上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（临床研究） • 上一篇    下一篇

基于靶向基因测序的原发性免疫缺陷病基因诊断方法

杨丽君1，李牛2，刘毅1，王剑2   

  1. 1. 上海健康医学院医学技术学院，上海 201318；2. 上海交通大学 医学院附属上海儿童医学中心分子诊断实验室，上海 200127
  • 出版日期:2017-03-28 发布日期:2017-03-30
  • 通讯作者: 王剑，电子信箱：wangjian@scmc.com.cn。
  • 作者简介:杨丽君（1980—），女，讲师，硕士；电子信箱：yanglj99@163.com。

Gene diagnostic method for primary immunodeficiency disease based on targeted panel sequencing

YANG Li-jun1, Li Niu2, LIU Yi1, WANG Jian2   

  1. 1. College of Medical Technology, Shanghai University of Medicine & Health Sciences, Shanghai 201318, China; 2. Molecular Diagnostic Laboratory, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
  • Online:2017-03-28 Published:2017-03-30

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摘要：

目的 ·设计并建立基于靶向基因测序（TPS）的适合原发性免疫缺陷病（PID）的高通量基因诊断方法。方法 ·阅读文献并查询相关数据库确定PID的已知致病基因，设计并定制针对这些基因所有外显子及侧翼序列的捕获探针，并通过该方法对1例疑似PID患儿进行分子诊断。结果 ·该PID测序panel共包含100个已知致病基因。该疑似PID患儿测序结果共产生读条数16 414 298（reads），平均覆盖深度为157 X，98.35%的目标区域测序深度大于20 X，99.97%的目标区域具有1 X以上的测序深度。最终在患儿的CXCR4基因第2号外显子区域发现一个杂合的无义突变（c.1000C>T，p.Arg334*）。Sanger测序结果验证了患儿CXCR4基因的变异并表明其父母在相应位点均为野生型，证实了患儿CXCR4基因的变异为新生突变（de novo）。结论 ·建立了PID高通量基因诊断方法，并借助该靶向基因测序技术成功诊断1例WHIM综合征患儿。

关键词: 原发性免疫缺陷病, 靶向基因测序, WHIM综合征, CXCR4基因, 新生突变

Abstract:

Objective · To design and build a high-throughput sequencing approach based on targeted panel sequencing (TPS) using for the primary immunodeficiency disease (PID) diagnosis. Methods · By reviewing the literature and querying the relevant databases to determine the known disease-causing genes of PID, capture probes using for the TPS were designed and customized for all exons and flanking sequences of these genes. A child suspected with PID was diagnosed by the customized TPS. Results · The PID sequencing panel contains a total of 100 known pathogenic genes. The sequencing data of the patient has 16 414 298 reads. The average coverage depth is 157 X, 98.35% of the target region sequencing depth is greater than 20 X, and 99.97% of the target region sequencing depth is greater than 1 X. Finally, a heterozygous nonsense mutation was found in the exon 2 of the CXCR4 gene (c.1000C>T, p.Arg334*) in the child. The results of Sanger sequencing confirmed the variation in the child and showed that his parents were wild-type at the corresponding sites, indicating the mutation is de novo. Conclusion · This study established a high-throughput sequencing diagnostic approach for PID, with which a case of WHIM syndrome was successfully diagnosed.

Key words: primary immunodeficiency disease, targeted panel sequencing, WHIM syndrome, CXCR4 gene, de novo