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• W2765109854 abstract "The efficiency of a novel targeted next-generation sequencing (NGS) test, the Devyser BRCA kit, for a comprehensive analysis of all 48 coding exons of the high-risk breast/ovarian cancer susceptibility genes BRCA1 and BRCA2 has been assessed. The new assay intended to detect nucleotide substitutions, small deletions/insertions, and large deletions/duplications. To document the false-negative and false-positive rates of the NGS assay in the hands of end users, 48 samples with previously identified 444 small variants and seven gross rearrangements were analyzed, showing 100% concordance with gold standards. Furthermore, all other 43 variants (42 single-nucleotide variation or insertion/deletion variation and one copy number variation, whose significance is or may be of clinical value), which were called by the NGS assay in a prospectively analyzed 179-sample set, were confirmed by Sanger sequencing or multiplex ligation probe amplification, according to their nature. We conclude that the Devyser BRCA kit performed satisfactorily for use in a clinical laboratory. The efficiency of a novel targeted next-generation sequencing (NGS) test, the Devyser BRCA kit, for a comprehensive analysis of all 48 coding exons of the high-risk breast/ovarian cancer susceptibility genes BRCA1 and BRCA2 has been assessed. The new assay intended to detect nucleotide substitutions, small deletions/insertions, and large deletions/duplications. To document the false-negative and false-positive rates of the NGS assay in the hands of end users, 48 samples with previously identified 444 small variants and seven gross rearrangements were analyzed, showing 100% concordance with gold standards. Furthermore, all other 43 variants (42 single-nucleotide variation or insertion/deletion variation and one copy number variation, whose significance is or may be of clinical value), which were called by the NGS assay in a prospectively analyzed 179-sample set, were confirmed by Sanger sequencing or multiplex ligation probe amplification, according to their nature. We conclude that the Devyser BRCA kit performed satisfactorily for use in a clinical laboratory. Constitutional mutations in the tumor suppressor genes BRCA1 and BRCA2 feature hereditary breast and ovarian cancer syndrome (HBOC). Risk assessment and management are warranted for individuals with a significant personal and/or family history of breast, ovarian, pancreatic, and/or prostate cancer. Hence, clinical criteria and practice guidelines for identifying individuals who may benefit from BRCA1 or BRCA2 mutation testing have been developed.1Gradishar W.J. Anderson B.O. Balassanian R. Blair S.L. Burstein H.J. Cyr A. Elias A.D. Farrar W.B. Forero A. Giordano S.H. Goetz M.P. Goldstein L.J. Isakoff S.J. Lyons J. Marcom P.K. Mayer I.A. McCormick B. Moran M.S. O'Regan R.M. Patel S.A. Pierce L.J. Reed E.C. Salerno K.E. Schwartzberg L.S. Sitapati A. Smith K.L. Smith M.L. Soliman H. Somlo G. Telli M. Ward J.H. Shead D.A. Kumar R. NCCN Guidelines insights: breast cancer, version 1.2017.J Natl Compr Canc Netw. 2017; 15: 433-451Crossref PubMed Scopus (282) Google Scholar Likewise, the challenging evidence-based characterization of the clinical significance of BRCA1/2 variants is under continuous monitoring and revision.2Richards 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 (14622) Google Scholar Individuals identified with BRCA1 or BRCA2 mutation have significantly increased risk of breast, ovarian, prostate, pancreatic, and possibly other cancers.3Cavanagh H. Rogers K.M. The role of BRCA1 and BRCA2 mutations in prostate, pancreatic and stomach cancers.Hered Cancer Clin Pract. 