Medicine (RMH) - Theses
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ItemGenotypic and phenotypic studies in hyperplastic polyposis syndrome( 2010)Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps and colorectal cancer (CRC) risk, and is related to a third pathway to human CRC known as “serrated neoplasia pathway”. Several genetic aberrations have been linked to HPS, including frequent BRAF and KRAS mutations and aberrant methyation known as CIMP. At least half of CRCs arising in HPS also show a CpG island methylator phenotype (CIMP). CIMP usually involves a wide panel of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). DNA methylation is one of many epigenetic factors that are important for cellular differentiation, gene regulation and genomic imprinting. The aberrant DNA methylation found in CIMP phenotype may be a consequence of dysfunctional machinery involved in establishing and maintaining normal DNA methylation. DNA methyltransferases DNMTs (DNMT1, -3A, -3B and -3L) are a family of proteins responsible for transfer of methyl groups specifically to cytosine in CpG dinucleotides of DNA. Given the role of DNMT’s in DNA methylation, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, together with variation in BRAF and KRAS genes, in the molecular pathogenesis of HPS. A candidate gene approach identified no novel sequence variants that showed significant association with HPS. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01), a finding deserving further study. The DNMT1, DNMT3A and DNMT3B promoters were un-methylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells, and this may be important in expression of the gene in vivo. Expression levels of DNMT3L in polyps and normal terminal ileum tissue varied inversely with methylation may be a functionally important defect in gut. BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps. In addition, DNMT3L promoter hypermethylation was more often found in polyps harbouring KRAS mutations (p=0.0053). Attempts with candidate gene DNA methylation approaches were unsuccessful or unconvincing in assigning a single responsible gene responsible for HPS. We therefore applied a hypothesis-free approach by studying serrated polyps with Infinium HumanMethylation27 BeadChip Illumina® genome-wide promoter methylation. This approach identified three pathways: homedomain and basic-helix-loop transcription factors, homeobox genes and the ERK pathway, demonstrating co-ordinate methylation of genes in the polyps compared to normal controls; we speculated that the functional consequences, could explain HPS pathogenesis. This study has found that DNMT3L may be of a functional importance in gut functioning and in particular is associated with polyps harbouring KRAS mutation. A non-candidate gene approach also identified important pathways that may explain the complex aetiology of HPS.