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Epigenetic Regulation in the Placenta and Its Role in Fetal Growth

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Epigenetic Regulation in the Placenta and Its Role in Fetal Growth Epigenetic regulation in the placenta and its role in fetal growth by Jose Carlos Pinto Barreto Ferreira A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by Jose Carlos Pinto Barreto Ferreira 2011 Epigenetic regulation in the placenta and its role in fetal growth Jose Carlos Pinto Barreto Ferreira Doctor of Philosophy Institute of Medical Science University of Toronto 2011 Abstract Fetal growth potential reflects a complex regulatory system delivered by genetic and environmental factors acting directly on the fetus or through the placenta. Compromise of this potential, as seen in intrauterine growth restriction (IUGR), is associated with increased perinatal mortality and short and long term morbidity. The expression of several genes has been shown to be disturbed in placentas of fetuses with growth restriction. However, the primary causes for these changes have not yet been elucidated. I proposed that epigenetic mechanisms, specifically DNA methylation, may be involved in placental development leading to modulation of the expression of specific genes, and that their altered regulation will impact fetal development and growth. My primary objective was to identify DNA methylation variation in placenta, in association with variation of gene expression and with poor fetal growth. I used a global genomic screening approach, with 24 selected placental samples, from newborns considered IUGR or normal controls, to identify candidate target genomic regions carrying epigenetic alterations. Candidate regions were followed up, by expression analysis of corresponding regulated genes, for associations with altered expression and by targeted ii methylation analysis in an expanded cohort of 170 samples, for associations with birthweight percentile. I analyzed methylation variation at imprinting centers (IC), gene promoters and CpG islands. In two genome-wide case control screening studies using distinct commercial microarray platforms I identified approximately 68 differentially methylated autosomal candidate genomic regions overlapping gene promoters. Hypomethylated CpGs mapping to gene promoters were found to be more abundant in placentas of growth restricted newborns than in controls. One of the most interesting candidates, WNT2, was analyzed in an extended sample cohort and showed an association of high promoter methylation to low expression as well as low birthweight percentile. This gene is involved in a pathway that diverts cells from programmed apoptosis. It is highly expressed in placenta, and in mice, targeted biallelic inactivation of Wnt2 has been shown to cause poor growth and perinatal death in 50% of the affected pups. These findings support the hypothesis that dysregulation of epigenetic mechanisms are involved in abnormal placental development and can impact fetal growth. iii Acknowledgments It is at this point in time, when I start thinking about the people that I have to acknowledge, that I realize how fortunate and how thankful I have to be for being able to engage so many people who have contributed, helped and/or supported all the work I have done during those past 4-5 years. So, you, the reader, prepare yourself for the big list that follows: I first would like to thank the patients who participated in this study and their physicians. They are the subject and inspiration for all human medical research. I thank my funding agency for the scholarship I was awarded - FCT (Science and Technology Foundation), Lisbon, Portugal - SFRH / BD / 28642 / 2006 - funded by POPH (Operational Program for Human Potential) co-participated by FSE (European Social Fund) and by national funds from MCTES (Ministry of Science, Technology and Higher Education) –the agency that funded the research – Canadian Institute for Health and Research (CIHR) - and the Center for Applied Genomics. I thank the staff from the Research Centre for Women’s and Infants’ Health BioBank program of the CIHR Group in Development and Fetal Health (CIHR #MGC-13299), the Samuel Lunenfeld Research Institute and the Department of Obstetrics & Gynecology of Mount Sinai Hospital/University Health Network for the human specimens used in my studies. In particular, I would like to mention Dragica Curovic, Melanie Henriques, Ruchita Uxa, Ljiljana Petkovic, Richard Sullivan, Nora Tsao, Dr. Lee Adamson, for their insights and support. I truly and sincerely appreciated the relationships I had with all staff members of this wonderful resource made available to me and other researchers. I thank all my friends, student colleagues, post-doctoral fellows and technicians from Dr. Rosanna Weksberg’s laboratory, past and present, for