2015; 13: 16Crossref PubMed Scopus (93) Google Scholar Therefore, screening for BRCA1/2 mutations is of great value for breast and ovarian cancer prevention and early detection as risk-reducing options, including increased screening, chemoprevention, and/or prophylactic surgery, can be acted on. Even though the benefits offered by genetic testing for BRCA1/2 mutation status are manifold, a large proportion of mutation carriers may not be identified through current eligibility criteria,4Yurgelun M.B. Hiller E. Garber J.E. Population-wide screening for germline BRCA1 and BRCA2 mutations: too much of a good thing?.J Clin Oncol. 2015; 33: 3092-3095Crossref PubMed Scopus (40) Google Scholar leading some authors to call for more inclusive standards or even population screening.5Metcalfe K.A. Poll A. Royer R. Nanda S. Llacuachaqui M. Sun P. Narod S.A. A comparison of the detection of BRCA mutation carriers through the provision of Jewish population-based genetic testing compared with clinic-based genetic testing.Br J Cancer. 2013; 109: 777-779Crossref PubMed Scopus (29) Google Scholar Moreover, an increasing number of low penetrance susceptibility genes have been discovered and included in test panels.6Lincoln S.E. Kobayashi Y. Anderson M.J. Yang S. Desmond A.J. Mills M.A. Nilsen G.B. Jacobs K.B. Monzon F.A. Kurian A.W. Ford J.M. Ellisen L.W. A systematic comparison of traditional and multigene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients.J Mol Diagn. 2015; 17: 533-544Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar However, because their added value in terms of clinical utility remains debated,7Thompson E.R. Rowley S.M. Li N. McInerny S. Devereux L. Wong-Brown M.W. Trainer A.H. Mitchell G. Scott R.J. James P.A. Campbell I.G. Panel testing for familial breast cancer: calibrating the tension between research and clinical Care.J Clin Oncol. 2016; 34: 1455-1459Crossref PubMed Scopus (134) Google Scholar several diagnostic laboratories stay on the paradigm less is more and limit the analysis to BRCA1 and BRCA2 genes. Great technological advances have allowed next-generation sequencing (NGS) platforms to become commonly available for high-throughput and low-cost per base genome analysis. Overlapping sequences generated by massive parallel sequencing of numerous small DNA fragments are aligned and assembled by different analysis software. Most of the variations are suited for quantification, including single-nucleotide variation (SNV), insertion/deletion variation (indel), and copy number variation (CNV).8Castéra L. Krieger S. Rousselin A. Legros A. Baumann J.J. Bruet O. Brault B. Fouillet R. Goardon N. Letac O. Baert-Desurmont S. Tinat J. Bera O. Dugast C. Berthet P. Polycarpe F. Layet V. Hardouin A. Frébourg T. Vaur D. Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes.Eur J Hum Genet. 2014; 22: 1305-1313Crossref PubMed Scopus (182) Google Scholar NGS approaches for diagnostic testing of mutations in the BRCA1/2 genes are being exploited by several laboratories.6Lincoln S.E. Kobayashi Y. Anderson M.J. Yang S. Desmond A.J. Mills M.A. Nilsen G.B. Jacobs K.B. Monzon F.A. Kurian A.W. Ford J.M. Ellisen L.W. A systematic comparison of traditional and multigene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients.J Mol Diagn. 2015; 17: 533-544Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 9Bosdet I.E. Docking T.R. Butterfield Y.S. Mungall A.J. Zeng T. Coope R.J. Yorida E. Chow K. Bala M. Young S.S. Hirst M. Birol I. Moore R.A. Jones S.J. Marra M.A. Holt R. Karsan A. A clinically validated diagnostic second-generation sequencing assay for detection of hereditary BRCA1 and BRCA2 mutations.J Mol Diagn. 