Multiplex ddPCR assay for screening copy number variations in BRCA1 gene

Area of Science:

• Genetics
• Molecular Biology
• Cancer Research

Background:

• Germinal and somatic rearrangements in the BRCA1 gene are critical in breast and ovarian cancer development.
• Accurate detection of these rearrangements is essential for clinical diagnosis and risk assessment.

Purpose of the Study:

• To develop a multiplex droplet digital PCR (ddPCR) assay for detecting large deletions and duplications in the BRCA1 gene.
• To establish a precise, cost-effective, and reliable method for BRCA1 rearrangement analysis.

Main Methods:

• Development of an in-house tetraplex ddPCR assay using 8 multiplexes, each targeting 3 BRCA1 exons and 1 reference gene (ALB or RPP30).
• Validation of the ddPCR assay against a commercial MLPA-based kit using DNA samples from 24 individuals.

Main Results:

• The developed multiplex ddPCR assay demonstrated 100% concordance with the commercial MLPA kit.
• The assay successfully identified 9 specimens with BRCA1 deletions, 1 with duplication, and 14 with wild-type BRCA1.
• The assay proved to be sensitive and specific for detecting large genomic alterations in the BRCA1 gene.

Conclusions:

• A simple, precise, and cost-effective ddPCR-based assay for BRCA1 rearrangement testing has been successfully designed.
• This novel multiplex ddPCR assay is the first of its kind to offer results comparable to MLPA.
• The developed assay is suitable for routine clinical screening of BRCA1 rearrangements in hereditary cancer syndromes.