A Reference System for BRCA Mutation Detection Based on Next-Generation Sequencing in the Chinese Population

The absence of interpretation guidelines and limited data on BRCA1/2 mutations in the Chinese population have impeded the detection of BRCA variants based on next-generation sequencing (NGS) in China. This study was performed to establish a reference system for performance evaluation of BRCA genetic testing and variant interpretation, which includes interpretation rules, reference materials (RMs), and a reference database (RD). BRCA1/2 mutations identified in cell lines and clinical cases were selected to establish RMs. All mutations were detected by NGS and validated by Sanger sequencing. Variant call format files and standard variant data sets were collected and annotated to build the RD. Participant laboratories were invited to validate this reference system. Interpretation rules for BRCA variants in the Chinese population were generated as a standard for BRCA variant interpretation. Mutational analysis demonstrated that BRCA2 mutations (55%) were more common than BRCA1 mutations (45%) in Chinese patients. Eliminating duplicates from 19,886 variants, the RD contained 750 unique BRCA mutations. Most BRCA1/2 mutations in the reference system were pathogenic or likely pathogenic (RMs, 77.5%; RD, 57%). In total, 91 novel pathogenic/likely pathogenic variants were identified in the RD. The reference system can contribute to NGS performance and high-quality interpretation to facilitate clinical decision making. It could also accelerate the development and application of BRCA mutation detection technologies in China. The absence of interpretation guidelines and limited data on BRCA1/2 mutations in the Chinese population have impeded the detection of BRCA variants based on next-generation sequencing (NGS) in China. This study was performed to establish a reference system for performance evaluation of BRCA genetic testing and variant interpretation, which includes interpretation rules, reference materials (RMs), and a reference database (RD). BRCA1/2 mutations identified in cell lines and clinical cases were selected to establish RMs. All mutations were detected by NGS and validated by Sanger sequencing. Variant call format files and standard variant data sets were collected and annotated to build the RD. Participant laboratories were invited to validate this reference system. Interpretation rules for BRCA variants in the Chinese population were generated as a standard for BRCA variant interpretation. Mutational analysis demonstrated that BRCA2 mutations (55%) were more common than BRCA1 mutations (45%) in Chinese patients. Eliminating duplicates from 19,886 variants, the RD contained 750 unique BRCA mutations. Most BRCA1/2 mutations in the reference system were pathogenic or likely pathogenic (RMs, 77.5%; RD, 57%). In total, 91 novel pathogenic/likely pathogenic variants were identified in the RD. The reference system can contribute to NGS performance and high-quality interpretation to facilitate clinical decision making. It could also accelerate the development and application of BRCA mutation detection technologies in China. Despite years of intensive study and substantial progress in understanding susceptibility to breast and ovarian cancers, these diseases remain a major public health problem and are an important cause of death in women. Approximately 5% to 10% of breast cancer cases and 10% to 15% of ovarian cancer cases are thought to be hereditary, caused by abnormal genes passed from parent to child.1Fackenthal J.D. Olopade O.I. Breast cancer risk associated with BRCA1 and BRCA2 in diverse populations.Nat Rev Cancer. 2007; 7: 937-948Crossref PubMed Scopus (361) Google Scholar, 2Ramus S.J. Gayther S.A. The contribution of BRCA1 and BRCA2 to ovarian cancer.Mol Oncol. 2009; 3: 138-150Crossref PubMed Scopus (156) Google Scholar The genes BRCA1 and BRCA2 have been associated with susceptibility to breast and ovarian cancers.3Ripperger T. Gadzicki D. Meindl A. Schlegelberger B. Breast cancer susceptibility: current knowledge and implications for genetic counselling.Eur J Hum Genet. 