Significance of rare variants in genes involved in the pathogenesis of Lynch syndrome

Abstract

The molecular characterization of patients with Lynch syndrome (LS) involves germline testing to detect a deleterious mutation in one of the genes of the mismatch repair (MMR ) pathway. To date, however, a large proportion of patients with a clinical suspicion of LS who undergo genetic testing do not show a germline pathogenetic variant in these genes. Germline DNA from 73 patients with a clinical suspicion of LS was examined with next-generation sequencing methods, using a multigene custom panel designed and standardized by our research group, that targets a set of 15 genes. Deleterious variants were identified in 5.6% of index cases, while unclassified variants were identified in 80.3% of probands. To evaluate the pathogenicity of these uncertain variants, the American College of Medical Genetics and Genomics criteria was used, also considering wherever possible the microsatellite instability (MSI) status detected on tumor tissues as pathogenic criterion. In this manner, 8 of these uncertain significance variants were classified as likely pathogenic variants. Notably, some of these likely pathogenetic variants were also identified in the MLH3 gene that is a gene not routinely analyzed for cases with a clinical suspicion of LS. The present study highlighted the importance of verifying the pathogenicity of the numerous variants of unknown significance identified in patients for whom heredity is already clinically confirmed suggesting the importance of considering the MSI-H status on the tumor of patients carrying an uncertain variant to evaluate its pathogenicity. Moreover, the present study also suggested analyzing other MMR genes, such as MLH3 , in panels used for the molecular screening of LS.

Functional characterization of MLH1 missense variants unveils mechanisms of pathogenicity and clarifies role in cancer

“…A significant proportion of germline variants are found in the MLH1 gene, followed by the MSH2 gene (50 and 40%, respectively), with only 10% found in the MSH6 and PMS2 genes [ 25 ]. The majority are missense variants with a high proportion whose clinical significance is unclear [ 26 , 27 ]. These variants are referred to as variants of unclear significance (VUS) or unclassified variants (UV) which require pathogenicity classification [ 28 ].…”

Germline Variants in MLH1 and ATM Genes in a Young Patient with MSI-H in a Precancerous Colonic Lesion

“…In silico analysis carried out using Human Splicing Finder ( , accessed on 5 March 2023) software version 3.1 showed that this variant could lead to an alteration of the canonical splicing acceptor site with the activation of a new cryptic splicing site, as described by our team previously [ 28 ]. These computational data were confirmed by PCR analysis of the MLH1 cDNA fragment, including exons 6–10 of this gene.…”

“…This variant was already reported in the international database of the InSiGHT Group ( , accessed on 5 March 2023) and is classified as a class 3 mutation, that is, of uncertain pathogenetic significance. In our recent work, this variant was classified as a likely pathogenic variant via bioinformatic analysis [ 28 ]…”

“…Thus, this variant was evaluated according to the criteria of the American College of Medical Genetics and Genomics (ACMG) [ 28 , 29 ]. Although this variant was already classified in our previously study as a variant likely pathogenic, in this study, we confirmed the prediction via computational analysis because this variant caused in vivo the skipping of exon 8, resulting in the formation of a truncated protein that was likely dysfunctional.…”

“…However, our analysis showed that this variant leads to the formation of abnormal mRNA that lacks the entire exon 8 and thus to a probably non-functional protein product, which is compatible with a defective repair system at the somatic level. Accordingly, reclassification with ACMG criteria [ 28 , 29 ] established that this VUS can be considered probably pathogenic and, therefore, likely to be responsible for the clinical phenotype of the index case. It is important to note that the MSI-H condition was found in DNA extracted from the precancerous lesion, an adenoma that the patient developed at a young age.…”