasma; the presence of four positive replicates for each of RHD exons 4, 5 and 10, and the lack of positive replicates for SRY, indicate that the fetus is D-positive (and female)

A meta-analysis published in 2006 by Geifman-Holtzman et al.26 analysed 37 publications describing 44 protocols for fetal D testing from DNA in maternal blood. Their overall conclusion was an accuracy of 2919/3078 (94.8%), when excluding the small studies and excluding samples for absence of DNA or lack of Rh confirmation. In reality, the level of accuracy is much higher than this in laboratories providing a routine service. At the International Blood Group Reference Laboratory (IBGRL) in Bristol, we have provided a non-invasive fetal RHD genotyping service since 2001 and have tested about 1400 pregnancies. As far as we can ascertain, we have reported only six erroneous results (two false negative and four false positive, two of which were from frozen plasma that could have been contaminated at source). Had amniocentesis been used to obtain fetal DNA for these tests, those errors might have been avoided

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but we can estimate that between seven and 14 of the fetuses would have been lost and antibody levels would have been boosted in over 100 of the pregnant women. In England, a routine fetal RHD genotyping service is provided by the IBGRL (part of NHS Blood and Transplant) for alloimmunised pregnant D-negative women who require non-invasive monitoring and potentially intrauterine transfusions if they are carrying a D-positive fetus. This would include mothers with a previous history of fetal or neonatal haemolysis regardless of current anti-D level and those with a significant level of anti-D, usually defined as 4 iu/ml or more. Several similar services are provided around Europe.19 Partly owing to intellectual property rights issues and partly owing to the absence of satisfactory internal controls, almost no non-invasive fetal RhD testing is carried out in North America, although at least one service is provided in the USA.27 One unusual potential source of error is a previous solid organ transplant in the mother. Tests on DNA isolated from the plasma of a D-negative pregnant woman predicted a D-positive male fetus, whereas DNA isolated from amniocytes gave a D-negative result. The woman, who had received a kidney transplant from a D-positive male, delivered a D-negative girl.28 The problem of including internal controls A problem in all tests on fetal DNA derived from maternal plasma arises from the large quantity of maternal DNA present in the DNA preparation, complicating the inclusion of satisfactory internal controls to test for successful amplification of fetal DNA. Without a control, an apparent D-negative result could arise from the presence of insufficient fetal DNA. One control that is commonly used is amplification of the Y-linked gene SRY but this is only effective as a control when the fetus is male. When a result suggesting a D-negative female fetus is obtained, some laboratories have incorporated tests for a selection of polymorphisms that involve insertion or deletion of a DNA sequence, in an attempt to obtain a positive result derived from fetal, but not maternal, DNA.29 Short tandem repeats may also be employed in a similar way.30,31 These methods have several drawbacks: n the tests are not true internal controls, as it is not possible to incorporate them as part of a multiplex with the RHD amplification n they are often not informative unless a large number of polymorphisms are employed n they are very labour intensive, making the test time-consuming and expensive n and they could give a false sense of security as the test for the polymorphism may be more sensitive than the test for RHD. RASSF1A is a tumour suppressor gene in which the promoter is hypermethylated in fetal DNA and hypomethylated in maternal DNA.32 Treatment of DNA with the methylation-sensitive restriction enzyme BstU1 results in digestion of an RASSF1A promoter sequence derived from maternal DNA, but not that from fetal DNA, which originates from the placenta. The undigested sequences could then be detected by RQPCR in a multiplex with the test for RHD. This has potential for providing a suitable control but so far there has been no report of this technology being incorporated into a diagnostic test. Incorporation of reactions to ‘housekeeping’ genes, such as CCR5, β-globin (HBB), β-actin (ACTB) or albumin (ALB), demonstrate that amplification has occurred but will amplify fragments from both maternal and fetal DNA and so provide no control

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for the presence of adequate fetal DNA. In addition, when quantitative PCR is used, amplification of a ‘housekeeping’ gene gives an estimate of the total amount of DNA present. If too much is detected, this probably arises from an excess of maternal DNA, which could compromise the sensitivity of fetal DNA detection. Spiking of the plasma with mouse DNA, maize DNA or Escherichia coli plasmid, followed by specific detection, provides a control for sensitivity of the test.23,33,34

Fetal testing to ascertain the requirement for antenatal anti-D immunoglobulin prophylaxis In 2002, the then National Institute for Clinical Excellence (NICE) in the UK recommended that all D-negative pregnant women should be offered anti-D immunoglobulin at 28 and at 34  weeks of gestation to reduce the incidence of antenatal alloimmunisation.4 It is now policy in many countries to offer one or two doses of anti-D immunoglobulin antenatally. In a predominantly white population, however, about 38% of these women would be carrying a D-negative fetus and will receive this treatment unnecessarily.12 So far, lack of a safe and reliable technique of sufficiently high throughput for routine fetal RHD genotyping has meant that, to protect all D-negative women, those with a D-negative fetus also receive prophylaxis. Two sets of trials have been described employing high-throughput methods, including robotic isolation of plasma DNA and RQPCR technology, with the potential for testing the fetal D  type for all D-negative pregnant women. In the Netherlands, 2359 samples taken at 30  weeks of gestation were tested for RHD exon  7.35 In the1257 cases in which molecular results could be compared with serological results, three false negative results and five false positive results were obtained, giving a diagnostic accuracy of 99.4%. In the UK, Finning et al.36 analysed RHD exons 5 (negative with RHDΨ) and 7 (positive with RHDΨ) from maternal plasma. CCR5 was included as an amplification control. Serologically determined RhD phenotypes were obtained. A correct fetal RhD phenotype was predicted by the genotyping tests in 95.7% of 1869 pregnancies in which a serological D phenotype was obtained from the cord blood samples. In 3.4%, results were either unobtainable or inconclusive. In 0.75% (14 samples), a false positive result was obtained, probably because of unexpressed or weakly expressed fetal RHD genes. In only three samples (0.16%) were false negative results obtained. It is these false negatives that cause most concern, as in a diagnostic setting anti-D immunoglobulin prophylaxis would have been withheld and the women would have been at risk from immunisation. In all three cases, however, the blood samples had been delayed in transport before plasma isolation and would not have been accepted for diagnostic testing. If the results of the UK trials had been applied as a guide to treatment, only 2% of the women would have received anti-D immunoglobulin unnecessarily, compared with 38% without the genotyping. A large validation of over 1000 D-negative pregnant women in Germany, in which DNA isolation was not performed by high-throughput technology, gave 99.5% accuracy.37 A similar level of accuracy was achieved in a French analysis of almost 900 pregnancies but this study required re-testing all negative results and so would be uneconomical for screening D-negative pregnant women.22 There will be many advantages to carrying out fetal D testing of all D-negative pregnant women: n It is likely to prove cost-effective, the cost of the test being less than the cost of anti-D immunoglobulin, although no thorough economic evaluation has

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been published. In addition, there will be no requirement to give anti-D immunoglobulin antenatally to women with D-negative fetuses after potential sensitising events such as amniocentesis or other trauma. If the test proves accurate enough, there will be no need to test cord red cells serologically for D. n Anti-D immunoglobulin is in short supply. It is produced from plasma of volunteers who have been immunised with D-positive red cells, so there is an ethical issue about immunising people with blood products to produce a drug that is used unnecessarily. n Most importantly, it eliminates unnecessary treatment of pregnant women with blood products and avoids the associated inconvenience, discomfort and perceived risks of infection from pooled donor blood products that such injections entail. Blood services worldwide are spending increasing sums of money to ensure the safety of the blood supply. Fetal RHD screening provides a way of significantly reducing the quantity of blood products given routinely to pregnant women.

Fetal genotyping for other blood groups After anti-D, the next most common causes of HDFN are anti-c (anti-RH4) of the Rh system and anti-K (anti-KEL1) of the Kell system.12 The Rh antigens C (RH2) and E (RH3) also occasionally cause severe HDFN. The molecular background to the c/C polymorphism is basically a 307C>T SNP in exon 2 of RHCE encoding Pro103Ser in the second extracellular loop of the RhCcEe protein.38 To recognise the presence of an RHCE*c allele, C307 can be detected. The situation for C is more complex as the sequences of exons  2 of RHCE*C and RHD are identical, making specific amplification of RHCE*C impossible in D-positive individuals. However, RHCE*C has a 109 bp insert in intron 2 that is not present in RHCE*c or RHD, and this can be used for C typing.39 The e/E polymorphism results from a 676G>C SNP in exon 5 of RHCE encoding Ala226Pro in the fourth extracellular loop of the protein.38 Typing for  E, therefore, involves detection of C676. The k/K polymorphism results from a 698C>T SNP in exon  6 of KEL encoding Thr193Met.40 There are only a few published reports of non-invasive fetal genotyping for  C, c and E.34,41–43 All involve RQPCR with allele-specific primers or probes. The genotyping results, compared with serological determinations following birth, show 100% accuracy. K typing presents more of a problem. Finning et al.42 were unable to obtain a satisfactory level of specificity by conventional RQPCR methods owing to mispriming of the K (KEL*1) allele-specific primer on the k (KEL*2) allele. This was overcome, with a sacrifice of reduced sensitivity, by employing locked nucleic acids (LNAs) and the introduction of a mismatch into the allele-specific primer. LNAs are nucleic acid analogues that lock the structure into a rigid bicyclic formation.44 Oligonucleotides that contain LNAs have exceptionally high affinity for complementary DNA strands and excellent mismatch discrimination. The only false negative result was obtained in a sample taken at 17 weeks of gestation that had a particularly low yield of total DNA. Consequently, it is a recommendation in England that a K-negative result obtained before 28 weeks of gestation should be followed up with a repeat test at or after 28 weeks.

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Li et al.45 obtained 94% accuracy in fetal K  detection by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)-based single allele-based extension reaction (SABER). Most K antibodies are stimulated by blood transfusion rather than pregnancy.46 Consequently, the partners of many women with anti-K are K-negative, indicating a K-negative fetus. It is valuable, therefore, to test the father for K before making the decision to carry out fetal testing. Development of a non-invasive genotyping method for determining fetal HPA-1a platelet antigen would be beneficial because anti-HPA-1a is the most common cause of neonatal alloimmune thrombocytopenia.

Quality assurance As an increasing number of laboratories internationally are introducing non-invasive fetal blood group testing, it is important that their performances are monitored through external quality assurance schemes. The International Society of Blood Transfusion (ISBT) organised international workshops in molecular blood group genotyping in 2004,17 200618 and 2008.19 In the most recent workshop, two plasma samples from pregnant D-negative women, one with a D-positive and one with a D-negative fetus, were distributed to 17  laboratories (14 from Europe and one each from Australia, Brazil and China). From a total of 31 results submitted, 27 were correct, two were inconclusive and two reported the D-positive fetus as D-negative (Table  12.1). All laboratories employed RQPCR.19 A European network of excellence on special non-invasive advances in fetal and neonatal evaluation (SAFE), funded by the European Union, has provided money for research, meetings and workshops, increasing communication between workers in the field.47 A SAFE workshop on extraction of fetal DNA from maternal plasma demonstrated that the highest yield was obtained by the QIAamp DSP virus kit.48

Conclusion To date, the enormous promise of the application of free fetal DNA in maternal plasma to prenatal diagnostics has only been realised in fetal blood grouping and fetal sexing. Fetal blood grouping often plays an important role in the avoidance of unnecessary procedures and is the standard of care in England for pregnant women with significant levels of anti-D. Since a reliable non-invasive test is available, it could be considered unethical to perform amniocenteses purely for fetal RhD typing. In the future, high-throughput fetal D typing will reduce wastage of anti-D immunoglobulin and avoid unnecessary treatment of pregnant women with blood products. As intellectual property rights have been granted for free fetal DNA testing worldwide, let us hope that progress in this field is not hampered by financial and legal issues.

Table 12.1 Fetal D type D-negative D-positive

Plasma fetal RHD typing results from the International Society of Blood Transfusion (ISBT) workshop in 2008; 17 laboratories received samples19 Correct 14 13

Incorrect 0 2

Inconclusive 1 1

No results reported 2 1

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Geifman-Holtzman O, Grotegut CA, Gaughan JP. Diagnostic accuracy of noninvasive fetal genotyping from maternal blood – a meta analysis. Am J Obstet Gynecol 2006;195:1163–73. Lenetix [www.lenetix.com]. Minon JM, Semterre JM, Schaaps JP, Foidart JP. An unusual false positive fetal RHD typing result using DNA derived from maternal plasma from a solid organ recipient. Transfusion 2006;46:1454. Page-Christiaens GC, Bossers B, van der Schoot CE, de Haas M. Use of bi-allelic insertion/ deletion polymorphisms as a positive control in maternal blood. First clinical experience. Ann N Y Acad Sci 2006;1075:123–9. Pertl B, Pieber D, Panzitt T, Haeusler MC, Winter R, Tului L, et al. RhD genotyping by quantitative fluorescent polymerase chain reaction: a new approach. Br J Obstet Gynaecol 2000;107:1498–502. Liu FM, Wang XY, Feng X, Wang W, Ye YX, Hong C. Feasibility study of using fetal DNA in maternal plasma for non-invasive prenatal diagnosis. Acta Obstet Gynecol Scand 2007;86:535–41. Chan KC, Ding C, Gerovassili A, Yeung SW, Chiu RW, Leung TN, et al. Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006;52:2211–18. Costa JM, Giovangrandi Y, Ernault P, Lohmann L, Nataf V, El Halali N, et al. Fetal RHD genotyping in maternal serum during the first trimester of pregnancy. Br J Haematol 2002;119:255–60. Legler TJ, Lynen R, Maas JH, Pindur G, Kulenkampff D, Suren A, et al. Prediction of fetal Rh D and Rh CcEe phenotype from maternal plasma with real-time polymerase chain reaction. Transfus Apher Sci 2002;27:217–223. Van der Schoot CE, Soussan AA, Koelewijn J, Bonsel G, Paget-Christiaens LG, de Haas M. Noninvasive antenatal RHD typing. Transfus Clin Biol 2006;13:53–7. Finning K, Martin P, Summers J, Massey E, Poole G, Daniels G. Effect of high throughput RHD typing of fetal DNA in maternal plasma on use of anti-RhD immunoglobulin in RhD negative pregnant women: prospective feasibility study. BMJ 2008;336:816–18. Müller SP, Bartels I, Stein W, Emons G, Gutensohn K, Köhler M, et al. The determination of the fetal D status from maternal plasma for decision making on Rh prophylaxis is feasible. Transfusion 2008;48:2292–301. Mouro I, Colin Y, Chérif-Zahar B, Cartron JP, Le Van Kim C. Molecular genetic basis of the human Rhesus blood group system. Nature Genet 1993;5:62–5. Poulter M, Kemp TJ, Carritt B. DNA-based Rhesus typing: simultaneous determination of RHC and RHD status using the polymerase chain reaction. Vox Sang 1996;70:164–8. Lee S, Wu X, Reid M, Zelinski T, Redman C. Molecular basis of the Kell (K1) phenotype. Blood 1995;85:912–16. Hromadnikova I, Vechetova L, Vesela K, Benesova B, Doucha J, Vlk R. Non-invasive fetal RHD and RHCE genotyping using real-time PCR testing of maternal plasma in RhD-negative pregnancies. J Histochem Cytochem 2005;53:301–5. Finning K, Martin P, Summers J, Daniels G. Fetal genotyping for the K (Kell) and Rh C, c, and E blood groups on cell-free fetal DNA from maternal plasma. Transfusion 2007;47:2126–33. Orzińska A, Guz K, Brojer E, Zupańska B. Preliminary results with fetal Rhc examination in plasma of pregnant women with anti-c. Prenat Diagn 2008;28:335–7. Petersen K, Vogel U, Rockenbauer E, Nielsen KV, Kølvraa S, Bolund L, et al. Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms. Mol Cell Probes 2004;18:117–22. Li Y, Finning K, Daniels G, Hahn S, Zhong X, Holzgreve W. Noninvasive genotyping fetal Kell blood group (KEL1) using cell-free fetal DNA in maternal plasma by MALDI-TOF mass spectometry. Prenat Diagn 2008;28:203–8. Klein HG, Anstee DJ. Blood Transfusion in Clinical Medicine. 11th ed. Oxford: Blackwell; 2005. Chitty LS, van der Schoot CE, Hahn S, Avent ND. SAFE – The Special Non-invasive Advances in Fetal and Neonatal Evaluation Network: aims and achievements. Prenat Diagn 2008;28:83–8. Legler TJ, Liu Z, Mavrou A, Finning K, Hromadnikova I, Galbiati S, et al. Workshop report on the extraction of foetal DNA from maternal plasma. Prenat Diagn 2007;27:824–9.

13 Chapter 13

Selective termination of pregnancy and preimplantation genetic diagnosis: some ethical issues in the interpretation of the legal criteria Rosamund Scott

Introduction This chapter considers some of the ethical issues at stake in the legal interpretation of the grounds for selective termination of pregnancy on the one hand and preimplantation genetic diagnosis (PGD) on the other.*1 Termination of pregnancy is legal under section 1(1)(d) of the Abortion Act 19672 (as amended by the Human Fertilisation and Embryology [HFE] Act 19903) if two doctors have formed an opinion in good faith that ‘there is a substantial risk that if the child were born it would suffer from such physical or mental abnormalities as to be seriously handicapped’.† These terms leave considerable scope for interpretation, particularly about what is meant by ‘seriously’, and to date there has been no direct judicial interpretation of this section. In a similar but not identical vein, PGD is legal if there is a significant risk ‘that a person … will have or develop a serious physical or mental disability, a serious illness or any other serious medical condition’.‡3,4 In both cases, then, great reliance is placed on the idea of seriousness. A certain degree of risk is also essential in both cases – a substantial risk in the context of termination of pregnancy and a significant risk in the case of PGD. In this chapter, I focus on some of the difficulties in interpreting seriousness in either context. My analysis will draw on a key and widely endorsed distinction in the bioethics literature between a life that may not be worth living and one that is worth living. Attention to this distinction shows that it is only where a person would have a condition that is so serious that he or she may not think his or her life worth living that selective termination or PGD can truly be said to be done for the sake of the person who would otherwise be born. This is likely to be the case in relation to surprisingly * Some parts of this chapter have been adapted from my work in Scott.1 † The relevant part of section 1(1) of the Abortion Act 19672 as amended by the HFE Act 19903 reads: ‘Subject to the provisions of this section, a person shall not be guilty of an offence under the law relating to abortion when a pregnancy is terminated by a registered medical practitioner if two registered medical practitioners are of the opinion, formed in good faith… (d) that there is a substantial risk that if the child were born it would suffer from such physical or mental abnormalities as to be seriously handicapped’. ‡ HFE Act 19903 as amended by the HFE Act 2008,4 section 1(Z)(A)(2). See also section 1(Z)(A)(1) and (3) for similar uses of the term ‘serious’.

