Functional analysis of BRCA1 and BRCA2 splicing variants using a minigene assay

Area of Science:

• Genetics
• Molecular Biology
• Oncology

Background:

• BRCA1 and BRCA2 are tumor suppressor genes linked to increased breast and ovarian cancer risk.
• Intronic variants can cause aberrant splicing, leading to variants of uncertain significance (VUS).
• Accurate classification of VUS is crucial for genetic counseling and patient management.

Purpose of the Study:

• To investigate the functional impact of intronic variants in BRCA1 and BRCA2.
• To reclassify the clinical significance of non-coding BRCA variants currently designated as VUS.
• To improve the interpretation of splicing abnormalities in cancer predisposition genes.

Main Methods:

• Utilized minigene assays to study splicing patterns of selected BRCA1 and BRCA2 variants.
• Designed wild-type and mutant minigene constructs for each variant.
• Employed patient samples from Seoul National University Hospital for experimental validation.

Main Results:

• Aberrant splicing patterns, including exon skipping and intron retention, were observed in three variants: BRCA1:c.80+3_80+5del, BRCA1:c.548-15G>A, and BRCA2:c.8755-19A>G.
• BRCA1:c.80+3_80+5del exhibited exon skipping.
• BRCA1:c.548-15G>A and BRCA2:c.8755-19A>G showed intron retention.

Conclusions:

• Experimental analysis confirmed aberrant splicing for BRCA1:c.80+3_80+5del, BRCA1:c.548-15G>A, and BRCA2:c.8755-19A>G.
• Reclassified BRCA1:c.80+3_80+5del from VUS to Likely Pathogenic (LP) based on observed splicing defects.
• Maintained BRCA1:c.548-15G>A and BRCA2:c.8755-19A>G as VUS pending further functional data, highlighting the complexity of variant interpretation.