2013; 15: 796-809Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 10Chong H.K. Wang T. Lu H.M. Seidler S. Lu H. Keiles S. Chao E.C. Stuenkel A.J. Li X. Elliott A.M. The validation and clinical implementation of BRCAplus: a comprehensive high-risk breast cancer diagnostic assay.PLoS One. 2014; 9: e97408Crossref PubMed Scopus (62) Google Scholar, 11Judkins T. Leclair B. Bowles K. Gutin N. Trost J. McCulloch J. Bhatnagar S. Murray A. Craft J. Wardell B. Bastian M. Mitchell J. Chen J. Tran T. Williams D. Potter J. Jammulapati S. Perry M. Morris B. Roa B. Timms K. Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk.BMC Cancer. 2015; 15: 215Crossref PubMed Scopus (87) Google Scholar, 12Strom C.M. Rivera S. Elzinga C. Angeloni T. Rosenthal S.H. Goos-Root D. Siaw M. Platt J. Braastadt C. Cheng L. Ross D. Sun W. Development and validation of a next-generation sequencing assay for BRCA1 and BRCA2 variants for the clinical laboratory.PLoS One. 2015; 10: e0136419Crossref PubMed Scopus (34) Google Scholar, 13Kang H.P. Maguire J.R. Chu C.S. Haque I.S. Lai H. Mar-Heyming R. Ready K. Vysotskaia V.S. Evans E.A. Design and validation of a next generation sequencing assay for hereditary BRCA1 and BRCA2 mutation testing.PeerJ. 2016; 4: e2162Crossref PubMed Scopus (15) Google Scholar As any other analytical procedure, NGS involves different processes, each of which requires proper validation before integration into a diagnostic workflow.14Gargis A.S. Kalman L. Berry M.W. Bick D.P. Dimmock D.P. Hambuch T. et al.Assuring the quality of next-generation sequencing in clinical laboratory practice.Nat Biotechnol. 2012; 30: 1033-1036Crossref PubMed Scopus (362) Google Scholar Critical parameters that should be externally validated include sensitivity and specificity. Because no detailed guidelines exist to determine both the number of characterized samples and types of DNA variants that are needed for validation, clinical laboratories have performed disparate comparisons of NGS versus the gold standard Sanger sequencing. The data analysis in these studies included independent and blind evaluations as well as power estimations, and the results showed that these new methods generally are highly accurate.6Lincoln S.E. Kobayashi Y. Anderson M.J. Yang S. Desmond A.J. Mills M.A. Nilsen G.B. Jacobs K.B. Monzon F.A. Kurian A.W. Ford J.M. Ellisen L.W. A systematic comparison of traditional and multigene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients.J Mol Diagn. 2015; 17: 533-544Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 9Bosdet I.E. Docking T.R. Butterfield Y.S. Mungall A.J. Zeng T. Coope R.J. Yorida E. Chow K. Bala M. Young S.S. Hirst M. Birol I. Moore R.A. Jones S.J. Marra M.A. Holt R. Karsan A. A clinically validated diagnostic second-generation sequencing assay for detection of hereditary BRCA1 and BRCA2 mutations.J Mol Diagn. 2013; 15: 796-809Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 10Chong H.K. Wang T. Lu H.M. Seidler S. Lu H. Keiles S. Chao E.C. Stuenkel A.J. Li X. Elliott A.M. The validation and clinical implementation of BRCAplus: a comprehensive high-risk breast cancer diagnostic assay.PLoS One. 2014; 9: e97408Crossref PubMed Scopus (62) Google Scholar, 11Judkins T. Leclair B. Bowles K. Gutin N. Trost J. McCulloch J. Bhatnagar S. Murray A. Craft J. Wardell B. Bastian M. Mitchell J. Chen J. Tran T. Williams D. Potter J. Jammulapati S. Perry M. Morris B. Roa B. Timms K. Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk.BMC Cancer. 