© Rosamund Scott. Volume compilation © RCOG

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few conditions. In all other cases, since it is not against the interests of a person with a life worth living to be born, we need to consider in whose potential interests prenatal diagnosis (PND), selective termination and PGD are. I shall argue that it is legitimate, to a certain extent at least, for parental (and wider family) interests to be taken into account in the interpretation of the notion of ‘serious’ in both contexts. I shall first briefly discuss the distinction between a life that someone may not think is worth living and one that someone will think is worth living and consider its implications for the question of whose interests may be at stake in PND and PGD. I then turn to consider the interpretation of the grounds for selective termination of pregnancy in English law. I conclude that parental interests have a legitimate place in the interpretation of ‘serious’ under the Act but that it is not clear that parental interests could justify termination up until birth. Rather, it may be that after 24 weeks of gestation, fetal interests – and so the protection of a future child from a so-called ‘wrongful life’ – provide the most legitimate grounds for termination. In the latter part of the chapter I briefly touch on the recommendations that laid the groundwork for the current criteria for PGD, which have been accepted by Parliament in the HFE Act 2008.4 I also raise some questions about the interpretation of the relevant provisions of the new Act and two aspects of the draft guidance in the forthcoming Human Fertilisation and Embryology Authority (HFEA) Eighth Code of Practice.

The severity of a condition: whose interests? When born, a child may be or become so severely impaired that we might ask whether his or her life is overall good or bad for him or her, in terms of the wellbeing it contains. Part of this question entails reflection on the severity of the condition that a child would have. Tay–Sachs disease appears to be one example of a very serious condition.5 Another might be Lesch–Nyhan syndrome.6 Judgements by third parties about the quality of life of possible others are obviously difficult and sensitive. As Jonathan Glover7 has suggested, it may be best to restrict ourselves to the question of whether there is a ‘serious risk’ that someone may have a life he or she does not think worth living, a life of very low quality. It may be that this could fairly be said about life with the two conditions noted here. When we move away from these extreme and rare conditions, judging the seriousness of a condition becomes more difficult and thus controversial. This has been acknowledged by the Human Genetics Commission (HGC):8 It has proved impossible to define what ‘serious’ should mean in this context. We have listed some factors that should be taken into account when considering seriousness, but perhaps the most important is that this technique should not be used for the purposes of trait selection or in a manner which could give rise to eugenic outcomes.

The HFEA and Advisory Committee on Genetic Testing (AGCT), in their Consultation Document on Preimplantation Genetic Diagnosis,9 observe that: … individual judgments on seriousness will vary depending on personal and family circumstances and on the nature and severity of the condition and the likelihood of transmission.

Parents, people with relevant conditions of some kind and the medical profession may well have diverging views about this issue. Although it is likely that we have some hold on these issues at the extremes – for instance Tay–Sachs versus the webbing of two toes – difficulties attach to the large range of ‘mid-spectrum’ conditions in

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between.*10 I am thinking here of conditions such as cystic fibrosis, spina bifida and Down syndrome, the seriousness of which (depending on the prognosis, especially in the first two cases) is very much a matter of debate. Despite the significant difficulties that will be experienced in the lives of people with Down syndrome or cystic fibrosis, on balance it may be fair to say that a person with one of these conditions will probably have a life he or she thinks is worth living, one that particularly he or she may also think is of reasonable quality. Indeed, there is evidence from positive psychological research on disability and disease that seems to suggest that in very many cases our subjective wellbeing is largely genetic, so that whether we have a condition of some kind matters a lot less than people may think.11 So, although the severity of these conditions varies, even at their worst it seems that it would not have been against the interests of someone with one of them to have been born.†12 This means that PND or PGD for conditions such as cystic fibrosis or Down syndrome could not be said to be conducted for the sake of the future person that a given fetus or embryo will become. Nevertheless, a child’s condition may still implicate his parents’ interests in reproduction in serious ways: he or she might have significant mental impairment or serious health problems requiring repeated hospitalisation, with an uncertain future. So, although people may reasonably disagree about this, and although the spectrum of severity of a given condition varies, it is arguable that some children born with a condition such as Down syndrome or cystic fibrosis may fairly be judged to have a serious condition, even though it was not against their own interests to be born, so that they do not have a ‘wrongful life’. In this light, it appears that testing and selection decisions can in fact be made for either of two principal reasons: first, where the child’s quality of life would be one of extremely low (sometimes called ‘subzero’) quality, as in the case of Tay–Sachs, primarily because of what that life would be like for the resulting child (here the parents’ interests will also be implicated); second, where the child would in fact have a reasonable quality of life but their condition may nevertheless have the potential adversely to impact on their parents, because of their interests in the kind of child who will be born. Selection against Down syndrome or cystic fibrosis (in some cases) may put special weight on the parents’ interests rather than those of the child. In such cases, prospective parents arguably do have a legitimate moral interest (at least in the earlier rather than later stages of pregnancy) in deciding whether to have a child with a serious condition that would nevertheless mean that a child had a life he or she thought worth living. Furthermore, this interest is one aspect of their broader interest in deciding whether to have any child. There are a number of writers who have noted the possible distinction between the interests of the future child on the one hand and those of the parents on the other in such decisions, including Jonathan Glover, Sally Sheldon, Stephen Wilkinson and Tom Shakespeare.13 It is not clear whether or to what extent the distinction between those rare cases in which birth might not be in the interests of the future child, given the severity of its impairment or condition on the one hand, and cases in which selection is really, potentially, in the parents’ interests or those of their wider family on the other, is explicitly acknowledged in the conduct of selection practices. In some ways, the idea that parents may prefer to avoid the birth of an impaired child for their own sake or that of their family is somewhat taboo. In this regard, one obstetrician observed * Page 263 onwards in Wertz10 discusses the difference of views among practitioners as to what is serious. † For interesting work on the views of people with conditions such as cystic fibrosis, Down syndrome and spina bifida, both on their lives and on screening practices, see Alderson.12

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that she considers that clinicians tend always to cite and use the disability ground of the Abortion Act in relation to termination for fetal disability because this is easier for parents.14 That those working in the field do reflect on this issue is evidenced by a research project in which I was involved that looked at the views and experience of those working in the field of PGD. We interviewed healthcare professionals and scientists at two London sites and conducted ethics discussion groups with them. In one discussion group, a scientist observed [my emphasis]:* I was at a conference … Parent Project UK, which is a charity which is aimed at … therapy for Duchenne muscular dystrophy people, and they were all parents. So one presented a talk actually which I found very interesting, and they looked at the quality of life for families with boys with Duchenne muscular dystrophy, which is a severe disease. The average lifespan now is about 19. And the quality of life of – the perception of the quality of life of the affected boy was rated differently by parents, by the clinicians looking after them and by the boys themselves. And the boys themselves … gave their rating of quality of life the same as any healthy controlled sample. And the parents gave them the lowest quality and the clinicians gave them somewhere in between the two, which was interesting, I thought. … So that implies we’re doing this for the parents and not for the child in some respects.

This person also said: Obviously people want to have children and when they have children with disability or handicap, to some extent that makes their life a bit more miserable compared to what they’re hoping for.

The potential impact of a child’s disability on parents was observed by a number of participants in this project. For instance, another scientist† referred to the ‘huge burden’ that parents may experience. More particularly, he or she also alluded to the idea of undoubtedly serious conditions on the one hand and conditions about which people may disagree on the other: I mean I think there are conditions which are under all circumstances, horrendous. And can be put very firmly on that list. But I completely agree with [participant], I mean I think there are lots of conditions which aren’t clearcut and which for some families might be considered serious and others not.

Can the requirement of seriousness in the statutory criteria for termination of pregnancy and PGD legitimately be interpreted in such a way as to mean that prospective parents’ interests can be taken into account in those cases which appear to form the majority of selection decisions? These are the cases in which birth would not in fact be against the interests of the child who would be born. I turn first to selective termination of pregnancy.

Interpreting the law on selective termination of pregnancy and PGD Termination of pregnancy A few years ago, Reverend Joanna Jepson brought the case of Jepson versus The Chief Constable of West Mercia Police Constabulary,15 which concerned an application * Ethics discussion group 2, Scientist 21, Wellcome Trust Biomedical Ethics Programme, grant no. 074935. The project was entitled ‘Facilitating Choice, Framing Choice: the Experience of Staff Working in PGD’, January 2005 to June 2007, and was conducted by Clare Williams (principal researcher), Kathryn Ehrich, Bobbie Farsides, Clare Sandall, Rosamund Scott and Peter Braude. † Scientist 8.

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for judicial review of the police’s decision not to prosecute doctors in relation to a termination of pregnancy for cleft lip and palate at 28 weeks of gestation. (I do not further discuss the facts of this case here.) The case did not progress beyond the initial hearing but at that hearing she contended that a key error of law in that case was ‘that the medical practitioners who signed the certificate … took into account the views of the parents involved’ and ‘that … in relation to the decision in question, the parents’ views, as a matter of law, could have no weight’. Certainly the Act states that two doctors must be ‘of the opinion, formed in good faith’ that the impairment is serious. Whether two doctors can decide in good faith that the condition is serious and mean (or also mean) in so doing ‘serious for this woman’ (their patient for the purposes of a legal termination) is a crucial question. Doctors could decide that a woman’s views as to seriousness are relevant to their opinion of whether the criterion of seriousness is satisfied if they consider that at least one purpose of the disability section of the Act is to give women (and couples) choice about the birth of a seriously disabled child and recognise that, in making this choice, women (together with their husbands or partners, family and wider social network) will have views about the degree of impairment in a child they feel able to take on. Some scholars have indeed suggested that, where the fetus could have a reasonable quality of life as a born child, it is in fact the woman’s or parents’ interests that underlie this section of the Abortion Act. Glanville Williams16 laid the ground for this argument some time ago and the basis of this section has been reconsidered by Sally Sheldon and Stephen Wilkinson.17 They argue that, because very few fetuses aborted under this ground would have lives of little or no quality if born, most terminations of pregnancy under this section cannot be seen as protecting the fetus from such a life. Rather, in most cases in which this section is invoked, the real concern is with the woman’s or parents’ interests. This is a moral argument as to the legal interpretation of this section of the Act and one I support. Whether or not parents’ interests could be relevant to the interpretation of the disability ground of the Act was set to be a key issue in the Jepson case. When the police investigated the matter, it contacted the Royal College of Obstetricians and Gynaecologists (RCOG). The original advice given to them by its Vice President was (in part) as follows:15 The decision to abort under Clause E would always ultimately be with the mother having taken into consideration the perception of the parents to the serious nature of the handicap following counselling and information from experts with special knowledge of the condition.

On one reading, this means the woman is to take into account her own views, but this cannot be what was intended. Another reading is that legally the woman is required to take account of her partner’s views, but this is legally incorrect. A further interpretation is that although discussions about the seriousness of the impairment will take place between the woman (perhaps her partner), practitioners and relevant specialists, at the end of the day, assuming two doctors think that the legal criteria for a termination of pregnancy are made out, it is the woman who decides whether to continue the pregnancy. This makes the most sense. In contrast, since the Act requires that two doctors consider the condition to be serious, the sentence cannot mean that the woman decides whether the criterion of seriousness is satisfied. Despite this, whether two doctors can certify in good faith that the condition is serious – and mean in so doing (at least to some degree) ‘serious for this woman or couple’ – is the critical issue. The claimant in the Jepson case was seeking to steer the law away from any such interpretation. A further interpretation of this important sentence could be

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that it contained a ‘typo’ and that it should have read [my emphasis]:14 ‘The decision to abort under Clause E would always ultimately be with the doctor having taken into consideration the perception of the parents to the serious nature of the handicap following counselling and information from experts with special knowledge of the condition.’ (Alternatively, the ‘typo’ may have entered the scene in the case report.) Indeed, this would be consistent with an earlier sentence in the Vice President’s letter that read: ‘There is no precise definition of “serious handicap” and the decision is therefore one for the practitioner to make in consultation with the parents and other interested parties.’ On this point, one clinician commented that ‘it would be normal good medical practice for a doctor to take account of parents’ views’.15 Given that the Vice President alluded to discussions between a woman and her doctor, her statement may be describing the practice that typically occurs and has built up over the years. This practice may partly be a response to the discomfort some practitioners evidently feel at being the gatekeepers to termination of pregnancy and the administrators of a system in which a pregnant woman’s autonomy is deeply at stake but lacks legal protection in the form of a right to terminate.*18 The question, however, is whether such a practice is lawful. One way to reflect on this would be to try to reconcile use of section 1(1)(a) of the Act in relation to fetal anomaly with section 1(1)(d), the disability ground. Section 1(1)(a) permits termination of pregnancy where two doctors are of a goodfaith opinion ‘that the pregnancy has not exceeded twenty-four weeks and that the continuance of the pregnancy would involve risk, greater than if the pregnancy were terminated, of injury to the physical or mental health of the pregnant woman or any existing children of her family’. The possible use of two different sections of the Abortion Act in relation to fetal impairment is not apparent from the terms of the Act itself. However, it seems right that section 1(1)(a) can be interpreted in this way given its concern with the mental (or physical) health of the woman and the RCOG has itself endorsed such use in some cases.19 Women vary in their reaction to being told that their fetus is, or may be, abnormal. Occasionally a woman feels strongly that she is unable to accept a probability of risk or a degree of handicap that her medical practitioners consider less than substantial or serious. Under such circumstances, and only when the gestation is less than 24  weeks, the practitioners may decide that abortion has become necessary to protect her mental health.

What can we glean from the use of section 1(1)(a) for our interpretation of the disability ground of the Act? When section 1(1)(a) is used in this way, essentially the thought is that, although doctors do not think either that the risk is substantial or the condition serious, a woman may be seriously concerned about what she is able to take on, to the extent that there are risks to her physical or mental health greater than those inherent in termination. Yet, since raising any child, but perhaps particularly a disabled one, has at least the potential to have considerable implications for the life of a woman (and her partner), any termination for serious fetal anomaly is likely seriously to implicate a woman’s (and her partner’s) interests, including where the termination is legally justified on the basis of section 1(1)(d). * In discussions between healthcare professionals, a philosopher and sociologists, one (unnamed) obstetrician said: ‘I think things are probably made more difficult because most obstetricians and gynaecologists know that terminations for social reasons, or whatever, are done effectively on demand, and one of the main reasons for that is because of anxiety generated about the pregnancy continuing … our sort of baseline has shifted because of shifts in how the Abortion Act has been applied over the past 20 years or so.’22

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On the one hand, then, section 1(1)(a) of the Act does not explicitly indicate that it might, in effect, be used in relation to fetal impairment, but arguably can be interpreted in this way to some degree, as evidenced in the RCOG guidelines; on the other hand, the disability ground of the Act does not state that prospective parents’ views about impairment can be a legitimate factor in the interpretation of this section but arguably can be interpreted in this way. Notably, this may particularly be where a ‘serious’ condition may nevertheless result in a life that a child would think worth living, so that termination cannot be claimed, morally speaking, to be in the interests of the fetus or the child it would become. Indeed, assuming section 1(1)(a) can be used in relation to fetal impairment (which must be right), it would be hard to assert that prospective parents’ views, particularly about the seriousness of an impairment, are legally irrelevant under section 1(1)(d), the disability ground, even though that section is not based on risks to the woman’s mental or physical health. Rather, the use of section 1(1)(a) in relation to fetal anomaly confirms that the question of the degree of risk or, particularly, the seriousness of an impairment cannot be a purely objective matter. As noted earlier, this has been acknowledged by others, including the HGC. Importantly, we therefore have good reason to think that doctors can, to some extent at least, take the views and interests of a woman (and her partner) into account when deciding whether the criteria of the disability ground of the Act are satisfied. If so, then the practice to which the Vice President alludes in her letter, which was also referred to by a clinician, is arguably defensible at law. Importantly, if parents’ views about raising a disabled child and their thoughts about the seriousness of its disability were irrelevant to the legality of termination on the basis of the disability ground of the Act, then it is likely that the legality of many terminations that are currently performed should at least be questioned, as should the purpose of certain kinds of screening and testing. Indeed, this would probably be in line with Reverend Jepson’s desires and beliefs in relation to selective termination of pregnancy. For instance, given that it is most unlikely to be against the interests of someone with Down syndrome to be born, screening, testing and termination for Down syndrome could rarely, perhaps never, be said to be based on the seriousness of the condition from the child’s point of view (although one in ten children do not survive the first year of life).*20,21 As regards the third-trimester fetus, the pressure for terminations of pregnancy after 24  weeks to be for particularly serious reasons is rightly strong. The RCOG guidelines on termination of pregnancy after this time assert that terminations must be for particularly serious reasons:22 As the protection due increases with embryonic development and fetal growth, reasons for termination, at no stage trivial, must be more pressing the longer pregnancy has progressed.

The guidelines thus endorse a gradualist approach to fetal moral status, which holds that the longer a pregnancy has been allowed to develop, the greater must be the justification for in any way compromising it. It is a position that I suggest is intuitively appealing and one I have explored and defended in detail elsewhere.23 Can the disability ground of the Act be invoked on behalf of the pregnant woman (and her partner) after 24 weeks? While I have argued that parental views about the seriousness of the disability that a child would have should be taken into account in the interpretation of the disability ground of the Act, at least where disabilities are the subject of reasonable disagreement, it does not follow that this means that their possible * There is also a high rate of stillbirth between screening and birth.

