Abstract P2-07-03: High-throughput germline mutation screening for hereditary breast cancer in southern Chinese patients by massively parallel DNA sequencing

Abstract

Abstract Introduction: Breast cancer is the most common malignancy and 3rd leading cause of deaths among the female population in Hong Kong. Since the establishment of The Hong Kong Hereditary Breast Cancer Family Registry in 2007, 1344 patients with breast and/or ovarian cancer who met the selection criteria were recruited for genetic testing in Hong Kong. Since 2011 we started to employ next-generation DNA sequencing (NGS) to expedite the analysis workflow and expand the panel of genes for sequencing. Aim: To evaluate the workflow of NGS in mutation screening of BRCA1, BRCA2, TP53 and PTEN genes, and compared with the sequence data obtained by Sanger sequencing. Methods: We sequenced BRCA1, BRCA2, TP53 and PTEN genes in peripheral blood samples of 410 patients, 53 positive controls and 107 healthy local individuals using 454 GS Junior System. Generation of barcoded amplicon libraries was streamlined by microfluidic PCR using Fluidigm Access Array System. Sequencing data were analyzed by an in-house developed fully automatic bioinformatics pipeline, which mainly consists of GS Amplicon Variant Analyzer, SAMtools and Ensembl Variant Effect Predictor. All putative mutations identified were validated by Sanger sequencing. Furthermore, the frequency of BRCA1, BRCA2 and PTEN missense variants of unknown significance (VUS) identified in the cohort were compared among 107 healthy local individuals and 1000 Genomes project samples. The VUS were also subjected to a panel of in silico prediction methods including PolyPhen and SIFT. Results: Among 410 patients, there were 7 in BRCA1, 6 in BRCA2 and 1 in TP53 mutations found, including 1 novel recurrent BRCA2 (c.7007G>T) and 1 novel founder BRCA2 (c.5164_5165delAG) mutations. Based on multiple criteria, 12 in BRCA1, 12 in BRCA2 and 1 in PTEN VUS could be prioritized for further investigation. The bioinformatics pipeline was extensively evaluated with Sanger-validated controls. The evaluation determined minimum sequencing coverage needed in this sequencing platform for accurate analysis. The pipeline accuracy was demonstrated by successful detecting mutations from 53 positive controls, including single nucleotide variants, insertions and deletions in different sequence context. Conclusion: BRCA1, BRCA2, TP53 and PTEN mutation screening of 410 patients were expedited by high-throughput DNA sequencing. This method could detect 14 positive cases, including recurrent mutations, in a shorter period of time when compared with Sanger full gene sequencing. High-risk patients who are negative for the gene panel may need further investigation other than screening for BRCA1/2. The in-house developed bioinformatics pipeline was validated to detect various types of mutations and potentially become a conventional platform for genetic screening. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-03.