2009; 17: 722-731Crossref PubMed Scopus (164) Google Scholar The carrier rate of BRCA1/2 mutations accounts for approximately 0.2% to 0.3% in the general population, approximately 3% in breast cancer, and 10% in ovarian cancer.4Levy-Lahad E. Friedman E. Cancer risks among BRCA1 and BRCA2 mutation carriers.Br J Cancer. 2007; 96: 11-15Crossref PubMed Scopus (243) Google Scholar Patients predisposed to breast and ovarian cancers exhibit hereditary breast and ovarian cancer syndrome, an autosomal dominantly inherited disease with variable penetrance, which has also been associated with deleterious mutations in BRCA1/2.5Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. et al.A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1.Science. 1994; 266: 66-71Crossref PubMed Scopus (5295) Google Scholar, 6Wooster R. Bignell G. Lancaster J. Swift S. Seal S. Mangion J. Collins N. Gregory S. Gumbs C. Micklem G. Identification of the breast cancer susceptibility gene BRCA2.Nature. 1995; 378: 789-792Crossref PubMed Scopus (2950) Google Scholar Mutations in either of these genes confer a 60% and 85% lifetime risk of breast cancer and a 15% to 40% lifetime risk of ovarian cancer.7Thompson D. Easton D.F. Breast Cancer Linkage ConsortiumCancer incidence in BRCA1 mutation carriers.J Natl Cancer Inst. 2002; 94: 1358-1365Crossref PubMed Scopus (920) Google Scholar, 8Brose M.S. Rebbeck T.R. Calzone K.A. Stopfer J.E. Nathanson K.L. Weber B.L. Cancer risk estimates for BRCA1 mutation carriers identified in a risk evaluation program.J Natl Cancer Inst. 2002; 94: 1365-1372Crossref PubMed Google Scholar Research has suggested that the peak breast cancer onset occurs at 30 to 40 and 40 to 50 years of age in BRCA1 and BRCA2 mutation carriers, respectively. After onset, the morbidity rate remains constant at 20% to 30% per year until 80 years of age.9Kuchenbaecker K.B. Hopper J.L. Barnes D.R. Phillips K.A. Mooij T.M. Roos-Blom M.J. et al.Risks of breast, ovarian, and contralateral breast cancer for BRCA1 and BRCA2 mutation carriers.JAMA. 2017; 317: 2402-2416Crossref PubMed Scopus (1352) Google Scholar Therefore, BRCA was developed as a biomarker for individual therapeutics. Previous studies have confirmed that patients with BRCA-mutant ovarian cancer are sensitive to platinum chemotherapy, and these patients exhibit a good prognosis after treatment with poly (ADP-ribose) polymerase inhibitors.10Taylor K.N. Eskander R.N. PARP inhibitors in epithelial ovarian cancer.Recent Pat Anticancer Drug Discov. 2018; 13: 145-158Crossref PubMed Scopus (37) Google Scholar, 11Morgan R.D. Clamp A.R. Evans D.G.R. Edmondson R.J. Jayson G.C. PARP inhibitors in platinum-sensitive high-grade serous ovarian cancer.Cancer Chemother Pharmacol. 2018; 81: 647-658Crossref PubMed Scopus (50) Google Scholar The poly (ADP-ribose) polymerase inhibitors olaparib (Lynparza), rucaparib (Rubraca), and niraparib (Zejula) have been approved by the Food and Drug Administration. BRCA1 is located on 17q21 and contains 1863 amino acids and 24 exons (NM_00,724.3),5Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. et al.A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1.Science. 1994; 266: 66-71Crossref PubMed Scopus (5295) Google Scholar whereas BRCA2 is located on 13q12–13 and contains 3418 amino acids and 27 exons (NM_000059.3).12Wooster R. Neuhausen S.L. Mangion J. Quirk Y. Ford D. Collins N. et al.Localization of a breast cancer susceptibility gene, BRCA2, to chromosome 13q12-13.Science. 1994; 265: 2088-2090Crossref PubMed Scopus (1621) Google Scholar There are no obvious mutation hot spots in BRCA except in the Ashkenazi population.13Janavicius R. Founder BRCA1/2 mutations in the Europe: implications for hereditary breast-ovarian cancer prevention and control.EPMA J. 2010; 1: 397-412Crossref PubMed Scopus (154) Google Scholar, 14Roa B.B. Boyd A.A. Volcik K. Richards C.S. Ashkenazi Jewish population frequencies for common mutations in BRCA1 and BRCA2.Nat Genet. 