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Clinical Chemistry 53: 1577-1584, 2007. First published July 18, 2007; 10.1373/clinchem.2007.087361
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Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2007;53:1577-1584.)
© 2007 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

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

Silvia Parajes1, Celsa Quinterio1, Fernando Domínguez1,2 and Lourdes Loidi1,a

1 Fundación Pública Gallega de Medicina Genómica (Unidad de Medicina Molecular), Santiago de Compostela, Spain.
2 Universidad de Santiago de Compostela, Departamento de Fisiología, Santiago de Compostela, Spain.

aAddress correspondence to this author at: Unidad de Medicina Molecular, Fundación Pública Gallega de Medicina Genómica, Hospital Clínico Universitario, 15706 Santiago de Compostela, Spain. Fax 34-981951473; e-mail lloidi{at}usc.es.

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 ({Delta}Ct) was used to assess CYP21A2 copy number.

Results: The {Delta}Ct 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.






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