Reclassification of Two MLH1 Variants of Uncertain Significance Utilizing Clinical and Functional Data.

Area of Science:

• Genetics
• Oncology
• Molecular Biology

Background:

• Pathogenic variants in mismatch repair genes increase colorectal cancer (CRC) risk.
• Two MLH1 gene variants (c.696_698del, p.(Cys233del) and c.1919C > G, p.(Pro640Arg)) were previously identified as variants of uncertain significance (VUS) in Danish families with high CRC incidence.

Purpose of the Study:

• To reclassify the VUS status of the two identified MLH1 variants.
• To improve genetic risk assessment for colorectal cancer in families carrying these variants.

Main Methods:

• Collected clinical data and initiated tumor and co-segregation analysis.
• Performed RNA splicing analysis, subcellular localization, and protein stability studies.
• Applied mismatch repair (MMR) gene-specific ACMG/AMP guidelines for variant reclassification.

Main Results:

• Functional analyses indicated that c.696_698del, p.(Cys233del) affects RNA, subcellular localization, and protein stability.
• The c.1919C > G, p.(Pro640Arg) variant demonstrated decreased expression and protein stability.
• Both variants were suggested to disrupt DNA mismatch repair processes.

Conclusions:

• The MLH1 variants c.696_698del, p.(Cys233del) and c.1919C > G, p.(Pro640Arg) are proposed for reclassification as likely pathogenic (class 4).
• This reclassification enables accurate risk assessment for variant carriers.
• Family members without these variants can be excluded from intensified cancer surveillance, optimizing healthcare resource allocation.