Functional characterization and molecular modeling of the mutations in CYP21A2 gene from patients with Congenital Adrenal Hyperplasia.

Steroid 21-Hydroxylase deficiency is an inherited autosomal recessive metabolic disorder of the adrenal steroidogenesis caused due to mutations in the CYP21A2 gene in 95% of CAH cases. Notably, the de novo mutations arise at the rate of 3-5%, therefore the functional characterization is of utmost importance for categorization of the novel mutations. Herein, we have functionally characterized the CYP21A2 missense mutations viz., p. F306V and p. H365N. Notably, both the mutations were harbored by the patients exhibiting the non classical phenotype. We followed the approach of in vitro characterization of the mutant proteins expressed in COS7 cells. Of note, all the mutant constructs exhibited reduced residual enzyme activity fraternized with altered kinetic constants accompanied by higher requirement for the activation energy. Further, there was reduced protein expression in the mutant constructs to that of the wild-type. Molecular modeling suggested alteration in the structure-function relationship of the protein due to mutations. The evidence suggested by the in vitro and the in silico characterization of mutations directed us to conclude that both, p. F306V and p. H365N should be considered as non classical CAH causing mutations. Conceivably, the knowledge about the functional consequences of the mutations is the basis for improved genetic counseling with respect to prognosis and therapeutic implications.