MLH1 c.27G>A (p.Arg9=) is a synonymous likely/pathogenic variant underlying variably mosaic constitutional MLH1 methylation in Lynch syndrome.

Abstract

The MLH1 synonymous variant c.27G>A (p.Arg9 =) has been reported in four index cases with suspected Lynch syndrome, but is variably classified as "likely pathogenic" or "variant of uncertain significance" due to insubstantial clinical and functional evidence. We report three new MLH1 c.27G>A index cases with family histories fulfilling Amsterdam criteria for Lynch syndrome and reassessed collective evidence for pathogenicity. Two are European families from the UK (two siblings) and Spain (three members spanning three generations), and the third is a proband from Mongolia, the first non-European reported with this variant. Blood-based constitutional MLH1 methylation testing in six heterozygotes from the three families revealed varying levels of mosaic methylation, even within the same family, ranging from extremely low (≤ 1%) to ~ 16%. Two heterozygotes with blood methylation ≤ 1% had elevated methylation (5-8%) in normal colon distant from their colon cancers. Mosaic constitutional MLH1 methylation was linked in cis to the variant c.27A allele in all six heterozygotes and segregated together across generations. Three archived early-onset colon cancers available from three heterozygotes (UK siblings, Mongolian proband) each displayed MLH1 loss, MLH1 hypermethylation, and loss-of-heterozygosity of the wild-type c.27G allele, consistent with methylated c.27A alleles within a fraction of colon cells predisposing to tumorigenesis. Nanopore sequencing in the two European families found no significant shared ancestry and no other candidate variants. Multifactorial data collated from these and prior observational studies now provide sufficient evidence for the classification of MLH1 c.27G>A as likely/pathogenic via a functional mechanism of variably mosaic "secondary" constitutional MLH1 epimutation.