High-resolution melting curve (HRM) analysis to establish CYP21A2 mutations converted from the CYP21A1P in congenital adrenal hyperplasia

Abstract

Background: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease of an inborn error of steroid metabolism in humans. More than 90% of CAH cases are caused by mutations of the steroid 21-hydroxylase (CYP21A2) gene, and approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene.

Methods: A high-resolution melting (HRM) curve analysis was designed to characterize 11 mutation sites of the CYP21A2 gene that commonly appeared in 21-hydroxylase deficiency. Among these 11 mutations, 9 were found in CAH patients, and 2 were mutations created from normal individuals.

Results: From the HRM analysis using 6 fragments of amplicons, we have successfully identified these 11 common disease-causing mutations of the CYP21A2 gene, among which 3 showed 3 distinguishable melting plots; the heteroduplexes showed an upcurved plot, a horizontal plot of homoduplexes of wild-type (WT), and a downcurved plot of homoduplexes of compound mutations.

Conclusions: The HRM analysis is a 1-step of non-gel resolution technique which saves time and is a low-cost method to undertake such a program for screening CAH patients with the 21-hydroxylase deficiency caused by intergenic conversions from the neighboring CYP21A1P pseudogene.