2015; 15: 215Crossref PubMed Scopus (87) Google Scholar, 12Strom C.M. Rivera S. Elzinga C. Angeloni T. Rosenthal S.H. Goos-Root D. Siaw M. Platt J. Braastadt C. Cheng L. Ross D. Sun W. Development and validation of a next-generation sequencing assay for BRCA1 and BRCA2 variants for the clinical laboratory.PLoS One. 2015; 10: e0136419Crossref PubMed Scopus (34) Google Scholar, 13Kang H.P. Maguire J.R. Chu C.S. Haque I.S. Lai H. Mar-Heyming R. Ready K. Vysotskaia V.S. Evans E.A. Design and validation of a next generation sequencing assay for hereditary BRCA1 and BRCA2 mutation testing.PeerJ. 2016; 4: e2162Crossref PubMed Scopus (15) Google Scholar Not only does integration of NGS into the clinical laboratory demand validation, but it also requires continuous monitoring through participation in external quality control exercises after the assay characteristics have been established.15Wallace A.J. New challenges for BRCA testing: a view from the diagnostic laboratory.Eur J Hum Genet. 2016; 24: S10-S18Crossref PubMed Scopus (63) Google Scholar Our laboratory has been involved in BRCA1/2 mutation detection since the beginning and has followed the methodologic evolution in the field until the NGS era. Because of the significant increase of patient referrals during the last few years, shortage of resources amenable to high-throughput are supposed to affect costs and waiting times accordingly, demanding straightforward NGS solutions. For instance, the possibility of directly performing a rapid and accurate search for large rearrangements based on NGS sequencing data, allowing simultaneous SNV/indels and CNV detection, would be of great value because of the considerably reduced time and cost for obtaining clinical results in diagnostic laboratories. Our aim was to evaluate analytical sensitivity and specificity of the new Devyser BRCA kit, an NGS-based mutation detection system for BRCA1/2 testing. Here, we report the results from a set of 227 patient DNA samples, in which we demonstrate 100% concordance with reference data. BRCA1 and BRCA2 analysis was performed in a total of 227 cases selected from families with suspected HBOC undergoing genetic counseling. The criteria for eligibility to diagnostic mutation screening of BRCA1/2 genes were according to the Genetic/Familial High-Risk Assessment: Breast and Ovarian, NCCN Clinical Practice Guidelines (https://www.nccn.org/professionals/physician_gls/f_guidelines.asp#genetics_screening, last accessed September 5, 2017). All patients provided a written informed consent for BRCA1/2 gene mutation testing and for the use of their biological samples for research purposes. DNA samples from 41 patients were previously tested using Sanger sequencing, and pathogenic SNVs or indels mutations were present in BRCA1 or BRCA2. Seven DNA samples were previously found to carry CNVs, leading to a deletion or duplication of one or more BRCA1 exons using multiplex ligation–dependent probe amplification (MLPA). An additional 179 samples were prospectively studied. Only variants with known or uncertain pathogenic significance discovered through the test assay underwent confirmation by Sanger sequencing to rule out false-positive results. Likewise, the samples with large genomic rearrangements of BRCA1 or BRCA2 through the test assay were reassessed with MLPA. Constitutional DNA samples were obtained from peripheral blood leukocytes, using a FlexiGene DNA Kit (Qiagen, Hilden, Germany). Full-sequence determination of all coding exons and all adjacent exon/intron boundaries of BRCA1 and BRCA2, at least 20 bp proximal to the 5′ end and 10 bp distal to the 3′ end of each exon boundary, was achieved using the