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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.

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