Bastida J M, Del Rey M, Lozano M L, Sarasquete M E, Benito R, Fontecha M E, Fisac R, García-Frade L J, Aguilar C, Martínez M P, Pardal E, Aguilera C, Pérez B, Ramos R, Cardesa M R, Martin-Antorán J M, Silvestre L A, Cebeira M J, Bermejo N, Riesco S, Mendoza M C, García-Sanz R, González-Díaz M, Hernández-Rivas J M, González-Porras J R
Department of Hematology, H. Universitario de Salamanca, IBSAL, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain.
Instituto de Investigación Biomédica de Salamanca, IBMCC, Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, Spain.
Haemophilia. 2016 Jul;22(4):590-7. doi: 10.1111/hae.12908. Epub 2016 Feb 15.
Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype-phenotype correlations are important for predicting the clinical course of the disease and to allow tailor-made follow-up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next-generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost.
The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs.
A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology.
In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant.
Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.
遗传性出血性凝血障碍（IBCDs）的分子检测不仅有助于确诊，还能辅助遗传咨询、产前诊断，在某些情况下，基因型 - 表型相关性对于预测疾病临床进程以及为个体提供量身定制的随访至关重要。直到最近，基因分型主要通过桑格测序进行，该技术耗时且昂贵。目前，新一代测序（NGS）提供了一种新的潜在方法，能够以可承受的成本同时检测多个基因。
本研究的目的是设计并分析一个包含23个基因的NGS检测板在IBCDs患者分子诊断中的适用性。
设计了一个定制的靶向富集文库，以捕获31个已知与IBCDs相关的基因。针对296个靶点生成探针，以覆盖这些基因的86.3 kb区域（所有外显子和侧翼区域）。使用NGS技术对20例具有IBCDs表型的患者进行了研究。
在所有患者中，我们的NGS方法检测到了致病突变。共发现21个致病变异；其中大多数为错义突变（18个），还鉴定出3个缺失突变。检测到6个影响F8、FGA、F11、FIO和VWF基因的新突变以及15个先前报道的变异。NGS和桑格测序结果100%一致。
我们的结果表明，这种方法可能是IBCDs快速基因诊断中一种准确、可重复且可靠的工具。
