Clinical and Genetic Characterization of Constitutional MLH1 Promoter Hypermethylation: Implications for Lynch Syndrome Diagnosis.

PURPOSE: Constitutional MLH1 promoter hypermethylation (CMPH) is a relatively rare cause of Lynch syndrome. While most cases appear sporadic, some result from secondary epimutations, mainly caused by germline variants in the MLH1 promoter region. This study describes the clinical phenotype and genetic etiology of CMPH in the largest clinical cohort to date. METHODS: A retrospective analysis was conducted for 422 individuals who underwent clinical CMPH testing. Promoter sequencing was used to identify the underlying variants. Long-read sequencing further characterized MLH1 promoter methylation. RESULTS: CMPH was identified in 15.6% of study cohort participants. Of these, 63 exhibited clinical features consistent with Lynch syndrome. The most common associated cancers were colorectal, followed by endometrial, breast, and sebaceous neoplasms. Mendelian inheritance of CMPH was observed in five families in the study cohort, indicating secondary epimutations. Promoter sequencing identified eight unique germline variants, including three novel variants. Methylation analysis by long-read sequencing demonstrated mutant allele-specific promoter methylation for these variants. CONCLUSION: Our findings provide the most comprehensive review of the clinical phenotype associated with CMPH and highlight the significant contribution of promoter variants to its etiology. These results underscore the need to include constitutional MLH1 promoter methylation assessment for Lynch syndrome diagnosis.