Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing.
Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing.
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DOI：
10.1007/s11568-010-9137-y
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发表时间：
2009-12
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影响因子：
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通讯作者：
Chen, Wei
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作者：
Hu, Hao;Wrogemann, Klaus;Kalscheuer, Vera;Tzschach, Andreas;Richard, Hugues;Haas, Stefan A;Menzel, Corinna;Bienek, Melanie;Froyen, Guy;Raynaud, Martine;Van Bokhoven, Hans;Chelly, Jamel;Ropers, Hilger;Chen, Wei
大规模并行测序已经彻底改变了对人类基因组中致病性变异的搜索，但对于常规诊断，对大量患者的完整人类基因组进行重新测序仍然过于昂贵。最近，已经开发了新的基因组划分方法，其允许将重测序靶向特定的基因组区室，但是这些方法的实践经验仍然有限。在这项研究中，我们结合了一种新的基于液滴的多重PCR方法和下一代测序，以筛选X连锁精神发育迟滞（XLMR）患者的86个先前确定的XLMR基因突变。总共分析了来自24个大型XLMR家族的受影响男性，其中包括三个已知突变的人。在Illumina/Solexa基因组分析仪II平台上对对应于这些基因的功能相关区域的扩增子进行测序。实现了高度特异性和均匀的富集：平均而言，67.9%的明确映射读数来自扩增子，对于88.5%的目标碱基，测序深度足以可靠地检测变异。在24名患者中，有10名确定了潜在的致病序列变异，包括已知的三种突变，所有这些都可以通过桑格测序来证实。这种方法的稳健性能证明了基于液滴的多重PCR用于数百个基因中的平行突变筛查的一般效用，这是诊断精神发育迟滞和可能由于多种基因缺陷引起的其他疾病的先决条件。本文的在线版本（doi：10.1007/s11568-010-9137-y）包含补充材料，可供授权用户使用。
Massive parallel sequencing has revolutionized the search for pathogenic variants in the human genome, but for routine diagnosis, re-sequencing of the complete human genome in a large cohort of patients is still far too expensive. Recently, novel genome partitioning methods have been developed that allow to target re-sequencing to specific genomic compartments, but practical experience with these methods is still limited. In this study, we have combined a novel droplet-based multiplex PCR method and next generation sequencing to screen patients with X-linked mental retardation (XLMR) for mutations in 86 previously identified XLMR genes. In total, affected males from 24 large XLMR families were analyzed, including three in whom the mutations were already known. Amplicons corresponding to functionally relevant regions of these genes were sequenced on an Illumina/Solexa Genome Analyzer II platform. Highly specific and uniform enrichment was achieved: on average, 67.9% unambiguously mapped reads were derived from amplicons, and for 88.5% of the targeted bases, the sequencing depth was sufficient to reliably detect variations. Potentially disease-causing sequence variants were identified in 10 out of 24 patients, including the three mutations that were already known, and all of these could be confirmed by Sanger sequencing. The robust performance of this approach demonstrates the general utility of droplet-based multiplex PCR for parallel mutation screening in hundreds of genes, which is a prerequisite for the diagnosis of mental retardation and other disorders that may be due to defects of a wide variety of genes. The online version of this article (doi:10.1007/s11568-010-9137-y) contains supplementary material, which is available to authorized users.