University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Genomic Approaches to Understanding Variable Expressivity in Alagille Syndrome and Genetic Susceptibility to Biliary Atresia Ellen Tsai University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Bioinformatics Commons, and the Genetics Commons Recommended Citation Tsai, Ellen, "Genomic Approaches to Understanding Variable Expressivity in Alagille Syndrome and Genetic Susceptibility to Biliary Atresia" (2013). Publicly Accessible Penn Dissertations. 932. https://repository.upenn.edu/edissertations/932 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/932 For more information, please contact [email protected]. Genomic Approaches to Understanding Variable Expressivity in Alagille Syndrome and Genetic Susceptibility to Biliary Atresia Abstract The biliary system facilitates transport of bile from the liver, where it is produced, to the gall bladder, where it is stored and later released to aid digestion. Obstruction and defects in the biliary system are a primary indication of liver transplantation in children. Alagille syndrome (ALGS) and biliary atresia (BA) are two cholangiopathies with different etiopathology, but both affect bile flow and can lead ot end-stage liver disease. ALGS is a dominant, multisystemic disease caused by mutations in JAG1 or NOTCH2 characterized by intrahepatic ductopenia and variable expressivity. BA is a multifactorial disease characterized by necroinflammatory obliteration of the extrahepatic biliary tree with unknown etiology. I used genomic tools including genome-wide gene expression analysis, genome-wide association (GWA) studies of single nucleotide polymorphisms (SNPs) and copy number variation (CNVs), and exome sequence analysis to uncover the genetic component to variable expressivity in ALGS and susceptibility to BA. In the first part of this thesis, I searched for genetic modifiers of the ALGS phenotype by investigating the effects of the non-mutated JAG1 allele, characterizing downstream gene expression of Notch signaling, and performing a SNP and CNV GWA studies of liver disease severity. This work revealed that cardiac defects are correlated with liver disease severity and the presence of butterfly erv tebrae. I uncovered an enhancer element upstream of THBS2 that may affect the progression of liver disease in ALGS patients. In the second part of this thesis, I looked for markers of genetic susceptibility to BA, using an association study, exome sequencing of unrelated patients, and exome sequencing and CNV analysis of familial recurrence of BA-associated phenotypes. This work suggested several BA candidate genes, including FOXA2, which we propose as a BA susceptibility gene. Additionally, fine mapping of a previously reported susceptibility locus demonstrated an intronic risk allele in ADD3. Exome sequencing of candidate genes and a pathway-enriched analysis revealed alterations in several other genes that support the hypothesis that BA is a genetically heterogeneous disorder. This work identifies multiple areas for future research to better understand the genetics of these two biliary disorders. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Genomics & Computational Biology First Advisor Nancy B. Spinner Second Advisor Marcella Devoto Keywords copy number variation, exome sequencing, genome-wide association study Subject Categories Bioinformatics | Genetics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/932 GENOMIC APPROACHES TO UNDERSTANDING VARIABLE EXPRESSIVITY IN ALAGILLE SYNDROME AND GENETIC SUSCEPTIBILITY TO BILIARY ATRESIA COPYRIGHT 2013 Ellen A. Tsai This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ To my father, Thank you for the sacrifices you have made for us. iii ACKNOWLEDGMENTS Each and every graduate experience is unique, and mine has been blessed with a wealth of resources and incredible opportunities. Foremost, I would like to thank my advisors, Nancy and Marcella, for their support and supervision. They have given me expert advice over the past five years not only in science but also in life. Their boundless positive energy helped carry me through the ups and downs of graduate research. I am truly grateful for the opportunity to conduct research in such nourishing environment. I would also like to thank members of the laboratory. Their kindness and friendship have created such a pleasant atmosphere. I am indebted to many colleagues for their help throughout the years: Laura C, Joanne, Rob, Jen, Melissa, Anne, Laura L, Grace, Henry, Chris, Debbie, Ramki, Ying, and Alex. I have also been fortunate