Detection of BRCA1/2 large genomic rearrangement including BRCA1 promoter-region deletions using next-generation sequencing
Introduction
Germline mutations in BRCA1 and BRCA2 (BRCA1/2) increase the risks of breast and ovarian cancer [1], [2]. Lifetime cancer risks in BRCA mutation carriers are 60–80% for breast cancer and 20–40% for ovarian cancer [3]. Mutations in BRCA1/2 can be detected by sequence analysis for single-nucleotide variations (SNVs) and small insertion or deletion events (InDels) (over 80% of pathogenic variants), and deletion/duplication analysis for large genomic rearrangements (LGRs) (about 10% of pathogenic variants) [4]. BRCA1/2 mutation testing is commonly done by Sanger sequencing, which is considered the ‘gold standard’ of DNA sequencing [5], and multiplex ligation-dependent probe amplification (MLPA), which has been a widely used and highly sensitive method for detecting the relative copy-number variations (CNVs) [6].
Nowadays, next-generation sequencing (NGS) has been used to provide relatively rapid, cost-effective, and accurate detection of BRCA1/2 mutations in clinical laboratories worldwide. NGS has replaced Sanger sequencing of the BRCA1/2 genes. Commercial NGS BRCA1/2 assays are widely used in clinics and have shown excellent analytical performance in identifying SNVs and InDels [7]. CNV analysis by NGS was challenging yet. Despite continuous efforts, they have not sufficed to confirm the usability of NGS for identifying clinically significant CNVs in the BRCA1/2 genes. In this study, we aimed to evaluate the performance of NGS BRCA1/2 assays by comparing them with the conventional method.
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Patients
We included 108 patients who were suspected of having a hereditary breast and ovarian cancer (HBOC) syndrome and were referred for BRCA1/2 analyses at Seoul St Mary‘s Hospital between December 2014 and April 2018. Sanger sequencing for all BRCA1/2 exons and MLPA were done to identify mutations and LGRs. Among them, 48 cases had SNVs or small InDels in the BRCA1 or BRCA2 gene. In addition, seven cases had LGRs in the BRCA1 (n = 6) or BRCA2 gene (n = 1). All participants gave informed consent,
Detection of SNVs and small indels by Truseq NGS BRCA1/2 assay
Truseq NGS BRCA1/2 assay ran paired-end NGS and generated an average of 4,385,195 reads. From these reads, 4,259,518 (97%) were mapped against the human genome (version GRCh37). The average coverage of target regions per patient was 13,538, ranging from 165 to 138,617. The average coverage per nucleotide per gene was 7988 (476–18,076) and 36,313 (1697–138,617) for BRCA1 and BRCA2, respectively.
The NGS BRCA1/2 assay successfully detected and described SNVs and small InDels. Of the results, 20
Discussion
In this study, we demonstrated that the Truseq BRCA1/2 NGS assay identified most SNVs and small InDels. When appropriate analytical algorithms are used, LGRs could be well detected. It has been shown that read-depth CNV detection algorithms based on normalized depth of coverage can be successfully applied to NGS data in a research context and clinical setting [15], [16]. The frequency of CNVs of BRCA1/2 differs am-ng populations. LGRs in the BRCA1 gene are responsible for up to 27% of all BRCA1
CRediT authorship contribution statement
Eunhee Han: Data curation, Writing - original draft. Jaeeun Yoo: Data curation. Hyojin Chae: Formal analysis, Validation. Seungok Lee: Investigation. Do-Hoon Kim: Investigation. Kwang Joong Kim: Methodology, Software. Yonggoo Kim: Project administration, Conceptualization, Supervision. Myungshin Kim: Supervision, Writing - review & editing.
Acknowledgement
This study was supported by Research Fund of Seoul St. Mary’s Hospital, The Catholic University of Korea.
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