1996; 14: 185-187Crossref PubMed Scopus (633) Google Scholar Consequently, the entire coding region of the BRCA sequence compared with the present large sample database was adopted to screen mutations in BRCA. Sanger sequencing, which is considered the gold standard of DNA sequencing, is commonly performed for BRCA testing. However, limited throughput and lower cost-effectiveness are drawbacks of Sanger sequencing, restricting the development of this technology in clinical genetic testing. Meanwhile, the development of next-generation sequencing (NGS) technologies has provided unprecedented opportunities for researchers to investigate the role of genetic variations in common diseases. With the obvious advantage of high-throughput screening, NGS has been applied in the clinical diagnosis of BRCA mutations. Early clinical studies on BRCA testing have shown that NGS offers high sensitivity, specificity, and cost-effectiveness compared with current approaches.15Walsh T. Lee M.K. Casadei S. Thornton A.M. Stray S.M. Pennil C. Nord A.S. Mandell J.B. Swisher E.M. King M.C. Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing.Proc Natl Acad Sci U S A. 2010; 107: 12629-12633Crossref PubMed Scopus (385) Google Scholar, 16Morgan J.E. Carr I.M. Sheridan E. Chu C.E. Hayward B. Camm N. Lindsay H.A. Mattocks C.J. Markham A.F. Bonthron D.T. Taylor G.R. Genetic diagnosis of familial breast cancer using clonal sequencing.Hum Mutat. 2010; 31: 484-491Crossref PubMed Scopus (75) Google Scholar, 17Feliubadalo L. Lopez-Doriga A. Castellsague E. del Valle J. Menendez M. Tornero E. Montes E. Cuesta R. Gomez C. Campos O. Pineda M. Gonzalez S. Moreno V. Brunet J. Blanco I. Serra E. Capella G. Lazaro C. Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes.Eur J Hum Genet. 2013; 21: 864-870Crossref PubMed Scopus (84) Google Scholar, 18Chan M. Ji S.M. Yeo Z.X. Gan L. Yap E. Yap Y.S. Ng R. Tan P.H. Ho G.H. Ang P. Lee A.S. Development of a next-generation sequencing method for BRCA mutation screening.J Mol Diagn. 2012; 14: 602-612Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar However, NGS introduces complexities arising from the choice of components of the BRCA testing workflow, such as the NGS platform, enrichment method, and bioinformatics analysis. Different NGS sequencing platforms have applied independent technologies in clinical diagnosis, such as sequencing by synthesis (Illumina, San Diego, CA), ion torrent (Thermo Fisher, Carlsbad, CA), and combinatorial probe–anchor synthesis (Beijing Genomics Institute, Shenzhen, Guangdong, China), which use different enrichment methods and produce different forms of sequencing data. The resulting sequencing data have different setting requirements for use in analysis software. As a result, it is challenging to assess the accuracy of interpreting complex BRCA sequencing data. Most BRCA1/2 variations do not increase the risk of cancer. Thus, the clinical significance of BRCA variants is judged by pathogenicity. The American College of Medical Genetics and Genomics (ACMG) classifies variants into five tiers: pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign. Only variants considered pathogenic or likely pathogenic have clinical significance. Although the ACMG has provided directive opinions and advice for this classification,19Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. Voelkerding K. Rehm H.L. ACMG Laboratory Quality Assurance CommitteeStandards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med. 2015; 17: 405-424Abstract Full Text Full Text PDF PubMed Scopus (14840) Google Scholar variant data interpretation should be evaluated synthetically with the use of information from population and disease databases, literature reports, and patient medical histories. Consequently, with the lack of a sophisticated evaluation system for BRCA variant detection and interpretation, analysis of the large-scale data obtained from NGS and defining the clinical significance of these variants remains challenging at present. To date, no large-scale study based on the mutation frequency and characteristics of BRCA in the Chinese population has been performed. It is estimated that the breast