A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• DNA sequencing mutation detection relies on laborious scanning methods requiring post-PCR manipulation.
• High-resolution melting (HRM) analysis is a rapid, cost-effective screening strategy for heterozygous variants.
• BRCA1 and BRCA2 germline pathogenic mutations are consistently heterozygous, making them suitable for HRM screening.

Purpose of the Study:

• To develop and optimize HRM assays for comprehensive screening of BRCA1 and BRCA2 coding regions and intron-exon boundaries.
• To establish a high-throughput, cost-effective method for detecting germline sequence variants in BRCA1 and BRCA2.

Main Methods:

• Designed and optimized 94 HRM assays (36 for BRCA1, 58 for BRCA2) for identical amplification conditions.
• Employed meticulous primer design, including deoxyinosine incorporation, to ensure reproducible mutation detection and minimize polymorphism interference.
• Utilized a multi-plate, 384-well format to facilitate high-throughput sample processing.

Main Results:

• Validated assays using 169 known BRCA1 and 239 BRCA2 sequence variants.
• Achieved 100% detection of all heterozygous variants in an extensive blinded validation with 384 patient DNAs.
• Demonstrated the efficiency of the optimized HRM protocol for accurate variant detection.

Conclusions:

• This study presents the first HRM approach for entire BRCA1 and BRCA2 coding region screening under unified conditions in a high-throughput format.
• The developed method significantly reduces the need for sequencing by over 90%, drastically lowering costs.
• This cost reduction is expected to increase the accessibility of BRCA1 and BRCA2 mutation testing for a wider population.