Objective: To study the procedure of CYP21A2 gene mutation detection in 21-hydroxylase deficiency (21-OHD) patients. Methods: The detail clinical and biochemical data of 51 patients with 21-OHD [18 males and 33 females, with an average age of (16.4±9.9) years] were collected between December 2016 and December 2017 at Department of Endocrinology, Peking Union Medical College Hospital. Multiplex ligation dependent probe amplification (MLPA) and Sanger sequencing of the CYP21A2 gene were used to clarify the cause of 21-OHD. The genotype-phenotype correlation was also analyzed. Results: The incidences of large deletion, 8 bp deletion, I2G, I172N and F306+T were 19.6% (20/102), 1.0% (1/102), 30.4% (31/102), 25.5% (26/102) and 1.0%(1/102), respectively, and the detection rate of gene mutation in 51 21-OHD patients was 77.5% (79/102) by MLPA test. Except large and 8 bp deletion, all above mutations contained in MLPA and other 8 mutations, including P31L, Q319X, R361L, R357W, V282L, R484Q, G425S and R342W were detected, and the detection rate was 79.4% (81/102) by Sanger sequencing of CYP21A2. MLPA combined with direct sequencing identified mutations in all patients. Genotype correlated well with clinical phenotype in 21-OHD patients. Conclusions: When MLPA or CYP21A2 gene sequencing were used alone to diagnose the cause of 21-OHD, gene mutations in all patients could not be detected. The combination of the two methods can complement each other and fully clarify the underlying causes of 21-OHD.

目的： 探究21-羟化酶缺陷症（21-OHD）的基因诊断流程。 方法： 纳入2016年12月至2017年12月于北京协和医院内分泌科就诊的51例21-OHD患者，男18例，女33例，年龄（16.4±9.9）岁，收集患者临床和血生化资料。利用多重连接探针扩增技术（MLPA）和Sanger测序方法检测CYP21A2基因突变，比较两种方法的检出率，并探究21-OHD基因型与临床表型的一致性。 结果： 经MLPA检测，51例患者的102个CYP21A2等位基因中大片段缺失、第三外显子8 bp缺失、I2G、I172N及F306+T的比例分别为19.6%（20/102）、1.0%（1/102）、30.4%（31/102）、25.5%（26/102）及1.0%（1/102），基因突变检出率为77.5%（79/102）。CYP21A2基因的PCR扩增和Sanger测序检测出除大片段和第三外显子8 bp缺失之外的MLPA方法中包含的所有突变，还检出其他8种突变，分别为：P31L、Q319X、R361L、R357W、V282L、R484Q、G425S和R342W，突变检出率为79.4%（81/102）。MLPA联合直接测序确定了全部患者的基因突变，且通过基因型预测表型与临床表型相符。 结论： 单用MLPA或测序方法诊断21-OHD病因时，都会漏诊部分21-OHD患者，联合应用两种方法，能优势互补，有助于明确21-OHD的病因。.

Keywords: 21-hydroxylase deficiency; CYP21A2 gene; Gene amplification; Genetic diagnosis.