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Clinical Chemistry 46: 606-611, 2000;
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(Clinical Chemistry. 2000;46:606-611.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Analysis of the Chimeric CYP21P/CYP21 Gene in Steroid 21-Hydroxylase Deficiency

Hsien-Hsiung Lee1,4,a, Jan-Gowth Chang1, Chang-Hai Tsai1, Fuu-Jen Tsai1, Hsiang-Tai Chao2, and Bon-chu Chung3

1 Department of Medical Research, Division of Molecular Medicine, China Medical College Hospital, No. 2, Yue-Der Road, Taichung 404, Taiwan, Republic of China.

2 Department of Obstetrics and Gynecology, Veterans General Hospital-Taipei, Taipei 11217, Taiwan, Republic of China.

3 Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan, Republic of China.

4 King Car Food Industrial Co., Ltd., Yuan-Shan Research Institute, No. 86, Chen-Hsiang Rd., Yuan-Shan I-Lan 264, Taiwan, Republic of China.
a Author for correspondence. Fax 886-3-9228030; e-mail hhlee{at}Kingcar.com.tw

Background: A single nonfunctional chimeric gene with its 5' and 3' ends corresponding to CYP21P and CYP21, respectively, is caused by unequal gene crossover in the CYP21 genes during meiosis. The presence of the chimeric CYP21P/CYP21 molecule can not be detected by conventional PCR methods and therefore may be lost in PCR amplification. This leads to a false result and diagnostic discordance.

Methods: We developed a rapid and direct method to detect a chimeric CYP21P/CYP21 gene that uses a 3'-specific primer for the CYP21 gene and two different 5' primers for both CYP21 and CYP21P to amplify the wild-type CYP21 and the chimeric CYP21P/CYP21 genes. A secondary PCR that can differentiate the chimeric from the wild-type gene was also performed. The PCR product was directly analyzed on agarose gel.

Results: After careful titration, we found that earlier failure to detect the chimeric CYP21P/CYP21 gene could be caused by unequal concentrations of two independent alleles as the PCR template or by the lack of primers to amplify chimeric molecules. We successfully amplified the chimeric gene using our improved method.

Conclusions: The chimeric CYP21P/CYP21 is present in a large portion of congenital adrenal hyperplasia patients. By adding a CYP21P/CYP21-specific primer, we were able to amplify and detect both homozygous and heterozygous chimeric genes. Therefore, our new PCR-based assay is a more effective way to analyze congenital adrenal hyperplasia mutations.




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


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S. Parajes, C. Quinterio, F. Dominguez, and L. Loidi
A Simple and Robust Quantitative PCR Assay to Determine CYP21A2 Gene Dose in the Diagnosis of 21-Hydroxylase Deficiency
Clin. Chem., September 1, 2007; 53(9): 1577 - 1584.
[Abstract] [Full Text] [PDF]

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 J. Clin. Endocrinol. Metab.Home page
H.-H. Lee, S.-F. Chang, F.-J. Tsai, L.-P. Tsai, and C.-Y. Lin
Mutation of IVS2 -12A/C>G in Combination with 707-714delGAGACTAC in the CYP21 Gene Is Caused by Deletion of the C4-CYP21 Repeat Module with Steroid 21-Hydroxylase Deficiency
J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2726 - 2729.
[Abstract] [Full Text] [PDF]

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 Hum Mol GenetHome page
P. F.J. Koppens, T. Hoogenboezem, and H. J. Degenhart
Carriership of a defective tenascin-X gene in steroid 21-hydroxylase deficiency patients: TNXB -TNXA hybrids in apparent large-scale gene conversions
Hum. Mol. Genet., October 2, 2002; 11(21): 2581 - 2590.
[Abstract] [Full Text] [PDF]

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 Clin. Chem.Home page
N. Krone, A. Braun, S. Weinert, M. Peter, A. A. Roscher, C.-J. Partsch, and W. G. Sippell
Multiplex Minisequencing of the 21-Hydroxylase Gene as a Rapid Strategy to Confirm Congenital Adrenal Hyperplasia
Clin. Chem., June 1, 2002; 48(6): 818 - 825.
[Abstract] [Full Text] [PDF]


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