Novel rapid molecular diagnosis methods for comprehensive genetic analysis of 21-hydroxylase deficiency

Abstract Background Molecular analysis of the CYP21A2 gene is highly important for understanding the aetiology of 21-hydroxylase deficiency (21-OHD). The aim of this study was to use a novel approach named CNVplex, together with the SNaPshot assay and direct sequencing, to identify CYP21A2 mutations efficiently and comprehensively. Targeted CYP21A2 mutation analysis was performed in 113 patients and 226 parents. Large rearrangements of CYP21A2 were characterized by CNVplex; twenty prevalent mutations, including nine common micro-conversions and eleven high-frequency mutations reported in the literature, were detected by SNaPshot; and rare mutations were investigated by direct sequencing. Results Among the 113 21-OHD patients, 95.6% of the affected alleles were detected accurately by SNaPshot and CNVplex. Prevalent mutations were detected in 69.5% of the alleles; 62.4% of alleles contained pseudogene-derived micro-conversions, 1.8% contained nonpseudogene-derived mutations, and 5.3% contained complex variations resulting from multiple recombinations between CYP21A2 and CYP21A1P. Large rearrangements were identified in 27.0% of the alleles, including five types (CH-1, CH-3, CH-4, CH-5 and CH-8) of chimeric CYP21A1P/CYP21A2 genes. Two novel CYP21A2 haplotypes and four de novo CYP21A2 mutations were characterized. A rare haplotype with a c.955 C > T mutation in the duplicated CYP21A2 gene was found in 0.9% of the probands and 33.3% of the parents. In addition, four parents were also diagnosed with 21-OHD. Conclusion CNVplex and SNaPshot appear to be highly efficient and reliable techniques for use in a molecular diagnosis laboratory, and combined with direct sequencing based on locus-specific PCR, they might constitute a definitive way to detect almost all common and rare 21-OHD-related alleles.