cancer information core database contains >1700 and >2000 distinct variants of BRCA1 and BRCA2, respectively, and these variants include only 79 BRCA1 (4.44%) and 67 BRCA2 (3.35%) mutations identified in the Chinese population (https://research.nhgri.nih.gov/projects/bic, last accessed May 8, 2017). The large sample database of BRCA variants consists primarily of data from European and American populations. This has increased the difficulty of annotating BRCA variants in China. Meanwhile, clinical applications of BRCA mutation detection are also under investigation in a large number of laboratories in China. Therefore, it will be important to adopt a standard to evaluate the performance of NGS-based BRCA variant screening that is clinically actionable. The primary aim in this study was to establish a reference system to evaluate the performance of BRCA genetic testing and variant interpretation. The reference system contained BRCA mutation reference materials (RMs), interpretation rules, and a reference database (RD). BRCA mutation RMs were DNA samples that harbored BRCA mutations, which are used to evaluate the performance of BRCA mutation testing, data analysis, and variant interpretation. The BRCA mutation RD, which supplements the RMs, includes NGS data from clinical cases, which are applied to evaluate the performances of data analysis and variant interpretation. Interpretation rules are developed based on the progress of RD preparation, which is suitable for interpreting BRCA1/2 variants in the Chinese population. To provide a standard of BRCA variant interpretation to build the RMs and RD, experts in the interpretation of sequence variants in China reached a consensus on BRCA variant interpretation in the Chinese population. The judgment criteria for the clinical significance of BRCA mutations were defined according to the guidelines of ACMG,19Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. Voelkerding K. Rehm H.L. ACMG Laboratory Quality Assurance CommitteeStandards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med. 2015; 17: 405-424Abstract Full Text Full Text PDF PubMed Scopus (14840) Google Scholar and the characteristics of BRCA variants in the Chinese population. From the information on BRCA single nucleotide polymorphisms from noninvasive prenatal testing of 100,000 samples, the characteristics of BRCA variants specific to the Chinese population were defined. BRCA variants were filtered with single nucleotide polymorphism >0.003, which were identified as normal in the Chinese population. Items in the ACMG that were not applicable for BRCA variant interpretation were also canceled. To establish the BRCA mutation RMs, eight cell lines that harbor multiple BRCA mutations, 16 clinical cases with BRCA1 or BRCA2 mutations, and one case without BRCA mutations were selected. Of the 16 clinical subjects, six were women from the pedigrees of BRCA families. Clinical sample collection was approved by the participating centers (Annoroad Gene Technology, Beijing, China; Darui Technology, Guangzhou, China; Top Gene Technology, Shanghai, China). Written informed consent was obtained from all subjects, and 40 mL of whole blood was collected from each participant and kept at −20°C. Genomic DNA was extracted from the enrolled subjects. All mutations were detected by NGS and validated by Sanger sequencing. DNA was added to 180 tubes (20-μL samples of 10 ng/μL) as the RMs of BRCA variants. Four participating centers (BGI-Shenzhen, Shenzhen, China; Darui Technology; Berry Genomics, Beijing, China; Annoroad) were invited to validate the performance of the RMs. Three sets of RMs were sent to each laboratory to assess the accuracy and specificity of detecting and interpreting BRCA variants and the consistency of repeat testing. Detection methods were performed according to the standard operating procedure and instructions of the BRCA variant detection kit of each laboratory. Detected variants were marked and interpreted according to the interpretation rules of BRCA genetic variants. Because pathogenic (class 5) and likely pathogenic (class 4) tiers had the same clinical significance in terms of the clinical decision, the detection results were judged by whether the variants were interpreted correctly, which will directly affect the clinical decisions. Table 1 shows the judgment standard for the interpretation errors. Undetected noncharacterized pathogenic and likely pathogenic genetic mutations were taken as the requirement of specificity. The RM assays were performed in triplicate to meet accuracy and specificity requirements.Table 1Judgment Rules of Interpretation ErrorClassificationExcluded interpretation errorInterpretation errorDeduction, pointsClass 5Class 4Class 35Class 5Class 4Class 2, class 15Class 4Class 5Class 35Class 4Class 5Class 2, class 15Class 3Class 2Class 5, class 45Class 3Class 2Class 12Class 2Class 3, class 1Class 5, class 45Class 1Class 2Class 5, class 45Class 1Class 2Class 32Class 5, pathogenic; class 4, likely pathogenic; class 3, variant of uncertain significance; class 2, likely benign; class 1, benign. Open table in a new tab Class 5, pathogenic; class 4, likely pathogenic; class 3, variant of uncertain significance; class 2, likely benign; class 1, benign. The DNA samples were kept at −20°C and transported on dry ice. To test DNA stability, two sets of RMs were kept for 3 or 7 days at 37°C, separately, and one set was frozen and thawed three times. An additional three sets were selected to verify the uniformity. To establish the BRCA mutation RD, variant call format (VCF) files and standard variant data sets (Excel format; Microsoft, Redman, WA) obtained by NGS sequencing of clinical cases were collected by five participating laboratories (BGI; Geneplus Technology, Beijing, China; Darui Technology; Berry Genomics; AmoyDx, Xiamen, China). Subjects eligible for the RD included at least one BRCA variant screened with the use of the entire coding region of BRCA obtained by NGS, without the limitations of clinical significance and testing samples (germline or somatic). Eligible VCF files were reviewed by five participating centers to assess the accuracy of detection of BRCA1/2 mutations and variant interpretation. VCF files were named according to the sample names, with VCF ver. 4.0 and coverage region of BRCA sequencing in CDS ±15. Each sample should match one VCF file, and the variant callings in the VCF files should be kept consistent in the variant data set. The following fields of the variant data sets were required to be submitted: sample ID, condition, origin, sample type, standard genome [hg19/(GRCh37)], Chr, Pos (1 base), Ref base, alter base, zygosity, minor allele frequency, depth, gene, ExIn_ID, function, transcript (BRCA1:NM_007294.3, BRCA2:NM_000059.3), changes in nucleotides (cHGVS), changes in amino acids (pHGVS), clinical significance, evidence level, interpretation, submitter, detection method, platform, validation, method of validation, and date of submission. Standard gene variant nomenclature published by the Human Genome Variation Society (HGVS; http://varnomen.hgvs.org/recommendations/checklist, last accessed July 2, 2018) was required in cHGVS and pHGVS. Annotation of the clinical significance based on the interpretation rule consensus was required to classify variants as pathogenic, likely pathogenic, VUS, likely benign, or benign. Evidence level of interpretation to the five-tier classification should be listed according to the ACMG guidelines. At present, 19,886 variants in 2843 cases have been enrolled to establish the RD. Of the 2843 cases, 1323 were tested with the Illumina sequencing platform, whereas 1032 and 488 were detected with the BGISEQ-500 sequencing platform (BGISEQ, Shenzhen, China) and Ion Torrent sequencing platform, respectively. In addition, 987 and 1856 cases were obtained by the NGS enrichment method of multiple PCR amplification and hybrid capture targeted technologies, respectively. To form the RD, duplicate variants were removed from the variant records. VCF files from the RD were imported into the analysis procedure of each participant enterprise and analyzed to obtain the interpretation results