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wish not to have a disabled child can carry weight up until birth. This is owing to the increasing moral claims of the fetus. Since the law has to have a clear line, legally speaking the shift in the strength of those claims occurs at the 24th week, after which the reasons for termination of pregnancy must be that much more stringent. Looking at the other grounds of the Act, we see this in the continuing relevance only of the risk to a woman’s life and the need to avoid grave permanent damage to her physical or mental health (in sections 1(1)(c) and (b), respectively).* It is therefore arguable that the disability ground of the Act should also be more stringently interpreted after this time. In this light, if termination on the grounds of fetal anomaly after 24 weeks is permissible in the interests of the pregnant woman, it may be that this can only be on the basis of section 1(1)(b). Although it seems unlikely that termination on the grounds of disability could be required, and therefore justified, to prevent grave permanent damage to her mental health under section 1(1)(b), a meritorious case cannot be categorically ruled out, particularly where there are other complicating factors. Moving now to consider the fetus, at any stage of pregnancy a termination could only be said to be in its interests when there is a real risk that a subsequently born child would not think his or her life worth living. As the RCOG report on third-trimester terminations notes, termination in the fetus’s interests will in effect be appropriate where as a neonate it would not be in its best interests to continue to receive lifesustaining medical treatment.22 It is beyond the scope of this chapter to say more about late-term terminations of pregnancy. PGD I now turn briefly to PGD. Elsewhere I have reviewed in detail the HFEA and HGC recommendations and discussions that led to the original formulation of the PGD criteria of ‘a significant risk of a serious genetic condition’.1 In essence, these are the criteria that have now been put on a statutory footing. How should they be interpreted? The original HFEA and HGC recommendations sought to recognise, at least to some extent, the personal nature of the issues at stake in PGD but also to observe limits to the legitimacy of prospective parents’ views. Essentially, this was to be achieved by requiring discussion between prospective parents and healthcare professionals and scientists about the degree of risk and the seriousness of any given condition. The HFEA and HGC also put these professionals in the position of being something of a ‘check’ on what these bodies saw as the potentially excessively wide views of those seeking treatment.† Under the new HFE Act, the HFEA must itself be satisfied as to the seriousness of the condition. In this regard, early in 2009 the HFEA sought views as to whether it should make two particular changes to its guidance to clinics. In the first case, the previous guidance stated:24 The use of PGD should be considered only where there is a significant risk of a serious genetic condition being present in the embryo. The perception of the level of risk by those seeking treatment is an important factor in the decision making process. The seriousness of the condition should be a matter for discussion between the people seeking treatment and the clinical team.

The HFEA proposed that the last sentence of the above should be dropped. In addition, among the list of factors that clinics are to consider in deciding whether * Section 1(1)(c) permits abortion where ‘the continuance of the pregnancy would involve risk to the life of the pregnant woman, greater than if the pregnancy were terminated’; section 1(1)(b) permits abortion where ‘necessary to prevent grave permanent injury to the physical or mental health of the pregnant woman’. † HFEA licence committees are of course a further possible check but, at least until now, it has been likely that if a clinic supports an application for PGD, the licence committee will usually agree.

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PGD is appropriate, the first item used to be ‘the view of the people seeking treatment of the condition’.* The HFEA consulted about whether the word ‘view’ should be changed to ‘experience’, with the thought that this might decrease any subjectivity in the decision-making process. In the documentation relating to these possible changes, the HFEA observed:25 Of particular relevance is the intent behind the updated legislation to ensure that the licensing process for PGD is set out within clear boundaries. This is a response to concerns that over time, PGD could be used to avoid less serious conditions. With this in mind, the law will now explicitly require that the HFEA, rather than the clinic in consultation with the people seeking treatment, be satisfied of the seriousness of that condition.

My research into the HFEA’s and HGC’s working groups that originally formulated the PGD criteria and accompanying guidance suggests that this would be contrary to the original intentions in the formulation of the guidance, which in fact allowed for at least some regard to be given to parental views.1 The proposed changes also appear unnecessary in that it is not clear that Parliament, in broadly confirming the original criteria in the HFE Act 2008, was seeking to make those grounds any more stringent.26 Moreover, in any event, we saw that the HGC, which worked with the HFEA in establishing the criteria for PGD, suggested that ‘objectivity’ in relation to serious disability is elusive. This thought is echoed in much of the literature.†27 At the time of writing, it is understood that the HFEA is likely to modify these proposed changes in the light of feedback from its consultation exercise.

Conclusion Looking to the future of PGD and PND, with regard to PGD there may be a tendency to assume that there are lower costs in choosing between embryos (at least once several embryos exist) than in terminating an existing pregnancy. Theoretically at least, there is greater scope to focus on less serious considerations, including ones that would be unlikely to influence prospective parents in deciding to terminate. However, the physical and emotional burdens of undergoing in vitro fertilisation will always be considerable. In this light, the concerns of people engaging in PGD are likely to be very serious indeed given their past reproductive experience and possible reproductive future. They will have in mind both the interests of their possible child and, particularly when a child’s impaired existence is nevertheless compatible with a reasonable quality of life but they remain concerned about the possible impact of such a birth on them, their own interests in reproduction. Turning to PND, despite the advances in testing, including in non-invasive testing, and the concerns that are sometimes expressed that a pregnancy will be terminated for trivial reasons, it seems unlikely that parents would seek a termination of pregnancy for a condition that they did not see as in some way seriously implicating their interests. Overall, both in PGD and PND, where a given child would have a life he or she thought worth living, parental interests may be the ones that are most often potentially invoked. In my view, it would be helpful to recognise and accept this * The original list in full, as formulated by the HGC and HFEA and then adopted by the HFEA in its Code of Practice at paragraph 12.3.3 stated: ‘In any particular situation the following factors should be considered when deciding the appropriateness of PGD: the view of the people seeking treatment of the condition to be avoided; their previous reproductive experience; the likely degree of suffering associated with the condition; the availability of effective therapy, now and in the future; the speed of degeneration in progressive disorders; the extent of any intellectual impairment; the extent of social support available; and the family circumstances of the people seeking treatment.’ † See, for example, comments to this effect in the Introduction by Parens and Asch to their Prenatal Testing and Disability Rights,27 which resulted from a Hastings Center Working Party.

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more clearly in our interpretation of the legal criteria in both contexts. This does not mean we have to accept parental views and reasons at face value. What it does mean is recognising that parents may be seriously affected by the condition of the child that is born and that ‘serious’ can therefore also mean serious for the parents and their family. This would more clearly enable their interests to be taken into account in the interpretation of the criterion of seriousness.

References 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Scott R. Choosing Between Possible Lives: Law and Ethics of Prenatal and Preimplantation Genetic Diagnosis. Oxford: Hart Publishing; 2007. Abortion Act 1967 [www.statutelaw.gov.uk/content.aspx?activeTextDocId=1181037]. Human Fertilisation and Embryology Act 1990 [www.opsi.gov.uk/acts/acts1990/ Ukpga_19900037_en_1.htm]. Human Fertilisation and Embryology Act 2008 [www.opsi.gov.uk/acts/acts2008/ ukpga_20080022_en_1]. National Tay-Sachs and Allied Diseases Association [www.ntsad.org]. National Institute of Neurological Disorders and Stroke [www.ninds.nih.gov/disorders/lesch_ nyhan/lesch_nyhan.htm]. Glover J. Choosing Children: Genes, Disability and Design. Oxford: Oxford University Press; 2006. Human Genetics Commission. Draft Response to the HFEA on the Outcome of the HFEA/ACGT Consultation on Preimplantation Genetic Diagnosis. Annex D in: Human Genetics Commission. Preimplantation Genetic Diagnosis. London: HGC; 2001 [www.hgc.gov.uk/UploadDocs/DocPub/ Document/hgc01-p2.pdf]. Human Fertilisation and Embryology Authority and Advisory Committee on Genetic Testing. Consultation Document on Preimplantation Genetic Diagnosis. November 1999 [www.hfea.gov.uk/cps/ rde/xbcr/hfea/PGD_document.pdf]. Wertz D. Drawing lines: notes for policymakers. In: Parens E, Asch A, editors. Prenatal Testing and Disability Rights. Washington, DC: Georgetown University Press; 2000. p. 261–87. Fave A, Massimini F. The relevance of subjective well-being to social policies: optimal experience and tailored intervention. In: Huppert F, Baylis N, Kaverne B. The Science of Wellbeing. Oxford: Oxford University Press; 2005. p. 379. Alderson P. Prenatal counselling and images of disability. In: Dickenson D, editor. Ethical Issues in Maternal–Fetal Medicine. Cambridge: Cambridge University Press; 2002. p. 195–212. Shakespeare T. Disability Rights and Wrongs. London: Routledge; 2006. S Bewley, personal communication. High Court of England and Wales [2003] E.W.H.C. 3318. Williams G. Textbook of Criminal Law. 1st ed. London: Stevens; 1978. Sheldon S, Wilkinson S. Termination of pregnancy for reason of foetal disability: are there grounds for a special exception in law? Med Law Rev 2001;9:85–109. Williams C, Alderson P, Farsides B. ‘Drawing the line’ in prenatal screening and testing: health practitioners’ discussions. Health Risk Soc 2002;4:61–75. Royal College of Obstetricians and Gynaecologists. Termination of Pregnancy for Fetal Abnormality in England, Wales and Scotland. London: RCOG Press; 1996. Julian-Reynier C, Aurran Y, Dumaret A, Maron A, Chabal F, Giraud F, et al. Attitudes towards Down’s syndrome: follow up of a cohort of 280 cases. J Med Genet 1995;32:597–9. Won RH, Currier RJ, Lorey F, Towner DR. The timing of demise in fetuses with trisomy 21 and trisomy 18. Prenat Diagn 2005;25:608–11. Royal College of Obstetricians and Gynaecologists Ethics Committee. A Consideration of the Law and Ethics in Relation to Late Termination of Pregnancy for Fetal Abnormality. London: RCOG Press; 1998. Scott R. Rights, Duties and the Body: Law and Ethics of the Maternal–Fetal Conflict. Oxford: Hart Publishing; 2002. Human Fertilisation and Embryology Authority. Code of Practice. 7th ed. London: HFEA; 2007. Human Fertilisation and Embryology Authority. Regulating Preimplantation Genetic Diagnosis for the Future: Listening to Your Views. London: HFEA; 2009. E Harris, MP, personal communication. Parens E, Asch A. Introduction. In: Parens E, Asch A, editors. Prenatal Testing and Disability Rights. Washington, DC: Georgetown University Press; 2000. p. 3–43

14 Chapter 14

Implementation and auditing of new genetics and tests: translating genetic tests into practice in the NHS Rob Elles and Ian Frayling

Background Genetic testing for single-gene disorders has been available in the NHS since the mid-1980s and for chromosomal disorders since the early 1970s. A mature network of multidisciplinary regional genetics centres has evolved combining clinical genetics and counselling, laboratory diagnostics and research.1 The link between research and diagnostics has proved to be an effective mechanism for rapid translation of tests and technologies into practice and the close involvement of clinical geneticists has meant that the introduction of new tests and gatekeeping of referrals to ensure appropriate use has been relatively effective. The professionally led network approach to test provision that avoided much duplication of service provision for single-gene disorders was formalised and supported by the Government through the formation of the UK Genetic Testing Network (UKGTN).2 A key part of the remit of the UKGTN is to evaluate new tests proposed for service and recommend them to specialist service commissioners for NHS funding. Our discussions will be limited to potentially heritable, germline genetic disorders.

The UK approach to genetic test evaluation To fulfil its evaluation role, UKGTN adopted the ACCE framework (‘analytic validity; clinical validity; clinical utility; and ethical, legal, and social implications’) developed by the US Centers for Disease Control and Prevention3 and adapted by Kroese et al.4 into a practical system for individual laboratories to submit a data set for peer group evaluation. This ‘gene dossier’ consists of a number of headings designed to define the test precisely in a clinical context and set out its characteristics referenced against the research literature and the results of an adequate series of in-house validation tests. This concise description of the test is generally sufficient for a small expert panel to make a judgement on whether a test should be recommended to the specialist genetic commissioners who meet as the Genetics Commissioning Advisory Group (GenCAG)5 for adoption on the UKGTN menu of tests (now over 400 in number). © Rob Elles and Ian Frayling. Volume compilation © RCOG

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International frameworks The UK experience has helped lead and influence international guidelines in genetic testing. NHS practice is largely compliant with international recommendations such as those adopted by the Organisation for Economic Co-operation and Development in 2007 for quality assurance in molecular genetic testing.6 The OECD guidelines make some important framework statements that are increasingly relevant as some UK-based private companies have become active in the ‘lifestyle genetics’ and ‘nutrigenomics’ areas and internet-ordered direct-to-consumer tests have become available. An example is a test for early fetal sexing from a maternal blood sample.7 The OECD recommendations relevant to the evaluation of new genetic tests and the context in which they are offered for service include:6 n ‘Molecular genetic testing should be delivered within the framework of health care.’ n ‘Pre and post test counselling should be available. It should be proportionate and appropriate to the characteristics of the test, the test limitations, the potential for harm, and the relevance of test results to individuals and their relatives.’ n ‘Laboratories should make available to service users current evidence concerning the clinical validity and utility of the tests they offer.’

The ACCE framework translated to a gene dossier Test definition The UKGTN gene dossier asks the service provider to define their proposed test in the context of the disease and associated gene(s) targeted, the mutation spectrum detectable by the set of assays used, the patient group targeted and, most importantly, those clinicians from whom referrals are acceptable. This may be defined by a set of symptoms (for example, the analysis of the FMR gene in the context of women with primary ovarian insufficiency (previously known as premature ovarian failure) or its application in boys with learning difficulty to exclude or establish a diagnosis of fragile X syndrome) or a risk (for example, the prior risk of carrying a mutation in a breast cancer predisposition gene calculated from the family history of the index case). Analytical validity The analytical validity of the technical assay (which is usually formulated by the service proposers) is documented by in-house trials. A trial programme is specifically designed to calculate the analytical sensitivity and specificity of the assay; in other words, its ability to accurately detect genetic variants in the sequence targeted. Assays may be complex, for example in a condition exhibiting genetic heterogeneity, a scan for genetic variants may employ a cascade strategy examining a series of genes, any one of which may be responsible for the condition. In this case, the overall yield of genetic variants detected (compared with reference sequences) is the measure required. This is established by using the assay in a baseline set of index cases (where the phenotype is ascertained by independent methods. An example might be congenital adrenal hyperplasia, where the presence of disease is defined by biochemical parameters. A second example would be a panel of patients in full compliance with formal diagnostic criteria (for example, in adult polycystic kidney disease the presence of bilateral cysts with more than one cyst in one kidney). Alternatively, the variant assay is referenced against a genotype established by statistically significant genetic linkage score using an independent set of markers.

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Clinical validity Establishing the clinical validity of the test requires a judgement of the strength of the evidence that a variant or set of variants in the gene(s) assayed are associated with the condition. These data are usually documented from research literature and include a discussion of the penetrance of the variants described (that is, the likelihood that a variant will manifest as a phenotype). The data are often incomplete as the research effort is unlikely to have surveilled all possible variants in a gene for a rare condition. The in-house validation trial may supplement this evidence and include a consideration of the data drawn from the local and clinically ascertained population (as opposed to patients ascertained for research purposes). As such, it is often a more accurate (and frequently lower than published) estimate of both the clinical sensitivity and the specificity of the test and its positive and negative predictive value. Clinical utility Clinical utility is the measure on which the evaluation of a test by UKGTN may stand or fall. Laboratories are asked to indicate the endpoint of the test in terms of its power to: n exclude or establish a diagnosis where the test indication is a sign or symptom; the ability of a test to provide a definitive diagnosis and end a diagnostic odyssey for a family is considered to be a significant utility n modify a carrier risk in an asymptomatic individual and in an autosomal recessive condition such as cystic fibrosis to open reproductive decision-making options n predict the onset of a condition and to improve prognostic information and the management and/or treatment of individuals. The gene dossier includes a discussion of the economic costs and benefits of the proposed test and laboratories are asked to use a standardised formula to calculate the cost of the test, including staff and materials costs and institutional overheads. In addition, the gene dossier asks for indicative details of savings to the NHS and to the time, effort and psychosocial costs to the patient and their family in terms of alternative investigations avoided by a genetic test outcome. The potential of a genetic test to end regular tumour surveillance under anaesthetic in children at risk of retinoblastoma is a good example of this type of argument. In contrast, laboratories are also asked to indicate alternative tests to a molecular genetic analysis that may be a cheaper, effective and more straightforward alternative. Adult polycystic kidney disease is an example of a condition where a simple and cheap abdominal ultrasound scan has been adopted in preference to the complex, difficult and expensive molecular genetic test for this condition. Ethical legal and social issues The gene dossier asks for any indication of significant ethical, legal and social issues associated with the test. Genetic testing within the UKGTN lies within the governance framework of the NHS and pre- and post-test counselling is generally considered to be accessible, adequate and appropriate to the clinical situation. Examples of infrequent requests, for example for prenatal diagnosis for variants indicative of adult-onset conditions such as familial cancers, or sensory conditions such as inherited forms of adult-onset blindness, are considered within the context of this counselling framework.

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Gatekeeping Service proposers are asked to outline their experience of the test in the validation phase and indicate the strength of connection to research and local clinical expertise. In addition, they are asked to indicate how gatekeeping is to be organised in terms of the categories of specialist clinicians from whom referrals would normally be accepted and, if necessary, how potentially inappropriate referrals are to be managed. Information for commissioning The gene dossier provides information on the potential national impact of the proposed test by detailing the incidence and prevalence of the condition, as well as any downstream costs that the NHS will incur. An example of this would be the cost of implantable defibrillators in at-risk family members ascertained by predictive genetic testing, consequent upon finding a familial mutation causing an inherited form of cardiac dysrhythmia. Service proposers are asked to estimate any backlog of cases, the national steady-state demand and what proportion of this caseload they will service on an annual basis. This informs local commissioners of the expected yearon-year funding level for UKGTN services in their region. In turn, commissioners can be satisfied that test requests will be made by clinicians, and likewise accepted by laboratories consistent with the referral criteria specified in the gene dossiers. The annual audit of activity carried out by UKGTN also enables the demand for and availability of testing across the UK to be assessed. Auditing effectiveness UKGTN’s aim is to provide equity of access to genetic tests and to ensure that they are performed to service quality standards. It audits reporting turnaround times against the targets specified in the genetics White Paper of 20038 and has begun to measure the uptake and estimate the costs of new tests evaluated and approved for commissioning by GenCAG. In addition, it has returned to consider long-established tests ‘grandfathered’ onto the UKGTN list when first assembled. Examples include tests for cystic fibrosis and fragile X syndrome. As a result, the normal clinical criteria for requesting these tests have been defined. Are the UKGTN systems future-proof? The UKGTN gene dossier-based system of genetic test evaluation has served providers, commissioners and end users of tests (clinicians and their patients) adequately over the past 5 years. The number of tests available through UKGTN has increased each year. This is despite complaints from service providers concerning the apparent gap in the National Institute for Health Research remit, which does not extend to genetic test evaluation studies. Resources to translate research findings to clinical services are limited and patient advocates, frustrated by the pace of translation, have frequently stepped in to support the assay design, test development and validation studies required to present a gene dossier for consideration for NHS commissioning. Despite these limitations, the UKGTN system has proved to be a practical solution in which individual service laboratories can present a limited data set for evaluation. The system works best where the arguments for utility are clear and qualitative arguments can be advanced based on the obvious effects of high-penetrance variants on phenotype. As more genes causing rare conditions are identified, this system will continue to be used

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and to be effective. The services considered by UKGTN to date are those in which the impacts on individuals and their families are explicit in decision making or influencing clinical management (even if treatment improvements are rarely demonstrable). The case for clinical utility will change as previously intractable genetic conditions become open to clinical trials, often using safe and well-characterised drugs (for example, prescription of losartan to moderate the effects of Marfan syndrome9). However, the gene dossier was designed to evaluate tests for rare single-gene disorders. It has become increasingly obvious that this framework will need to be adapted and evaluation mechanisms strengthened as genetic testing moves towards mainstream medicine. Genetic tests for common complex disorders have been proposed. These envisage tests for genetic variants representing low-penetrance biomarkers that raise the risk of late-onset conditions such as type  2 diabetes, coronary disease and age-related macular degeneration. The genetic test result may interact with less defined but important genetic factors (family history) and environmental factors (body mass index, diet, smoking, etc). Evaluation of this type of test will be essential to avoid their adoption in an ad hoc way and a consequent waste of resources, risking tests being discredited as expensive and ineffective as tools in NHS clinical care. Other major challenges to the evaluation system include the potential impact of the next-generation technologies that allow a parallel scan for sequence and copy number variants in many genes and chromosomal regions relevant to a phenotype or disease pathway. In addition, technologies allowing non-invasive prenatal diagnosis to screen for common aneuploidies may require as careful a consideration of their wide social impact and impact on the NHS as a service delivery system as their technical features if they are to be accepted by the public.

