The Genetics of Atrial Septal Defect and Patent Foramen Ovale
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The Genetics of Atrial Septal Defect and Patent Foramen Ovale EDWIN PHILIP ENFIELD KIRK A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy December, 2007 School of Women’s and Children’s Health University of New South Wales ORIGINALITY STATEMENT I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged. Signed …………………………………………….............. Date …………………………………………….............. COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Coyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation’ Signed……………………………………………………. Date………………………………………………………. Authenticity Statement ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed……………………………………………………. Date………………………………………………………. DEDICATION To my dear wife, Sue. With gratitude, and with all my love. i Abstract Congenital heart disease is the most common form of birth defect, affecting approximately 1% of liveborn babies. Secundum atrial septal defect (ASD) is the second most common form of congenital heart disease (CHD). Most cases have no known cause. Chromosomal, syndromal and teratogenic causes account for a minority of cases. The hypothesis that mutations in the ASD genes NKX2-5 and GATA4 may cause apparently sporadic ASD was tested by sequencing them in unrelated probands with ASD. In this study, 1/102 individuals with ASD had an NKX2-5 mutation, and 1/129 had a deletion of the GATA4 gene. The cardiac transcription factor TBX20 interacts with other ASD genes but had not previously been associated with human disease. Of 352 individuals with CHD, including 175 with ASD, 2 individuals, each with a family history of CHD, had pathogenic mutations in TBX20. Phenotypes included ASD, VSD, valvular abnormalities and dilated cardiomyopathy. These studies of NKX2-5, GATA4 and TBX20 indicate that dominant ASD genes account for a small minority of cases of ASD, and emphasize the considerable genetic heterogeneity in dominant ASD (also caused by mutations in MYH6 and ACTC). A new syndrome of dominant ASD and the Marcus Gunn jaw winking phenomenon is reported. Linkage to known loci was excluded, extending this heterogeneity, but a whole genome scan did not identify a candidate locus for this disorder. Previous studies of inbred laboratory mice showed an association between patent foramen ovale (PFO) and measures of atrial septal morphology, particularly septum primum length (“flap valve length” or FVL). In humans, PFO is associated with cryptogenic stroke and migraine, and is regarded as being in a pathological contiuum with ASD. Twelve inbred strains, including 129T2/SvEms and QSi5, were studied, with generation of [129T2/SvEms x ii QSi5] F1, F2 and F14 mice. Studies of atrial morphology in 3017 mice confirmed the relationship between FVL and PFO but revealed considerable complexity. An F2 mapping study identified 7 significant and 6 suggestive quantitative trait loci (QTL), affecting FVL and two other traits, foramen ovale width (FOW) and crescent width (CRW). Binary analysis of PFO supported four of these. iii Acknowledgements During the course of an 8 year candidature, I have become indebted to a great number of people for help of many kinds. Above all, I am grateful to my wife, Susan O’Regan, for her love, support, encouragement and forbearance. My children, Seamus, Yasmin and Finn are starting to understand what I’m up to and have been cheering me on in recent months. I followed my father into medicine and he has been an inspiration to me. My mother’s love and encouragement have been very important to me. In addition, she has given me help with graphics (a talent I managed not to inherit from her). Academically, my greatest debts are, of course, to my supervisor, Prof Richard Harvey and co-supervisor, Dr Michael Buckley, for their patient guidance and friendship over the years. I’ve learned a great deal from both of them, and in particular I think I’m starting to get the hang of Richard’s lessons on structure (of manuscripts, as well as hearts). Prof Richard Henry was initially my supervisor. Over time the research moved in a different direction, but he continued to provide support and encouragement. Many members of Richard Harvey’s lab have helped me and I am grateful to all of them. I want especially to thank Dr Christine Biben for teaching me to dissect mouse hearts, and for providing ongoing advice and occasional second opinions; Dr David Elliott for advice and guidance with molecular methods; and Dr Changbaig Hyun for his contributions to the QTL study and the studies of GATA4 and TBX20. Others at the Victor Chang Cardiac Research Institute who have helped include Leticia Castro, Louise Lynagh, Haley Crotty, Milena Furtado, and Drs Mauro Costa, Robyn Otway, Thomas Yeoh, Guanglan Guo, Owen Prall, Orit Wolstein, Daniel Schaft, Mark Solloway, Aaron Schindeler, Suchitra Chander, Fiona Stennard, and Donna Lai have all helped me in various ways and have made the lab an enjoyable place to work. A/Prof Diane Fatkin has been generous with her time and advice. At Sydney University, Prof Chris Moran has been a major guiding force, generous with his time and experience. A better collaborator could not be found. Dr Ian Martin taught me everything I have learned about care and breeding of laboratory mice, including how to dissect them. He also bred the F1 and F2 mice (Chapters 5 and 6), a large undertaking. Dr Peter Thomson has been unstinting in giving of his time and expertise in matters mathematical. All of the animal house staff, but especially Matt Jones and Mamdouh Nessiem, have been unfailingly helpful, often going well beyond the scope of iv their duties to make sure that things ran well. Noelia Lopez ordered and bred the Hapmap mice, and did a number of dissections as well as co-measuring a subset of these mice. Kim Dilati provided assistance in the last few weeks of the advanced intercross line breeding and dissection, when things were very busy. At Sydney Children’s Hospital, members of the Department of Medical Genetics have been unfailingly supportive and helpful. Dr Fiona McKenzie worked as a part time research assistant during the first year of the project, and I doubt it would have got off the ground at all without her kickstarting things. Dr Owen Jones was invaluable in recruitment of children with ASD. At the South Eastern Area Laboratory Service, Peter Taylor, George Eliakis and Glenda Mullan have been especially helpful, but many other members of staff have given advice or help over the years. At the Children’s Hospital at Westmead, A/Prof David Winlaw and members of his lab have contributed greatly to the human studies. Prof Ian Glass first identified the ASD + Marcus Gunn family and contributed greatly to recruitment of family members; he continues to advise and encourage on that project. A/Prof Jenny Donald provided guidance on mapping, and Dr Kyall Zenger taught me to drive LINKAGE, as well as doing a good deal of analysis towards the project himself. Dr Carol Cheung did a similar QTL study and taught me to use Mapmaker/QTL, along with a great deal of other advice and help. Many cardiologists have contributed their time and expertise, especially Dr Rob Justo, Dr Michael Tsicalis (and his wonderful secretary, Dianne Reddell) and Prof Michael Feneley. My thanks to them. Every co- author on the papers arising from this project has helped me, and my thanks are due to all. There have also been many others, such as the private laboratory services who never failed to help with specimen collection and shipping, whom I’m unable to name but would like to acknowledge. Likewise, I am immensely grateful to but unable to name the many people who volunteered as research subjects, particularly those whose families I studied and into whose homes and workplaces I was invited. If there are others I should have named but have omitted, my apologies as well as thanks to them. Lastly, I am deeply indebted to the bodies which funded this research. The National Heart Foundation of Australia awarded me a scholarship as well as providing research grant support.