Applying Massive Parallel Sequencing to Molecular Diagnosis of Marfan and Loeys-Dietz Syndromes
Applying Massive Parallel Sequencing to Molecular Diagnosis of Marfan and Loeys-Dietz Syndromes
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DOI：
10.1002/humu.21525
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发表时间：
2011-09-01
期刊：
影响因子：
3.9
通讯作者：
Coucke, Paul J.
中科院分区：
文献类型：
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作者：
Baetens, Machteld;Van Laer, Lut;Coucke, Paul J.
Marfan(MFS)和Loeys-Dietz(LDS)综合征分别由纤维蛋白-1(FBN1)和转化生长因子β受体1和2(TGFBR1和TGFBR2)基因突变引起。在目前的常规突变筛选技术下，对这组基因的分析既耗时又昂贵。我们利用多重聚合酶链式反应和下一代测序技术，量身定做了一种经济有效且可靠的突变发现策略。在第一阶段，对五个先前发现FBN1和TGFBR1和2突变和/或多态的MFS或LDS患者样本的基因组DNA进行了分析，并揭示了所有预期的变异。在第二阶段，我们对87例符合根特标准的MFS患者的样本进行了验证。这导致鉴定出75个FBN1突变，其中67个是独一无二的。随后对其余阴性样本的多重连接依赖探针扩增(MLPA)分析发现了四个大的缺失/插入。最后，Sanger测序确定了FBN1外显子1中的一个错义突变，该突变没有包括在NGS工作流程中。总体而言，总的突变识别率为92%，这与之前发表的数据一致。我们的结论是，对FBN1和TGFBR1/2的所有编码外显子进行多重聚合酶链式反应，然后进行NGS分析和MLPA分析，是一种在时间和成本效益上鉴定突变的可靠策略。嗡嗡作响32：1053-1062,2011。(C)2011年Wiley-Liss，Inc.
The Marfan (MFS) and Loeys-Dietz (LDS) syndromes are caused by mutations in the fibrillin-1 (FBN1) and Transforming Growth Factor Beta Receptor 1 and 2 (TGFBR1 and TGFBR2) genes, respectively. With the current conventional mutation screening technologies, analysis of this set of genes is time consuming and expensive. We have tailored a cost-effective and reliable mutation discovery strategy using multiplex PCR followed by Next Generation Sequencing (NGS). In a first stage, genomic DNA from five MFS or LDS patient samples with previously identified mutations and/or polymorphisms in FBN1 and TGFBR1 and 2 were analyzed and revealed all expected variants. In a second stage, we validated the technique on 87 samples from MFS patients fulfilling the Ghent criteria. This resulted in the identification of 75 FBN1 mutations, of which 67 were unique. Subsequent Multiplex Ligation-dependent Probe Amplification (MLPA) analysis of the remaining negative samples identified four large deletions/insertions. Finally, Sanger sequencing identified a missense mutation in FBN1 exon 1 that was not included in the NGS workflow. In total, there was an overall mutation identification rate of 92%, which is in agreement with data published previously. We conclude that multiplex PCR of all coding exons of FBN1 and TGFBR1/2 followed by NGS analysis and MLPA is a robust strategy for time-and cost-effective identification of mutations. Hum Mutat 32:1053-1062, 2011. (C) 2011 Wiley-Liss, Inc.