DYSMORPHOLOGY, ABNORMAL GROWTH AND COPY NUMBER VARIANTS IN AUTISM SPECTRUM DISORDER by Norazlin Kamal Nor A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy. Baltimore, Maryland December 2017 © Norazlin Kamal Nor 2017 All rights reserved Abstract Background and Purpose: Autism Spectrum Disorders (ASD) is a complex persistent neurodevelopmental disorder characterized by impairment in social interaction, communication difficulties and repetitive or stereotypic behaviors. In the past four decades, the prevalence of ASD has increased dramatically. The risk factors associated with ASD include genetic, environmental and possibly gene-environment interactions. Although the core features of ASD are well characterized, ASD presents heterogeneously with a wide spectrum of manifestations. These overlapping features or phenotypes are co- morbidities that occur in ASD and span developmental, medical, behavioral and psychiatric conditions. These co-morbid features can include dysmorphology and growth abnormalities. It is postulated that children with significant dysmorphology are more likely to have an underlying genetic etiology and may have a higher load of genes controlling risk to ASD, including a higher copy number variant (CNV) burden and more single location variants. CNVs are alterations of the DNA resulting in structural variants, including deletion and duplication of genome sequence. ASD sub-phenotypes (such as dysmorphology) offer the potential of determining distinct genetic etiologies and enhancing genotype-phenotype correlations in ASD. In this study, we first characterized growth abnormalities in children with ASD in the Study to Explore Early Development (SEED) study. To investigate genotype-phenotype associations in ASD, we determined the association between genome-wide CNV burden with dysmorphology and abnormal growth in SEED children and tested for association between ASD-associated CNVs with dysmorphology and growth abnormalities in children in the SEED study. ii Methods: The study population was drawn from the SEED Study, which was developed to identify risk factors for ASD in the prenatal and early post-natal period. To characterize abnormal growth patterns associated with ASD, we assessed growth abnormalities for all available anthropometric measures of growth (height, weight and head circumference), the bi-dimensional measure of body mass index (BMI), and a tri- dimensional growth measure of growth phenotype assessing the symmetry of growth involving all three modalities in a single individual. We examined genotype-phenotype associations between genome-wide estimated CNV burden with dysmorphology and abnormal growth. Finally, we investigated the association of specific CNVs reportedly associated with ASD for possible association with dysmorphology and abnormal growth in children in the SEED study. Results: Assessment of growth abnormalities in SEED 1 study showed females with ASD had short stature and a combination of short stature, microcephaly and normal weight compared to typically developing or control females. We found genome-wide CNV burden was negatively associated with dysmorphology, and CNV burden in recognized ASD genes was negatively associated with tall stature and macrocephaly; these associations varied by sex. Investigation of association between CNVs associated with ASD and abnormal growth revealed a potential shared genetic risk for ASD and short stature at CNV region 15p11.2, which was significantly associated only in females. iii Conclusions: These findings demonstrate the importance of taking into consideration the influence of sex in characterizing any association between growth abnormalities and/or dysmorphology and risk of ASD, as well as variability of reported genetic risk factors in ASD. There is potential shared genetic risk for ASD and growth abnormality that differs by sex, and this may lead to potential future clinical application in diagnosing of ASD that could be tailored to the child’s needs. Future directions: These results should be replicated in a different population, while expanding measurments of growth assessment to incorporate longitudinal change to better characterize growth abnormalities in ASD. Using a larger sample size and with parental genotyping information would enable CNV burden for de novo and rare CNVs to be considered. Whole exome sequencing would be a useful in excluding chromosomal abnormalities and non-chromosomal genetic syndromes when considering CNV burden associations. iv Thesis Readers: Margaret Daniele Fallin, Ph.D. (Co-Advisor) Terri H. Beaty, Ph.D. (Co-Advisor) Christine Ladd-Acosta, Ph.D. Julie Hoover-Fong, M.D., Ph.D. Brion Maher, Ph.D. v Abbreviations ADOS – Autism Diagnostic Observation Schedule