The Pennsylvania State University The Graduate School STUDYING DIVERTICULAR DISEASE USING FAMILIAL BASED NEXT- GENERATION SEQUENCING TO IDENTIFY POTENTIAL CAUSATIVE VARIANTS A Dissertation in Cellular and Molecular Biology by Joel Leonard Coble © 2020 Joel Leonard Coble Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2020 The dissertation of Joel Coble was reviewed and approved by the following: James Broach Chair and Professor of the department of Biochemistry and Molecular Biology Director of the Penn State Institute for Personalized Medicine Dissertation Advisor Co-Chair of Committee Glenn Gerhard Chair and Professor of the department of Medical Genetics and Molecular Biochemistry Co-Chair of Committee Professor of Pathology and Laboratory Medicine Walter Koltun Chief, Division of Colon and Rectal Surgery Professor, Biomedical Sciences Gregory Yochum Associate Professor, Department of Biochemistry and Molecular Biology Milena Bogunovic Assistant Professor, Department of Microbiology and Immunology ii Abstract Diverticular disease (DD) is a multistage gastrointestinal disorder of the colon. DD begins with colonic outpouching, creating diverticula. This stage of disease is called diverticulosis and is asymptomatic. Approximately 32.6% of the population between 50-59 years of age have diverticulosis, and that number increases to 71.4% of individuals ≥ 80. When individual diverticula become inflamed, the disease has progressed to diverticulitis. Approximately 20% of individuals with diverticulosis will develop diverticulitis. Only 1-4% of those individuals will require surgical treatment. Naturally occurring genetic variants can predispose a person to a particular disease. This concept has yet to be thoroughly investigated for DD. Familial and population-based studies suggest 40-53% of DD is a result of genetics, while the remainder is a result of environmental factors such as diet or smoking. The overarching goal of this research was to use next-generation sequencing (NGS) to identify possible causative variants in families with DD and further investigate the mechanistic role of those variants and genes in the pathogenesis of DD. We recruited two families with DD at the Penn State Health Inflammatory Bowel Disease Center biorepository. We then performed exome sequencing on each family and assessed rare variants (<1% minor allele frequency (MAF)) matching the segregation pattern of disease for a possible biological role in the disorders. These data led to the identification of genes LAMB4 and COL1A1, which each contain variants in the two families with DD. iii LAMB4 and COL1A1 are critical for a functional extracellular matrix. LAMB4 is a member of the laminin family of genes, and COL1A1 is a member of the collagen family of genes. This led to the hypothesis where structurally or functionally damaging variants in these genes may decrease the integrity of the basement membrane in the colon, leading to greater susceptibility for outpouching to occur. We then performed targeted sequencing for LAMB4 or COL1A1 in 148 non-familial cases. This sequencing allowed for a comparison of patients with DD, also having a variant in one of the genes, to patients with DD that have no variants in these genes. Immunohistochemistry was then performed on colon tissue samples from those individuals to look at protein localization and expression. LAMB4 localizes to the myenteric plexus of the colon, and DD patients with LAMB4 variants have significantly decreased expression of LAMB4 compared to healthy control patients without variants in LAMB4 or compared to patients with DD but no variants in LAMB4. The localization and reduced expression in patients with both DD and variants in LAMB4 provide additional supporting evidence for the role of the enteric nervous system in the pathogenesis of DD. COL1A1 showed no discernible change in the pattern of expression or localization between and within similar groups. This research has identified potential causative variants for DD and assessed the functional role of the LAMB4 and COL1A1 proteins in DD. Although this data has helped to shed light on the pathogenesis of DD, further research is iv needed to fully elucidate the exact mechanistic role of these genes in DD to provide new and novel pathways to be the focus of treatment or intervention. v Table of Contents List of Figures…………....………………………………………………..….…ix List of Tables………………………………………………………………........xi List of Abbreviations………...………...…………………………………........xii Acknowledgments……………………...……………………………….....