Identification of the COL2A1 Mutation in Patients with Type I Stickler Syndrome Using RNA from Freshly Isolated Peripheral White Blood Cells
Identification of the COL2A1 Mutation in Patients with Type I Stickler Syndrome Using RNA from Freshly Isolated Peripheral White Blood Cells
复制标题
使用新鲜分离的外周血白细胞中的 RNA 鉴定 I 型 Stickler 综合征患者的 COL2A1 突变
DOI：
10.1089/gtmb.2010.0138
复制
发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Yonekura H
中科院分区：
文献类型：
--
作者：
Yaguchi H;Ikeda T;Osada H;Yoshitake Y;Sasaki H;Yonekura H
I型Stickler综合征是由II型胶原蛋白基因（COL2A1）突变引起的，该基因在软骨和玻璃体中特异性表达。我们开发了一种简单且无创的策略，利用新分离的外周血细胞中的RNA来鉴定ol2a1突变，并在一个患有Stickler综合征的日本家庭中鉴定了一个新的3 '剪接位点突变。RNA是从患者外周血白细胞中分离出来的，这些白细胞与环己亚胺（一种无义介导的mRNA衰变抑制剂）孵育。利用高保真DNA聚合酶进行rt -聚合酶链反应克隆，获得覆盖整个编码区的COL2A1cDNA片段，并对其进行测序。对患者的cDNA进行全测序，发现在18号外显子对应的区域有49bp的缺失。该缺失引入了一个过早终止密码子。靶向基因组测序发现在内含子17的3 '剪接受体位点的a（−2）位置有碱基替换。这种突变导致利用位于正常剪接位点下游49个碱基的隐剪接位点，导致mRNA剪接异常，导致患者mRNA中49个碱基缺失。我们的方法比传统的基因组筛查更容易，为Stickler综合征患者提供了一种简单且无创的诊断测试。
Stickler syndrome type I is caused by mutations in the type II collagen gene (COL2A1), which is specifically expressed in cartilage and vitreous humor. We developed a simple and noninvasive strategy for identifying theCOL2A1mutation using RNA from freshly isolated peripheral white blood cells and identified a new 3′ splice site mutation in a Japanese family with Stickler syndrome. RNA was isolated from a patient's peripheral white blood cells that had been incubated with cycloheximide, an inhibitor of nonsense-mediated mRNA decay.COL2A1cDNA fragments covering the entire coding region were obtained by RT-polymerase chain reaction cloning using a high-fidelity DNA polymerase and sequenced. Whole sequencing of the patient's cDNA resulted in identification of a 49-bp deletion in the region corresponding to exon 18. The deletion introduced a premature termination codon. Targeted genome sequencing identified a base substitution at the A (−2) position of the 3′ splice acceptor site of intron 17. This mutation led to utilization of the cryptic splice site, which is located 49 bases downstream of the normal splice site and causes aberrant mRNA splicing, resulting in a 49-base deletion in the patient's mRNA. Our method is much easier than conventional genomic screening and provides a simple and noninvasive diagnostic test for patients with Stickler syndrome.