基于二代测序原始数据的拷贝数变异分析在原发性免疫缺陷病分子诊断中的应用

Application of copy number variation analysis based on raw data of next-generation sequencing in the molecular diagnosis for primary immunodeficiency disease

目的 探讨基于二代测序原始数据的拷贝数变异(CNV)分析在原发性免疫缺陷病(PID)分子诊断中的应用.方法 回顾性研究.对于2014年9月至2017年3月深圳市儿童医院风湿免疫科165例(男110例、女55例)疑诊PID患儿的二代测序原始数据进行常规生物信息分析,对未发现致病或可疑致病突变的患儿采用CNVkit软件进行基于二代测序原始数据的CNV分析.以344种PID致病基因名称及别名,检索亚洲原发性免疫缺陷病数据库、人类基因变异数据库、与疾病相关的人类基因组变异数据库查找致病性CNV.以"primary immunodeficiency disease""copy number variation""next generation sequencing"为关键词检索2010年1月至2019年5月Pubmed数据库;以"原发性免疫缺陷病""拷贝数变异"为关键词检索2010年1月至2019年5月维普、万方、中国知网数据库查找PID相关基因致病性CNV文献情况.结果 165例患儿中95例(57.6%)经常规生物信息分析未发现致病或可疑致病突变,免疫失调性疾病的阴性率最高(68.6%,24/35).95例患儿中经CNV分析发现大片段缺失12例,其中X连锁遗传7例、常染色体隐性遗传3例、常染色体显性遗传2例;部分外显子缺失4例,所有外显子缺失8例.经检索文献及数据库,共有51个PID基因有CNV致病性变异(14.8%,51/344),以基因组内部的部分外显子缺失为主(70.6%,36/51),常染色体隐性遗传(56.9%, 29/51)是致病性CNV最常见的遗传方式.结论 CNV在PID患者中并不少见,当临床及免疫学表现典型但通过常规生物信息分析未见可疑致病突变时,需考虑CNV因素.基于二代测序原始数据的CNV分析是常规生物信息分析的有力补充.

Objective To study the application of copy number variation (CNV) analysis based on the raw data of next?generation sequencing (NGS) in diagnosing primary immunodeficiency disease (PID). Methods One hundred sixty?five patients with suspicious PID were tested by NGS in the Department of Rheumatology and Immunology, Shenzhen Children's Hospital during September 2014 and Mary 2017. The raw data of the patients who got negative result were further analyzed for the CNV with CNVkit software. The pathogenic CNV were identified in the databases including Resource of Asian Primary Immunodeficiency Diseases (RAPID), Human Gene Mutation Database (HGMD) and ClinVar with the known 344 pathogenic genes of PID. The associated literature from January 2010 to May 2019 were searched in Pubmed, Weip, Wanfang and CNKI database with key words as "primary immunodeficiency disease" "copy number variation" and "next generation sequencing". Results Ninety?five out of 165 patients (57.6%) had negative result of the NGS test, among whom the patients with immune dysregulation had the highest negative rate (68.6%, 24/35). CNV analysis found large fragment deletion in 12 patients, within which 7 was X?linked inheritance, 3 was autosomal recessive inheritance, 2 was autosomal dominant inheritance. Partial exon deletion was found in 4 patients while whole gene deletion in 8 patients. According to the review of literature, CNV was reported in 51 pathogenic genes of PID (14.8%,51/344), mainly intern deletion (70.6%, 36/51), while autosomal recessive inheritance (56.9%,29/51) was the most common pattern. Conclusions CNV is not rare in PID. When the phenotype is clear in the patients who have negative NGS test, CNV should be considered.