Endocrine Abstracts

JOINT699

Congenital adrenal hyperplasia (CAH) is a genetic disease inherited in an autosomal recessive manner. Over 90% of CAH cases are caused by reduced activity of 21-hydroxylase encoded by CYP21A2, a cytochrome P450 protein which catalyzes the conversion of 17-hydroxyprogensterone (17-OHP) to 11-deoxycortisol and progesterone to 11-deoxycorticosterone (11-DOC), leading to the production of cortisol and aldosterone. Clinically, three distinct phenotypes are identified: the salt-wasting, the simple virilizing, and the nonclassical (NC) phenotype. Typically, the phenotype is dictated by the specific variant present in the milder allele. Therefore, the presence of a severe mutation cannot always be inferred from the phenotype in some cases of NC-CAH, which may lead to the severe symptom associated with significant neonatal mortality and morbidity. Furthermore, discordance between the phenotype and the identical genotype can be observed within families. Protein stability, enzymatic function, and gene expression regulation may contribute to this mechanism. It is necessary to obtain sufficient experimental evidence using computational and functional analysis of mutants to elucidate the molecular pathogenic mechanisms and improve clinical diagnosis. Five novel disease-associated mutants in CYP21A2 were selected from ClinVar by analyzing their positions on the surface of protein structure in PyMOL. Conservation and chemical properties of these amino acid substitutions were predicted using ConSurf, PolyPhen-2, SNAP2, Meta-SNP, Predict SNP, and MutPred2. The structure stability of CYP21A2 variant was calculated by the FoldX tool. Changes in enzymatic activity were measured by the conversion of 17-OHP to 11-deoxycortisol and progesterone to 11-DOC in HEK293T cells. The expression levels of CYP21A2 were determined by Western Blot. Three selected mutations showed less than 50% of WT activity: L308V, R401G, and R436C exhibited 30%, 40%, and 26% of WT activity, respectively. These three mutations showed similar reductions in enzyme activity for both 17-OHP and progesterone conversion: for 17-OHP conversion, L308V, R401G, and R436C exhibited 11%, 43%, and 12% activity, respectively; and for progesterone conversion, L308V, R401G, and R436C exhibited 20%, 27%, and 12% activity, respectively. Mutations E162G and S373N showed 58% and 99% of WT activity, respectively. E162G showed increased expression compared to WT, while the other mutations showed decreased expression. The reduced activity observed for variants L308V, R401G, and R436C shows their potential pathogenicity in CAH. The observed differences in protein expression may be related to gene expression regulation, mRNA degradation, or protein stability. These findings contribute to our understanding of the molecular mechanisms underlying CAH caused by CYP21A2 deficiency.