Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform.
Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform.
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DOI：
10.1186/1755-8794-5-17
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发表时间：
2012-05-18
影响因子：
2.7
通讯作者：
Coucke P
中科院分区：
文献类型：
--
作者：
De Keulenaer S;Hellemans J;Lefever S;Renard JP;De Schrijver J;Van de Voorde H;Tabatabaiefar MA;Van Nieuwerburgh F;Flamez D;Pattyn F;Scharlaken B;Deforce D;Bekaert S;Van Criekinge W;Vandesompele J;Van Camp G;Coucke P
遗传性听力损失（HL）可能起源于参与听力复杂过程的众多基因之一的突变。目前，鉴定患者的遗传缺陷是一项劳动密集型工作，而且费用昂贵。虽然用Sanger测序筛查GJB2突变是很常见的，但对于其他已知的耳聋基因（bbb60）却不是这样。下一代测序技术（NGS）具有成本效益更高的潜力。已发表的方法主要使用基于杂交的目标富集程序，节省时间和效率，但导致灵敏度的损失。在这项研究中，我们使用了半自动PCR扩增和NGS，以结合高灵敏度，速度和成本效益。在这个概念验证研究中，我们筛选了5例先天性遗传性耳聋患者的15个常染色体隐性耳聋基因。使用primerXL设计了所有外显子的646对特异性引物和15个选定基因的大部分UTR。使用患者特异性标识符，将所有扩增子汇总并使用罗氏454 NGS技术进行分析。其中三名患者的家庭成员，其感兴趣的区域以前已通过连锁研究表征。在这些基因中，我们能够鉴定出CDH23和OTOF的两个新突变。另一位患者耳聋的病因不明，未发现病因突变。在第五名患者中，作为阳性对照，我们可以确认TMC1已知突变。我们已经开发了一种检测方法，作为筛查家族性常染色体隐性非综合征性听力损失（ARNSHL）患者的工具，它具有很大的前景。基于聚合酶链反应富集的一种高效、可靠和经济有效的基因检测方法首次为未确诊耳聋的新生儿提供了可能。
Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency. In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1. We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.
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影响因子：
3
作者：
De Schrijver JM;De Leeneer K;Lefever S;Sabbe N;Pattyn F;Van Nieuwerburgh F;Coucke P;Deforce D;Vandesompele J;Bekaert S;Hellemans J;Van Criekinge W
通讯作者：
Van Criekinge W
影响因子：
9.8
作者：
Rehman, Atteeq Ur;Morell, Robert J.;Friedman, Thomas B.
通讯作者：
Friedman, Thomas B.
影响因子：
12.3
作者：
Brownstein Z;Friedman LM;Shahin H;Oron-Karni V;Kol N;Abu Rayyan A;Parzefall T;Lev D;Shalev S;Frydman M;Davidov B;Shohat M;Rahile M;Lieberman S;Levy-Lahad E;Lee MK;Shomron N;King MC;Walsh T;Kanaan M;Avraham KB
通讯作者：
Avraham KB
影响因子：
5.3
作者：
Hilgert, Nele;Smith, Richard J. H.;Van Camp, Guy
通讯作者：
Van Camp, Guy
影响因子：
9.8
作者：
Schraders, Margit;Haas, Stefan A.;Kremer, Hannie
通讯作者：
Kremer, Hannie