to mentor some incredibly talented students, Carly, Matt, and Andrew: I have learned much from you as well. Because of the interdisciplinary nature of genomics research, I have been fortunate to work with many clinicians and scientists. I would like to thank Barbara Haber, Kathy Loomes, Randy Matthews, Pierre Russo, Ian Krantz, and Li-San Wang for their scientific discussions and collaboration. In addition, I would like to also thank the members of my committee, Shane Jensen, Paul Gadue, and Mingyao Li, for their advice throughout the progress of my dissertation work. At Penn, I have made so many amazing friends who challenged me to grow as a person. Last but not least, I am grateful for the support from my wonderful family. They believed in me even during the times when I doubted myself. This dissertation would have impossible without all of these wonderful people who supported and motivated me throughout this entire process. They have my unyielding gratitude. This research was supported by NIH grants, R01-DK081702 and R01-DK090045, the Fred and Suzanne Biesecker Foundation, and a NHGRI training grant, T32-HG000046. iv ABSTRACT GENOMIC APPROACHES TO UNDERSTANDING VARIABLE EXPRESSIVITY IN ALAGILLE SYNDROME AND GENETIC SUSCEPTIBILITY TO BILIARY ATRESIA Ellen A. Tsai Nancy B. Spinner, PhD Marcella Devoto, PhD The biliary system facilitates transport of bile from the liver, where it is produced, to the gall bladder, where it is stored and later released to aid digestion. Obstruction and defects in the biliary system are a primary indication of liver transplantation in children. Alagille syndrome (ALGS) and biliary atresia (BA) are two cholangiopathies with different etiopathology, but both affect bile flow and can lead to end-stage liver disease. ALGS is a dominant, multisystemic disease caused by mutations in JAG1 or NOTCH2 characterized by intrahepatic ductopenia and variable expressivity. BA is a multifactorial disease characterized by necroinflammatory obliteration of the extrahepatic biliary tree with unknown etiology. I used genomic tools including genome-wide gene expression analysis, genome-wide association (GWA) studies of single nucleotide polymorphisms (SNPs) and copy number variation (CNVs), and exome sequence analysis to uncover the genetic component to variable expressivity in ALGS and susceptibility to BA. In the first part of this thesis, I searched for genetic modifiers of the ALGS phenotype by investigating the effects of the non-mutated JAG1 allele, characterizing downstream gene expression of Notch signaling, and performing a SNP and CNV GWA studies of liver disease severity. This work revealed that cardiac defects are correlated with liver disease severity and the presence of butterfly vertebrae. I uncovered an enhancer element upstream of THBS2 that may affect the progression of liver disease in ALGS patients. In the second part of this thesis, I looked for markers of genetic susceptibility to BA, using an association study, exome sequencing of unrelated patients, and exome sequencing and CNV analysis of familial recurrence of BA- v associated phenotypes. This work suggested several BA candidate genes, including FOXA2, which we propose as a BA susceptibility gene. Additionally, fine mapping of a previously reported susceptibility locus demonstrated an intronic risk allele in ADD3. Exome sequencing of candidate genes and a pathway-enriched analysis revealed alterations in several other genes that support the hypothesis that BA is a genetically heterogeneous disorder. This work identifies multiple areas for future research to better understand the genetics of these two biliary disorders. vi TABLE OF CONTENTS ACKNOWLEDGMENTS ....................................................................................... iv! ABSTRACT ........................................................................................................... v! LIST OF TABLES ................................................................................................. xi! LIST OF ILLUSTRATIONS ................................................................................ xiii! CHAPTER 1: Introduction ................................................................................... 1! 1.1! Approaches to Understanding the Complexity of Genetic Disorders .................................... 1! 1.1.1! Genetic Models for Modifier and Susceptibility Genes ................................................... 1! 1.1.2! Methods to Detect Genes for Complex Disorders .......................................................... 4! 1.2 The Hepatobiliary System