Quantifying BRCA1 and BRCA2 mRNA Isoform Expression Levels in Single Cells

Area of Science:

• Genetics
• Molecular Biology
• Cancer Research

Background:

• BRCA1 and BRCA2 spliceogenic variants are linked to increased breast and ovarian cancer risks.
• Traditional analyses of splicing patterns lack single-cell resolution, potentially obscuring cellular variation.
• Understanding variant impact on mRNA expression is crucial for accurate cancer risk assessment.

Purpose of the Study:

• To develop and validate a single-cell RNA in situ hybridization method for analyzing BRCA1 and BRCA2 mRNA splicing events.
• To investigate the expression patterns of specific spliceogenic variants (BRCA1c.671-2 A>G and BRCA2c.7988 A>T) at the single-cell level.
• To compare single-cell findings with traditional population-based analyses.

Main Methods:

• Utilized RNA in situ hybridization to quantify absolute mRNA splicing events in single lymphoblastoid cells.
• Analyzed cells with known spliceogenic variants (BRCA1c.671-2 A>G, BRCA2c.7988 A>T) and control cells.
• Measured expression levels and proportions of targeted BRCA1 and BRCA2 mRNA isoforms.

Main Results:

• A significant proportion of cells (>42%) showed no detectable mRNA expression for the targeted genes.
• Increased levels of BRCA2 ∆17_18 mRNA were observed in cells with the BRCA2c.7988 A>T variant.
• Cells with BRCA1c.671-2 A>G did not show significantly increased BRCA1 ∆11 levels, but a higher proportion expressed the targeted splicing event compared to controls.

Conclusions:

• BRCA1/2 mRNA expression is stochastic at the single-cell level, challenging previous RT-PCR findings.
• Single-cell analysis provides a more nuanced understanding of spliceogenic variant effects on mRNA isoforms.
• Further research is needed to establish this method for diagnostic applications in spliceogenic variant assessment.