Multiple sequence variants of BRCA2 exon 7 alter splicing regulation

Area of Science:

• Genetics and Molecular Biology
• Cancer Genomics
• RNA Splicing Mechanisms

Background:

• Exonic variants of unknown significance (VUS) can disrupt mRNA splicing by altering splice sites or regulatory elements.
• Accurate prediction of splicing effects for VUS remains challenging, with limited functional data for BRCA2.
• Eight VUS in BRCA2 exon 7 were selected for functional splicing analysis.

Purpose of the Study:

• To investigate the impact of eight BRCA2 exon 7 variants of unknown significance on mRNA splicing.
• To functionally characterize splicing alterations caused by these variants using in vitro and patient-derived RNA.
• To evaluate the utility of quantitative RNA analysis and minigene assays for assessing splicing defects.

Main Methods:

• Splicing minigene assays were performed to assess the functional impact of variants.
• Patient RNA was analyzed to measure allelic imbalance in transcript production.
• An exonic splicing enhancer (ESE)-dependent minigene assay was used to evaluate regulatory element disruption.
• Pyrosequencing was employed for quantitative measurement of exon 7-containing transcripts.

Main Results:

• Six of the eight studied variants demonstrated significant splicing defects.
• Specific variants altered natural splice sites (c.517G>T, c.631G>A) or created new ones (c.572A>G).
• Exon 7 skipping was observed for variants c.520C>T (66%), c.587G>A (25%), and c.617C>G (46%), confirmed by pyrosequencing.
• Variants c.520C>T and c.587G>A were found to disrupt ESE activity.

Conclusions:

• BRCA2 exon 7 splicing is regulated by multiple exonic elements and is sensitive to sequence variations.
• Quantitative allelic imbalance in patient RNA and minigene assays effectively estimate partial splicing defects.
• Determining the pathogenicity of variants with partial splicing effects requires further evidence, including segregation analyses.