Identification of constitutional MLH1 epimutations and promoter variants in colorectal cancer patients from the Colon Cancer Family Registry

Abstract

Purpose:Constitutional MLH1 epimutations manifest as promoter methylation and silencing of the affected allele in normal tissues, predisposing to Lynch syndrome–associated cancers. This study investigated their frequency and inheritance.Methods:A total of 416 individuals with a colorectal cancer showing loss of MLH1 expression and without deleterious germline mutations in MLH1 were ascertained from the Colon Cancer Family Registry (C-CFR). Constitutive DNA samples were screened for MLH1 methylation in all 416 subjects and for promoter sequence changes in 357 individuals.Results:Constitutional MLH1 epimutations were identified in 16 subjects. Of these, seven (1.7%) had mono- or hemi-allelic methylation and eight had low-level methylation (2%). In one subject the epimutation was linked to the c.-27C>A promoter variant. Testing of 37 relatives from nine probands revealed paternal transmission of low-level methylation segregating with a c.+27G>A variant in one case. Five additional probands had a promoter variant without an MLH1 epimutation, with three showing diminished promoter activity in functional assays.Conclusion:Although rare, sequence changes in the regulatory region of MLH1 and aberrant methylation may alone or together predispose to the development of cancer. Screening for these changes is warranted in individuals who have a negative germline sequence screen of MLH1 and loss of MLH1 expression in their tumor.

The MLH1 c.-27C>A and c.85G>T variants are linked to dominantly inherited MLH1 epimutation and are borne on a European ancestral haplotype

“…Four of the cases reported to date (USA Family 1, Netherlands Family 3, WA Family 16 and Proband H) met the Amsterdam I criteria due to a significant family history of syndromal cancers that included CRC below 50 years of age in at least one member. Of the cancers tested, both herein and previously, 10,17 MSI and MLH1 loss were consistently observed. Furthermore, the finding of LOH as the 'second hit' in the CRCs from the Dutch and WA probands 17 is consistent with the c.[ À27C4A; 85G4T] germline haplotype serving as the 'first hit' .…”

“…Thus these three families and the two previously reported Australian cases were of European origin. 10,17 The occurrence of two distinct SNVs on the same MLH1 haplotype in association with MLH1 epimutation in five ostensibly unrelated cancer-affected European families raised the question of whether they share common ancestry. If so, the carriers in each family would be predicted to share a larger founder haplotype on chromosome 3 extending beyond MLH1 itself.…”

“…In support of this, artificial promoter reporter assays have shown this substitution results in significantly diminished transcriptional output. 10,17 However, a clear mechanistic link between this SNV and epigenetic modification of the MLH1 promoter remains to be demonstrated. Irrespective of the precise molecular mechanism via which the MLH1 epimutation is induced on this founder haplotype, or whether the c. À27C4A variant is indeed responsible, the c. À27C4A and c.85G4T SNVs can serve as markers for this disease-causing genetic haplotype.…”

“…3 Classification of such variants as pathogenic or benign is based upon a compilation of clinical, genetic and epidemiological factors, as well as functional analyses. 6 A cluster of sequence variants within the promoter regions of the mismatch repair genes has also been identified in cancer-affected families; [7][8][9][10] however, the pathogenic significance of most of these has remained largely uninterpreted.…”

“…17 Two other index cases have also been reported as carriers of the MLH1 c. À27C4A and c.85G4T SNVs. 7,10 In the first familial case ever reported (Family 1744), 7 no methylation or allelic expression studies were performed and so this allele was not linked to constitutional epimutation at the time. Instead, that study focused on the potential role of the c.85G4T SNV as the disease-causing mutation in this family.…”