21羟化酶缺陷症患者CYP21A2基因拷贝数变异及CYP21A1P/CYP21A2融合基因类型

Analysis of copy number variation of CYP21A2 gene and the type of CYP21A1P/CYP21A2 fused gene in patients with 21-hydroxylase deficiency

目的分析21羟化酶缺陷症(21-OHD)患者的CYP21A2基因拷贝数变异情况,明确CYP21A2基因3拷贝重复、单拷贝缺失及CYP21A1P/CYP21A2融合基因的分型及所占比例。方法纳入2015年1月至2018年1月于北京协和医院就诊的424例21-OHD患者,年龄(17.1±12.4)岁,其中男140例,女284例,收集其临床和生化资料。提取外周血白细胞DNA,利用Sanger测序方法和多重连接探针扩增技术(MLPA)分别检测CYP21A2基因突变及拷贝数变异。结果424例21-OHD患者中有287例(67.7%)的CYP21A2基因为2拷贝;137例患者(32.3%)存在拷贝数变异,其中1例患者(0.2%)的CYP21A2基因为3拷贝,136例(32.1%)携带CYP21A2基因大片段缺失/转换突变。存在3拷贝CYP21A2基因的患者检测到3个致病突变,包含1个Q319X蛋白截短突变。携带CYP21A2基因大片段缺失/转换突变的患者中,82例(60.3%)携带CYP21A1P/CYP21A2融合基因,其余54例为CYP21A2全基因单拷贝缺失。最常见的CYP21A1P/CYP21A2融合基因类型是CH-5型、1型和2型,各占31.7%(26例)、26.8%(22例)和19.5%(16例);其次是CH-4型和7型,各占8.5%(7例)和4.9%(4例);此外发现CH-3型、6型和8型各2例,CH-9型1例。结论本研究首次在较大样本的21-OHD患者中检测了CYP21A2基因拷贝数及CYP21A1P/CYP21A2融合基因的发生情况。对于21-OHD患者遗传学特征的进一步研究应在常规突变检测的基础上结合拷贝数及融合基因的筛查。

Objective To analyze the copy number variation of CYP21A2 gene in 21-hydroxylase deficiency(21-OHD)patients,and identify the three copy repetition,single copy deletion of CYP21A2 gene and the type and proportion of CYP21A1P/CYP21A2 fused gene in 21-OHD patients.Methods A total of 424 patients(140 males and 284 females)with 21-OHD who visited Peking Union Medical College Hospital from January 2015 to January 2018 were enrolled and the average age was(17.1±12.4)years.All clinical and biochemical data were collected.DNAs were extracted from peripheral blood leukocytes,and CYP21A2 gene mutation and copy number variation were detected by Sanger sequencing and multiple ligation probe amplification(MLPA).Results Of 42421-OHD patients,287(67.7%)had two copies of CYP21A2 gene,137(32.3%)had copy number variation,of which 1 patients(0.2%)had 3 copies of CYP21A2 gene and 136(32.1%)were carriers of large deletion/rearrangement mutation of CYP21A2 gene.Three pathogenic mutations including a truncated Q319X protein mutation were detected in the patient with 3 copies of CYP21A2 gene.Of 136 patients with large deletion/rearrangement mutation of CYP21A2 gene,82(60.3%)carried fused CYP21A1P/CYP21A2 gene,and the remaining 54 harbored the one allele deletion of CYP21A2.The most common types of fused CYP21A1P/CYP21A2 gene were CH-5,CH-1 and CH-2,with the frequency being 31.7%(26 cases),26.8%(22 cases)and 19.5%(16 cases),respectively,and followed by CH-4 and CH-7,with the incidence being 8.5%(7 cases)and 4.9%(4 cases),respectively.In addition,two cases of CH-3,CH-6 and CH-8 and one case of CH-9 were detected.Conclusions This is the first study to detect the occurrence of CYP21A2 gene copy number variation and fused CYP21A1P/CYP21A2 gene in a large cohort of 21-OHD patients.The number of CYP21A2 gene copies in 21-OHD patients includes 2 copies,1 copy deletion and 3 copies duplication.One copy deletion of CYP21A2 includes one allele deletion of CYP21A2 gene and fused CYP21A1P/CYP21A2 gene.In patients with 3 copies of CYP21A2 gene,pathogenic mutations should be verified in all 3 copies of CYP21A2 gene to make the precise diagnosis.Therefore,the accurate molecular diagnosis of 21-OHD patients should take both genotype and copy number variation of CYP21A2 into account.