Devyser BRCA kit (Devyser AB, Stockholm, Sweden) according to the manufacturer's protocol. A total of 188 amplicons with a mean amplicon target length of 196 bp (range, 120 to 278 bp) were amplified to create sequencing libraries of the complete BRCA1/2 genes in a single tube. Briefly, 10 ng (2 ng/μL) of genomic DNA was used to amplify the BRCA1 and BRCA2 genes in a single-tube multiplex reaction; this PCR-based library was diluted and used to incorporate the molecular barcodes and adapter sequences into each amplicon by a second PCR reaction. Amplicon libraries were pooled to generate a sequencing library that was purified using the Devyser Library Clean kit (Devyser AB) and quantified using the High Sensitivity Qubit quantification kit (Life Technologies, Carlsbad, CA). The final library was normalized to a concentration of 2 nM and prepared for sequencing using Illumina MiSeq with MiSeq Reagent Kit version 2 (300 cycles) (Illumina, San Diego, CA) according to the manufacturer's instructions to generate single-end reads. Primary data processing (base calling, demultiplexing, and FastQ file generation) was conducted directly in the MiSeq analysis pipeline. Secondary data analysis was performed using two different bioinformatic platforms: the SeqNext module of the Sequence Pilot software version 4.3.1 (JSI Medical Systems, Ettenheim, Germany) and the Sophia DDM software-as-a-service tool (Sophia Genetics, Lausanne, Switzerland). This analysis included read alignment to the human reference genome (Genome Reference Consortium GRCh37), variant calling, visualization of the sequence reads, and report generation. The reference transcript sequences used were NM_007294.3 for BRCA1 and NM_000059.3 for BRCA2. A high mean read quality score (Q score >35) for each amplicon and a 100× read coverage per amplicon were considered to ensure high confidence variant calling. However, variants with lower read quality (Q score of 20 to 35) and variant fraction >20% were listed apart; any low coverage regions, if present, were also reported. In the 48 previously characterized samples, all BRCA1 and BRCA2 variant calls were annotated as true- or false-positive results compared with Sanger sequencing data, whereas any missing variant was flagged as false-negative results. In the prospective series, all candidate pathogenic changes and all variants of uncertain significance (VUS) were confirmed by direct Sanger sequencing to allow false-positive detection. This step was performed manually and consisted of a preliminary check of the quality metrics of the individual runs followed by the search of the called variants in publicly available databases, such as Breast Cancer Information (National Human Genome Research Institute, https://research.nhgri.nih.gov/bic/), Leiden Open Variation Database–International Agency for Research on Cancer (Leiden University Medical Center, http://hci-exlovd.hci.utah.edu/home.php), ClinVar (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/clinvar), and BRCA Exchange (BRCA Exchange of the Global Alliance for Genomics and Health, http://brcaexchange.org). Population variant frequencies were retrieved from the Exome Variant Server (National Heart, Lung, and Blood Institute, http://evs.gs.washington.edu/EVS), Exome Aggregation Consortium (Broad Institute, http://exac.broadinstitute.org), and dbSNP (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/projects/SNP), whereas in silico functional predictions were made by SIFT (J. Craig Venter Institute, http://sift.jcvi.org), MutationTaster (http://www.mutationtaster.org), Provean (J. Craig Venter Institute, http://provean.jcvi.org/index.php), Polyphen2 (Harvard University, http://genetics.bwh.harvard.edu/pph2), and Human Splicing Finder (UMD Website, http://www.umd.be/HSF3/). A literature search through Google and PubMed was eventually performed to identify further supporting data on the variants under assessment. All variants were classified according to ENIGMA BRCA1/2 Gene Variant Classification Criteria (ENIGMA Consortium, https://enigmaconsortium.org/wp-content/uploads/2016/01/ENIGMA_Rules_2017-05-09.pdf) as benign, likely benign, VUS, likely pathogenic, and pathogenic (classes 1 to 5). Although all variant calls from the retrospective 48-sample set were evaluated for concordance with gold standards, only those classed ≥3 from the prospective series were forwarded to Sanger sequencing confirmation. All websites in this section last accessed June 15, 2017. Briefly, the entire coding sequence of BRCA1 and BRCA2 along with flanking intron boundaries was amplified from blood DNA, and direct sequencing of the PCR products was performed on both strands using the Big Dye Terminator version 1.1 Cycle Sequencing kit (Applied Biosystems, Warrington, Cheshire, UK) according to the manufacturer's instructions. Raw and analyzed sequence results were visualized on Sequence Scanner version 1.0 (Life Technologies). Nonreference base positions were reported according to the recommendations of the Human Genome Variation Society (http://varnomen.hgvs.org, last accessed June 15, 2017). MLPA analysis (MRC Holland, Amsterdam, The Netherlands) was used to detect genomic rearrangements in the coding and in the 5′-UTR of BRCA1 (P002 kit) and BRCA2 (P045 kit). To evaluate the Devyser BRCA kit, a total of 227 samples were sequenced in three different runs using the Illumina MiSeq system. The mean on-target read alignment for all samples was >99%. True-positive, true-negative, false-positive, and false-negative variant calls were annotated (Figure 1), with respect to the gold standard method Sanger sequencing. To allow calculation of false-positive and false-negative rates, the total reportable range of 1,185,312 bp (resulting from 24,694 bp sequenced in the 48 samples) was used for run 1, whereas only the interrogated 14,676 bp (resulting from the sequenced exons to confirm variants) were used for runs 2 and 3. The first run was performed using 48 samples previously characterized by Sanger sequencing and MLPA for the occurrence of BRCA1 and BRCA2 variations and large genomic rearrangements, respectively. All different types of variants were represented in these samples. Specifically, 41 samples were used as positive controls for the presence of 37 different SNVs/indels (Table 1). In addition, 35 common single-nucleotide polymorphisms, detected 403 times at a heterozygous or homozygous status in these 41 samples, were taken into account (Table 2). Seven other samples were used as positive controls for the presence of BRCA1 CNVs (Table 3). The SeqNext module of the Sequence Pilot software version 4.3.1 correctly identified all known variants, SNV/indels, and CNV in the 48 samples and correctly assigned homozygosity or heterozygosity, with no false-negative or false-positive predictions, ie, 100% sensitivity [TruePositive/(TruePositive+FalseNegative)] (95% CI, 98.93%–100%) and 100% specificity [TrueNegative/(TrueNegative+FalsePositive)] (95% CI, 99.9996%–100%).Table 1BRCA1/2 Single-Nucleotide and Insertion/Deletion Variants Found by Full-Exon Sanger Sequencing in 41 Samples Used for In-House Validation of the Devyser BRCA Kit as Analyzed by SeqNextBRCA1Samples, nBRCA2Samples, nNucleotide variantProtein variantNucleotide variantProtein