Novel and Rapid Molecular Diagnosis Methods in Comprehensive Genetic Analysis of 21-hydroxylase Deficiency

Abstract

Abstract Background Molecular analysis of CYP21A2 gene is of great importance to understanding the etiology of 21-hydroxylase deficiency. The aim of the study was to introduce a novel approach named CNVplex, together with 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 microconversions and eleven high-frequency mutations reported in the literature were detected by SNaPshot, and rare mutations were investigated by direct sequencing. Results Of the 113 21-OHD patients, 95.6% of affected alleles could be detected accurately by SNaPshot and CNVplex. Prevalent mutations were detected in 69.5% alleles, with 62.4% were pseudogene-derived microconversions, 1.8% were non pseudogene-derived mutations, and the rest 5.3% were complex variations resulting from multiple recombination between CYP21A2 and CYP21A1P. Large rearrangements were identified in 27.0% 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. Rare haplotype with a c.955C > T mutation on the duplicated CYP21A2 gene was found in 0.9% of proband and 33.3% of parents. In addition, four parents were also diagnosed for 21-OHD patients. Conclusion CNVplex and SNaPshot appears to be a highly efficient and reliable technique to use in a molecular diagnosis laboratory and together with direct sequencing based on locus-specific PCR might be a definitive way to detect almost all, common as well as rare, 21-OHD alleles.