What changes or new systems should be considered? The ACCE framework is a useful starting point and Burke and Zimmern10 have used it in considering the issue of evaluating genetic testing for common complex disorders. They emphasise the difference between the technical ‘assay’ for one or more genetic variants and the ‘test’ that is designed to identify a set of genotypes for a particular condition in a defined clinical population and, importantly, for a specific purpose. They propose expanded dimensions of the ACCE headings, in particular considering the clinical validity of the test and distinguishing between closed assays (for a specific set of variants) and open assays (for any variant associated with a phenotypic indicator). Burke and Zimmern disaggregate the concept of clinical utility into a consideration of the defined purpose of the test. They differentiate between high- and low-penetrance variants contributing to a disease phenotype or risk. They consider measures of health quality including legitimacy (for example, questioning whether a prediction contributes to individual wellbeing in the absence of treatment) and how utility is considered and regulated differently when preimplantation genetic diagnosis or prenatal diagnosis are the proposed applications of the genetic test. Genetic test proposals with significant public health impact and implications for NHS policy, resources and service configuration require commensurately more and better-quality data. In-depth evaluation will involve multidisciplinary expertise (epidemiologists, health economists and public engagement) and policy decisions involving commissioners and service providers at all levels of NHS care. The Public Health Genetics Foundation workshop report and National Institute for Health Research-funded RAPID study to facilitate the translation into service of non-invasive

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prenatal diagnosis using cell-free fetal DNA in maternal circulation is an example of such a multidisciplinary evaluation study. More generally, Burke and Zimmern10 propose prioritising genetic test evaluations according to their likely scale of impact and removing the responsibility for data generation from commercial interests and service providers that inevitably have a vested interest in the outcome. These issues were considered more broadly at a diagnostic summit organised by the Royal College of Pathologists in January 2008, which recommended that a new body (that has been called a NICE [National Institute for Health and Clinical Excellence] for diagnostic tests) be established for laboratory-based diagnostic tests taking responsibility for evaluation and making data on the validity and utility of all tests, new or historical, publicly available. The evaluation of new genetic tests cannot be separated from the enabling technologies that will allow them to be realised. Next-generation genomic technologies (such as microarrays and parallel DNA sequencing) will require full health technology and economic assessment. These studies should pool the valid requirements of the genetic service network, laboratory medicine and mainstream medical disciplines. They should consider the rising numbers of clinically useful tests against falling unit costs. They will recognise that the implementation of new technology will incur an increased global cost for genomic diagnostics and ask whether this spending will be offset by the health benefits to patients and savings in diagnosis and treatment elsewhere in the NHS.

References 1. 2. 3. 4.

5.

6. 7. 8.

9. 10.

Donnai D, Elles R. Integrated regional genetic services: current and future provision. BMJ 2001;322:1048–52. UK Genetic Testing Network [www.ukgtn.nhs.uk]. US Centers for Disease Control and Prevention. Genomic Translation: ACCE Model Process for Evaluating Genetic Tests [www.cdc.gov/genomics/gtesting/ACCE/index.htm]. Kroese M, Zimmern RL, Farndon P, Stewart F, Whittaker J. How can genetic tests be evaluated for clinical use? Experience of the UK Genetic Testing Network. Eur J Hum Genet 2007;15:917–21. Department of Health. Genetics Commissioning Advisory Group (GenCAG) [www. dh.gov.uk/en/Publichealth/Scientificdevelopmentgeneticsandbioethics/Genetics/ Geneticsgeneralinformation/DH_4117687]. Organisation for Economic Co-operation and Development. OECD Guidelines for Quality Assurance in Molecular Genetic Testing [www.oecd.org/dataoecd/43/6/38839788.pdf]. Baby Gender Mentor [babygendermentor.com/information.php?information_id=2]. Department of Health. Our Inheritance, Our Future: Realising The Potential of Genetics in the NHS. London: Department of Health; 2003 [www.dh.gov.uk/en/Publicationsandstatistics/Publications/ PublicationsPolicyAndGuidance/DH_4006538]. Marfan syndrome trial example [clinicaltrials.gov/ct2/show/NCT00593710]. Burke W, Zimmern R. Moving Beyond ACCE: an Expanded Framework for Genetic Test Evaluation. UKGTN; 2007 [www.phgfoundation.org/pages/work7.htm#acce].

15 Chapter 15

New advances in prenatal genetic testing: the parent perspective Jane Fisher

Introduction Antenatal Results and Choices (ARC), which was established in 1988, is the only UK charity providing non-directive information and specialised support to parents before, during and after antenatal testing and when an abnormality is diagnosed in an unborn baby. Continuing support is offered for as long as required, whatever decision is made about the future of the pregnancy. ARC also runs a well-established training programme for healthcare professionals in communication skills, breaking bad news and supporting parent decision making, all in the context of antenatal testing For more than 20 years, ARC’s core support service has been its national helpline. We take calls from across the UK on all aspects of antenatal testing and its aftermath. This chapter will use ARC’s extensive experience with expectant parents to explore the potential implications for them of new techniques in prenatal genetic testing, particularly advances in non-invasive prenatal diagnosis (NIPD). Throughout the chapter I will refer to women and their partners as parents and the fetus as a baby, as this is how the vast majority of callers to ARC conceptualise themselves and their fetus from the earliest stages of the pregnancy.

ARC’s contact with parents making decisions about invasive diagnostic tests By far the most common call on the ARC helpline is from parents after a screening result or ultrasound finding, when they are deciding whether to have a diagnostic procedure, either chorionic villus sampling (CVS) or amniocentesis. Approximately half of all helpline contacts by phone or email are of this kind. Most hospitals quote a procedure-associated miscarriage risk of 1% and so a large number of contacts are from parents who have been given a Down syndrome screening result that means they have the chance of having an affected pregnancy of between one in 100 and one in 250. In other words, they are told that they have a higher than average risk of their baby having Down syndrome and then offered a procedure that carries a higher or at least comparable risk of miscarriage. This can make the decision-making process particularly onerous. © Jane Fisher. Volume compilation © RCOG

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ARC’s specialised helpline team helps parents work out how to proceed in a way that is right for them in their individual circumstances. We have no agenda other than to enable them to make the decision they can best live with. It is often a very distressing process for parents as they weigh up the importance to them of having a confirmed diagnosis (or gaining reassurance) against the small but significant chance of losing a wanted pregnancy as a result of the procedure. We are fortunate at ARC to be able to spend as long as individual parents need to explore potential outcomes. Different parents bring different factors to their decision making, including how they feel about the condition being tested for and whether they would consider termination of pregnancy if their baby were affected. For many, a major concern is whether they will be able to manage their anxieties for what is usually over half the pregnancy without having a definitive answer. Conversations will almost always include discussion of how the procedure is performed and what, if anything, can minimise the risk of miscarriage. While some will consider CVS because it can provide an earlier diagnosis, others perceive it as a riskier procedure and prefer to wait to undergo an amniocentesis. Many parents are desperate to know whether there is a risk-free alternative. Some want to know what more information can be gained from ultrasound scans, and the media coverage of research into NIPD using cell-free fetal DNA (cffDNA) has led some to ask us whether they can access the test privately or as part of a clinical trial.

Implications for parents of the future implementation of NIPD From our experience, we can confidently predict that a risk-free genetic diagnostic test will be welcomed by parents; with the proviso that the test is robust and reliable. There will be a wait of some years before the sensitivity and specificity of cffDNA testing or competing non-invasive techniques are such that it can be routinely used in practice as a diagnostic tool for aneuploidy but research is currently making strides, with the added spur of huge potential demand. The fact that NIPD is likely to be offered well within the first trimester will also be welcome to many parents as potentially providing earlier reassurance. However, we must avoid making assumptions that an earlier diagnosis will necessarily be easier for parents to cope with or that decisions about the future of the pregnancy will be any less emotionally painful. The diagnosis of a genetic condition in their baby will have a significant emotional impact on parents whatever the gestation. A choice sounded better than not having a choice. Yet, the idea of choosing was overwhelming and confusing. We were devastated at the thought of either course of action. It wasn’t until much later that I realised the real tragedy was the loss of the healthy child and that I had not had a choice about. The loss had to be accepted and mourned; and before that could take place we had to make an irreversible decision.1

Making difficult decisions about the future of what is most often a wanted pregnancy is distressing regardless of gestation. Furthermore, there is no evidence that earlier terminations for fetal abnormality have substantially less emotional impact on women and couples than those carried out later in the pregnancy.2 I went in there twelve and a half weeks pregnant, and woke after a brief sleep, no longer carrying a baby. I don’t know what I dreamed of while asleep, but I woke crying silently. I know it must have been so much easier than a later induced termination – but the loss is less tangible. We never even saw our baby, except kicking around on that fateful scan.3

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In England and Wales, approximately 400 000 women undergo Down syndrome screening annually and around 25 000 diagnostic procedures are carried out.4 Much of this screening is currently done in the second trimester. In 2006, 1132 prenatal diagnoses of Down syndrome were made. In approximately 92% of cases, parents decided to terminate.5 If a comparable number of women who currently have screening should opt for first-trimester non-invasive diagnostic testing for Down syndrome and the proportion ending an affected pregnancy remains consistent, we can predict that there will be a rise in the diagnoses of affected pregnancies, as many cases will be diagnosed that would have otherwise been lost spontaneously before the second trimester. Indeed, there may even be a higher uptake, as the procedure itself does not put the pregnancy at risk. This will have implications for the provision of first-trimester termination of pregnancy services. At present within the NHS, women who decide to have a termination after a diagnosis of fetal abnormality before 13  weeks of gestation are offered a surgical procedure to end the pregnancy and medical induction for diagnoses after 13 weeks. If, as expected, the roll-out of NIPD gives rise to an increase in early diagnoses of aneuploidy, this in turn could result in more surgical procedures. It is likely that most will take place on gynaecological wards or be provided by independent service providers such as the British Pregnancy Advisory Service or Marie Stopes International. This will require staff training in providing individualised care for women having terminations for abnormality as within these settings most women are ending unwanted pregnancies for nonmedical reasons. Even if it remains the case that most women choose to have a termination, it will also be essential that carefully coordinated multidisciplinary care pathways are in place for women who want to continue after a diagnosis of aneuploidy.

NIPD and the challenge of informed consent Even though there has been a national programme in place for over 5 years, there is evidence that pre-Down syndrome screening discussions are limited and that many women perceive the tests as routine rather than requiring them to make an informed choice to opt in.6 I wasn’t aware I could have a choice not to have the test. I was just invited and turned up. I was invited by letter with an appointment saying please come for test. Nothing on [the] letter said it was optional. If I’d known I’d have still had the test but they should make it clear it’s optional. Other people would choose not to. I needed to know everything was okay.*

With the current roll-out throughout England of the combined Down syndrome screening programme and haemoglobinapathy screening, which are both offered in the first trimester, there is pressure on women to book ever earlier and pressure on staff to accommodate both early booking and time for screening discussions. If diagnostic genetic tests are to be offered earlier, there will need to be careful consideration of how best to ensure women have time to explore the implications of taking up the offer. It will no longer be a two-stage process with the opportunity for parents to reflect on whether they want diagnostic procedures after the screening stage and to discuss this in consultation with their healthcare professionals.

* Quote taken from a questionnaire distributed for ARC’s evaluation of combined screening for Down syndrome in four units in South West London, March 2009 (qualitative data Q11–07).

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A further concern from the parental perspective is the form NIPD testing takes, being a simple blood test. Women are very accustomed to having blood taken in pregnancy and, while holding out an arm for a needle to be inserted is not always pleasant, it is something with which every pregnant woman is familiar. The ‘routine’ nature of the procedure could mean that some women embark on it without considering the possible outcomes and so will be particularly distressed if test results bring unexpected news about the pregnancy. Healthcare professionals may also perceive it as being more ‘routine’, which may impair pre-test counselling.7 As is currently the case with antenatal screening programmes, by far the majority of women who will undergo future NIPD testing will be reassured by a negative result. It is a continuing challenge to maintain a careful balance in order that informed consent is achieved without raising anxieties in all pregnant women to unmanageable levels.

Lessons to be learned from antenatal ultrasound scanning There is a precedent for a non-invasive diagnostic tool in routine use in antenatal care, namely ultrasound scanning. Every time an ultrasound probe is placed on the abdomen of a pregnant woman, there is the possibility that an abnormality will be detected. Although information provision to women about the purpose of antenatal ultrasound is improving, we cannot overestimate the profound impact when the scan shows that there may be something wrong with an unborn baby. However wellinformed a woman may be, such news will always come as a shock and will generate considerable anxiety and distress. This will also be the case for a diagnosis made from cffDNA testing, even if it comes earlier in pregnancy. Just as we do now in the context of ultrasound, ARC would advocate the implementation of planned care pathways in the instance of the diagnosis of an affected pregnancy. This will help ensure that parents have the information and support they need to decide how to proceed.

New techniques in preimplantation genetic screening and diagnosis Parents undergoing in vitro fertilisation (IVF) treatment are desperate for a successful outcome. It is more than 30 years since the birth of Louise Brown and great strides have been made: in 2006 the success rate per treatment rose to 23%, with a total of 12 956 babies born.8 However, the process remains physically and emotionally demanding for women. Therefore tests that can be carried out before implantation with the goal of improving success rates will often be attractive to parents undergoing IVF. They will also often be particularly anxious to avoid invasive testing because of its associated miscarriage risk. Many clinics offer preimplantation genetic screening (PGS) of embryos for chromosomal abnormalities and media interest in these techniques is high. In one of the most recent advances, a fertility clinic in Nottingham announced their first pregnancy after using comparative genomic hybridisation on an unfertilised egg, which was covered on BBC News.9 At the same time, it is worth noting that the British Fertility Society (BFS) updated its guidelines on PGS in July 2008, stating the following:10 It remains possible that PGS may be of benefit under certain circumstances, but the BFS currently recommends that PGS should therefore preferably be offered within the context of well-designed randomised trials performed in suitably experienced centres. Patients should be made aware that there is no robust evidence that PGS for advanced maternal age improves live birth rate; indeed from the evidence currently available the live birth rate may be significantly reduced following PGS.

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Here is an example of how the excitement and hype surrounding a new technology can mask its actual success in practice. Another topic that regularly attracts media interest is preimplantation genetic diagnosis (PGD). The reality is that very small numbers of parents undergo PGD. The latest figures from the Human Fertilisation and Embryology Authority state that there were 183 cycles that included PGD in 2006 (0.41% of all IVF cycles). These cycles led to 39 live births and a total of 46 babies (0.38% of the total of IVF-conceived babies).11 PGD is no easy option and is part of an IVF process that is still more likely to fail than not. It is undertaken by a small number of parents for whom it represents at least a chance of having a baby without a debilitating medical condition.

Ethical concerns: ‘eugenics’ and ‘designer babies’ Media interest in advances in prenatal genetic testing is high and news stories almost always spark debates about ethical issues. There are those who feel strongly that there is a state-endorsed eugenic purpose behind any form of prenatal test that leads to the offer of terminating an affected pregnancy.12 Media coverage has included claims that clinicians put pressure on parents to terminate affected pregnancies,13 which adds further fuel to the eugenic argument. Our two decades of experience of parental and professional attitudes to prenatal genetic diagnosis bears out a different reality. Parents never take ending a pregnancy lightly after a diagnosis; it is an agonising process as they work out whether to end their wanted unborn baby’s life before it has begun. They are very conscious of the ethical dimension; a major contributor to the pain of the process is their attempt to predict which decision they will best be able to live with. Healthcare professionals caring for the parents are most often extremely wary of appearing directive, which can mean parents feel very alone, struggling with one of the most harrowing decisions they are ever likely to face. Other familiar phrases that pepper media articles about prenatal testing are ‘designer babies’ and parents’ ‘pursuit of perfection’. Certain commentators accuse parents of terminating pregnancies for ‘minor’ or rectifiable conditions14 or warn that wider use of PGD will lead to parents seeking ‘the perfect baby’.15 All such sensationalist coverage is offensive to the parents who take their responsibilities very seriously and want nothing more than to deliver a healthy baby.

Private services As outlined previously, we at ARC can confidently predict that most parents will welcome the introduction of early non-invasive prenatal genetic diagnosis. Indeed, many are impatient for its arrival in practice and we must be careful not to raise unrealistic expectations as to how soon it might be widely available within the NHS. This impatience will lead some parents to the internet to access private provision from other parts of the world. This will be impossible to prohibit and it will therefore be important to provide accessible information to help parents to assess the quality of such services and to consider the possible consequences of this route.

Conclusion From ARC’s perspective, advances in NIPD and other forms of genetic testing that can offer a safe way to obtain diagnostic information will be of significant benefit to

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parents and families. For some parents who carry genetic conditions, the availability of such tests may enable them to start or complete their families, which they may consider to be an untenable prospect without such provision. We would want to see new tests implemented into practice in a considered and entirely equitable way, with the excitement surrounding such new developments not blinding us to their consequences. High-quality pre-test information and, perhaps even more importantly, the opportunity to discuss testing with well-informed healthcare professionals are both crucial. ARC would also strongly advocate an emphasis on ensuring that individualised care and support pathways are firmly in place for the parents for whom test results bring difficult news about their pregnancy.

References 1.

2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15.

