Exploring Genetic Susceptibility: Using a combined systems biology, in vitro and ex vivo approach to understand the pathology of ulcerative colitis

Brooks, Johanne (2018) Exploring Genetic Susceptibility: Using a combined systems biology, in vitro and ex vivo approach to understand the pathology of ulcerative colitis. Doctoral thesis, University of East Anglia.

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Abstract

The overall aim of this PhD is to use a multidisciplinary approach to determine the
function of Ulcerative Colitis (UC) associated SNPs, to help understand the role of SNPs in
the pathogenesis of UC in general and in a patient specific context.
UC is a chronic, relapsing inflammatory disease of the large bowel for which the aetiology
is thought to be a trifecta of 1) dysregulation of the immune system in response to 2) an
environmental trigger in a 3) genetically susceptible host. Genetic susceptibility or
susceptibility loci for UC have been identified by Genome Wide Associations Scanning and
subsequent fine mapping and deep sequencing.
This work intended to further characterise these susceptibility loci at a global level and a
patient specific level using both a systems biology approach and experimental validation
of the in-silico work. Using publicly available datasets non exonic UC associated SNPs were
functionally annotated to regulatory regions within the genome. Exonic SNPs were also
analysed looking at impacts in protein linear motifs and splice enhancement motifs.
Bioinformatics was used to identify interacting proteins and create a UC-interactome
network. This suggested that UC was a disease of fine regulators as opposed to a disease
of specific target proteins.
Analysis of the UC-interactome identified the focal adhesion complex (FAC) that is
involved in regulating wound healing as major component of the network. One member
of the FAC, Leupaxin (LPXN), was identified as a potential target for validation. Using
CRISPR-Cas9 technology, LPXN overexpressing cell lines and knock out cell lines were
created. Wound healing assays and cytokine analysis identified that overexpression of
LPXN impaired wound healing and reduced the secretion of MCP-1. In addition, using
genotyped colonic biopsies from UC patients and control patients in a polarised in vitro
organ culture (pIVOC) system we show that the LPXN risk allele may impact on cytokine
production.
Finally, UKIBD genetics consortium data was used to access a pilot dataset of 58 patients’
SNP profiles from Immunochip data who were patients at the Norfolk and Norwich
University Hospital to create patient-specific UC-interactomes. Analysis of these
footprints identified convergent interacting proteins affected by multiple SNPs and novel
pathogenic pathways.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User:	Jackie Webb
Date Deposited:	18 May 2018 12:30
Last Modified:	31 Mar 2021 00:38
URI:	https://ueaeprints.uea.ac.uk/id/eprint/66935
DOI:

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