….xiv Chapter 1: Introduction…………………………………………………........1 1.1.1 Epidemiology and financial burden of gastrointestinal disorders……1 1.1.2 Epidemiology and financial burden of diverticular disease…………..1 1.2.1 Gastrointestinal and colonic anatomy………………………………….2 1.2.2 Enteric Nervous System anatomy………………………………………6 1.2.3 Colonic microbiome………………………………………………………7 1.2.4 Extracellular matrix…………………………………………………........8 1.3.1 Pathogenesis of diverticular disease…………………………………...9 1.3.2 Genetics of diverticular disease……………………………………….11 1.4.1 Disorders with DD comorbidity……………………………………..….14 1.4.2 Colonic wall changes in conditions with diverticular disease as comorbidity…………………………………………………………………......15 1.4.3 Neuronal/motility changes in diseases with diverticular disease comorbidity………………………………………………………………......…21 1.5 Next-generation sequencing……………………………………………..24 1.6 Summary…………………………………………………………………...24 Chapter 2: Identification of a rare LAMB4 variant associated with familial diverticulitis through exome sequencing……………………...26 2.1 Abstract…………………………………………………………………….27 2.2 Introduction………………………………………………………………...28 2.3 Results……………………………………………………………………...30 2.3.1 Exome sequencing identified a rare variant in the LAMB4 gene that segregated with diverticulitis………………………………………………….30 2.3.2 LAMB4 variants are associated with decreased LAMB4 protein expression in colonic myenteric plexus……………………………………..33 2.3.3 Additional rare LAMB4 variants are present in sporadic diverticulitis patients………………………………………………………………………….37 2.3.4 Patients with diverticulitis and variants in LAMB4 showed decreased expression of LAMB4 in the myenteric plexus……………………………...39 2.4 Discussion………………………………………………………………….43 2.5 Materials and Methods……………………………………………………47 2.5.1 Patients and samples……………………………………………...…...47 2.5.2 Whole-exome sequencing…………………………………………......48 2.5.3 Bioinformatics pipeline………………………………………………….48 2.5.4 Variant filtering…………………………………………………………..49 2.5.5 Targeted and confirmation sequencing……………………………….49 vi 2.5.6 Immunohistochemistry of LAMB4……………………………………..50 2.5.7 Quantification of images………………………………………………..51 2.6 Accession Number………………………………………………………..52 2.7 Funding……………………………………………………………………..52 Chapter 3: Identification of a rare COL1A1 variant associated with familial diverticulitis through exome sequencing…………......………53 3.1 Abstract…………………………………………………………………….54 3.2 Introduction……………………………………………………………...…55 3.3 Results……………………………………………………………………...58 3.3.1 Exome sequencing of recruited family identified two variants in COL1A1…………………………………………………………………………58 3.3.2 Non-familial sporadic diverticulitis patients…………………………..59 3.3.3 COL1A1 localization and abundance did not correlate with disease status or presence of variant…………………………………………………61 3.4 Discussion………………………………………………………………….63 3.5 Materials and Methods……………..…………………………………….68 3.5.1 Patients and samples…………………………………………………..68 3.5.2 Whole-exome sequencing…………………………………………......69 3.5.3 Bioinformatics pipeline………………………………………………….69 3.5.4 Variant filtering…………………………………………………………..69 3.5.5 Targeted and confirmation sequencing……………………………….69 3.5.6 Immunohistochemistry of COL1A1..…………………………………..69 Chapter 4: Conclusion……………………………....………………………70 4.1.1 Genomics of colorectal disorders……………………………………..71 4.1.2 Identification of the LAMB4 variant……………………………….......71 4.1.3 Identification of the COL1A1 variant…………………………………..73 4.2.1 Limitations of this research…………………………………………….74 4.2.2 Value and benefits of this research…………………………...………79 4.3 Future directions……………………………………………………...…...80 4.4 Conclusion…………………………………………………………………84 Appendix: Identification of two rare variants associated with familial Crohn’s Disease through exome sequencing…………..…….86 5.1 Background………………………………………………………………...87 5.1.1 Epidemiology and financial burden of Crohn’s Disease…..………..87 5.1.2 The pathogenesis of Crohn’s Disease………………………………..87 5.1.3 Genetics of Crohn’s Disease…………………………………………..90 5.2 Introduction………………………………………………………………...92 5.3 Results………………………………………………………………...……94 5.4 Discussion………………………………………………………………….95 5.5 Materials and Methods……………………………………………….…103 vii 5.5.1 Recruitment of the affected family…………………………………...103 5.5.2 Exome sequencing of the recruited family………………………….104 5.6 Conclusion………………………………………………….…………….106 References……………………………………………………….…………..107 viii List of Figures 1-1 Schematic representation of the human colon…………………………………..3 1-2 Diagram of the gastrointestinal layers and innervation of the colon…………..4 1-3 Colonic crypts……………………………………………………………………….6 1-4 Schematic illustration of some of the possible mechanisms that may contribute to the development of diverticular disease……………………………..10 1-5 Schematic illustration of the observation that a diverticulum often occurs near sites of vascularization………………………………………………………………..11 1-6 Relative risk (standardized incidence ratio) in siblings according to the age at diagnosis of the index case…………………………………………………………..12