[Diagnostic value of multiplex ligation dependent probe amplification combined with Sanger sequencing in...

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.