Genetic modulation of RNA splicing rescues BRCA2 function in mutant cells

Area of Science:

• Genetics
• Molecular Biology
• Cancer Research

Background:

• Hereditary cancer is often linked to mutations in BRCA1 and BRCA2 genes.
• Gene variants can disrupt RNA splicing, leading to truncated proteins.
• Quantitative analysis of variant-specific splicing isoforms is limited.

Purpose of the Study:

• To investigate the splicing consequences of the BRCA2:c.681+5G>C variant.
• To analyze the functional impact of variant-specific BRCA2 isoforms on DNA repair.
• To explore therapeutic strategies for restoring BRCA2 function through splicing modulation.

Main Methods:

• Droplet digital RT-PCR was used to identify and quantify mRNA isoforms.
• CRISPR-Cas9 gene editing was employed to induce in-frame transcripts.
• DNA repair capacity was assessed by measuring RAD51 focus formation and chromosomal breaks.

Main Results:

• Two variant-specific mRNA isoforms were identified for BRCA2:c.681+5G>C.
• The predominant isoform was out-of-frame, leading to nonsense-mediated decay.
• Homozygous cells showed reduced BRCA2 protein, DNA repair defects, and increased chromosomal instability.
• CRISPR-Cas9 editing restored in-frame transcripts, increasing protein levels and DNA repair efficiency.

Conclusions:

• The BRCA2:c.681+5G>C variant significantly impacts RNA splicing and protein function.
• Splicing modulation represents a promising therapeutic approach to restore BRCA2 function.
• Targeting splicing defects could offer a novel strategy for hereditary cancer prevention.