Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants.

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Cancer Research

Background:

• Disrupted pre-mRNA splicing is a key mechanism in hereditary cancers.
• The breast cancer susceptibility gene CHEK2 is frequently implicated.

Purpose of the Study:

• To functionally analyze candidate spliceogenic variants in the CHEK2 gene.
• To assess the impact of these variants on splicing using reporter minigenes.

Main Methods:

• Analyzed 128 CHEK2 splice-site variants from the BRIDGES project.
• Sub-selected 52 variants predicted to impact splicing.
• Constructed and validated CHEK2 minigenes, engineered variants, and assayed in MCF-7 cells.

Main Results:

• 46 out of 52 variants (88.5%) impaired CHEK2 splicing.
• Observed complex splicing patterns, including exon skipping and intron retention, generating 89 transcripts.
• 34 variants led to loss of full-length transcript; 59 transcripts predicted premature termination codons.

Conclusions:

• Minigene assays facilitated classification of 32 CHEK2 variants (27 pathogenic/likely pathogenic, 5 likely benign) using ACMG/AMP criteria.
• 38% of variants remained of uncertain significance due to complex splicing patterns.