Background:

Lynch syndrome is a hereditary cancer predisposition affecting the colon, rectum, and endometrium. It is characterized by inherited monoallelic genetic alterations in genes involved in the mismatch repair system: MLH1, MSH2, MSH6, or PMS2. However, a constitutional epigenetic alteration of MLH1, i.e.promoter hypermethylation leading to transcriptional silencing, can also be the cause of the disease. Two types of epimutations have been described: primary (pure epigenetic events labile in the germline) and secondary (linked to a cis-genetic alteration inherited in an autosomal dominant manner). MLH1 epimutations can be primary or secondary and remain poorly characterized due to their heterogeneity. Several families with transmission of the MLH1 promoter hypermethylation (secondary epimutations) were diagnosed in the clinical lab, and different genetic variants segregating with the epimutation were identified. Our objective is to introduce these variants into cellular models to study the dynamics of de novo MLH1 promoter methylation.

Methods:

We used induced pluripotent stem cells (iPSCs) to develop cellular models of MLH1 secondary epimutations. Unlike differentiated cells, iPSCs retain the de novo DNA methylation machinery and have unique properties enabling the study of epigenetic processes. Using CRISPR-Cas9 technique, we introduced single-nucleotide variants identified in epimutation-carriers into the MLH1 gene of iPSCs derived from a healthy donor. This approach aimed to recreate the conditions leading to de novo promoter methylation in a controlled cellular context.

Results:

Our iPSC-based models demonstrated that specific variants induce de novo MLH1 promoter methylation and transcriptional silencing, recapitulating the epigenetic defect observed in patients. These results establish for the first time a direct causal link between specific genetic variants and the onset of constitutional MLH1 epimutations, and demonstrate the relevance of iPSC as cellular models of epimutations.

Conclusion:

These iPSC-based models provide a unique system to investigate the dynamics and underlying molecular mechanisms of MLH1 epimutations. Future work will focus on identifying key factors involved in de novo methylation and exploring targeted demethylation strategies to reverse epimutations, potentially reducing cancer risk in Lynch syndrome.