Next generation massively parallel sequencing of targeted exomes to identify genetic mutations in primary ciliary dyskinesia: implications for application to clinical testing.
Next generation massively parallel sequencing of targeted exomes to identify genetic mutations in primary ciliary dyskinesia: implications for application to clinical testing.
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DOI：
10.1097/gim.0b013e318203cff2
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发表时间：
2011-03
期刊：
影响因子：
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通讯作者：
Zariwala MA
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文献类型：
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作者：
Berg JS;Evans JP;Leigh MW;Omran H;Bizon C;Mane K;Knowles MR;Weck KE;Zariwala MA
基因测序技术的进步有可能加强与遗传异质性临床综合征相关的基因检测，如原发性睫状体运动障碍(PCD)。本研究的目的是研究外显子捕获技术结合大规模并行测序技术在临床诊断评估中的性能特征。我们对四个具有先前发现的各种PCD突变的个体进行了初步研究。我们设计了一种定制的阵列(NimbleGen)来捕获79个与PCD或睫毛功能相关的基因的2089个外显子，并使用GS Flx钛(罗氏454)平台对丰富的材料进行了测序。生物信息学分析是以盲法进行的，试图检测之前发现的突变并验证这一过程。使用这种方法很容易识别出三个替换突变中的三个和三个小的插入/缺失突变中的一个。在调整前面描述的SNPs的生物信息学处理后，明显观察到一个小的插入突变。这一过程未能检测到两个已知的突变：一个单核苷酸插入和一个完整外显子缺失。另外的生物信息学回溯性分析显示，插入的基于序列的证据很强，但未能检测到整个外显子的缺失。还检测到许多其他变异，可能代表PCD表型的潜在遗传修饰物。我们得出结论，大规模并行测序在研究和临床诊断方面都有相当大的潜力，但在临床环境中广泛采用之前，还需要进一步的发展。
Advances in genetic sequencing technology have the potential to enhance testing for genes associated with genetically heterogeneous clinical syndromes, such as primary ciliary dyskinesia (PCD). The objective of this study was to investigate the performance characteristics of exon-capture technology coupled with massively parallel sequencing for clinical diagnostic evaluation. We performed a pilot study of four individuals with a variety of previously identified PCD mutations. We designed a custom array (NimbleGen) to capture 2089 exons from 79 genes associated with PCD or ciliary function and sequenced the enriched material using the GS FLX Titanium (Roche 454) platform. Bioinformatics analysis was performed in a blinded fashion in an attempt to detect the previously identified mutations and validate the process. Three of three substitution mutations and one of three small insertion/deletion mutations were readily identified using this methodology. One small insertion mutation was clearly observed after adjusting the bioinformatics handling of previously described SNPs. This process failed to detect two known mutations: one single nucleotide insertion and a whole exon deletion. Additional retrospective bioinformatics analysis revealed strong sequence-based evidence for the insertion but failed to detect the whole exon deletion. Numerous other variants were also detected, which may represent potential genetic modifiers of the PCD phenotype. We conclude that massively parallel sequencing has considerable potential for both research and clinical diagnostics, but further development is required before widespread adoption in a clinical setting.