Integrated Functional and Structural Analysis of Eleven <em>CYP21A2 </em>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 &gt;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 &lt;20% of wild-type activity for 17-OHP. Structural modeling revealed that p.L308V causes steric clashes in the conserved I-helix, while p.P387L induces core destabilization and kinetic instability. p.R436C disrupts the surface hydrogen-bond network essential for P450 Oxidoreductase interaction. These variants are reclassified as Pathogenic, likely associated with Simple Virilizing CAH. Variants p.H393Q and p.R401G showed moderate impairment (~40–45% activity), consistent with a Likely Pathogenic status and Non-Classic CAH phenotype. Conclusions: This study resolves the diagnostic status of eleven CYP21A2 variants. We provide definitive evidence that p.L308V is a severe pathogenic missense mutation distinct from frameshift alleles at the same locus, and that surface mutations like p.R436C can be as deleterious as core mutations due to the disruption of redox partner interfaces. These findings refine the genetic classification of CAH, enabling precise diagnosis and personalized management for affected families.