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Title: Investigating the mechanisms of pathogenesis underlying inherited colorectal adenomatous polyposis
Author: Azzopardi, Duncan
ISNI:       0000 0004 2749 1065
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2009
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In order to investigate the role of nonsynonymous variants of the APC gene in inherited predisposition to colorectal adenomas (CRAs) the entire APC ORF was sequenced in 691 unrelated North American patients with CRAs and 969 healthy controls. There was significant over representation of rare inherited nonsynonymous variants in patients who did not carry conventional pathogenic mutations in APC or MUTYH (P = 0.0113) when compared to patients with familial adenomatous polyposis (FAP) and MUTYH associated polyposis (MAP). The over representation was highest in non-FAP non-MAP patients with 11 to 99 CRAs (P = 0.0103). More non-FAP non-MAP patients carried rare nonsynonymous variants within the functionally important p-catenin down-regulating domain compared with healthy controls (P = 0.0166). In silico analyses predicted that approximately 46% of the variants identified were expected to affect function. Functional characterisation in vitro showed that 7 of 16 nonsynonymous variants altered p-catenin-regulated transcription consistent with a role in predisposition to CRAs. An optimum level of p-catenin signalling is proposed to drive colorectal tumourigenesis, mediated by selection for APC genotypes retaining one, or rarely two, 20 amino acid p-catenin down regulating repeats (20AARs). We investigated the mechanism through which the APC variant E1317Q contributes to colorectal tumourigenesis. We compared the somatic mutation spectra of APC in tumours from AFAP patients that did (Family B) or did not (Family S) co-inherit E1317Q. Significant differences were identified between these tumours, 8.2% of tumours carrying E1317Q had somatic mutations predicted to result in mutant polypeptides retaining a single 20AAR, as compared to 62.1% of those which did not carry this variant (P=5.64x10 9). In vitro assays showed that E1317Q significantly impaired p-catenin regulated transcription when expressed with 'weak' truncating mutations (P<0.05) suggesting that E1317Q relaxes the target for tumourigenic somatic APC mutations through its own effects on p-catenin-associated signalling. Inherited mutations in the MUTYH gene predispose to an autosomal recessive disorder characterise by multiple CRAs and carcinomas (MAP). MUTYH is a DNA glycosylase which removes adenines that are misincorporated opposite 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG), one of the most stable products of oxidative DNA damage. Tumours from patients with MAP display a high proportion of somatic G:C->T:A mutations due to a failure to repair these mismatches, and it is presumed that this mutator phenotype drives tumourigenesis. We studied the response of primary MUTYH-deficient fibroblasts to oxidative stress and found that significantly more of these cells survived exposure to hydrogen peroxide and f-butyl-hydroperoxide as compared to wild type cells. We found that MUTYH-deficient cells failed to enter apoptosis and showed that this may be mediated via an inability to recruit Rad9 to the correct nuclear position, indicating a failure to engage the 9-1-1 DNA damage sensor complex. Consistent with this, we found that MUTYH-deficient cells failed to activate the downstream checkpoint protein Chk1, after exposure to oxidative stress. We propose that MAP-associated tumourigenesis is driven by failure to undergo apoptosis in conjunction with an underlying mutator phenotype.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available