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Validation of an NGS Approach for Diagnostic BRCA1/BRCA2 Mutation Testing

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Abstract

Background and Objective

Pathogenic mutations in BRCA1/2 tumor suppressor genes increase the lifetime risk for developing breast and ovarian cancer. The aim of the present study was to evaluate the sensitivity and specificity of the Ion Torrent PGM™ for diagnostic mutation screening of BRCA1/2 genes.

Methods

In the current study we included a cohort of 58 Bulgarian high-risk breast cancer patients to validate a next-generation sequencing approach for diagnostic mutation screening of the BRCA1/2 genes using the Ion Torrent Personal Genome Machine® (PGM™) platform. We have also optimized the workflow by comparing two different library preparation methods and using three software packages: NextGENE, Torrent Suite variantCaller, and Samtools/BCFtools to achieve detection of all variants with high specificity and sensitivity.

Results

We have validated a quick and accurate diagnostic test, with an overall specificity of 95.9 % and sensitivity of up to 100 %, which can be the first method of choice, followed by confirmation of the identified variants by Sanger sequencing. Our results prove that the Ion AmpliSeq™ BRCA1/2 Community Panel used with the PGM™ platform, and coupled with our variant selection pipeline, is able to detect all sequence variants discovered by Sanger sequencing.

Conclusion

The application of the new test which outperforms the classical approach in turn-around time and price will have great impact in the clinical practice to identify the mutation carriers and guide the better personalized treatment of the patients, as well as to contribute for the improved prophylaxis in hereditary breast and ovarian cancer families.

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Acknowledgments

DD, RD, IP, TG, AM, VM and RK declare no conflicts of interest.

This study was supported by Grant DUNK 01/2/2009 “National University Complex in Biomedical and Translational Research”, funded by the National Science Fund, Ministry of Education, Youth and Science, Bulgaria and Grant 48, funded by the Science Fund of the Medical University of Sofia, Bulgaria. We are grateful to all our patients and clinical teams of Prof. Svetlana Christova and Prof. Konstanta Timcheva.

Declaration of ethical conduct

This study and all Grants that provided funding for it have been approved by the Medical Ethics Committee of the Medical University of Sofia. All patients have been properly informed and have signed an informed consent form prior inclusion in the study.

Author contributions

Study concept—RK and AM; molecular genetic analysis—DD, RD and TG; sequence alignment and data analysis—IP and TG; clinical and molecular interpretations of results—DD, IP, AM and RK; manuscript preparation—DD and IP; final critical revision—AM, RK and VM; design and coordination of the study—DD and RK; administrative supervision and support—VM; the guarantee for the overall content—DD and RK.

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Authors and Affiliations

  1. Department of Medical Chemistry and Biochemistry, Molecular Medicine Center, Faculty of Medicine, Medical University of Sofia, 2 Zdrave Str., 1431, Sofia, Bulgaria

    Daniela Dacheva, Rumyana Dodova, Ivan Popov, Teodora Goranova, Atanaska Mitkova, Vanio Mitev & Radka Kaneva

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  1. Daniela Dacheva
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  2. Rumyana Dodova
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  3. Ivan Popov
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  4. Teodora Goranova
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  5. Atanaska Mitkova
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  6. Vanio Mitev
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  7. Radka Kaneva
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Correspondence to Daniela Dacheva.

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Dacheva, D., Dodova, R., Popov, I. et al. Validation of an NGS Approach for Diagnostic BRCA1/BRCA2 Mutation Testing. Mol Diagn Ther 19, 119–130 (2015). https://doi.org/10.1007/s40291-015-0136-5

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  • DOI: https://doi.org/10.1007/s40291-015-0136-5

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