Oliveira Théo G M, Mitne-Neto Miguel, Cerdeira Louise T, Marsiglia Julia D C, Arteaga-Fernandez Edmundo, Krieger José E, Pereira Alexandre C
Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo, São Paulo, Brazil.
Department of Research and Development, Fleury Group, São Paulo, Brazil.
J Mol Diagn. 2015 Jul;17(4):420-30. doi: 10.1016/j.jmoldx.2015.02.003. Epub 2015 Apr 30.
In inherited cardiomyopathies, genetic testing is recognized as an enriching procedure in the diagnostic closure of a cardiac condition. Many genetic mutations have been described as pathogenically related to cardiomyopathies, turning next-generation sequencing into an extremely reliable scenario. Here we describe the validation process of a pipeline constructed with a target panel of 74 cardiomyopathy-related genes sequenced using a next-generation sequencing system. Fifty-two samples from a hypertrophic cardiomyopathy casuistic with previous molecular diagnostics (Sanger-sequenced for MYH7, MYBCP3, and TNNT2; 19 positives and 33 negatives) were processed in parallel with a HapMap reference sample (NA12878) applied for a complete panel assessment. Sequencing coverage values were satisfactory, with a mean of 250× (95% CI, 226.03-273.91) and 95.2% of target bases with a coverage of ≥10×. With a total of 567 variants, variant call sensitivity was tested in five scenarios of coverage and variant allele frequency cutoffs. Maximum achieved sensitivity was 96.7% for single-nucleotide variants and 28.5% for indels, and positive predictive values remained above 0.959 during the whole process. Inter- and intra-assay reproducibility values were 89.5% and 87.3%, respectively. After a careful assessment of analytical performance, we infer that the assay presents potential feasibility for application in diagnostic routines, with minimal time requirements and a simple bioinformatics structure.
在遗传性心肌病中，基因检测被认为是完善心脏病诊断过程的一项重要程序。许多基因突变已被描述为与心肌病存在致病相关性，这使得下一代测序成为一种极为可靠的手段。在此，我们描述了一个流程的验证过程，该流程采用一个包含74个与心肌病相关基因的靶向捕获测序板，通过下一代测序系统进行测序。从肥厚型心肌病病例中选取了52个样本（此前已进行分子诊断，对MYH7、MYBCP3和TNNT2进行了桑格测序，其中19个阳性和33个阴性），与用于全面评估测序板的HapMap参考样本（NA12878）并行处理。测序覆盖值令人满意，平均为250×（95%置信区间，226.03 - 273.91），95.2%的目标碱基覆盖率≥10×。共检测到567个变异，在五种覆盖度和变异等位基因频率截断值的情况下测试了变异检出敏感性。单核苷酸变异的最高敏感性为96.7%，插入缺失变异为28.5%，在整个过程中阳性预测值均保持在0.959以上。批间和批内重复性值分别为89.5%和87.3%。在仔细评估分析性能后，我们推断该检测方法在诊断常规应用中具有潜在可行性，所需时间最少且生物信息学结构简单。