Support after Termination for Abnormality (SATFA). SATFA News May 1991. [The woman quoted had a termination after the diagnosis of a chromosomal abnormality. SATFA was ARC’s original name. A newsletter now called ARC News is sent out to ARC members three times a year.] Statham H. Prenatal diagnosis of fetal abnormality: the decision to terminate the pregnancy and the psychological consequences. Fetal Matern Med Rev 2002;13:213–47. Healthtalkonline [www.healthtalkonline.org/Pregnancy_children/ Ending_a_pregnancy_for_fetal_abnormality/Topic/2001]. Association for Clinical Cytogenetics. UK National Audit Data 2006/7 [www.cytogenetics.org.uk]. Wolfson Institute of Preventive Medicine. The National Down Syndrome Cytogenetic Register Annual Report 2006. [www.wolfson.qmul.ac.uk/ndscr/reports/NDSCRreport06.pdf]. Marteau TM, Dormandy E. Facilitating informed choice in prenatal testing: how well are we doing? Am J Med Genet 2001;106:185–90. van den Heuvel A, Chitty L, Dormandy E, Newson A, Deans Z, Attwood S, et al. Will the introduction of non-invasive prenatal diagnostic testing erode informed choices? An experimental study of health care professionals. Patient Educ Couns 2009 [Epub ahead of print]. Human Fertilisation and Embryology Authority. Latest UK IVF Figures – 2006 [www.hfea.gov. uk/1269.html#1276]. Walsh F. IVF pregnancy from screened egg. BBC News website 26 January 2009 [news.bbc. co.uk/1/hi/health/7852292.stm]. Anderson RA, Pickering S. The current status of preimplantation genetic screening: British Fertility Society Policy and Practice Guidelines. Hum Fertil (Camb) 2008;11:71–5. Human Fertilisation and Embryology Authority. Pre-implantation genetic diagnosis (PGD) [www. hfea.gov.uk/69.html]. Lawson D. Shame on the doctors prejudiced against Down Syndrome. The Independent 25 November 2008. Salkeld L. Doctors told us to abort our disabled baby – but our son is proof that we were right to say no. Daily Mail 28 December 2007. Templeton SK. Babies aborted for minor disabilities. The Sunday Times 21 October 2007. Outcry as clinic offers ‘designer baby’ embryo screening for 200 diseases. London Evening Standard 13 November 2006.

16 Chapter 16

Informed consent: what should we be doing? Jenny Hewison and Louise Bryant

Introduction: consent, choices and decisions The concept of informed consent is central to the delivery of ethical healthcare services. Informed consent starts from the assumption that, following the provision of appropriate information, and based on his or her expertise, a clinician recommends a course of action to which the individual is asked to consent. Informing patients about the potential risks and consequences of a medical procedure is now considered essential to good practice and psychological preparation for such procedures has been shown to be linked to improved patient outcomes.1 Within reproductive genetic services, however, the term informed choice has come to be preferred over informed consent. Choosing to have prenatal screening, for example for Down syndrome, is not directly equivalent to consenting to a surgical procedure or even screening for conditions such as breast cancer, although many of the issues are related. In contrast to informed consent, an informed choice starts from the assumption that, once appropriate information has been provided, the course of action should be chosen by the patient rather than the clinician. In most cases, the only therapeutic intervention on offer following a diagnosis of abnormality via prenatal testing is termination of pregnancy. For this reason, most clinicians accept that prenatal testing choices should reflect the values of the individual woman and her partner. Testing policy and related guidelines reflect this viewpoint and require that information about the testing process should support individual choice by being balanced and nondirective.2,3 The emphasis on informed choice rather than consent reflects, among other things, the desire to distance prenatal testing programmes from unwanted eugenic associations.4 While informed choice and informed decision are often used interchangeably, a choice tends to refer to the outcome whereas a decision reflects the process of choosing between alternatives.5 As we shall discuss later, this is an important distinction when considering how to evaluate prenatal screening programmes. For these reasons, the term ‘informed decision’ is preferred. There are a number of definitions of informed decision making but one by Briss et al.6 works well within the prenatal screening context: An informed decision occurs when an individual understands the nature of the disease or condition being addressed; understands the clinical service © Jenny Hewison and Louise Bryant. Volume compilation © RCOG

206  |  JENNY HEWISON AND LOUISE BRYANT and its likely consequences, including risks, limitations, benefits, alternatives, and uncertainties; has considered his or her preferences as appropriate; has participated in decision making at a personally desirable level; and makes a decision consistent with his or her preferences and values.

This chapter will consider how healthcare services can support informed decision making within prenatal screening programmes. It then will then go on to discuss potential approaches to evaluating screening programmes that use ‘informed choice’ rather than test uptake as the outcome measure and discuss how these approaches cannot yet be considered satisfactory. Alternative ways that prenatal screening programmes could be evaluated will be suggested.

Practicalities: facilitating informed decisions To facilitate informed decision making in practice, both the constituent parts of this somewhat well-worn phrase need to be addressed. Information In the prenatal testing context, it is known that women and their partners welcome up-to-date, timely, accurate information delivered in a format that is understandable. For a screening decision to be informed, the following are considered essential areas of knowledge:7–9 n the purpose of the test n what the testing procedure involves n any risks associated with the test n the implications of the possible test results n the main features of the target condition(s). Therefore, as a minimum, information on these topics should be made available to pregnant women. In addition, a recent collaboration of members of the public, people with personal experience of genetic conditions, clinicians and information providers has suggested extensions to this list:10 n more information about the condition being tested for, including an explanation of the background and effects of the condition and any treatment and management options n an explanation of risk in simple terminology n the accuracy of test results n what happens after the test and potential follow-up tests n who will have access to test results, especially in relation to genetic information n an indication that decision making can be shared with clinicians or family. Information about the tested-for condition has often had a lower priority than information about the testing process and any such information provided has tended to have a medical or clinical focus. However, parents want to know what parenting a child with the testedfor conditions may be like and the need for balanced information on potential quality of life for a child with the tested-for condition has been found to be important.11 In reality, resource constraints make it unrealistic for healthcare professionals to provide in person all the information that a woman and her partner may need and

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those delivering prenatal screening may also not be best placed to provide information about the conditions being tested for. Language barriers may also reduce the ability of healthcare professionals to convey adequate information to support an informed decision. The UK National Screening Committee (NSC) prenatal screening leaflet for Down syndrome (now subsumed into the booklet Screening Tests for You and Your Baby) has been translated into 17 languages and is available through the NSC’s Screening Portal website.12 Healthtalkonline,13 the website of UK-based charity DIPEx, is supported by the NSC and provides information on prenatal screening, including carrier screening for sickle cell disease and thalassaemia. The modules contain interviews with women and couples who have undergone prenatal screening, diagnostic testing and termination. Website users can watch, listen to or read these interviews, some of which are in other languages (French, Mirpuri, Portuguese, Sylheti, Urdu). The AnSWeR website14 (Antenatal Screening Web Resource) was funded by the Wellcome Trust and provides information about the lives of people with cystic fibrosis, Down syndrome, neural tube defects, Klinefelter syndrome and Turner syndrome. It contains interviews with affected individuals and family members. Website users can listen to or read these interviews. Decision making There may be an assumption that if good-quality pre-test information is provided, an informed decision has been facilitated. While it is true that the provision of goodquality information is essential to support informed decisions, on its own this is insufficient.15 To meet the definition given earlier, the decision maker needs to be sufficiently engaged with the process so that their values and preferences can inform their screening choice. The evidence suggests that simple information giving does not guarantee this kind of engagement.16,17 In the situation where people are being asked to choose between different options using information containing unfamiliar terminology and complex ideas such as risk, the likelihood increases that their decisions will be influenced by the way the options themselves are presented rather than be the outcome of a deliberative decision process.18,19 For example, seemingly neutral interventions such as offering screening within an opt-out rather than an opt-in model or offering the test ‘today’ rather than at a future appointment are likely to increase screening uptake.20,21 This is because making difficult decisions is effortful and can produce emotional conflict. People thus prefer to make decisions using cognitive short cuts or heuristics: making one option ‘easier’ than another may override value-led decision making. Research into the psychology of decision making provides evidence that even presenting decisions as ‘choices’ can enhance the perceived value of whatever is on offer because choice is inherently attractive to humans.22 Therefore, it can be concluded that the way in which the offer of screening is presented, as well as the information people are given, can have a significant impact on the outcome in a way that may not always relate to a person’s underlying values about parenting a child with a disability, for example. Three simple strategies can be employed in any situation where the patient is involved in making decisions about their health care, including decisions about prenatal screening, to reduce these framing or biasing effects and to increase the likelihood that a more ‘thoughtful’ and value-consistent decision is made:22 n Change the language with which screening decisions are presented. Healthcare professionals should use language that emphasises decisions and potential consequences rather than choices and options. Instead of ‘offering’

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the choice of having screening, be explicit that there is a decision to be made between not having screening and having screening. n Slow down the decision-making process. Heuristics are more likely to come into play in situations where the time for making a decision is constrained, or there appears to be pressure to make a quick decision.23 This means slowing down the decision-making process wherever appropriate. One way of doing this would be to state, where gestation permits, that the screening choice does not have to be made immediately. Ensuring that women are not only given the NHS screening information booklet but are shown the appropriate pages is important so that they can take time to read and discuss the options with their partner and family at home before reaching a decision. n Make explicit the need for women to ‘think actively’. Many women are unfamiliar with having to take responsibility for healthcare decisions, particularly in the antenatal setting.24 It is useful, therefore, to reinforce written information with verbal confirmation that there is a decision to be made with potential consequences, and to raise the idea that different consequences have different values for different people. One way to do this is to suggest that women write down the pros and cons of each option to help them see which option is the best decision for them and their family. Making decisions in this way is effortful, can be emotionally uncomfortable and may not always lead to greater satisfaction with the encounter, particularly if the woman has a preference for ‘physician-led’ decisions. However, if the goal of screening policy is to increase informed choice, rather than ease of choice, this is a trade-off that may have to be accepted. In other areas of health care, use of strategies such as these has been shown to lead to more informed decisions because they promote attention to relevant information, help reduce decisional conflict and increase value-consistent decisions.25

Evaluating the performance of prenatal screening and testing programmes The emphasis on increasing informed decisions rather than promoting test uptake brings an additional challenge to the evaluation of fetal anomaly screening programmes. Outcome measurement is a cornerstone of evidence-based practice because of its role in establishing the effectiveness of different kinds of care. In addition to providing the best evidence of an intervention’s effectiveness, outcome measures have the distinct advantage that they allow a focus on the service user rather than the service provider. All other things being equal, therefore, it would be desirable for the various strands of NSC activity to be measured in the same way; that is, tables or graphs showing the proportion of eligible individuals having a screening test in any one year, and how that proportion changes over time. An evaluation of a screening programme aimed at reducing deaths from breast cancer, for example, would in the long term need to examine outcome data to see whether a fall in mortality had occurred. In the short term, however, an acceptable proxy outcome may be figures demonstrating an increasing proportion of eligible women attending for mammograms. Since it can be assumed that avoiding death from cancer is universally desirable, it has become acceptable shorthand to assume that having a cancer screening test is the ‘right’ behaviour, provided of course that test performance has been formally examined and

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judged to be of an adequate standard. However, as we have already identified, it is axiomatic in prenatal testing that there is no such thing as the ‘right’ behaviour. How then is the ‘right’ outcome for a particular woman to be determined in relation to prenatal screening? Can a measure be found that acts as a proxy outcome for effectiveness purposes? In a response to this, Marteau and colleagues26 developed the Multi-dimensional Measure of Informed Choice (MMIC), its aim being to allow informed choice to be treated as an alternative to test uptake in terms of a patientrelated outcome. The MMIC is based on the premise is that it is possible to work out what an individual woman ‘should’ do, using a psychological theory called the Theory of Planned Behaviour.27 This theory, which is widely used in health psychology, aims to understand, predict and explain behaviour, particularly in terms of the relationship between a person’s attitudes towards engaging in a behaviour (for example, having a screening test) and actually engaging in that behaviour. According to the MMIC, a person’s test choice can be considered informed (a) if their test-related knowledge is good (dimension 1), they hold a favourable attitude towards undergoing the test (dimension 2) and then undergo that test (dimension 3); or (b) if their knowledge is good, they hold an unfavourable attitude towards undergoing the test and then do not undergo that test. Any other combination, that is, low knowledge and/or an inconsistent attitude–behaviour relationship, would constitute an uninformed choice. This three-dimensional definition of informed choice is usually represented diagrammatically as a 2 × 2 × 2 cube.26 In research studies using the MMIC, knowledge and attitudes have been captured using a self-completion questionnaire given to women following consultation with their healthcare provider; test behaviour is then captured via the patient notes. The developers of the measure have suggested that, by using the measure, screening services can be ‘classified in terms of the proportions of populations making informed choices’, in other words, informed choice can be used as an outcome instead of test uptake.28 However, while this is an important first step in the move away from the ‘high uptake equals success’ approach, the MMIC and similar measures developed within the same theoretical framework are limited in some important ways that are sufficient to make them inappropriate for measuring the outcomes of fetal anomaly screening programmes. Knowledge In the latest version of the MMIC, ten knowledge items are measured.29 Of these ten, five are concerned with understanding the screening risk result and two with assessing knowledge of Down syndrome (association with learning disability and lower life expectancy). The other three items assess whether the woman knows the conditions the test screens for, whether she knows the ‘possible risks’ associated with an invasive test and whether that woman would be offered a termination of pregnancy following a positive diagnosis. A measure based on the MMIC and developed by van den Berg and colleagues5,30 for research in the Netherlands uses a similar knowledge measure but has fewer risk-related items and does not make any reference to the potential outcome of the offer of termination. There are a number of potential difficulties with these knowledge measures as they stand. Altering the content of the items has significant implications for assessing whether or not a woman is considered informed or not. For example, numerical risk is known to be widely misunderstood, so scores will be higher if the measure includes fewer risk items – as in the van den Berg measure. If the scale contains a higher proportion of risk items – as in the MMIC – numbers of informed women

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will decrease. Although various instruments measuring test-related knowledge exist within the literature, there is no agreement about the knowledge items that are necessary and sufficient to support informed choice.31 For example, women who are intending to decline a test may pay less attention to, or retain less information about, the meaning of different risk cut-off figures (which are not salient for them), making them more likely to appear in the ‘less informed’ group. In fact, women who actively decline tests have been found to be more likely to have thought about the decisionrelevant issues than many women who accept an offer of testing.5 There are also problems with the way in which that level of knowledge is operationalised in this type of measure. Knowledge scores are dichotomised, enabling women to be classified as ‘informed’ and ‘not informed’. Different studies have used the sample median, the scale midpoint of five, or a scale midpoint figure corrected for guessing, but in each of these cases it is possible to be classified as informed while having major gaps in understanding. Although a median split identifies those who have an above-average level of knowledge, this is not equivalent to being informed if the average is low. Using a midpoint score of five, or even a guess-corrected score of seven, still allows for the possibility that women considered to be well informed may not understand some key information that most would consider essential in any meaningful definition of informed choice. It is argued, therefore, that while current measures of knowledge in relation to the measurement of informed choice may be of value in a research setting, they cannot yet be considered satisfactory in relation to assessing the outcome of screening programmes. Knowledge measures could be improved by further research to establish how best to capture the knowledge most important for informed test decisions, and by addressing the issues with measurement. Values and attitudes Central to the notion of informed choice, and to the definition on which the MMIC is based, are personal values. Personal values are psychological constructs that are fundamental to the belief systems of an individual. A person’s underlying values will influence their specific attitudes – their evaluations of a particular social object or act. For example, values about the relative worth of people with disabilities will be reflected in attitudes, say towards the rights to employment, housing and education of those people. Prenatal testing can give potential parents information about the health status of their baby – information that can, if they wish, be used to justify a termination of a pregnancy under current UK law (with the exception of Northern Ireland), in some cases up until full term. The values that underlie attitudes towards termination of pregnancy for fetal abnormality, or indeed continuation of such a pregnancy, can be subtle, differentiated and complex, and they are very difficult to measure. Because of this complexity, the developers of the MMIC have chosen to measure attitudes towards a specific action – that of undergoing a screening test, for example for Down syndrome. This attitude was selected, in line with the theoretical model on which it is based, on the assumption that, as attitudes reflect values, measurement of this specific attitude ‘will encompass one or more salient values’, for example a person’s perceived ability to parent a disabled child.26 The version of the measure used by van den Berg et al.30 is less specific and measures attitudes towards ‘testing for congenital defects during my pregnancy’. Depending on the version of the measure, around six items are used to measure attitude towards undergoing the screening test. The items ask women to use a rating scale to indicate whether ‘having the screening

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test’ will be ‘a bad thing’ or ‘not a bad thing’, ‘beneficial’ or ‘not beneficial’, for example. The median score is used by the MMIC developers as the cut-off between a favourable attitude and an unfavourable attitude, although van den Berg et al.5,30 split attitudes into three groups (favourable, neutral and unfavourable). However, a screening test is a means not an end. Different testing technologies (for example, serum screening and ultrasound) can produce the same risk information, while the same technology can produce information about different conditions (for example, risk figures for Down syndrome and neural tube defect). People may hold different attitudes towards undergoing different testing procedures despite the same risk information they provide, for example ultrasound scanning is viewed very favourably by most pregnant women and their families who often look forward to the opportunity of ‘seeing their baby’.32 Of course, one test may not be enough: another procedure may need to be offered, and, even then, definitive information may not be obtained. Attitudes towards undergoing screening do not necessarily reflect attitudes towards undergoing invasive procedures or termination of pregnancy, the latter being most closely linked with the underlying values and attitudes associated with parenting a disabled child.33 The question must therefore be asked: does an attitude towards undergoing a specific test – or to undergoing tests more generally – act as an adequate proxy for underlying values for parenting a child with Down syndrome? For these measures to work as planned, the answer must be a convincing ‘yes’. In reality, the picture is more complicated. First, it is of course necessary that a woman consents to a procedure (for example, a scan or a blood test). Second, it is accepted that choosing to have a screening test does not imply that a woman will choose to have other tests that may subsequently be offered to her: each test is recognised as a separate choice. The important question is whether or not a woman offered a test is aware of and understands the potential consequences – harms as well as benefits – of both of the options available to her, that is, not undergoing a test as well as undergoing a test, and understands the relevance of the information to herself and her personal values. Because screening tests pose no risk to the pregnancy, it could perhaps be argued that the decision to have one is not really a decision at all. Potential subsequent decisions can then be rationalised as ‘we’ll cross that bridge when we come to it’. If the screening procedure involves an ultrasound scan then it becomes even less likely that attitudes towards undergoing a particular procedure can ever be treated as a proxy for underlying values about reproductive choice and the purposes of prenatal testing. It is therefore argued that the very specific measurement of attitudes towards undergoing a particular type of screening cannot be said to be a satisfactory measure of the decision maker’s values. Behaviour and the predictive power of attitudes The third dimension within the MMIC and related measures is behavioural implementation: did the woman have the offered screening test or not? Information from the three dimensions, all in binary form, is combined to produce a new binary outcome classification: the choice was informed, or it was uninformed. As described earlier, there are two different ways in which women can be classified as having made an uninformed choice. In the first of these, the underlying argument is that people whose knowledge scores fall below a certain level should always be classified as having made an uninformed choice, whatever their values and whatever the choice. There are no problems in principle with this approach, although it does depend on the quality of the method used to assess whether knowledge is adequate.

