The exon regions of the BRCA1 and BRCA2 genes were amplified by 31 pair and 41 pair primers, respectively (Supplementary Tables 1 and 2 ), using the SureCycler 8800 (Agilent, Penang, Malaysia). Direct DNA Sanger sequencing was carried out by ABI PRISM BigDye Sequencing Kits and ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The obtained sequences of BRCA1 and BRCA2 were aligned and analyzed using MEGA 5.0 software compared with GenBank accession numbers NM_007294.3 and NM_000059.3, respectively. We observed 48 variants in the discovery stage and selected candidate variants in validation stage with following criterions: (1) frameshift, nonsense, and missense variants; (2) minor allele frequency (MAF) less than 1% according to the Asian samples from 1000 Genomes Project [14 (link)]; and (3) the clinical classification identified by ENIGMA [15 (link)] was ascertained from BRCA exchange (https://brcaexchange.org/ ) ranging from Class 3 to Class 5. Then, we performed subsequent variant screening on the MassARRAY System (Agena Bioscience, San Diego, CA) among multicancers and the corresponding controls. For quality control, positive controls of candidate variants were used in each chip. Each recurrent variant identified from screening was doubly validated by Sanger sequencing.
Free full text: Click here
