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. H365 N. 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. H365 N 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.

类固醇21羟化酶缺乏症是一种由于95％的CAH病例中CYP21A2基因突变引起的肾上腺类固醇生成的遗传性常染色体隐性代谢紊乱。值得注意的是，从头突变的发生率为3-5％，因此功能表征对于新突变的分类至关重要。本文中，我们已在功能上表征了CYP21A2错义突变，即p。F306V和p。H365N。值得注意的是，两个突变都由表现出非经典表型的患者所掩盖。我们遵循了体外方法COS7细胞中表达的突变蛋白的特征。值得注意的是，所有突变体构建物均表现出降低的残留酶活性，其以改变的动力学常数为代表，同时伴随着对活化能的更高要求。此外，突变体构建体中的蛋白质表达降低至野生型。分子建模表明，由于突变，蛋白质的结构-功能关系发生了变化。体外和计算机上提出的证据突变的特征使我们得出结论，两者。F306V和p。H365 N应被视为非经典CAH引起的突变。可以想象，有关突变功能后果的知识是就预后和治疗意义进行改进的遗传咨询的基础。