基于靶向基因测序的原发性免疫缺陷病基因诊断方法  

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

作　　者：杨丽君[1] 李牛[2] 刘毅[1] 王剑[2] YANG Li-jun Li Niu LIU Yi WANG Jian(College of Medical Technology, Shanghai University of Medicine ＆ Health Sciences, Shanghai 201318, China Molecular Diagnostic Laboratory, Shanghai Children＇s Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China)

机构地区：[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　　处：《上海交通大学学报（医学版）》2017年第3期390-393,共4页Journal of Shanghai Jiao tong University:Medical Science

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。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.

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 

分 类 号：R725.9[医药卫生—儿科] R440[医药卫生—临床医学]