Molecular analysis of the CYP21A2 gene in Chinese patients with steroid 21-hydroxylase deficiency

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Objective21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), a family of autosomal recessive disorders involving impaired cortisol synthesis. This study aimed to design a reliable and rational approach for identifying mutations in the CYP21A2 gene and to characterize the molecular basis of 21-OHD in 30 Chinese patients. Design and methodsCopy number variations were investigated by multiplex ligation-dependent probe amplification (MLPA). Locus-specific polymerase chain reaction (PCR)/restriction endonuclease analysis was then used to verify CYP21A2 rearrangement products and prevent allele dropout. Direct sequencing of rearrangement products was performed to further refine recombination breakpoint locations. Direct sequencing of the entire CYP21A2 gene was used to detect microconversions. ResultsWe successfully characterized 60 CYP21A2 alleles from 30 patients with genetic defects. The most common one was intron 2 splice mutation (38.3%). Eighteen alleles with large gene deletions/conversions were identified, which accounted for nearly one-third (30.0%) of the genetic defects. Among these, three types of CYP21A1P/CYP21A2 chimeric genes (CH-1, CH-2, and CH-4) were characterized. Two novel CYP21A2 rearrangement genes were revealed and further demonstrated to be located downstream of the TNXB gene. ConclusionsOur results indicate that the stepwise diagnostic procedure involving MLPA analysis, locus-specific PCR/restriction endonuclease analysis, and direct DNA sequencing can provide detailed genetic information about Chinese 21-OHD patients, which is helpful for characterizing structural rearrangements of CYP21A2.