Functional analysis of 11 CYP21A2 variants reveals greater loss of enzyme activity in cis variants and improves genotype-phenotype correlation in 21-hydroxylase deficiency.

PURPOSE: Clinical severity of 21-hydroxylase deficiency (21-OHD) is primarily determined by the CYP21A2 genotype and residual enzymatic activity of mutant CYP21A2. However, CYP21A2 variants often lack functional characterization, and the pathogenic contribution of multiple variants arranged in cis remains poorly understood. We aimed to determine the residual 21-hydroxylase activity of 11 rare CYP21A2 variants, assess the combined functional impact of cis-configured double variants, and refine genotype-phenotype correlations. METHODS: Among 713 patients with 21-OHD, 14 carried rare CYP21A2 variants. Targeted long-read sequencing was used to resolve allele structures and phase variants. Pathogenicity was evaluated using conservation analysis, in silico prediction, enzyme activity assays, and minigene-based splicing analysis. Clinical data were reviewed to examine genotype-phenotype correlations. RESULTS: We identified 11 CYP21A2 variants in 14 patients, including 10 missense variants and one deep intronic variant (c.738 + 75 C > T); four were novel (p.Ile173Phe, p.Gly179Glu, p.Asp259Val, p.Pro361Leu). Two cis-configured double-variant alleles were confirmed: p.[His63Leu; Val70Leu] and p.[Thr124Ile; Ile173Asn]. Functional assays showed that all 10 missense variants exhibited reduced activity across both substrate conversions. Both cis-configured double-variant alleles showed greater functional impairment than their corresponding single variants, a pattern consistent with a cumulative functional effect. The intronic variant c.738 + 75C > T caused partial intron 6 retention (73-bp insertion), with a predicted frameshift and likely nonsense-mediated decay. Overall concordance between genotype and phenotype was 71.4%. CONCLUSION: These findings expand the functional spectrum of CYP21A2 variants in 21-OHD and support clinical interpretation of rare alleles. Integration of long-read phasing with enzyme and splicing assays improves allele-level pathogenicity assessment, clarifies the greater functional impairment associated with cis-configured double-variant alleles, and enhances risk stratification and counseling.