HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) 087361.Supplemental data All Versions of this Article: clinchem.2007.087361v1 53/9/1577    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Parajes, S. Articles by Loidi, L. Search for Related Content PubMed PubMed Citation Articles by Parajes, S. Articles by Loidi, L. Related Collections Molecular Diagnostics and Genetics

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

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 (Ct) was used to assess CYP21A2 copy number.

Results: The 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.

The following articles in journals at HighWire Press have cited this article:

				S. Kleinle, R. Lang, G. F. Fischer, H. Vierhapper, F. Waldhauser, M. Fodinger, and S. M. Baumgartner-Parzer Duplications of the Functional CYP21A2 Gene Are Primarily Restricted to Q318X Alleles: Evidence for a Founder Effect J. Clin. Endocrinol. Metab., October 1, 2009; 94(10): 3954 - 3958. [Abstract] [Full Text] [PDF]