ADI-R – Autism Diagnostic Interview- Revised ASD – Autism Spectrum Disorder BMI – Body Mass Index CAGS – Chromosomal Abnormalities and Non-chromosomal Genetic Syndromes CGI – Caregiver’s Interview CNV – Copy Number Variants CNV – Copy Number Variant DD – Children with Developmental Delay DRF – Dysmorphology Review Form DSM – Diagnostic and Statistical Manual of Mental Disorders HC – Head Circumference ID – Intellectual disability MCA – Multiple Congenital Anomalies POP – Typically developing children in the SEED Study SCQ – Social Communication Questionnaire SEED – Study to Explore Early Development SFARI – Simons Foundation Autism Research Initiative TGP – Trivariate Growth Phenotype vi ACKNOWLEDGEMENTS In the name of God, Most Gracious and Most Merciful, firstly I give my deepest gratitude to the Almighty, without whom none of this would be possible. I also give my greatest thanks to my wonderful parents Kamal Nor Mohd Yusof and Latifah Norma Mohamed, whose love and unwavering support carries me through the darkest hours. The one person who has helped me the most in giving encouragement, support and unwavering belief is my beloved son, Majid Abouzid, who has cajoled, rallied, bullied and cheered me in equal measure. I also give thanks to my husband, whose kindness and help has made the task of trying to juggle academic pursuit, work and family life lighter. I would like to thank my lovely siblings, Farah and Norazlan, and their families, for their love and for always being there. I would also like to thank my sponsors, the Malaysian Government Higher Education Ministry, and my university in Malaysia, the National University of Malaysia, for their financial support throughout these doctoral years. I need to say a special thanks to my department, the Paediatrics Department at the Faculty of Medicine at the National University, for supporting my academic endeavours. I am grateful for friends and extended family across continents from Malaysia to the United States. In this doctoral journey, a great many people have made an impression on me, and have been integral to any success I hope to achieve. Many of these people I met throughout the doctoral program at the Johns Hopkins Bloomberg School of Public Health. While it may not be possible to adequately thank and even name everyone who has provided support and encouragement throughout this program, I would like to highlight certain individuals to whom I owe immeasurable debt. Firstly, I would like to vii give my sincerest thanks to Dr. Dani Fallin, my advisor and mentor, who has, from the beginning of my journey at Hopkins, been a source of inspiration and admiration. She has taught with wisdom and kindness, with fairness and integrity, and if there is one thing I hope to take away from this experience, it is her lesson of reaching for the highest of scientific standards while developing and maintaining a supportive and collaborative research network. In addition, Dr. Terri Beaty has been an invaluable source of support, pragmatic wisdom and endless patience, and has made me feel able to straighten my spine and get through another day even when the challenges were daunting. In addition, I have learnt a lot of valuable lessons and tools from Dr. Julie Hoover-Fong, who is an inspiring advisor always ready to give her encouragement and support. I am also very grateful to have Dr. Chris Ladd-Acosta as a committee member and frequent advisor, and she has been unwavering in her help, kindness and willingness to guide. I also would like to thank the staff at both the Epidemiology and Mental Health departments at the Johns Hopkins School of Public Health, as well as the Institute of Genetic Medicine at the School of Medicine. Fran Burman and Matt Miller from the Epidemiology department are always ever helpful and supportive, and Michelle Maffett from the Mental Health department has been extremely kind and efficient in helping organize scheduling. I am deeply grateful to the SEED Study for the privilege of working on SEED data, and to participants and their families for their contribution to the advancement of research in the field of autism. I would like to extend my thanks also to the SEED Dysmorphology group and Dr. Stuart Shapira, for helping me better understand the dysmorphology study within SEED and for the use of dysmorphology data generated by the group. I would also like to viii thank Brooke Sheppard, who has since graduated from the school, for the painstaking groundwork she undertook that later became part of the foundation for my own work. I have met an amazing group of people at Hopkins, who have helped me immeasurably not only to survive the doctoral program, by giving invaluable advise and support, but also in making the