Next Generation Sequencing of Pooled Samples Reveals New SNRNP200 Mutations Associated with Retinitis Pigmentosa
Next Generation Sequencing of Pooled Samples Reveals New SNRNP200 Mutations Associated with Retinitis Pigmentosa
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DOI：
10.1002/humu.21485
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发表时间：
2011-06-01
期刊：
影响因子：
3.9
通讯作者：
Rivolta, Carlo
中科院分区：
文献类型：
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作者：
Benaglio, Paola;McGee, Terri L.;Rivolta, Carlo
SNRNP200基因由45个外显子组成，编码200 kDa解旋酶hBrr2，是Pre-mRNA剪接所必需的蛋白质。最近，在中国的两个家系中，SNRNP200的两个突变被发现与常染色体显性遗传性视网膜色素变性疾病(ADRP)相关。在这项工作中，我们分析了96名无关的北美ADRP患者的整个35kb的SNRNP200基因组区域。为了完成这一大规模测序项目，我们对汇集的、未标记的PCR产物进行了超高通量测序。然后，我们通过对该队列和95名患者中另外一名患者的个体样本进行桑格测序，验证了检测到的DNA变化。已知的两个突变之一(p.S1087L)在3例患者中被发现，而在6名无关个体中发现了影响高度保守密码子的4个新的错义突变(p.R681C，p.R681H，p.V683L，p.Y689C)，这表明SNRNP200相关的adrp的患病率较高。我们还利用这项研究对池和序列方法进行了评估，特别是在产生假阳性和阴性结果方面。我们的结论是，虽然这一策略可以用于快速发现新的疾病相关变异，但它仍然需要广泛的验证才能用于常规的DNA筛查。(C)2011年Wiley-Liss，Inc.
The gene SNRNP200 is composed of 45 exons and encodes a protein essential for pre-mRNA splicing, the 200 kDa helicase hBrr2. Two mutations in SNRNP200 have recently been associated with autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease, in two families from China. In this work we analyzed the entire 35-Kb SNRNP200 genomic region in a cohort of 96 unrelated North American patients with adRP. To complete this large-scale sequencing project, we performed ultra high-throughput sequencing of pooled, untagged PCR products. We then validated the detected DNA changes by Sanger sequencing of individual samples from this cohort and from an additional one of 95 patients. One of the two previously known mutations (p.S1087L) was identified in 3 patients, while 4 new missense changes (p.R681C, p.R681H, p.V683L, p.Y689C) affecting highly conserved codons were identified in 6 unrelated individuals, indicating that the prevalence of SNRNP200-associated adRP is relatively high. We also took advantage of this research to evaluate the pool-and-sequence method, especially with respect to the generation of false positive and negative results. We conclude that, although this strategy can be adopted for rapid discovery of new disease-associated variants, it still requires extensive validation to be used in routine DNA screenings. (C) 2011 Wiley-Liss, Inc.