Dacheva Daniela, Dodova Rumyana, Popov Ivan, Goranova Teodora, Mitkova Atanaska, Mitev Vanio, Kaneva Radka
Department of Medical Chemistry and Biochemistry, Molecular Medicine Center, Faculty of Medicine, Medical University of Sofia, 2 Zdrave Str., 1431, Sofia, Bulgaria,
Mol Diagn Ther. 2015 Apr;19(2):119-30. doi: 10.1007/s40291-015-0136-5.
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.
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.
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.
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.
BRCA1/2肿瘤抑制基因中的致病性突变会增加患乳腺癌和卵巢癌的终生风险。本研究的目的是评估Ion Torrent PGM™对BRCA1/2基因诊断性突变筛查的敏感性和特异性。
在本研究中，我们纳入了58名保加利亚高危乳腺癌患者队列，以验证使用Ion Torrent个人基因组测序仪®（PGM™）平台对BRCA1/2基因进行诊断性突变筛查的新一代测序方法。我们还通过比较两种不同的文库制备方法并使用三个软件包：NextGENE、Torrent Suite变异检测软件和Samtools/BCFtools来优化工作流程，以实现高特异性和敏感性地检测所有变异。
我们验证了一种快速准确的诊断测试，总体特异性为95.9%，敏感性高达100%，这可以作为首选方法，随后通过桑格测序法对鉴定出的变异进行确认。我们的结果证明，与PGM™平台一起使用的Ion AmpliSeq™ BRCA1/2社区检测试剂盒，再结合我们的变异选择流程，能够检测出桑格测序法发现的所有序列变异。
新测试在周转时间和价格方面优于传统方法，其应用将对临床实践产生重大影响，有助于识别突变携带者，指导患者进行更好的个性化治疗，并为改善遗传性乳腺癌和卵巢癌家族的预防工作做出贡献。
