SEQUENCE ANALYSIS OF FAMILIAL NEURODEVELOPMENTAL DISORDERS by Joseph Mark Tilghman A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland December 2020 © 2020 Joseph Tilghman All Rights Reserved Abstract: In the practice of human genetics, there is a gulf between the study of Mendelian and complex inheritance. When diagnosis of families affected by presumed monogenic syndromes is undertaken by genomic sequencing, these families are typically considered to have been solved only when a single gene or variant showing apparently Mendelian inheritance is discovered. However, about half of such families remain unexplained through this approach. On the other hand, common regulatory variants conferring low risk of disease still predominate our understanding of individual disease risk in complex disorders, despite rapidly increasing access to rare variant genotypes through sequencing. This dissertation utilizes primarily exome sequencing across several developmental disorders (having different levels of genetic complexity) to investigate how to best use an individual’s combination of rare and common variants to explain genetic risk, phenotypic heterogeneity, and the molecular bases of disorders ranging from those presumed to be monogenic to those known to be highly complex. The study described in Chapter 2 addresses putatively monogenic syndromes, where we used exome sequencing of four probands having syndromic neurodevelopmental disorders from an Israeli-Arab founder population to diagnose recessive and dominant disorders, highlighting the need to consider diverse modes of inheritance and phenotypic heterogeneity. In the study described in Chapter 3, we address the case of a relatively tractable multifactorial disorder, Hirschsprung disease. We identified new risk genes in a relatively small cohort (190 probands) by combining statistical genetics with functional assays. We then used both known and novel genes ii and loci for quantifying individual genetic risk for Hirschsprung disease, on the basis of common and rare variant genotypes. In the fourth and final chapter, we address the case of a highly complex and heterogeneous disorder, autism spectrum disorder. We investigated the basis for exceptionally high genetic risk in 99 families having multiple females affected with autism, showing their risk originates in part from the same genes responsible for de novo risk in simplex families. However lack of significant results in gene discovery indicates that functional and external validation is needed for definitive gene finding even in this cohort characterized by high genetic risk. Thesis readers: Dr. Aravinda Chakravarti, PhD (advisor) Dr. Sarah Wheelan, MD, PhD iii Preface: In my graduate work, I have relied primarily datasets and patient cohorts that predated the beginning of my time in graduate school, and the quality and design of these has been fundamental to my success. I am exceedingly grateful not only to my former and current coworkers in the Chakravarti Lab and external collaborators, but to many others who preceded me. I am of course also very grateful to the many affected families, physicians, and genetic counselors who have furnished the collections and clinical phenotyping upon which our work depends. Foremost, I would like to specifically thank my mentor, Aravinda, who has given me many great opportunities and from whom I have learned a great deal. I especially appreciate the lengths to which Aravinda has gone to provide guidance following my transition to being a remote lab member following his move to New York University. In my time in the Chakravarti lab, I have valued the advice and friendship of many. I worked with Dr. Ashish Kapoor in my rotation project when I first began at Hopkins; he was part of the reason I joined the lab, and he was a valuable source of advice for the several years that we worked together in the lab. I have also especially valued the friendships of Drs. Sumantra Chatterjee and Michael Chou and of Nan Hu. The Human Genetics program has provided me with an excellent setting in which to complete my thesis, for which I am very grateful to Sandy Muscelli the program administrator, the program head Dr. Dave Valle, the rest of the executive committee, and the many preceptors who helped me feel welcome and enriched my time at iv Hopkins. I also count myself fortunate to have benefited from such an excellent peer group in the Human Genetics program and in the School of Medicine as a whole. I would like to thank Drs. Anthony Leung, Dan Arking, and Rick Huganir for their helpful input and support as members of my thesis committee, and I thank Sarah Wheelan for her support, for serving as chair of my thesis committee, and for serving as a reader for my thesis. I would also be remiss not to acknowledge the critical role that my undergraduate mentors played in the formation of my identity as a scientist. The combined support and scientific responsibility that I was given as an undergraduate by Drs. David Marsh and Fiona Watson is something that I had never imagined possible as I undertook my undergraduate career. The support I had in the Biology department and Washington and Lee was truly amazing, even outside of my research mentors. And my great experience in Dr. Stephen DiFazio’s lab at West Virginia University, doing genetics research for the first time, was what inspired me to embrace genetics as lens through which to better understand biology. Of course, I am also very grateful for the support and constant arguing of my brothers, parents and the rest of my family, which prepared me well for a career in science. As I have worked to complete my thesis, the support of Dr. Yiwen Dai, my wife, has been critical. And, in the current crisis, I am eternally grateful for my son’s several hour naps each afternoon. v Table of Contents ABSTRACT: ............................................................................................................................................... II PREFACE: ................................................................................................................................................. IV LIST OF TABLES ...................................................................................................................................... VIII LIST OF FIGURES ....................................................................................................................................... X CHAPTER 1: INTRODUCTION .................................................................................................................... 1 CHAPTER 2: IDENTIFICATION OF MENDELIAN DISEASE GENES IN AN ISRAELI-ARAB COMMUNITY CHARACTERIZED BY CONSANGUINITY .................................................................................................... 11 2.1 CHAPTER INTRODUCTION ........................................................................................................................... 11 2.2 EXOME SEQUENCING AND ANALYSIS METHODS ............................................................................................. 13 2.3 CASE 1: A PIGN MUTATION RESPONSIBLE FOR MULTIPLE CONGENITAL ANOMALIES–HYPOTONIA–SEIZURES SYNDROME 1 (MCAHS1) - REPRINTED WITH PERMISSION FROM AM J MED GENET A ................................................................ 14 Introduction: ......................................................................................................................................... 14 Clinical Summary: ................................................................................................................................. 16 RESULTS: ...................................................................................................................................................... 18 Discussion: ............................................................................................................................................ 19 2.4 CASE 2: AN EDAR MUTATION RESPONSIBLE FOR HYPOHIDROTIC ECTODERMAL DYSPLASIA .................................... 22 Introduction: ......................................................................................................................................... 22 Clinical Summary: ................................................................................................................................. 23 Results: ................................................................................................................................................. 24 Discussion: ............................................................................................................................................ 25 2.5 CASE 3: AN INTERSTITIAL 3P26 DELETION RESULTING IN TERMINAL 3P DELETION SYNDROME WITH INCOMPLETE PENETRANCE ................................................................................................................................................. 28 Introduction: ......................................................................................................................................... 28 Clinical Summary: ................................................................................................................................. 29 Results: ................................................................................................................................................. 30 Discussion: ...........................................................................................................................................