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A woman whose knowledge is judged adequate can also be classified as having made an uninformed choice if her behaviour seems to be discrepant with her attitudes. The behaviour is uptake of the screening test on offer and it is measured unambiguously from clinic records. It follows, therefore, that the robustness of the discrepancy/no discrepancy conclusion depends on the quality and adequacy of the attitude measure as a predictor of who ‘should’ and ‘should not’ have the screening test. In effect, the attitude measure is used to ‘diagnose’ those who should and should not have Down syndrome screening, so its performance in that role needs to be assessed according to the usual methods and criteria. No test is ever 100% accurate, so test developers are required to calculate indicators such as the false positive and false negative rates and demonstrate their relationship to different scale cut-off points so that the best cut-off point can be chosen. Receiver operating characteristic (ROC) curves are frequently drawn up to illustrate and compare test performance. ROC curves can be constructed from clinical prediction rules or their psychological equivalents and provide a full picture of test accuracy and the possible sensitivity/specificity trade-offs that underlie it. The NSC requires that a test reaches certain standards of accuracy, with explicit levels of sensitivity and specificity, before it can be recommended for use. Within the current UK Down syndrome screening programme, screening methods are intended to achieve a detection rate greater than 75% with a relatively low level of false positives. Measures such as the MMIC use attitude questions and apply them, together with a cut-off point chosen by the authors, to classify women into two groups: those who ‘should’ and ‘should not’ have such a test. Actual uptake is then compared with the results of this classification rule. In another setting, such discrepancies would be characterised as false positives and false negatives; in the MMIC context, however, failures of prediction are all described as uninformed choices. This logic is not stated explicitly but it is clear: given adequate knowledge, if a woman’s choice is not explicable using the attitude assessment component, then that choice will be classified as uninformed. A corollary of the above is that improvements in attitude measurement or selection of settings that result in better prediction of behaviour will apparently lead to improved levels of informed choice, because the numbers of false positives and false negatives will have been reduced. Most psychological models such as those underlying the MMIC that use attitudes to predict behaviour have relatively modest predictive power and that power tends to be variable across samples and settings.34 Work continues on the development of the models but one of the better ways to improve predictive power seems to be to ask more-specific questions, for example how well can attitudes towards having a particular test, measured today, predict uptake of that same test, today? While having theoretical appeal in terms of behaviour prediction, these developments can leave values about parenting a child with a disability behind. Reliance on the individual patient’s attitude–behaviour relationship to determine informed choice (once knowledge has been established) can mean that important differences in service delivery can also be obscured. A number of studies have identified that offering a test at a routine visit is likely to increase uptake compared with sites where a separate appointment is required.21,35 However, other factors may also affect uptake across services. For example, in some sites, very active, testoriented staff may stress the benefits of testing to women or at least make sure the test opportunity is clearly signposted. In other sites, a more passive or ‘aloof ’ stance may be the cultural norm. These different service cultures may result in women holding different attitudes towards undergoing prenatal testing. There is very little research in

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this area, however, as most has focused on the individual attitudes of health providers. In either of these scenarios, as long as uptake (high or low) is ‘explicable’ in terms of attitude–behaviour consistency, levels of informed choice can be apparently high and important contributions to between-site differences will remain unscrutinised. Including a measure of decision making As discussed previously, some authors have drawn attention to the distinction between a choice and a decision, defining a decision as a process of choosing between two or more options and a choice as the outcome of a decision.5 In this scenario, measuring only the choice (the outcome) does not take account of whether or not people use their knowledge to think about the alternatives before making their final choice.36 Van den berg and colleagues5 have suggested that attitude–behaviour consistency may be achieved because some women believe testing is a ‘self-evident’ behaviour and not one that requires a decision; in this case, the outcome cannot be said to be an informed decision. As a result, they have added a measure of deliberation to their existing measures of knowledge and attitudes. The measure of deliberation assesses how well women have visualised and thought through the potential consequences of undergoing and not undergoing screening. In the one published study using this measure, substantially fewer women were classified as having made an informed decision than would have been classified as having made an ‘informed choice’ using measures of knowledge and attitude–behaviour consistency alone. While the addition of a measure of the deliberation process is an interesting development, it requires further work to test its reliability and validity in this setting. In addition, because van den Berg and colleagues’ measure incorporates the problematic elements discussed previously, it cannot be considered appropriate as a measure of the effectiveness of a screening programme.

Evaluating screening programmes: what else can we do? It is suggested that informed choice, as operationalised within existing measures, is not an adequate outcome measure for prenatal testing screening programmes, either at individual or clinic level. The ‘right’ choice for a particular woman and her family will be determined by a complex mixture of individual, social and serviceprovision factors. Future research should, therefore, increase understanding of how these interact. Theoretically derived instruments will continue to have a place in the research context but it must always be borne in mind that increasing the ability to predict what people will do is not at all the same as increasing understanding of what they should do. For the foreseeable future, programmes offering tests for which there can be no single right course of action should not be evaluated in terms of uptake or other proxy outcome indicator but rather in terms of the quality of their processes. Crucially, measures of service effectiveness or quality should be unrelated to the choices made because people who accept and decline tests should both have received the same high-quality support in making their choices. We should therefore be measuring quality of care at the site level and quality of decision making at the individual level. There are three main areas that can be targeted to achieve this: n The staff level. Providing good-quality written information, whether through a leaflet or the internet, is only one of the elements necessary to facilitate an informed decision. We have seen that the healthcare professionals with whom women interact have a crucial role to play, as the way in which the offer of

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screening is framed can bias the decisions women make. The simple strategies outlined above can help in this respect. However, while healthcare professionals must guard against influencing individual choice, it is acknowledged that many women would prefer a more shared supportive approach to decision making.37 There is growing evidence on shared decision making within healthcare settings, mostly from the USA and Canada, showing that decision support can be given in a way that also facilitates value-consistent individual choice. Staff education and training programmes, together with quality monitoring in relation to decision support protocols, are already operating in some areas of healthcare.38 n The service level. There are many settings and circumstances outside of the healthcare context in which there can be no pre-specified ‘right answer’ and no single best outcome of the service being provided. Arbitration services and custody hearings are two obvious examples. The quality of these kinds of service can only be measured in process terms, for example have the staff been properly trained? Are they adequately supervised? Are all clients given the time and opportunity to express their views? Are agreed procedures followed? Are there systems for ensuring comparability between different service providers? It is suggested that the concept of providing services fairly to people with different needs and points of view also applies to prenatal screening and testing programmes. The objective is ‘fairness’ to all and it is important to the reputation of the service that it is both known, and shown, to be fair. It is suggested that this concept could also be profitably investigated with respect to prenatal screening and testing. n The individual level. There are now a number of well-validated measures of decision making, for example the Decisional Conflict Scale, that may be suitable for evaluating quality of decision making in prenatal screening programmes at the individual level.39 These, together with measures of satisfaction with the fairness of the process, could be administered to a sample of women and used, in conjunction with the other approaches outlined, to assess whether a prenatal screening service is achieving high-quality decision support and fairness of process.

References 1. 2. 3. 4. 5. 6. 7. 8.

Johnston M, Vogele C. Benefits of psychological preparation for surgery: a meta-analysis. Ann Behav Med 1993;15:245–56. NHS Antenatal and Newborn Screening Programmes. Down’s Syndrome Screening Programme Objectives. London: NHS; 2006. National Institute for Health and Clinical Excellence. Antenatal Care: Routine Care for the Healthy Pregnant Woman. NICE clinical guideline 62. London: NICE; 2008. Human Genetics Commission. Making Babies: Reproductive Decisions and Genetic Technologies. London: Human Genetics Commission; 2006. van den Berg M, Timmermans DRM, ten Kate LP, van Vugt JMG, van der Wal LKG. Informed decision making in the context or prenatal screening. Patient Educ Couns 2006;63:110–17. Briss P, Rimer B, Reilley B, Coates RC, Lee NC, Mullen P, et al. Promoting informed decisions about cancer screening in communities and healthcare systems. Am J Prev Med 2004;26:67–80. Reid M. Consumer-oriented studies in relation to prenatal screening tests. Eur J Obstet Gynecol Reprod Biol 1988;28 Suppl:79–92. Royal College of Physicians. Prenatal Diagnosis and Genetic Screening: Community and Service Implications. London: Royal College of Physicians; 1989.

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Royal College of Obstetricians and Gynaecologists. Termination of Pregnancy for Fetal Abnormality in England, Wales and Scotland. London: RCOG Press; 1996. Shepperd S, Farndon P, Grainge V, Oliver S, Parker M, Perera R, et al. DISCERN-Genetics: quality criteria for information on genetic testing. Eur J Hum Genet 2006;14:1179–88. Ahmed S, Atkin K, Hewison J, Green JM. The influence of faith and religion and the role of religious and community leaders in prenatal decisions for sickle cell disorders and thalassaemia major. Prenat Diagn 2006;26:801–9. UK National Screening Committee. UK Screening Portal [www.screening.nhs.uk/]. DIPEx. Healthtalkonline [www.healthtalkonline.org/]. Antenatal Screening Web Resource. AnSWeR website [www.antenataltesting.info/]. Bryant LD, Ahmed S, Hewison J. Conveying information about screening. In: Rodeck C, Whittle M, editors. Fetal Medicine: Basic Science and Clinical Practice. 2nd ed. Edinburgh: Churchill Livingstone; 2008. Seror V, Ville Y. Prenatal screening for Down syndrome: women’s involvement in decisionmaking and their attitudes to screening. Prenat Diagn 2009;29:120–8. Stapleton H, Kirkham M, Curtis P, Thomas G. Framing information in antenatal care. Br J Midwifery 2002;10:197–201. Tversky A, Kahneman D. The framing of decisions and the psychology of choice. Science 1981;211:453–58. Jones SK, Frisch D, Yurak TJ, Kim E. Choices and opportunities: another effect of framing on decisions. J Behav Decis Making 1998;11:211–26. Johnson EJ, Goldstein D. Do defaults save lives? Science 2003;302:1339. Bekker H, Modell M, Denniss G, Silver A, Mathew C, Bobrow M, et al. Uptake of cystic fibrosis testing in primary care: supply push or demand pull? BMJ 1993;306:1584–6. Bryant LD, Bown N, Bekker HL, House A. The lure of ‘patient choice’. Br J Gen Pract 2007;57:822–6. Maule AJ, Edland AC. The effects of time pressure on judgement and decision making. In: Ranyard R, Crozier WR, Svenson O, editors. Decision Making: Cognitive Models and Explanation. London: Routledge; 1997. p. 189–204. Ahmed S, Green J, Hewison J. Antenatal thalassaemia carrier testing: women’s perceptions of “information” and “consent”. J Med Screen 2005;12:69–77. Bekker HL. Using decision making theory to inform clinical practice. In: Elwyn G, Edwards A, editors. Shared Decision Making – Achieving Evidence-based Patient Choice. London: Open University Press; 2009. Marteau TM, Dormandy E, Michie S. A measure of informed choice. Health Expect 2001;4:99–108. Ajzen I. The theory of planned behavior. Organ Behav Hum Decis Process 1991;50:179–211. Marteau TM. Informed choice: a construct in search of a name. In: Edwards A, Elwyn G, editors. Shared Decision-Making in Health Care: Achieving Evidence-based Patient Choice. 2nd ed. Oxford: Oxford University Press; 2009. p. 87–94. Dormandy E, Michie S, Hooper R, Marteau TM. Informed choice in antenatal Down syndrome screening: A cluster-randomised trial of combined versus separate visit testing. Patient Educ Couns 2006;62:56–64. Van den Berg M, Timmermans DR, ten Kate LP, van Vugt JMG, van der Wal G. Are pregnant women making informed choices about prenatal screening? Genet Med 2005;7:332–8. Green JM, Hewison J, Bekker HL, Bryant LD, Cuckle HS. Psychosocial aspects of genetic screening of pregnant women and newborns: a systematic review. Health Technol Assess 2004;8:iii,ix–x,1–109. Garcia J, Bricker L, Henderson J, Martin MA, Mugford M, Nielson J, et al. Women’s views of pregnancy ultrasound: a systematic review. Birth 2002;29:225–50. Bryant LD, Green JM, Hewison J. The role of attitudes towards the targets of behaviour in predicting and informing prenatal testing choices Psychol Health 2009 (in press). Cooke R, French DP. How well do the theory of reasoned action and theory of planned behaviour predict intentions and attendance at screening programmes? A meta-analysis. Psychol Health 2008;23:745–65.

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Dormandy E, Hooper R, Michie S, Marteau TM. Informed choice to undergo prenatal screening: a comparison of two hospitals conducting testing either as part of a routine visit or requiring a separate visit. J Med Screen 2002;9:109–14. Bekker HL. Genetic screening: facilitating informed choices. In: Cooper DN, Thomas N, editors. Encyclopaedia of the Human Genome. New York: Nature Publishing Group – Macmillan Publishers Ltd; 2003. p. 926–30. Garcia E, Timmermans DRM, van Leeuwen E. Rethinking autonomy in the context of prenatal screening. Prenat Diagn 2008;28:115–20. Stacey D, Pomey MP, O’Connor AM, Graham ID. Adoption and sustainability of decision support for patients facing health decisions: an implementation case study in nursing. Implement Sci 2006;1:17. O’Connor AM. Validation of a decisional conflict scale. Med Decis Making 1995;15:25–30.

17 Chapter 17

Consensus views arising from the 57th Study Group: Reproductive Genetics

Clinical practice 1. Genetic testing should be delivered within a framework of health care and appropriate pre- and post-test counselling should be available. 2. Laboratories should make available to service users evidence-based information on the analytical and clinical validity and utility of tests offered. 3. There should be increased capacity for improved high-throughput diagnostics for karyotyping, single-gene disorders and complex disorders (multigenic/copy number variants) with the ability to transition research genetic tests into the clinical arena as rapidly as possible when deemed appropriate. 4. Complex disorders of sex development should be managed by a multidisciplinary team with experience of relevant issues throughout life (fetal/newborn/ childhood/transition/adulthood). 5. Carefully coordinated care pathways need to be in place when diagnoses are made. 6. It should be acknowledged that prenatal gene transfer may be the only therapeutic approach for treatment of certain severe life-threatening diseases. 7. Fetal karyotyping should remain the ‘gold standard’ test following invasive prenatal diagnosis until appropriately tested and evaluated higher-resolution whole-genome analytical methods can be introduced.

Health policy 9. The National Institute for Health Research’s remit should be extended to genetic test evaluation studies and funding for translation of tests into NHS service (for example, test development and validation studies) should be more readily available through this route. 10. Tests for low-penetrance biomarkers should be subjected to a similar, though not necessarily identical, process of scrutiny to those rare high-penetrance genetic conditions currently dealt with by the UK Genetic Testing Network.

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11. Assuming the successful introduction of non-invasive prenatal diagnosis for the detection of Down syndrome and other common aneuploidies, future fetal karyotyping will focus on cases with ultrasound-detected structural abnormalities, in which case: n The use of prenatal array comparative genomic hybridisation (aCGH) should be focused in rapid-turnaround and high-throughput laboratories with the proven infrastructure to cope with the high levels of fast parental follow-up studies that will be required. n Cytogenetic laboratories need to change training infrastructure now to reflect the significant bioinformatic and molecular components involved with aCGH and other whole-genome analytical methodologies (for example, shotgun sequencing). n aCGH should replace fetal kayotyping as soon as the data proving its effectiveness are available. 12. Programmes offering tests for which there can be no recommended ‘right’ course of action should be measured not by test uptake or other outcome indicator but by the quality of their processes. 13. There is a need for a comprehensive programme of education for professionals and the public with regard to novel genetic tests and technologies. 14. Access to and the optimum service configuration of genomic technologies in consolidated specialised laboratory services should be specifically addressed by the new national clinical director for pathology services. 15. Fetal D testing of alloimmunised women should be extended to the whole of the UK and not just be for those with high levels of anti-D. 16. Fetal D testing for all D-negative pregnant women should be introduced as soon as the best time has been identified. 17. The public, healthcare professionals and policy makers need to be kept informed of the progress and limitations of fetal D testing, both to avoid raising unreasonable expectations and to enable preparation for expeditious implementation into NHS service when it is safe and appropriate to do so. 18. Couples should be seen by a genetics expert before PGD. 19. Guidelines need to be developed to define the criteria for referral for screening for Y chromosome microdeletions in male infertility. Saviour siblings 20. Preimplantation genetic diagnosis (PGD) for saviour siblings should be permitted or denied based on the permissibility or impermissibility of live-organ donation by children. 21. Prospective PGD and selection may be warranted in some cases. 22. PGD centres should closely integrate the services of assisted reproductive technology (ART) and genetics, and be accredited by appropriate organisations. 23. Paediatric follow-up should be a mandatory part of a PGD service. 24. PGD and preimplantation genetic screening (PGS) should be recognised as separate clinical services and PGS should only be offered as part of a randomised controlled trial.

CONSENSUS VIEWS  |  219

25. Future developments should continue to involve medical professionals, user groups, regulatory bodies and ethicists. 26. Fetal medicine units need to have a genetics team that is available to discuss atrisk pregnancies on a daily basis. 27. Teaching programmes should endeavour to include mutidisciplinary aspects whereby joint teaching between genetic and fetal medicine specialties occurs. Fetal gene and stem cell therapy 28. Policy development is needed in relation to procedures that do not fall neatly into either the research or ‘normal clinical practice’ categories. 29. There is a need to consider consent policy in relation to termination of pregnancy if an attempt at therapy fails. 30. There is a need to consider consent policy for highly uncertain new therapies.

Research Breakthrough technologies Embryonic stem cell-derived germ cells 31. Focused basic research is needed to understand the normal genetic and epigenetic events that occur during primordial germ cell specification from embryonic stem (ES) cells and in the differentiation of primordial germ cells. Fetal stem cell and gene therapy 32. Improvements in vector design and safety will be needed before safe targeted delivery to the fetus can be achieved; studies into long-term safety in large animal models (non-human primates) should be supported 33. There is a need for research into the acceptability of prenatal gene therapy. 34. Fetal stem cell therapy requires considerable preclinical development and research before clinical introduction, although rescue treatment may be justified in continuing pregnancies with osteogenesis imperfecta on a case-by-case basis subject to ethical, safety and oversight restrictions. Epigenetic regulation 35. Further research is needed into understanding changes in methylation and in histone modifications in the genome. Development of existing technology 36. Existing technology should be developed to: n promote the development of bioinformatics resources for data analysis n promote the development of biobanks into reproductive disease to facilitate appropriate research.