of the corresponding variants according to their bioinformatics analysis protocol. The interpretation reports were to indicate all mutations within CDS ±15 bp, and include the following information: sample ID, transcript, HGVS cDNA, HGVS protein, mutation frequency, clinical significance, and variant interpretation. The final conclusion about the BRCA mutation was classified as positive if detected as pathogenic or likely pathogenic and negative if detected as VUS, benign, or likely benign. The final results were judged by whether the variants were interpreted correctly and are presented as a penalty point. Table 2 shows the judgment standards and deduction rules. The interpretation results of BRCA sequencing variants from RD reports should be consistent with the RD results. If not, 5 or 2 points were deducted for each variant interpretation error. The assessment of qualified interpretation was defined as a total deduction ≤10.Table 2The Specific Differences between the Chinese Developed Rules and ACMG RulesBRCA variant interpretationChinese-developed rulesACMG rulesDeleted itemsNot applicablePVS1 Caveats: Beware of genes in which LOF is not a known disease mechanism (eg, GFAP, MYH7); use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intactPM1: Located in a mutational hot spot and/or critical and well-established functional domain (eg, active site of an enzyme) without benign variationPM3: For recessive disorders, detected in trans with a pathogenic variantNote: This requires testing of parents (or offspring) to determine phase.PM6: Assumed de novo but without confirmation of paternity and maternityPP2: Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of diseaseBS2: Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early ageBP5: Variant found in a case with an alternate molecular basis for diseaseDetailed itemsPVS1 Caveats: As for the variants at the extreme 3′ end of a gene, if there is no report at this variant and none of null pathogenic variants were reported after this variant, it is not formed as PVS1 evidence.PP3: Only all predictive results could be used as an evidence. Predictive results could not be used as the unique evidence for clinical classification and clinical decision.PP4 use condition: High clinical sensitivity; there is less overlap between phenotype and other disease; benign variant could be distinguished by the large population frequency library; family history keep the same with inheritance patterns.BA1: Allele frequency is >5% in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium, Allele frequency is ≥ 1% for BRCA in 1000 Genomes Project, or Exome Aggregation ConsortiumPVS1 Caveats: Use caution interpreting LOF variants at the extreme 3′ end of a gene.PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.) Caveat: Because many in silico algorithms use the same or similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.PP4: Patient's phenotype or family history is highly specific for a disease with a single genetic cause.BA1: Allele frequency is >5% in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation ConsortiumACMG, American College of Medical Genetics and Genomics; LOF, loss of function. Open table in a new tab ACMG, American College of Medical Genetics and Genomics; LOF, loss of function. Combined with the standards and guidelines of ACMG and the characteristics of BRCA mutations in the Chinese population, experts in the interpretation of sequence variants in China developed interpretation rules for BRCA variants in China. The differences between the ACMG guidelines and the interpretation rules for BRCA variants in China focused on the judgment of pathogenic, likely pathogenic, and VUS (Table 2). To make a judgment of pathogenic, compared with the ACMG guidelines, some of the deleterious mutations lacking case reports and database records that are outside the significant functional domain will be upgraded from likely pathogenic variants; some of the nondeleterious mutations lacking sufficient moderate evidence of pathogenicity or supporting evidence of pathogenicity will also be