BACKGROUND: More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by a steroid 21-hydroxylase deficiency. Approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. These 2 duplicated genes share a 98% nucleotide sequence homology. Therefore, precisely identifying the CYP21A2 gene in CAH patients is absolutely necessary. METHODS: We describe an established PCR-based amplification method, a denaturing high-performance liquid chromatography (DHPLC) analysis, to directly identify 11 different mutations commonly appearing in the CYP21A1P gene. Among these 11 mutations, 9 are found in CAH patients and 2 created mutations were from normal individuals. RESULTS: From the DHPLC analysis using 6 fragments of amplicons, the elution profiles of the 11 mutation sites were successfully used to distinguish these common disease-causing mutations of the CYP21A2 gene. Based on this resolution, we were able to rapidly search existing sequences of mutations in the CYP21A1P gene for this malady. CONCLUSION: DHPLC is an efficient and specific means to undertake such a program for screening patients with CAH caused by defects of the CYP21A2 gene resulting from the neighboring CYP21A1P pseudogene.
BACKGROUND: More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by a steroid 21-hydroxylase deficiency. Approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. These 2 duplicated genes share a 98% nucleotide sequence homology. Therefore, precisely identifying the CYP21A2 gene in CAH patients is absolutely necessary. METHODS: We describe an established PCR-based amplification method, a denaturing high-performance liquid chromatography (DHPLC) analysis, to directly identify 11 different mutations commonly appearing in the CYP21A1P gene. Among these 11 mutations, 9 are found in CAH patients and 2 created mutations were from normal individuals. RESULTS: From the DHPLC analysis using 6 fragments of amplicons, the elution profiles of the 11 mutation sites were successfully used to distinguish these common disease-causing mutations of the CYP21A2 gene. Based on this resolution, we were able to rapidly search existing sequences of mutations in the CYP21A1P gene for this malady. CONCLUSION:DHPLC is an efficient and specific means to undertake such a program for screening patients with CAH caused by defects of the CYP21A2 gene resulting from the neighboring CYP21A1P pseudogene.
Authors: Christopher N Greene; Suzanne K Cordovado; Daniel P Turner; Lisa M Keong; Dorothy Shulman; Patricia W Mueller Journal: Mol Genet Metab Rep Date: 2014-08-08