Studying Diverticular Disease Using Familial Based Next-Generation Sequencing to Identify Potential Causative Variants
Open Access
- Author:
- Coble, Joel Leonard
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 31, 2018
- Committee Members:
- James Riley Broach, Dissertation Advisor/Co-Advisor
Glenn Stephen Gerhard, Committee Chair/Co-Chair
Gregory Steven Yochum, Committee Member
Walter Alex Koltun, Committee Member
Milena Bogunovic, Outside Member
James Riley Broach, Program Head/Chair
James Riley Broach, Committee Chair/Co-Chair - Keywords:
- Genetics
Diverticulitis
diverticular disease
Next-Generation Sequencing
Familial based sequencing
LAMB4
Laminin
Collagen
COL1A1 - 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. 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 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.