Integrated Functional and Structural Analysis of Eleven CYP21A2 Variants of Uncertain Significance Resolves Their Pathogenic Role in Congenital Adrenal Hyperplasia

Context: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is a common autosomal recessive disorder characterized by impaired cortisol and aldosterone synthesis. While genotype-phenotype correlations are well-established for common CYP21A2 mutations, the clinical significance of rare missense variants remains a challenge, often leading to their classification as Variants of Uncertain Significance (VUS) and complicating genetic counseling. Objective: To determine the functional impact and pathogenicity of eleven CYP21A2 variants (p.L10del, p.R76K, p.E162G, p.S274Y, p.L308V, p.S373N, p.P387L, p.H393Q, p.R401G, p.R436C, and p.S494N) utilizing a comprehensive approach integrating population genetics, in silico structural modeling, and in vitro functional assays. Methods: Allele frequencies were analyzed using global databases (gnomAD, 1000G). Evolutionary conservation was assessed via ConSurf, and thermodynamic stability was predicted using FoldX and DUET. Structural dynamics were simulated using Molecular Dynamics (MD) (100 ns). Variants were expressed in HEK293T cells, and enzymatic activity was quantified using radiolabeled progesterone and 17-hydroxyprogesterone (17-OHP) as substrates, normalized to protein expression levels determined by Western blot. Results: Population analysis identified p.L10del and p.S494N as common polymorphisms (allele frequencies >5% in specific populations) with near-normal enzymatic activity (~99% and ~67% for 17-OHP, respectively), supporting a Benign classification. Conversely, p.L308V, p.P387L, and p.R436C were ultra-rare and exhibited severe loss of function, retaining