The identification of novel genetic and epigenetic mechanisms in Lynch syndrome

Abstract

Lynch syndrome is an inherited disorder that mainly predisposes to early-onset colorectal and endometrial cancer. It is most commonly caused by the autosomal dominant inheritance of a loss-of-function mutation in one of the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 or PMS2). About 75 % of pathogenic variants in Lynch syndrome occur in the MLH1 and MSH2 genes, with a much lower frequency in other MMR genes. However, in up to 30 % of patients with a clinical suspicion of Lynch syndrome, a definitive pathogenic cause has not been identified. The aims of this thesis were to identify novel genetic and epigenetic mechanisms in Lynch syndrome. First, the pathogenicity of promoter variants in the DNA MMR genes was reviewed, and an experimental framework was developed to assess their effect on gene expression. Next, cDNA analysis and an inversion PCR strategy were designed to investigate an MSH2 inversion as a possible cause of early onset CRC. A novel paracentric inversion in MSH2 was identified and a diagnostic PCR test was developed. A recently identified DNA helicase in the DNA MMR pathway (MCM9) was also screened for germline variants in a cohort of suspected Lynch syndrome patients with unexplained MLH1 or MSH2 loss. In silico analysis identified variants of uncertain significance. Finally, a 3C strategy was optimised to identify a known enhancer-promoter interaction at the β-globin locus and then applied to the MLH1 locus to search for novel cis-regulatory regions in this gene. Functional assays including luciferase reporter assays and CRISPR-Cas 9 identified the first known enhancer of MLH1. A potentially pathogenic variant in the enhancer was also identified in a cohort of suspected Lynch syndrome patients with MLH1 loss in their tumours. In conclusion, the findings of this thesis highlight that non-coding variants, that remain undetected by routine clinical genetic tests, account for suspected cases of Lynch syndrome. Future research should aim to identify other regulatory elements of the DNA MMR genes and to develop internationally accepted experimental guidelines to assess their pathogenicity. This will ultimately facilitate the clinical management of these patients.