variantc.134+2T>C—1c.196_198dupCAAp.66_67insGln1c.181T>Gp.Cys61Gly1c.67+1 G>A—1c.342delTp.Pro115fs1c.289G>Tp.Glu97∗1c.874delCp.Leu292fs1c.658_659delGTp.Val220fs1c.2071delAp.Arg691fs1c.1496_1497delAGp.Gln499fs1c.2188delGp.Glu730fs1c.3847_3848delGTp.Val1283fs1c.2269delGp.Val757fs1c.4284dupTp.Gln1429fs1c.3228_3229delAGp.Gly1077fs1c.5681dupAp.Tyr1894∗1c.3285delAp.Lys1095fs2c.5796_5797delTAp.His1932fs1c.3477_3480delAAAGp.Ile1159fs1c.6037A>Tp.Lys2013∗1c.3534delCp.Ser1178fs1c.6039delAp.Val2014fs1c.3553G>Tp.Glu1185∗1c.6468_6469delTCp.Gln2157fs2c.4161_4162delTCp.Gln1388fs1c.7180A>Tp.Arg2394∗1c.4964_4982del19p.Ser1655fs2c.7618-2A>G—1c.5035_5039delCTAATp.Leu1679fs1c.7877G>Ap.Trp2626∗1c.5062_5064delGTTp.Val1688del1c.8247_8248delGAp.Lys2750fs1c.5123C>Ap.Ala1708Glu1c.8487+1 G>A—1c.5266dupCp.Gln1756fs2c.8754+4A>G—1c.9455_9456delAGp.Glu3152fs1—, not applicable. Open table in a new tab Table 2BRCA1/2 Common Single-Nucleotide Polymorphisms Found by Full-Exon Sanger Sequencing in 41 Samples Used for In-House Validation of the Devyser BRCA Kit as Analyzed by SeqNextBRCA1n (het/hom)BRCA2n (het/hom)Nucleotide variantProtein variantNucleotide variantProtein variantc.591C>Tp.Cys197=2 (2/0)c.-11C>T1 (1/0)c.1067A>Gp.Gln356Arg8 (8/0)c.-26G>A21 (17/2)c.2077G>Ap.Asp693Asn6 (6/0)c.68-7delT1 (1/0)c.2082C>Tp.Ser694=27 (19/4)c.856T>Cp.Ser286Pro1 (1/0)c.2311T>Cp.Leu771=27 (19/4)c.865A>Cp.Asn289His6 (6/0)c.2596C>Tp.Arg866Cys1 (1/0)c.1114A>Cp.Asn372His27 (11/8)c.2612C>Tp.Pro871Leu29 (21/4)c.1365A>Gp.Ser455=5 (5/0)c.3113A>Gp.Glu1038Gly27 (19/4)c.1792A>Gp.Thr598Ala1 (1/0)c.3119G>Ap.Ser1040Asn2 (2/0)c.2229T>Cp.His743=6 (6/0)c.3548A>Gp.Lys1183Arg27 (19/4)c.2971A>Gp.Asn991Asp6 (6/0)c.4039A>Gp.Arg1347Gly1 (1/0)c.3396A>Gp.Lys1132=27 (19/4)c.4308T>Cp.Ser1436=27 (19/4)c.3807T>Cp.Val1269=12 (12/0)c.4535G>Tp.Ser1512Ile1 (1/0)c.4068G>Ap.Leu1356=1 (1/0)c.4837A>Gp.Ser1613Gly27 (19/4)c.5312G>Ap.Gly1771Asp1 (1/0)c.4956G>Ap.Met1652Ile2 (2/0)c.5744C>Tp.Thr1915Met2 (2/0)Total: 214 (158/28)c.6131G>Cp.Gly2044Ala1 (1/0)c.6322C>Tp.Arg2108Cys1 (1/0)c.7242A>Gp.Ser2414=22 (16/3)c.7544C>Tp.Thr2515Ile1 (1/0)c.7806-14T>C46 (24/11)Total: 189 (133/28)het, heterozygote; hom, homozygote. Open table in a new tab Table 3BRCA1 Copy Number Variations Found by Full-Exon Multiplex Ligation–Dependent Probe Amplification in Seven Samples Used for In-House Validation of the Devyser BRCA Kit as Analyzed by SeqNextSampleHGVS nomenclatureDescriptionS1c.(?_-2129)_(80+1_81-1)delDeletion 5′UTR_exon 2S2c.(5074+1_5075-1)_(5193+1_5194-1)dupDuplication exons 17_18S3c.(5074+1_5075-1)_(5193+1_5194-1)delDeletion exons 17_18S4c.(?_-2129)_(4185+1_4186-1)delDeletion 5′UTR_exon 11S5c.(5467+1_5468-1)_(*5320_?)delDeletion exon 23_3′UTRS6c.(5193+1_5194-1)_(5277+1_5278-1)delDeletion exon 19S7c.(?_-2129)_(5193+1_5194-1)delDeletion 5′UTR_exon 18HGVS, Human Genome Variation Society; UTR, untranslated region. Open table in a new tab —, not applicable. het, heterozygote; hom, homozygote. HGVS, Human Genome Variation Society; UTR, untranslated region. In the second run, we sequenced 96 unknown samples, whereas the third run included 83 unknown samples along with the same seven CNV positive control samples used in the first run. Data were analyzed with two different bioinformatic platforms, the SeqNext module of the Sequence Pilot software version 4.3.1 and Sophia DDM. A total of 42 clinically significant variants (19 pathogenic SNVs/indels and 23 VUS) were found in the 179 samples analyzed in the two runs, and all were confirmed with Sanger sequencing; besides, the resequencing step identified six common single-nucleotide polymorphisms in heterozygosity (five) or in homozygosity (one) (Supplemental Table S1).16Augello C. Bruno L. Bazan V. Calò V. Agnese V. Corsale S. Cascio S. Gargano G. Terrasi M. Barbera F. Fricano S. Adamo B. Valerio M.R. Colucci G. Sumarcz E. Russo A. Gruppo Oncologico del’Italia MeridionaleY179C, F486L and N550H are BRCA1 variants that may be associated with breast cancer in a Sicilian family: results of a 5-year GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study.Ann Oncol. 