MLH1 Constitutional Epimutation Screening Requires Highly Sensitive Assays to Identify Lynch Syndrome Patients With...

Constitutional epimutations of the MLH1 gene are an alternative cause of Lynch syndrome, in which inactivation of an allele of a mismatch repair (MMR) gene results from MLH1 promoter methylation, rather than a pathogenic genetic variant. These epimutations are often mosaic, and methylation levels ranging from ~50% monoallelic methylation to low-level methylation (1%-5%) are observed in the blood of MLH1 epimutation carriers. Using a specific and highly sensitive droplet digital methyl-specific PCR (ddMSP) assay, six patients with very low methylation levels (< 1%) were identified in a series of 142 patients with a MLH1-methylated tumor diagnosed before age 61, who had been referred to the clinical lab between 2020 and 2024. These patients were initially missed by standard pyrosequencing assay, emphasizing the need for highly sensitive assays for constitutional epimutation screening. To confirm that methylated DNA molecules detected by ddMSP did not correspond to circulating tumor DNA rather than germline DNA, multiple validation analyses were performed, including validation of the constitutional origin of methylation on other sources of germline DNA and tumoral analysis. Taking into account the other patients identified as epimutation carriers by pyrosequencing during the same 5-year period, 13.1% of patients with a MLH1-methylated tumor before age 61 were diagnosed as Lynch syndrome patients, which changed their clinical follow-up. These findings highlight the relevance of recommendations for systematic MLH1 epimutation screening using highly sensitive assays in patients with MLH1-methylated tumors diagnosed before age 61. Such screening will increase the number of patients diagnosed with Lynch syndrome caused by a MLH1 constitutional epimutation, improving patient care and outcomes, as well as genetic counseling.