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Direct Analysis of CYP21B Genes in 21-Hydroxylase Deficiency using Polymerase Chain Reaction Amplification

Endocrinology and Metabolism Section, Department of Pediatrics, Baylor College of Medicine Houston, Texas 77030

Address requests for reprints to: Dr. David Owerbach, Department of Pediatrics, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030.

Abstract

Steroid 21-hydroxylase deficiency is the leading cause of impaired cortisol synthesis in congenital adrenal hyperplasia (CAH). We have studied the structure of the CYP21B gene in 30 unrelated CAH patients using the polymerase chain reaction (PCR) to differentiate the active CYP21B gene from its highly related CYP21A pseudogene. The PCR approach obviates the need to distinguish the CYP21A and CYP21B genes by restriction endonuclease digestion and electrophoresis before analysis with labeled probes. Furthermore, direct nucleotide sequence analysis of CYP21B genes is demonstrated on the PCR-amplified DNA. Gene deletion of CYP21B, gene conversion of the entire CYP21B gene to CYP21A, frame shift mutations in exon 3, an intron 2 mutation that causes abnormal RNA splicing, and a mutation leading to a stop codon in exon 8 appear to be the major abnormalities of the CYP21B gene in our patients. These mutations appear to account for 21-hydroxylase deficiency in 22 of 26 of our saltwasting CAH patients.

FOOTNOTES

This work was supported in part by NIH Grant DK-39965 (to D.O.).

Received for publication June 29, 1989. Revision received September 26, 1989. Accepted for publication October 17, 1989.

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