all their help and support, for passing on their knowledge, for the fruitful and challenging discussions (academic, politic, etc.). Listed in alphabetic order, here are their names – Adam Smith, Andrea Stachon, Chunhua Zhao, Darci Butcher, Daria Grafodatskaya, Jonathan Shapiro, Lin Guo, Richard McCurdy (my Espresso buddy), Sanaa Choufani, Yi-An Chen, Youliang Lou. I also want to thank the summer students Sarah Ickowicz for her collaboration, and Rageen Ragendram for his collaboration and for the nice academic and entertaining political and technical conversations. iv I thank the administrative assistants of my supervisor, Kamalina Gupta and Khadine Wiltshire, for all their help and support. I also want to thank Dr. David Chitayat, Cheryl Shuman and Micki Thomas for all the help in the organization of the collection of samples, on consent and ethics issues and their friendship and support. I thank the friends from other labs for their friendship, in special, Elena Samiltchouk, Dalila Pinto, Pedro Castelo-Branco and their spouses. I also want to thank Schin-Itchi Horike for his collaboration. I also thank my colleagues from the Obstetrics and Gynecology Department and from the Pathology Department of Mount Sinai Hospital, including Dr. John Kingdom, Dr. Sarah Keating, Dr. Greg Ryan, Dr. Alan Bocking, for all the help, support, teaching and friendship. I also want to mention Dr. Ariadna Grigoriu for the wonderful collaborative work and friendship. I thank Theodore Chiang, Shuye Pu and Dr. Shoshana Wodak from the Center for Computational Biology, for being always available to help with my analytical and statistical questions. I thank the members of my thesis supervisory committee: Dr. Johanna Rommens, Dr. Lucy Osborne and Dr. Lee Adamson for their guidance and support. Their ideas and suggestions were precious for the development of my research. To my supervisor, Dr. Rosanna Weksberg, I thank for all the knowledge, the SUPPORT, help, friendship and effort she made to make me reach my goals. In spite of her many commitments in the last months she has been always available and I am sincerely thankful for that. I also want to thank my teachers at the courses I took and the friends I made – Carlos Ruiz, Gwen Schwartz – while taking those courses. Finally, I want to thank my family for the support in spite of my absence and, in special, my wife Maria Amélia Paiva, for her love, support and for having to stand one year of separation so that I could accomplish this work. v Contributors Dr. Sanaa Choufani, a research associate in Dr. Rosanna Weksberg’s laboratory, contributed with clarification of the epigenetic and imprinting concepts, experimental design, pyrosequencing technical support and data analysis. Dr. Daria Grafodatskaya, a current post-doctoral fellow in Dr. Rosanna Weksberg’s laboratory, contributed to the work described in Chapter 8 and to the development of the array technology in the laboratory and data analysis. Dr. John Kingdom, a physician specialist in Obstetrics and Maternal-Fetal Medicine, contributed with clarification of the concepts of intrauterine growth restriction, small for gestational age and placenta insufficiency, experimental design and facilitation of sampling process. Dr. Sarah Keating, a pathologist in the Pathology Department of Mount Sinai Hospital, contributed with clarification of placenta pathology concepts and experimental design, did the pathology assessments of the placentas and provided all the pathology images used in this thesis. Dr. Shin-Itchi Horike and Dr. M. Meguro-Horike, former postdoctoral fellows in Dr. Steve Scherer’s Laboratory, contributed to the work described in Chapter 4 Sarah Ickowicz, a former summer student in Dr. Rosanna Weksberg’s laboratory, contributed to the work described in Chapter 5. Yi-An Chen, a current MSc student in Dr. Rosanna Weksberg’s laboratory, contributed to the work described in Chapter 5. Dr. Ariadna Grigoriu, a former Maternal-Fetal Medicine fellow in the Obstetrics and Gynecology Department of Mount Sinai Hospital, contributed to the work described in Chapter 6. Rageen Ragendram, a summer student in Dr. Rosanna Weksberg’s laboratory, contributed to the work described in Chapter 8. Theodore Chiang, a Bioinformatics Analyst in the Center for Computational Biology of the Hospital for Sick Children, contributed to the work described in Chapter 8. vi Table of Contents Acknowledgments .......................................................................................................................... iv Contributors ................................................................................................................................... vi Table of Contents .........................................................................................................................
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