220  |  CONSENSUS VIEWS

Implementation of technologies 37. The effectiveness of aCGH should be formally evaluated in the UK with a large prospective trial. 38. Full health technology and economic assessments should be carried out on the emerging new genetic and genomic technologies, including the potential benefits and savings in other areas of healthcare spending. 39. Research into the development and implementation of technologies associated with the analysis of free fetal nucleic acids for diagnostic purposes is recommended. 40. Research is needed into the long-term outcomes of novel therapeutic interventions.

Other recommendations 41. It would be helpful and appropriate if the impossibility of objectively defining ‘serious’, at least in relation to disabilities, illnesses or conditions of ‘midspectrum’ severity, were recognised and reflected upon in the prenatal diagnosis (PND) and PGD contexts. This is complicated by uncertainties in relation to prognosis. 42. It would be helpful and appropriate if possible parental and family interests were recognised and reflected upon as being of particular relevance in the interpretation of ‘serious’ in the criteria for PND and PGD in the many cases where a condition would nevertheless mean that a future child will have a life he or she thinks worth living. 43. Measures of service quality should be unrelated to decisions made because people who accept and decline tests should both have received the same highquality support in making their choices. 44. There is a need to be more explicit about the challenges for parents and professionals in dealing with uncertain outcomes. .

i Index

Abortion Act 1967 183, 186, 187–90 ACCE framework 193, 194–7 achondrogenesis 136, 137 achondroplasia PGD selection for 52 prenatal diagnosis 132, 135, 137, 164 adeno-associated virus (AAV) vectors 105, 106 immune responses 102, 113 prenatal gene therapy 108, 109 safety 114 specific-organ targeting 109 adeno-retro hybrid vectors 106 adenovirus vectors 106 immune response 113 prenatal gene therapy 105, 108–9 safety 114 Advisory Committee on Genetic Testing (AGCT) 184 African populations, Rh D polymorphism 174, 175 AIRE mutations 3 alkaline phosphatase 137 allele drop-out (ADO) 43 Alzheimer’s disease, PGD 51–2 ambiguous genitalia 15, 26, 27–8 antenatal detection 140–1 early postnatal management 29–30 American Association for Reproductive Medicine 50 amniocentesis 159 decision making by parents 199–200 fetal karyotyping 147 fetal Rh D typing 173–4 rapid aneuploidy testing 148–9 amniotic fluid, mesenchymal stem cells 86–7 androgen insensitivity syndrome 3, 6, 27 antenatal detection 140 complete (CAIS) 6, 15, 21, 27 partial (PAIS) 6, 27 androgenotes 71, 72 androgen receptors 17 gene (AR) mutations 3, 6, 25

androgens disorders of synthesis and action 24–5, 27 excess states 25, 28 aneuploidies see chromosomal anomalies; Down syndrome; trisomies Angelman syndrome 77–8 animal models of human disease fetal stem cell therapy 89–91, 92–3 prenatal gene therapy 107–9 anovulation 2 AnSWeR (Antenatal Screening Web Resource) 207 Antenatal Results and Choices (ARC) 199–200, 202, 203–4 antenatal screening 131–2 Down syndrome 149–50, 201 evaluating programme outcomes 208–13, 220 informed consent, choices and decisions 205–14 ultrasound scanning 131–43, 202, 211 anti-D immunoglobulin prophylaxis 173, 178–9 antimüllerian hormone (AMH) 17, 20 impaired secretion 26 ‘anti-testis’ genes 17 Antley–Bixler syndrome 27 Apert syndrome 165 aromatase insufficiency 25, 28 array comparative genomic hybridisation (aCGH) 150–7, 218, 220 role in prenatal diagnosis 152–7 technique 150–2 ART see assisted reproductive technologies arthrogryposis 138–9 ARX/X-linked lissencephaly 23, 24 assisted reproductive technologies (ART) childhood growth and development 76 genomic imprinting and 73–6 genomic imprinting syndromes and 77–8 see also in vitro fertilisation atelosteogenesis type II 136

222  |  INDEX ATRX deletion 24 attitudes measuring 210–11 predicting behaviour 211–13 auditing, genetic test effectiveness 196 AURKC gene mutations 6 autonomy, reproductive, right to 61–2, 63–5 AZF microdeletions 5–6 bacterial artificial chromosomes (BACs) 150, 151 Bardet–Biedel syndrome 143 Batten disease 111 Beckwith–Wiedemann syndrome 77–8, 134–5 behaviour, predictive power of attitudes 211–13 bilirubin uridine diphosphate (UDP) glucuronosyltransferase 108, 113 biobanks 219 bioinformatics 219 Blackfan–Diamond anaemia 59 blastomere biopsy 38–9 blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) 3 blood groups, non-invasive fetal typing 173–80 BMP15 gene mutations 3 bone marrow adult stem cells 83–4 fetal stem cells 84–6 bone marrow transplantation (BMT) 84, 87, 91, 92 bowel obstruction, antenatal diagnosis 141–2 bradycardias, fetal 139–40 brain malformations 133–4, 135 branchio-oto-renal syndrome 132, 143 BRCA1/BRCA2 mutations, PGD 51–2 British Fertility Society (BFS) 50, 202 CAG triplet repeats, male infertility 6 campomelic dysplasia 23, 24, 26, 140 carbohydrate-deficient glycoprotein syndrome 140 cardiac abnormalities, fetal 132, 135, 137–8 cardiac dysrhythmias fetal hydrops 139–40 inherited 196 care pathways 201, 202, 217 cat eye syndrome 138 CBX2 gene 24 CCR5 gene 177, 178 CDY gene 5 cell-free fetal DNA (cffDNA) 159, 173, 198 diagnosis of aneuploidy 163–6

diagnosis of single-gene disorders 163, 164–5 fetal genotyping for other blood groups 179–80 fetal rhesus D genotyping 174, 175–9, 180 fetal sex determination 160–3 limitations 168 see also non-invasive prenatal diagnosis cell-free fetal mRNA 166, 167 cffDNA see cell-free fetal DNA CFTR gene mutations 6 prenatal transfer 105, 114 CGH see comparative genomic hybridisation CHARGE syndrome 140–1, 142 children live organ donation by 66–7, 68 proxy consent 127–8 saviour see saviour siblings choice informed see informed choice uninformed 209, 211–12 cholesterol side-chain cleavage deficiency (CYP11A1) 18, 24, 27 chorioamnionitis 114 chorionic villus sampling (CVS) 159, 168 decision making by parents 199–200 fetal karyotyping 147 fetal mesenchymal stem cells 86 fetal Rh D typing 173–4 rapid aneuploidy testing methods 148–9 chromosomal anomalies infertility 4–5, 8 PGD 44, 47, 48–9 prenatal diagnosis 147–57 array CGH and 152–4, 155, 156 current service delivery models 149–50 karyotyping reporting times 147, 148 new technologies 150–2 non-invasive 154–7, 163–7 potential service reconfiguration 154–7 rapid aneuploidy testing 147–9 ultrasound scan 138 see also fetal karyotyping recurrent miscarriage 4 chromosome rearrangements, sex selection 47–50 cleft lip and palate 138, 187 clinical geneticists PGD services 36

INDEX  |  223 role in fetal medicine unit 131–43, 218 clinical practice, consensus views 217 clitoromegaly 26 collagenopathies, type II 137 common complex disorders, genetic testing 197 comparative genomic hybridisation (CGH) 202 array (aCGH), prenatal diagnosis 150–7, 218, 220 PGD 35, 37, 47 complete androgen insensitivity syndrome (CAIS) 6, 15, 21, 27 confined placental mosaicism (CPM) 168 conflict theory, fetal growth 76 congenital abnormalities see fetal abnormalities congenital adrenal hyperplasia (CAH) 23, 27, 28 cffDNA for fetal sex determination 160, 162–3 genetic test evaluation 194 lipoid (StAR deficiency) 24, 27, 140 non-invasive prenatal diagnosis 165 prenatal diagnosis and treatment 28–9, 140 steroidogenesis defects causing 18 see also 21-hydroxylase deficiency congenital bilateral absence of vas deferens (CBAVD) 6, 36 consensus views 217–20 consent (informed) 205–14 fetal/early childhood stem cell and gene therapies 127–8 live organ donation by children 66–7 non-invasive prenatal diagnosis 201–2 contractures, fetal 138–9 copy number changes 150, 152–3 copy number variants (CNVs) 150, 151, 153 cord blood haematopoietic stem cells 84 mesenchymal stem cells 85, 86, 87 Cornelia de Lange syndrome 141, 142 cost-effectiveness fetal Rh D testing 178–9 new genetic tests 195 stem cell and gene therapies 128 Costello syndrome 133 costs, array CGH 153 counselling genetic testing 194 invasive prenatal diagnosis 199–200 non-invasive prenatal diagnosis 168, 201–2

Crigler–Najjar type 1 syndrome 108, 113 Crouzon syndrome 165 CYP11A1 deficiency 18, 24, 27 CYP17A1 see 17α-hydroxylase cystic fibrosis antenatal ultrasound detection 141 genetic testing 196 information provision 207 non-invasive prenatal diagnosis 163, 165 preimplantation genetic diagnosis 36, 38 prenatal gene therapy 104 severity of illness 185 cytogenetic laboratories 147, 155–7, 218 Database of Genomic Variants 150, 153 DAX1 gene 17, 24 DAZ gene 5–6 deafness, PGD selection for 52 Decisional Conflict Scale 214 decision making facilitation in practice 207–8 invasive prenatal diagnostic tests 199–200 measuring 213 quality of 213–14 shared 214 termination of pregnancy, legal aspects 187–90 see also informed decisions Denys–Drash syndrome 24, 26 desert hedgehog (DHH) gene defects 24 designer babies 203 development ART children 76 PGD/PGS children 41 developmental delay, prenatal diagnosis 150, 153, 155 dexamethasone, prenatal treatment 28–9, 162 diaphragmatic hernia, congenital 104, 105, 133 diastrophic dysplasia 136 differentially methylated regions (DMRs) 72–3, 74 DiGeorge/velocardiofacial 22q11.2 microdeletion 132, 138, 153 dihydrotestosterone (DHT) 17, 18 DIPEx 207 disability/impairment ground for termination of pregnancy 187–90 impact on parents 186 PGD for selection for 52

224  |  INDEX disorders of sex development (DSD) 15–16, 20–31 46,XX 21, 22, 25, 27–8 46,XY 21, 22, 24–5, 26–7 classification and diagnosis 21–3 consensus views 217 early postnatal management 29 fertility management 30–1 long-term outcome 30 molecular diagnosis and highthroughput analysis 30 multidisciplinary team (MDT) approach 15, 29–30 nomenclature 21 ovotesticular 26, 27–8 prenatal diagnosis 28–9 sex chromosome 21, 22, 23–6 testicular 27–8 DMRT1 deletion 24 DNA cell-free fetal see cell-free fetal DNA vectors 106, 107 DNA methylation controlling genomic imprinting 72–3 differential, fetal/maternal genes 160–1, 166–7, 177 effects of ART 74–6 male and female germlines 73, 74 male infertility and 7–8 Down syndrome (trisomy 21) antenatal screening 149–50, 201 decision making by parents 199–200 evaluating screening programmes 209, 210–11, 212 information provision 207 invasive prenatal diagnosis 147–50 non-invasive prenatal diagnosis 154–7, 163–7, 168, 201 severity of illness 185 termination of pregnancy 189 Duchenne muscular dystrophy 89, 104 ductus venosus, absent 140 duodenal atresia 141 echocardiography, fetal 137–8 ectrodactyly 141, 142 education non-invasive prenatal diagnosis 168, 218 see also counselling Ehlers–Danlos syndrome type IV 132 EIF2B gene mutations 3 Ellis–van Creveld syndrome 135–6 embryonic stem (ES) cells 83, 84 differentiation into germ cells 219 ethical issues 124–5

embryos biopsy 38–9, 50 genomic imprinting of cultured 74–5 number transferred 40 transfer 39 endocrine disruptors 7 endometriosis 1, 2 epidermolysis bullosa 104 epigenetics 71–9, 219 ART and 73–9 male infertility 7–8 see also DNA methylation; genomic imprinting equine infectious anaemia virus (EIAV) vector 107, 112–13 ESR1 gene 8 estrogens 20 ethical aspects eugenics and designer babies 203 genetic test evaluation 195 legal criteria for termination of pregnancy and PGD 183–92 non-invasive prenatal diagnosis 168, 201–2 prenatal gene therapy 114–15 saviour siblings 59–68 stem cell therapy and gene therapy 123–9 ethnic variations, causes of female infertility 2, 3 eugenics 203, 205 European Society of Human Reproduction and Embryology (ESHRE) 36, 40–1, 43, 47, 50 evil acts, benefiting from 124–5 factor VII deficiency 103, 104, 111 factor IX gene transfer 102, 107–8, 112 Fanconi anaemia 84, 141 female infertility 1–4 femur fibula ulna (FFU) syndrome 136 fertility treatment, disorders of sex development 30–1 fetal abnormalities 11–13 week ultrasound scan 133–5 18–20 week ultrasound scan 135–43 after PGD/PGS 41 invasive prenatal diagnosis 150, 153 termination of pregnancy for 183–90, 191–2, 200–1 see also prenatal diagnosis fetal autologous stem cell gene therapy 102 fetal cells, maternal circulation 159 fetal dysmorphology 131–43, 150 fetal growth restriction, prenatal gene therapy 103–5, 111–12

INDEX  |  225 fetal karyotyping 147–57 comparison with array CGH 153, 156 consensus views 217, 218 current service delivery models 149–50 future role 154–7 potential new technologies replacing 150–3, 154 rapid aneuploidy tests 147–9 fetal medicine units, role of clinical geneticists 131–43, 218–19 fetal somatic gene therapy 102 see also prenatal gene therapy fetal stem cells ethical issues 124–5 sources 84–7, 88 fetal stem cell therapy 83–94, 102 consensus views 218–19 consent issues 127–8 rescue therapy 93–4 routes to clinical translation 92–4 sources of stem cells 83–7, 88 see also in utero transplantation fetus interests, termination in 190 moral status 189–90 fluorescence in situ hybridisation (FISH) PGD 37, 47, 48–9, 50 prenatal diagnosis 147–9, 157 foamy virus vectors 107 follicle-stimulating hormone (FSH) 20 follicle-stimulating hormone (FSH) receptor mutations 3 FOXL2 gene mutations 3 fractures, intrauterine 136–7 fragile X mental retardation (FMR) gene 3, 194 fragile X syndrome 168, 194, 196 Frasier syndrome 24, 26 Free DNA Fetal Kit RhD® 175 GALT gene mutations 3 gastrointestinal disorders, antenatal diagnosis 141–2 gender assignment 26 gene dossiers 193, 194–7 gene therapy adult and neonatal 102 consent for fetuses and children 127–8 cost-effectiveness and resource allocation 128 ethical aspects 123–9 prenatal 101–17 reproductive effects 126 research–treatment overlap 126–7 stem cell-based 83

treatment–enhancement overlap 127 genetic association studies, infertility 2–3, 6–7, 8 Genetic Commissioning Advisory Group (GenCAG) 193, 196 genetic test evaluation 193–8, 217 ACCE framework 193, 194–7 changes/new systems required 197–8 common complex disorders 197 international frameworks 194 UK approach 193 genetic tests analytical validity 194 auditing effectiveness 196 clinical utility 195 clinical validity 195 consensus views 217, 220 counselling before and after 194 definition 194 ethical, legal and social issues 195 gatekeeping 196 information for commissioning 196 service provision 193, 217–19 genomic imprinting 71–9 effects of ART 73–6 growth and 76–8 mechanism 72–3, 74 placenta 76–7 postnatal, significance 78 syndromes 77–8 germ cells 18–19 embryonic stem cell-derived 219 see also primordial germ cells germline changes after prenatal gene therapy 111 ethical issues 126 Glover, Jonathan 184, 185 glucocorticoid resistance 25 α-glucosidase (GAA) 109 glycogen storage disease type II 109 gonadal dysgenesis 24, 26 complete 26 mixed 22, 24, 26 partial 26 gonads, development 17, 19–20 growth ART children 76 genomic imprinting and 76–8 PGD/PGS children 41 gynogenotes 71, 72 H19 gene 75–6, 77 haematopoietic stem cells (HSC) in utero transplantation (IUT) 88–9, 91 sources 84

226  |  INDEX haemoglobinopathies antenatal screening 201 fetal stem cell therapy 91 non-invasive prenatal diagnosis 164, 165 haemolytic disease of fetus and newborn (HDFN) 173, 179 haemophilia postnatal gene therapy 102, 105 prenatal gene therapy 103, 107–8, 111 harm principle 62, 63–4, 65 Hashmi saviour sibling case 59 healthcare professionals attitudes to screening tests 212–13 measuring quality of care 213–14 presentation of screening decisions 207–8 health policy, consensus views 217–19 health risk, ground for termination of pregnancy 188, 190 Healthtalkonline 207 herpes simplex virus vectors 106 HLA-matched siblings see saviour siblings holoprosencephaly 133–4 Holt–Oram syndrome 141 HPA-1a platelet antigen, fetal genotyping 180 HRAS mutations 133 human chorionic gonadotrophin (hCG) 17, 18 Human Fertilisation and Embryology (HFE) Act 1990 42, 183 Human Fertilisation and Embryology (HFE) Act 2008 47, 52, 183, 184, 191 Human Fertilisation and Embryology Authority (HFEA) 42, 59, 184, 190–1 Human Genetics Commission (HGC) 184, 189, 190, 191 Huntington disease non-invasive prenatal diagnosis 163, 165 PGD 51 Hurler syndrome 91 hydrocephalus 135 hydrops, fetal 139–40 11β-hydroxylase deficiency 18, 28 17α-hydroxylase (CYP17A1) deficiency 18, 24, 27 21-hydroxylase deficiency 15, 18, 25, 28 prenatal diagnosis 140 17-hydroxyprogesterone (17-OHP) 28 3β-hydroxysteroid dehydrogenase 2 (3β-HSD 2) (HSD3B2) deficiency 18, 24, 28 17β-hydroxysteroid dehydrogenase 3 (17β-HSD 3) deficiency 18, 25, 27 hypophosphatasia 137 hypospadias 23, 26

hypothalamic–pituitary–gonadal axis 17, 20 hypoxic ischaemic encephalopathy 104 Igf2 gene 75–6, 77 IGF2R/Igf2r gene 75, 76, 77 immune responses, vectors and transgenes 102, 108, 113 immune tolerance fetal stem cell therapy 85–6, 88 gene therapy 102–3 impairment see disability/impairment imprint control regions (ICRs) 73, 75–6 imprinting, genomic see genomic imprinting inducible pluripotent stem cells (iPS) 83, 84 infertility disorders of sex development 15 genetic aetiology 1–8 information, provision 206–7 informed choice definition 209 measurement 209–13 vs informed decisions 205, 213 informed consent see consent informed decisions definition 205–6 facilitation in practice 205–8 measuring outcomes 208–13 vs informed choice 205, 213 see also decision making inhibin A/B 20 insertional mutagenesis, integrating gene vectors 112–13 Ins gene 76 Institut de Biotechnologies Jacques Boy 175 insulin-like 3 (INSL 3) 20 interests child’s 184–5 parent’s 185–6 International Blood Group Reference Laboratory (IBGRL) 176–7 International Society of Blood Transfusion (ISBT) 180 International Standard Cytogenomic Array (ISCA) Consortium 151–2 intersex disorders see disorders of sex development intracytoplasmic sperm injection (ICSI) 38, 73–4, 78 in utero transplantation (IUT) of stem cells 87–94, 102 clinical development 91–2 preclinical development 88–91 rationale 88

