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A reliable method for the detection of BRCA1 and BRCA2 mutations in fixed tumour tissue utilising multiplex PCR-based targeted next generation sequencing.

ABSTRACT: BACKGROUND:Germline mutations in BRCA1 or BRCA2 lead to a high lifetime probability of developing ovarian or breast cancer. These genes can also be involved in the development of non-hereditary tumours as somatic BRCA1/2 pathogenic variants are found in some of these cancers. Since patients with somatic BRCA pathogenic variants may benefit from treatment with poly ADP ribose polymerase inhibitors, it is important to be able to test for somatic changes in routinely available tumour samples. Such samples are typically formalin-fixed paraffin-embedded (FFPE) tissue, where the extracted DNA tends to be highly fragmented and of limited quantity, making analysis of large genes such as BRCA1 and BRCA2 challenging. This is made more difficult as somatic changes may be evident in only part of the sample, due to the presence of normal tissue. METHODS:We examined the feasibility of analysing DNA extracted from FFPE ovarian and breast tumour tissue to identify significant DNA variants in BRCA1/ BRCA2 using next generation sequencing methods that were sensitive enough to detect low level mutations, multiplexed to reduce the amount of DNA required and had short amplicon design. The utility of two GeneRead DNAseq Targeted Exon Enrichment Panels with different designs targeting only BRCA1/2 exons, and the Ion AmpliSeq BRCA community panel, followed by library preparation and adaptor ligation using the TruSeq DNA PCR-Free HT Sample Preparation Kit and NGS analysis on the MiSeq were investigated. RESULTS:Using the GeneRead method, we successfully analysed over 76% of samples, with >95% coverage of BRCA1/2 coding regions and a mean average read depth of >1000-fold. All mutations identified were confirmed where possible by Sanger sequencing or replication to eliminate the risk of false positive results due to artefacts within FFPE material. Admixture experiments demonstrated that BRCA1/2 variants could be detected if present in >10% of the sample. A sample subset was evaluated using the Ion AmpliSeq BRCA panel, achieving >99% coverage and sufficient read depth for a proportion of the samples. CONCLUSIONS:Detection of BRCA1/2 variants in fixed tissue is feasible, and could be performed prospectively to facilitate optimum treatment decisions for ovarian or breast cancer patients.

SUBMITTER: Ellison G

PROVIDER: S-EPMC4391122 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A reliable method for the detection of BRCA1 and BRCA2 mutations in fixed tumour tissue utilising multiplex PCR-based targeted next generation sequencing.

Ellison Gillian G Huang Shuwen S Carr Hedley H Wallace Andrew A Ahdesmaki Miika M Bhaskar Sanjeev S Mills John J

BMC clinical pathology 20150324

<h4>Background</h4>Germline mutations in BRCA1 or BRCA2 lead to a high lifetime probability of developing ovarian or breast cancer. These genes can also be involved in the development of non-hereditary tumours as somatic BRCA1/2 pathogenic variants are found in some of these cancers. Since patients with somatic BRCA pathogenic variants may benefit from treatment with poly ADP ribose polymerase inhibitors, it is important to be able to test for somatic changes in routinely available tumour sample ...[more]

PMID: 25859162

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Project description:BackgroundDuring the last decade, remarkable progress with massive sequencing has been made in the identification of disease-associated genes for AIDs using next-generation sequencing technologies (NGS). An international group of experts described the ideal genetic screening method which should give information about SNVs, InDels, Copy Number Variations (CNVs), GC rich regions. We aimed to develop and validate a molecular diagnostic method in conjunction with the NGS platform as an inexpensive, extended and uniform coverage and fast screening tool which consists of nine genes known to be associated with various AIDs.MethodsFor the validation of basic and expanded panels, long-range multiplex models were setup on healthy samples without any known variations for MEFV, MVK, TNFRSF1A, NLRP3, PSTPIP1, IL1RN, NOD2, NLRP12 and LPIN2 genes. Patients with AIDs who had already known causative variants in these genes were sequenced for analytical validation. As a last step, multiplex models were validated on patients with pre-diagnosis of AIDs. All sequencing steps were performed on the Illumina NGS platform. Validity steps included the selection of related candidate genes, primer design, development of screening methods, validation and verification of the product. The GDPE (Gentera) bioinformatics pipeline was followed.ResultsAlthough there was no nonsynonymous variation in 21 healthy samples, 107 synonymous variant alleles and some intronic and UTR variants were detected. In 10 patients who underwent analytical validation, besides the 11 known nonsynonymous variant alleles, 11 additional nonsynonymous variant alleles and a total of 81 synonymous variants were found. In the clinical validation phase, 46 patients sequenced with multiplex panels, genetic and clinical findings were combined for diagnosis.ConclusionIn this study, we describe the development and validation of an NGS-based multiplex array enabling the "long-amplicon" approach for targeted sequencing of nine genes associated with common AIDs. This screening tool is less expensive and more comprehensive compared to other methods and more informative than traditional sequencing. The proposed panel offers advantages to WES or hybridization probe equivalents in terms of CNV analysis, high sensitivity and uniformity, GC-rich region sequencing, InDel detection and intron covering.

Dataset Information

A reliable method for the detection of BRCA1 and BRCA2 mutations in fixed tumour tissue utilising multiplex PCR-based targeted next generation sequencing.

Publications

A reliable method for the detection of BRCA1 and BRCA2 mutations in fixed tumour tissue utilising multiplex PCR-based targeted next generation sequencing.

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