A Simple and Robust Quantitative PCR Assay to Determine CYP21A2 Gene Dose in the Diagnosis of 21-Hydroxylase Deficiency

摘要

Background: Correct diagnosis of 21-hydroxylase deficiency (21OHD) requires the identification of CYP21A2 gene deletions and CYP21A1P / CYP21A2 chimeric genes, which are disease-causing alleles, and gene duplications, which can lead to false-positive 21OHD allele results. Because lack of suitable CYP21A2 dosage assessment methods hampers correct 21OHD diagnosis, we developed a new assay based on the relative quantification of the CYP21A2 gene using the DSP gene as a reference.Methods: The assay to determine CYP21A2 copy number is based on real-time PCR. The method also detects the presence of the CYP21A1P / CYP21A2 chimeric gene. We used a duplex PCR to coamplify the DSP gene, included as an internal control, along with CYP21A2 . The difference in threshold cycles between CYP21A2 and DSP genes (ΔCt) was used to assess CYP21A2 copy number.Results: The ΔCt values obtained from 24 samples used to set up the method clearly differentiated 3 nonoverlapping intervals, which corresponded to the number of CYP21A2 copies: ?1.35 to ?0.25 defined 2 gene copies, +0.20 to +2.00 defined 1 copy, and ?2.50 to ?1.50 defined 3 copies. With these intervals we were able to assess the gene copy number in 24 additional samples.Conclusions: This new method for gene copy assessment detects homozygous and heterozygous CYP21A2 gene deletions, CYP21A1P / CYP21A2 chimeric genes, and gene duplications. Moreover, the method is robust, fast, and easy to use in a molecular diagnosis laboratory. This method together with CYP21A2 gene sequencing can provide a definitive system for the detection of almost all, common as well as rare, 21OHD alleles.