Eunhee Han1, Jaeeun Yoo2, Hyojin Chae2, Seungok Lee1, Do-Hoon Kim3, Kwang Joong Kim4, Yonggoo Kim2, Myungshin Kim5. 1. Department of Laboratory Medicine, Incheon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Republic of Korea; Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Republic of Korea. 2. Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Republic of Korea; Department of Laboratory Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Republic of Korea. 3. Department of Laboratory Medicine, Keimyung University School of Medicine, Daegu, Republic of Korea. 4. R&D Center, NGeneBio Inc., Republic of Korea. 5. Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Republic of Korea; Department of Laboratory Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Republic of Korea. Electronic address: microkim@catholic.ac.kr.
Abstract
BACKGROUND: Germline mutations in BRCA1 and BRCA2 (BRCA1/2) have been conventionally analyzed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Nowadays, next-generation sequencing (NGS) is increasingly being used in clinical genetics. The aim of this study was to evaluate the performance of NGS BRCA1/2 assays by comparing them with the conventional method. MATERIALS AND METHODS: We did BRCA1/2 NGS assays of 108 breast and/or ovarian cancer patients whose BRCA1/2 mutation had been previously analyzed by Sanger sequencing and MLPA using TruSeq Custom Amplicon Design AFP2. Single-nucleotide variations (SNVs) and small insertions or deletions (InDels) were evaluated. In addition, we analyzed large genomic rearrangements (LGRs) using a coverage-based algorithm as well as a revised BRCA1/2 NGS assay (BRCAaccuTest PLUS), which additionally covered a BRCA1 promoter region. RESULTS: The NGS BRCA1/2 assay detected all 20 SNVs and 21 small InDels in 56 patients. Among seven LGRs detected by MLPA, six exonic LGRs were well identified by both NGS BRCA1/2 assays. One pathogenic LGR, located on a BRCA1 promoter region, was successfully identified using revised BRCAaccuTestPLUS. CONCLUSIONS: These results indicated that an NGS BRCA1/2 assay could detect most LGRs including BRCA1 promoter-region deletion as well as SNVs and small InDels. Therefore, it was applicable to clinical BRCA1/2 mutation tests.
BACKGROUND: Germline mutations in BRCA1 and BRCA2 (BRCA1/2) have been conventionally analyzed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Nowadays, next-generation sequencing (NGS) is increasingly being used in clinical genetics. The aim of this study was to evaluate the performance of NGS BRCA1/2 assays by comparing them with the conventional method. MATERIALS AND METHODS: We did BRCA1/2 NGS assays of 108 breast and/or ovarian cancerpatients whose BRCA1/2 mutation had been previously analyzed by Sanger sequencing and MLPA using TruSeq Custom Amplicon Design AFP2. Single-nucleotide variations (SNVs) and small insertions or deletions (InDels) were evaluated. In addition, we analyzed large genomic rearrangements (LGRs) using a coverage-based algorithm as well as a revised BRCA1/2 NGS assay (BRCAaccuTest PLUS), which additionally covered a BRCA1 promoter region. RESULTS: The NGS BRCA1/2 assay detected all 20 SNVs and 21 small InDels in 56 patients. Among seven LGRs detected by MLPA, six exonic LGRs were well identified by both NGS BRCA1/2 assays. One pathogenic LGR, located on a BRCA1 promoter region, was successfully identified using revised BRCAaccuTestPLUS. CONCLUSIONS: These results indicated that an NGS BRCA1/2 assay could detect most LGRs including BRCA1 promoter-region deletion as well as SNVs and small InDels. Therefore, it was applicable to clinical BRCA1/2 mutation tests.
Authors: Rosina De Cario; Ada Kura; Samuele Suraci; Alberto Magi; Andrea Volta; Rossella Marcucci; Anna Maria Gori; Guglielmina Pepe; Betti Giusti; Elena Sticchi Journal: Front Genet Date: 2020-12-02 Impact factor: 4.599