Targeted Next-Generation Sequencing Effectively Analyzed the Cystic Fibrosis Transmembrane Conductance Regulator Gene in Pancreatitis
Targeted Next-Generation Sequencing Effectively Analyzed the Cystic Fibrosis Transmembrane Conductance Regulator Gene in Pancreatitis
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DOI：
10.1007/s10620-014-3476-9
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发表时间：
2015-05-01
影响因子：
3.1
通讯作者：
Shimosegawa, Tooru
中科院分区：
文献类型：
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作者：
Nakano, Eriko;Masamune, Atsushi;Shimosegawa, Tooru
囊性纤维化跨膜传导调节因子（CFTR）基因是胰腺炎的易感基因，与囊性纤维化的发生有关。PCR扩增的CFTR基因片段的直接DNA测序是检测未知突变的一线方法，但鉴于基因的大小（27个外显子，1，480个氨基酸），这是一项繁琐且劳动密集型的奋进。下一代测序（NGS）正在变得标准化，降低了DNA测序的成本，并使每次运行能够产生数百万个读数。我们在这里报告了一个综合性的分析CFTR变异在日本慢性胰腺炎患者使用NGS耦合靶捕获。（121例特发性，46例酒精性，17例遗传性和9例家族性）通过HaloGlycoprotein靶富集技术捕获，结果测序数据覆盖了CFTR基因编码区的91.6%，平均读取深度为449。共鉴定出12个非同义变异体，其中包括3个新变异体[c.A1231G（p.K411E），c.1753G>T（p.E585X）和c.2869delC（p.L957fs）]和7个同义变异体，其中3个新变异体位于外显子区域。结论靶序列捕获结合NGS技术是一种有效的胰腺炎易感基因分析方法。
Background The cystic fibrosis transmembrane conductance regulator (CFTR) gene, responsible for the development of cystic fibrosis, is known as a pancreatitis susceptibility gene. Direct DNA sequencing of PCR-amplified CFTR gene segments is a first-line method to detect unknown mutations, but it is a tedious and labor-intensive endeavor given the large size of the gene (27 exons, 1,480 amino acids). Next-generation sequencing (NGS) is becoming standardized, reducing the cost of DNA sequencing, and enabling the generation of millions of reads per run. We here report a comprehensive analysis of CFTR variants in Japanese patients with chronic pancreatitis using NGS coupling with target capture.Methods Exon sequences of the CFTR gene from 193 patients with chronic pancreatitis (121 idiopathic, 46 alcoholic, 17 hereditary, and nine familial) were captured by HaloPlex target enrichment technology, followed by NGS.Results The sequencing data covered 91.6 % of the coding regions of the CFTR gene by >= 20 reads with a mean read depth of 449. We could identify 12 non-synonymous variants including three novel ones [c.A1231G (p.K411E), c.1753G>T (p.E585X) and c.2869delC (p.L957fs)] and seven synonymous variants including three novel ones in the exonic regions. The frequencies of the c.4056G>C (p.Q1352H) and the c.3468G>T (p.L1156F) variants were higher in patients with chronic pancreatitis than those in controls.Conclusions Target sequence capture combined with NGS is an effective method for the analysis of pancreatitis susceptibility genes.