INDEX  |  227 routes to clinical translation 92–4 see also fetal stem cell therapy; mesenchymal stem cells in vitro fertilisation (IVF) 38, 73 childhood growth and development 76 genomic imprinting syndromes and 78 PGD 203 PGS 50, 202 saviour siblings see saviour siblings in vitro maturation of oocytes 74 Jepson v. the Chief Constable of West Mercia Police Constabulary 186–8, 189 Jeune’s asphyxiating dystrophy 136 joint dislocations, fetal 139 karyotype/phenotype discordance 29 karyotyping disorders of sex development 21 fetal see fetal karyotyping Kell (K) antigen, fetal typing 179–80 Kennedy disease 6 kidney donation, live, by children 67 Klinefelter syndrome (XXY) 4, 22, 23, 207 fertility treatment 30–1 Kniest dysplasia 137 knowledge, measurement 209–10 language barriers 207 large offspring syndrome (LOS) 74, 75 Larsen syndrome 139 Leber congenital amaurosis (LCA) 108, 165 lentivirus vectors 106, 107, 108 reversion to wild type 113–14 silencing 112 Lesch–Nyhan syndrome 184 Leydig cells 17, 20 life, worth/not worth living 183–6 lifestyle genetics 194 limb abnormalities, isolated 141, 142 lissencephaly, X-linked 23, 24 live organ donation by children 66–7, 68 saviour siblings 65–6 lobster claw hand 141, 142 locked nucleic acids (LNAs) 179 long QT syndrome 140 luteinising hormone (LH) 17, 18, 20 receptor mutations 3 resistance 24 lysosomal storage disorders fetal stem cell therapy 91 prenatal gene therapy 104, 108–9 male infertility 4–8, 218

Marfan syndrome 197 MASPIN gene 160–1 MassARRAY® system 175 mass spectrometry 166, 167, 175, 180 Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome 1, 3 Meckel–Gruber syndrome 143 membranes, stem cells 86 menopause genetic influences 3 premature see primary ovarian insufficiency mental health, ground for termination of pregnancy 188, 190 mesenchymal stem cells (MSCs) 84–7, 88 adipose tissue (AT-MSCs) 85, 86 adult 84, 85, 88 amniotic fluid (AF-MSCs) 86–7 barriers to engraftment 92–3 cord blood 85, 86, 87 fetal (fMSCs) 84–7, 88 in utero transplantation 88, 89–92 production of clinical grade 93 Wharton’s jelly (WJ-MSCs) 87 see also fetal stem cell therapy; in utero transplantation (IUT) of stem cells MEST/Mest gene 75–6, 77 microarrays, PGD 35, 47, 52 microtubule-associated protein (MAPT) 155 Mill, John Stuart 62, 63–4 miscarriage recurrent 4 risk, and decision making 199–200 mixed gonadal dysgenesis 22, 24, 26 monosomy X see Turner syndrome mucopolysaccharidosis (MPS) type VII 108–9 Müllerian inhibiting substance (MIS) see antimüllerian hormone Multi-dimensional Measure of Informed Choice (MMIC) 209–13 multiple pregnancy non-invasive prenatal diagnosis 163, 168 PGD cycles 40 murine leukaemia virus (MLV) vectors 107, 112 muscular dystrophy 104 congenital 139 fetal stem cell therapy 89 myasthenia gravis 139 myeloablation, prior to stem cell therapy 92 myotonic dystrophy PGD 37–8 prenatal diagnosis 139, 165 myotubular myopathy 139

228  |  INDEX nail patella syndrome 139 nano-carriers 107, 117 National Institute for Health and Clinical Excellence (NICE) 178 National Institute for Health Research 196, 217 National Screening Committee (NSC), UK 147, 148–9 evaluation of screening programmes 208, 212 information provision 207 neural tube defects 207, 211 neuronal ceroid lipofuscinosis 111 next-generation technologies, evaluation 197, 198 Niemann–Pick disease 91 NOBOX mutations 3 non-invasive prenatal diagnosis (NIPD) 159–69, 197–8 aneuploidies 154–7, 163–7 consensus views 218, 220 ethics, education and counselling 168, 201–2 fetal blood group status 173–80 fetal sex determination 160–3, 168 limitations 168 parent perspective 200–1, 203–4 private services 203 single-gene disorders 163, 164–5 see also cell-free fetal DNA Noonan syndrome 133, 140 NR5A1 gene defects 24, 26 nuchal translucency (NT), increased, with normal karyotype 133, 136 nutrigenomics 194 oesophageal atresia 142 off-label treatment 127 oligohydramnios 139 oligonucleotides, array CGH 151–2 omphalocele 134–5 O’Neill, Onora 64 oocytes 19 superovulated, imprinting defects 75–6 in vitro maturation 74 oogonia 19 organ donation, live see live organ donation Organisation for Economic Co-operation and Development (OECD) 194 organ transplant recipients, fetal rhesus D typing 177 ornithine transcarbamylase deficiency 114 osteogenesis imperfecta (OI) antenatal diagnosis 136–7 fetal stem cell therapy 90–2, 93–4

postnatal stem cell therapy 91 osteopetrosis, fetal stem cell therapy 89 outcome measurement 208–9 ovarian stimulation 38 ovaries development 17, 19 development defects 25, 27–8 germ cell fate 19 primary insufficiency see primary ovarian insufficiency ovotesticular disorder of sex development (DSD) 26, 27–8 ovulatory infertility 1–2 P450 oxidoreductase (POR) deficiency 18, 25, 27, 28 P450 side-chain cleavage (P450scc) (CYP11A1) 18, 24, 27 palmarplantar hyperkeratosis 25 parents consideration of views on PGD 190–1 deciding on termination of pregnancy 187–90 freedom to determine child’s life 66 impact of child’s disability on 186 informed consent, choices and decisions 205–14 interests, termination of pregnancy and PGD 185–6, 191–2 perspective on prenatal diagnosis 199–204 reasons for having children 60, 61–5 partial androgen insensitivity syndrome (PAIS) 6, 27 paternity testing, prenatal 168 Peg3 gene 76 Pennings, G 60, 65–6, 68 PGD see preimplantation genetic diagnosis PGS see preimplantation genetic screening PHLDA2 gene 76, 77 phthalates 7 PLAC4 mRNA 166, 167 placenta confined mosaicism (CPM) 168 genomic imprinting and 76–7 stem cells from 84, 86 pleural effusions, fetal 140 pluripotent stem cells, inducible (iPS) 83, 84 POF1, 2 and 3 loci 3 polar body biopsy 38, 50 polycystic kidney disease adult 194, 195 prenatal diagnosis 143 polycystic ovary syndrome (PCOS) 2–3 polyhydramnios 139

INDEX  |  229 polymerase chain reaction (PCR) 37 digital 163, 166 non-invasive prenatal diagnosis 160, 164, 165 quantitative fluorescence (QF-PCR) 148–9, 154, 155 real-time fluorescence (f-PCR) 43, 160 real-time quantitative (RQPCR), fetal D testing 175, 176 postnatal test (Pennings), saviour siblings 60, 65–6 Prader–Willi syndrome 78 pregnancy-associated plasma protein A (PAPP-A) 141 preimplantation genetic diagnosis (PGD) 35–52, 203 chromosome rearrangements 47, 48–9 clinical service 35–6 consensus views 218–20 disability selection 52 ethical aspects of legal criteria 183–6, 190–2 funding 42–3 future prospects 52 late-onset disorders 51 non fully penetrant disorders 51–2 paediatric outcomes 41 process 37–40 reasons for requesting 36–7 regulation 41–2 selection of tissue-matched siblings 50–1, 59 sex selection 47–50 single-gene disorders 43–7 success rates 40–1 suitable conditions 42, 44–5 Preimplantation Genetic Diagnosis International Society (PGDIS) 36, 42, 43 preimplantation genetic haplotyping (PGH) 43–7, 50 preimplantation genetic screening (PGS) 35, 50, 202–3 consensus views 218 distinction from PGD 47, 50 paediatric outcomes 41 prenatal diagnosis (PND) chromosomal anomalies see chromosomal anomalies, prenatal diagnosis consensus views 217, 220 disorders of sex development 28–9 ethical concerns 203 evaluating outcomes 208–13, 220 legal criteria for termination of pregnancy 183–90, 191–2

parent perspective 199–204 PGD as alternative to 37 structural fetal anomalies 131–43 prenatal gene therapy 101–17 advantages 102–3 candidate diseases 103–5, 111–12 consensus views 217, 218–19 consent issues 127–8 delivery techniques 110–11, 114 duration of expression 112–13 ethical concerns 114–15 fetal and maternal immune responses 113 future 116–17 human application 115 in practice 115, 116 preclinical studies 107–9 reversion to wild type vector 113–14 safety of fetus, mother and future progeny 114 targeting to correct organ 109–11 vectors 105–7, 116–17 prenatal stem cell therapy see fetal stem cell therapy prenatal treatment congenital adrenal hyperplasia (CAH) 28–9, 162 consent issues 127–8 primary ovarian insufficiency (POS) 1–2, 3, 194 primordial germ cells (PGCs) 17, 18–19 genomic imprints 73 private services, non-invasive prenatal diagnosis 203 propionic acidaemia 165 psychological harms, saviour children 63 PTPN11 mutations 133 puberty 20 quality assurance, non-invasive fetal blood group testing 180 quality of care, evaluating 213–14 quality of life, judging future child’s 184–6 radial ray defects 141 RASSF1A gene 160–1, 177 receiver operating characteristic (ROC) curves 212 5α-reductase type 2 17 deficiency 25, 27 regional genetics centres, multidisciplinary 193 relative mutation dosage (RMD), digital 163 renal agenesis 142–3 renal anomalies, antenatal diagnosis 142–3

230  |  INDEX research consensus views 219–20 overlap with treatment 126–7 resource allocation, stem cell and gene therapies 128 resource inhibiting (RI) and enhancing (RE) genes 78 retinal guanylate cyclase-1 gene 108 retinal pigment epithelium-specific 65 kD protein (RPE65) gene 108 retinitis pigmentosa 165 retinoblastoma 195 RET mutations 142–3 retrovirus vectors 106, 107, 112 rhabdomyomas, cardiac 138 RHCE gene 174, 175, 179 RHD gene 174–5 rhesus (Rh) C (RH2) antigen, fetal typing 179 rhesus (Rh) c (RH4) antigen, fetal typing 179 rhesus (Rh) D antigen alloimmunisation 173, 174 invasive fetal testing 173–4 molecular basis of polymorphism 174–5 non-invasive fetal testing 174, 175–9, 218 alloimmunised pregnant women 175–7 assessing need for antenatal anti-D prophylaxis 178–9 internal controls 177–8 quality assurance 180 rhesus (Rh) E (RH3) antigen, fetal typing 179 risk, evaluating knowledge 209–10 Royal College of Obstetricians and Gynaecologists (RCOG) 187–8, 189, 190 RSPO1WNT4 gene defects 17, 24, 28 saviour siblings (HLA-matched siblings) 59–68 consensus views 218 harms to 62, 63, 67–8 Pennings’ postnatal test 60, 65–6 reasons for having 60, 61–5 selection using PGD 50–1, 59 third-party intervention requirement 60–1 treatment of 60, 65–7 value to parents 62–3 Sendai virus 105 Sequenom 175 serious condition criterion 183–4, 192 consensus views 220 interests of child vs parent 184–6

PGD 183, 190–1 termination of pregnancy 183, 187–90 Sertoli cells 17, 20 serum autologous 93 fetal bovine/calf 74, 93 severe combined immunodeficiency (SCID) fetal stem cell therapy 91 postnatal gene therapy 84, 107, 112 prenatal gene therapy 103, 104 sex ‘brain’ 16 chromosomal 16 fetal, determination 160–3, 168, 194 gonadal and phenotypic 17 reversal 140–1 selection, PGD 47–50 sex chromosome disorders of sex development (DSD) 21, 22, 23–6 sex development, human 16–20 disorders see disorders of sex development early postnatal changes 20 germ cell fate and gonad position 18–20 sheep, prenatal gene therapy 103, 108, 109, 110–11 Sheldon, Sally 185, 187 short-rib polydactyly syndromes 133, 136 SIDDT syndrome 24 single-gene disorders genetic test evaluation 194–7 non-invasive prenatal diagnosis 163, 164–5 PGD 43–7 single-nucleotide polymorphisms (SNPs) 151–2 Down syndrome 166, 167 single-gene disorders 164, 165 skeletal dysplasias 132, 133, 135–7 Slc22 genes 77 sleeping beauty (SB) transposons 107 SMCY gene 5 Smith–Lemli–Opitz syndrome 24, 140 Snrpn gene 75–6 SOX9 gene 17 defects see campomelic dysplasia duplication 4, 25 SPATA16 gene mutations 6 special non-invasive advances in fetal and neonatal evaluation (SAFE) network 180 spina bifida 185 spinal and bulbar muscular atrophy (SBMA) 6 spinal muscular atrophy 104 spondyloepiphyseal dysplasia congenita 137

INDEX  |  231 SRY gene 4, 16, 17 defects 24 fetal sex determination 160 translocation 25, 28 staff see healthcare professionals StAR see steroidogenic acute regulatory protein stem cell-based gene therapy 83 stem cells 83–7 stem cell therapy benefiting from an evil act 124–5 consent for fetuses and children 127–8 cost-effectiveness and resource allocation 128 ethical aspects 123–9 osteogenesis imperfecta 91 prenatal see fetal stem cell therapy reproductive effects 126 research–treatment overlap 126–7 treatment–enhancement overlap 127 see also bone marrow transplantation steroidogenesis 18 steroidogenic acute regulatory protein (StAR) 18, 24, 27, 140 steroidogenic factor 1 deficiency 24, 26 superovulation, genomic imprinting defects 75–6 talipes 136, 137, 138–9 Tay–Sachs disease 184–5 teratogenicity, virus vectors 114 teratomas ovarian 71 stem cell-related 83, 84 termination of pregnancy after 24 weeks 189–90 ethical aspects of legal criteria 183–90, 191–2 measuring attitudes to 210 parent perspective 200–1 PGD as means of avoiding 36–7 testes descent 19–20 determination 16 development 17 germ cell fate 18–19 postnatal function 20 steroidogenesis 18 undescended 23 testicular disorders of sex development (DSD) 27–8 testicular dysgenesis 7, 26 testicular germ cell cancer 7 testosterone 17, 18, 20 Tet-dependent transgene expression 113

tetralogy of Fallot 132 thalassaemia non-invasive prenatal diagnosis 163, 164 prenatal therapy 91, 103, 104 saviour sibling 59 thanatophoric dwarfism 137 Theory of Planned Behaviour 209 thrombocytopenia, neonatal alloimmune 180 tissue-matched siblings see saviour siblings torsion dystonia 165 total anomalous pulmonary venous drainage 138 tracheo-oesophageal fistula 142 transgenes immune responses 108, 113 regulation of expression 113, 117 see also gene therapy translocations, chromosome infertility 5 PGD 47, 48–9 Treacher Collins syndrome 132 trinucleotide repeats, infertility 3, 6 trisomies invasive prenatal diagnosis 147–57 non-invasive prenatal diagnosis 163–7 trisomy 4p 138 trisomy 13 133, 138 trisomy 18 134, 141 trisomy 21 see Down syndrome trophectoderm biopsy 38 TSPY gene, DSY14 marker sequence 159, 160 tubal infertility 1 tuberous sclerosis 138 Turner syndrome (monosomy X) 2, 3, 22, 23 fertility treatment 30–1 information provision 207 ultrasound detection 140 22q11.2 microdeletion syndrome 132, 138, 153 UK Genetic Testing Network (UKGTN) 193, 194–7, 217 ultrasound antenatal screening 131–43, 202, 211 11–13 week scan 133–5 18–20 week scan 135–43 guided injection, fetal gene therapy 110–11, 114 umbilical cord blood see cord blood mesenchymal stem cells 87 uniparental disomy (UPD) 71–2 USP26 gene variants 6–7

232  |  INDEX uterine abnormalities 1, 3, 139 UTY gene 5 VACTERL association 141, 142 values, measuring personal 210 vascular endothelial growth factor (VEGF), prenatal gene therapy 103–5, 111–12 vas deferens, congenital bilateral absence (CBAVD) 6, 36 vectors, gene immune responses 102, 113 insertional mutagenesis risk 112–13 maternal and fetal safety 114 organ-specific targeting 109 prenatal gene therapy 105–7, 116–17 reversion to wild type 113–14 silencing 112 WAGR syndrome 24 Wharton’s jelly mesenchymal stem cells (WJ-MSCs) 87 Whitaker saviour sibling case 59, 61, 63 Wilkinson, Stephen 185, 187 Williams, Glanville 187 Wilms’ tumour 135 WNT4/RSPO1 gene defects 17, 24, 28 women deciding on termination of pregnancy 187–90 risk to, as ground for termination of pregnancy 188, 190 see also parents WT1 gene defects 24, 26 45,X/46,XY mosaicism 24, 26 xenotransplantation 126 X-linked disorders cffDNA for fetal sex determination 160, 161–3 PGD 43, 44, 47–50 46,XX/46,XY karyotype 26 46,XX disorders of sex development (DSD) 21, 22, 25, 27–8 XX males 4, 25 46,XY disorders of sex development (DSD) 21, 22, 24–5, 26–7 Y chromosome 16 haplogroups 5 microdeletions 5–6, 218