upgraded to pathogenic from likely pathogenic variants. Regarding the judgment of likely pathogenic, compared with the ACMG guidelines, some of the missense or synonymous variants with database records and several case reports that indicated that these variants could change the splicing in experiments at the mRNA level will be downgraded to likely pathogenic from pathogenic, and some of the missense variants with database records and several case reports in which mutations with the same amino acid changes are known as pathogenic will also be downgraded to likely pathogenic from pathogenic. Regarding the judgment for VUS, similar differences were found as for the judgment of likely pathogenic. The RMs consisted of 25 DNA samples, which included 1 case of wild-type BRCA1/2, 10 cases of mutated BRCA1, and 14 cases of mutated BRCA2. Thirty-one variants were identified in the RMs, which contained 14 BRCA1 variants (45%) and 17 BRCA2 variants (55%) (Table 3). Among these mutations, 22 were pathogenic, 2 were likely pathogenic, 3 were VUS, 1 was likely benign, and 4 were benign. Mutational analysis indicated that BRCA1 variants were located in exons 2, 10, 11, 12, 18, and 23 and in intervening sequence 19, whereas BRCA2 variants were located in exons 2, 3, 10, 11, 13, 14, 18, 21_27, 23, 25, and 27. Most BRCA1 and BRCA2 variants were located in exons 10 and 11, respectively. Of the 25 RM samples, five exhibited multiple mutations: two with three variants and three with two variants. BRCA2_25 (EX21_27 Del) could not be tested with NGS kits that used library-building methods based on AmpliSeq. When the failure rate of RM detection was <2%, the NGS kit could be used to evaluate accuracy, specificity, and repeatability.Table 3The Variants Information of BRCA1/2 in the Reference MaterialsSamplescHGVSpHGVSFunctionDistributionVariant interpretationBRCA1_1c.3548A>Gp.K1183RMissenseEX10Benignc.1303 G>Tp.D435YMissenseEX10VUSc.5351delp.N1784fsFrameshiftEX11PathogenicBRCA2_2c.7397T>Cp.V2466AMissenseEX14Benignc.8021dupp.I2675fsFrameshiftEX18PathogenicBRCA1_3c.4327C>Tp.R1443*NonsenseEX12Pathogenicc.5351delp.N1784fsFrameshiftEX11Pathogenicc.5073delp.K1691fsFrameshiftEX11PathogenicBRCA2_4c.9097delp.T3033fsFrameshiftEX23Pathogenicc.2429A>Cp.N810TMissenseEX10VUSBRCA1_5c.5277+1 G>ANASpliceIVS19PathogenicBRCA2_6c.1744A>Cp.T582PMissenseEX10Likely benignBRCA2_7c.6952C>Tp.R2318*NonsenseEX13PathogenicBRCA2_8c.10070C>Gp.T3357RMissenseEX27VUSBRCA1_9c.981_982delAT_9p.C328*FrameshiftEX10PathogenicBRCA2_10c.5722_5723delp.L1908fsFrameshiftEX11PathogenicBRCA2_11c.3109C>Tp.Q1037*NonsenseEX11PathogenicBRCA1_12c.3333_3333del Ap.E1112fsFrameshiftEX10PathogenicBRCA2_13c.5171delTp.I1724fsFrameshiftEX11PathogenicBRCA2_14c.9275_9278delp.Y3092fsFrameshiftEX25PathogenicBRCA2_15c.6445_6446delATp.I2149*FrameshiftEX11Pathogenicc.52A>Gp.M18VMissenseEX2VUSBRCA2_16c.3883C>Tp.Q1295*NonsenseEX11PathogenicBRCA1_17c.5470_5477delATTGGGCAp.I1824fsFrameshiftEX23PathogenicBRCA1_18c.3800T>Gp.L1267*NonsenseEX10Likely pathogenicNormal _19BRCA1/2 wild typeNANANABenignBRCA2_20c.994delp.I332fsFrameshiftEX10PathogenicBRCA1_21c.5156delTp.V1719fsFrameshiftEX18Likely pathogenicBRCA2_22c.276dupAp.S93fsFrameshiftEX3PathogenicBRCA1_23c.4013delAp.K1338fsFrameshiftEX10PathogenicBRCA1_24c.68_69delCTp.E23fsFrameshiftEX2PathogenicBRCA2_25EX21_27 DelDeletionEX21_27PathogeniccHGVS, changes in nucleotides; EX, exon; NA, not applicable; pHGVS, changes in amino acids; VUS, variant of uncertain significance. Open table in a new tab cHGVS, changes in nucleotides; EX, exon; NA, not applicable; pHGVS, changes in amino acids; VUS, variant of uncertain significance. Four participant laboratories were invited to validate the accuracy and precision of the RMs. Analysis of common errors revealed that most multiple mutations in BRCA1/2 were interpreted in error by the participating laboratories. Ten error interpretations were reported among the four laboratories, which included three error reports from three laboratories and only one error report from one laboratory. Among the 10 error interpretations, three were reported by three di