Integrated mapping resolves pathogenicity of 11 CYP21A2 variants in congenital adrenal hyperplasia

Genotype-phenotype correlations in congenital adrenal hyperplasia (CAH) are often complicated by rare CYP21A2 missense variants classified as variants of uncertain significance.

To resolve the pathogenicity and structural mechanisms of 11 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).

We integrated population allele frequency analysis (gnomAD, 1000G, and 38KJPN) with in silico structural modeling (ConSurf, FoldX, and DUET) and molecular dynamics simulations. In vitro functional assays were performed in HEK293T cells using radiolabeled progesterone and 17-hydroxyprogesterone, with results normalized to CYP21A2 protein expression.

The variants p.L10del and p.S494N were identified as common polymorphisms (allele frequencies >5% in specific cohorts) retaining near-wild-type activity (∼67-99%), supporting a benign classification. Variants p.R76K and p.S373N showed higher activities (∼72-92%), supporting a likely benign classification. Conversely, p.L308V, p.P387L, and p.R436C exhibited severe loss of function (<20% activity for 17-hydroxyprogesterone) and were reclassified as pathogenic (simple virilizing CAH). Structural modeling revealed that p.L308V causes steric clashes in the conserved I-helix, while p.R436C disrupts the surface hydrogen-bond network essential for redox partner docking. Variants p.E162G, p.H393Q, and p.R401G showed moderate impairment (∼40-45% activity), consistent with likely pathogenic status and nonclassic CAH.

This study provides a potential diagnostic map for 11 CYP21A2 variants, distinguishing severe pathogenic mutations from benign polymorphisms. Our findings refine the molecular diagnosis of CAH.