Application of the DHPLC method for mutational detection of the CYP21A2 gene in congenital adrenal hyperplasia

BackgroundMore 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.MethodsWe 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.ResultsFrom 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.ConclusionDHPLC 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.