The role and molecular characteristics of constitutional MLH1 epimutation in young-onset cancer.

Lynch syndrome, the commonest form of familial young-onset cancer, is typically caused by germline sequence mutations within the DNA mismatch repair genes, usually MLH1 or MSH2. Constitutional MLH1 epimutation, characterised by monoallelic promoter methylation and transcriptional silencing throughout normal somatic tissues, is an alternative cause of cancer predisposition in mutation-negative cases.This study aimed to further define the role of MLH1 epimutations in cancer causation and their molecular basis. Mutation-negative Lynch syndrome cases with MLH1-deficient tumours were screened for constitutional epimutation and allelic expression imbalance (AEI) of MLH1 using new CpG and allele-quantification pyrosequencing assays. This led to the identification of a novel intronic splice mutation in one patient demonstrating AEI, and new cases with an MLH1 epimutation. Loss-of-heterozygosity of the normal allele was demonstrated in their tumours. In one sporadic case, the epimutation arose de novo. The other cases were familial, and their epimutations demonstrated dominant inheritance linked to a particular ancestral founder genetic haplotype, indicative of a cis genetic-based defect. A single nucleotide variant within the 5'UTR, c.-27C>A, was investigated as the underlying cause. This epimutation was mosaic, as indicated by variable AEI among carriers. Furthermore, it was erased in spermatozoa and reset in the next generation.The molecular characteristics of constitutional MLH1 epimutations were examined in the cell lines from carriers to better characterise the epigenetic modifications associated with this defect. Allele-specific chromatin immunoprecipitation assays were developed at polymorphic sites within the MLH1 promoter to determine the histone profile associated with each allele. Elevated levels of repressive modifications were associated with the epimutant alleles, whereas the unmethylated alleles retained transcriptionally permissive moieties. Treatment with epigenetic reversal drugs partially up-regulated the epimutant alleles, indicating transcriptional activity is dependent on the presence of repressive chromatin modifications.This study provided evidence for two distinct forms of MLH1 epimutation associated with different inheritance patterns, reflecting different underlying mechanisms. A cis genetic alteration likely causes the dominant form, whereas trans-acting factors may be involved in non-Mendelian epimutations