2006; 17: vii30-vii33Crossref PubMed Scopus (10) Google Scholar, 17Jalkh N. Nasse-Slaba J. Chouery E. Salem N. Urchammer N. Golmard L. Stoppa-Lyonnet D. Bignon Y.-J. Mégarbané A. Prevalance of BRCA1 and BRCA2 mutations in familial breast cancer patients in Lebanon.Hered Cancer Clin Pract. 2012; 10: 7Crossref PubMed Scopus (35) Google Scholar All 48 variants in the interrogated range of 14,676 bp were confirmed, resulting in a sensitivity of 100% (95% CI, 90.77%–100%) and in a specificity of 100% (95% CI, 99.97%–100%). Concerning CNV analysis, 12 of 96 samples (12.5%) in run 2 and 7 of 83 samples (8.4%) in run 3 were not analyzed by Sophia DDM because they did not reach the minimum amplicon coverage requested for CNV analysis. On the other hand, in the same samples, SeqNext performed CNV analysis and did not find any rearrangement, although it showed a higher than normal variability among amplicons. All these samples underwent MLPA and in none of them were CNVs detected (data not shown). In run 3, we reincluded the seven known CNVs to understand to what extent, under suboptimal read coverage, the method was still able to detect CNV. Although SeqNext identified the entire set of rearrangements, only six were reconfirmed by Sophia DDM (85.7%) (Figure 2). Moreover, Figure 2 shows a slightly lower confidence level for CNV scoring, because of poorer coverage, for BRCA1 exon 2 amplicons in samples S2 and S6 (with known rearrangements in other regions) and in sample S1, with a genuine exon 2 deletion. This problem was signaled by both platforms in the other eight unknown samples in run 3. All these samples were negative to MLPA, and one of them, with the worst exon 2 ratio relative coverage, is shown in Supplemental Figure S1. These findings made us confident, based on NGS analysis only, of the true nature of the BRCA1 exon 2 deletion called by both platforms in an unknown sample of run 3 (Figure 3, A and B ). The CNV was confirmed by MLPA (Figure 3C), and although the exact breakpoints have not been determined, the deletion spans a region between 5′-UTR and intron 2 of the BRCA1 gene.Figure 3A: Copy number variation (CNV) analysis by Sophia DDM showing BRCA1 exon 2 deletion (red squares). Normal copy number is denoted by blue squares (high confidence), but E7-2 (reduced confidence) is marked by a hollow square. B: CNV analysis by the Sequence Pilot module SeqNext. Relative coverage of the patient sample is in green, and mean relative coverage of the control samples is in blue; the histogram below shows the ratio relative coverage within the limits 75% to 125% in light blue and when it exceeds the limits in darker blue (red line indicates limit was exceeded). C: Coffalyser ratio chart using P002 MLPA kit. Results indicate a heterozygous deletion spanning from probe BRCA1-2-178 nt to probe BRCA1-up-324 nt. Probe ratios (red dots) are beyond the lower arbitrary border of 0.7 (red line). Probe ratio error bars do not overlap with the 95% CI of the same probe in the reference" @default.
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• W2765109854 title "Evaluation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Mutation Detection" @default.
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