MLH1 intronic variants mapping to + 5 position of splice donor sites lead to deleterious effects on RNA splicing

Area of Science:

• Genetics and Genomics
• Oncology
• Molecular Biology

Background:

• Lynch syndrome (LS) is caused by germline pathogenic variants in DNA mismatch repair (MMR) genes.
• Variants outside canonical splice sites are often classified as variants of unknown significance (VUS).
• Accurate classification of VUS is crucial for genetic counseling and patient management.

Purpose of the Study:

• To investigate the pathogenicity of three MLH1 intronic variants (c.588+5G>C, c.588+5G>T, and c.677+5G>A) previously classified as VUS.
• To assess the clinical significance of these variants in families with suspected Lynch syndrome.
• To provide evidence for reclassifying these variants and improving genetic diagnostics.

Main Methods:

• Collected clinical data from 8 unrelated Lynch syndrome families across Argentina, Brazil, and Chile.
• Performed segregation analysis to track variant inheritance within families.
• Conducted RNA splicing studies to determine the molecular effect of the variants on MLH1 gene expression.
• Assessed tumor characteristics including MMR protein expression and microsatellite instability (MSI).

Main Results:

• Segregation analysis demonstrated a clear co-occurrence of the variants with colorectal cancer and other LS-associated malignancies.
• Tumors from affected families showed deficient MLH1-PMS2 protein expression and MSI-high status.
• RNA analyses confirmed that MLH1 c.588+5G>C and c.588+5G>T induce exon 7 skipping, while c.677+5G>A causes exon 8 skipping.
• The variants MLH1 c.588+5G>C and c.677+5G>A were classified as pathogenic (Class 5), and c.588+5G>T as likely pathogenic (Class 4).

Conclusions:

• The combined clinical and molecular data provide strong evidence that the three studied MLH1 variants are causative of Lynch syndrome.
• RNA splicing assays are essential for determining the clinical significance of intronic variants.
• Reclassification of these variants will improve genetic counseling and clinical management for families affected by Lynch syndrome.