A Study On Genetic Diagnostic Platform For BRCA1/2 Genes Based On Multiplex-pcr And Next-Generation Sequencing

Background Human breast cancer susceptibility genes BRCA1/2 are risk factors for hereditary breast/ovarian cancer. The morbidity and mortality of breast/ovarian cancer are increasing year by year in recent years. About 60% to 70% and 80% to 90% of hereditary breast and ovarian cancer is due to BRCA1/2 gene mutations.Patients who carry the pathogenic mutations got significantly higher cumulative risk of breast/ovarian cancer than normal. The development of high-throughput sequencing technologies provides a powerful tool to BRCA1/2 gene diagnosis method which can realize high-throughput, rapid, accurate and less cost. Multiplx PCR technology can complete genetic signal amplification accurately, and with the combination of NGS, genetic testing for BRCA1/2 will be more accuracy, fast, simple and easy method.Objective To establish the Genetic Testing Platform for BRCA1/2 carrier based on Multiplex-PCR and Next-Generation Sequencing, and verify its sensitivity and specificity.Method 1, To build multiplx PCR technique to amplify BRCA1/2 all gene exon and exon introns splicing. Then, through the platform of multiplx-PCR NGS technology to detect BRCA1/2 gene of 3 breast cancer patients from Beijing university tumor hospital, who had already been known disease-causing mutations. And, compared with general Sanger sequencing and target-capture sequencing to validate the results of our study.2, We collect 7 Chinese high-risk BRCA1/2 gene mutation carriers from gynecology of people’s liberation army general hospital. Four of them diagnosis of ovarian cancer, three of them are high-risk carriers. All participants have no clear genetic diagnosis before study. After taking Multiplex-PCR NGS, we use Sanger sequencing to verify found mutations or low frequency cSNP.Results:1, The results of Multiplex-PCR NGS sequencing are correspond with the Sanger sequencing, Target-capture sequencing.2. After detect 7 high-risk BRCA1/2 gene mutation carriers by Multiplex-PCR NGS. we have not found pathogenic mutations, but found the nonsynonymous cSNPs and verified by Sanger sequencing, the results are consistent.Conclusion:1, Multiplex-PCR NGS platform has high Sensitivity and Specificity in detecting BRCA1/2 gene mutations.2, Compared with the Sanger sequencing and target capture sequencing, Multiplex-PCR NGS platform with the advantages of easy operation, shorter time and less costly.