Germline MLH1 c.-42 C > T is a likely pathogenic variant predisposing to a reduced-penetrance/modified Lynch syndrome phenotype featuring MLH1-methylated cancers.

The germline MLH1 c.-42 C > T (rs41285097) promoter variant has been identified in cases with MLH1-deficient colorectal or endometrial cancers but remains a variant of uncertain significance. Genetic testing identified two new MLH1 c.-42 C > T index cases from Australia and the USA. Clinicopathologic and molecular characterisation of tumour and non-neoplastic tissues was performed to investigate the potential mechanism of pathogenesis of this variant. The male Australian proband developed MLH1-deficient, BRAF p.V600 wildtype, CIMP-negative colon cancer at 61 years. MLH1 monoallelic methylation and a somatic pathogenic mutation, MLH1 c.1122_1126dup p.Asp376Valfs*27, were identified in his tumour. Droplet digital PCR (ddPCR) detected mosaic MLH1 methylation in normal colonic mucosa adjacent to the cancer (3.7%) with lower levels in blood (0.07%) and saliva (0.09%). The USA proband developed MLH1-deficient endometrial cancer at 38 years with MLH1 monoallelic methylation and loss-of-heterozygosity of the wildtype c.-42 C allele. No evidence of MLH1 methylation was found by ddPCR in normal tissues. MLH1 c.-42 C > T heterozygous relatives from both families had either no or extremely low levels of MLH1 methylation within blood or saliva. Allelic expression from MLH1 c.-42T was reduced to 70% relative to the wild-type allele in saliva from three heterozygotes. Three additional pedigrees were identified from the Colon Cancer Family Registry. Evaluation of combined multifactorial data from pooled informative index cases supports reclassification of this variant as “likely pathogenic” according to current ACMG/AMP mismatch repair gene-specific guidelines, though with likely reduced penetrance and/or modified phenotype. These findings highlight the clinical importance of identifying MLH1 c.-42 C > T to inform cancer risk management.