Enrichment of CYP21A2 Promoter Variant rs150223621 in Congenital Adrenal Hyperplasia Cohorts from the 100 000 Genomes Project

To determine whether non‑coding variation in the CYP21A2 promoter contributes to congenital adrenal hyperplasia (CAH) and to test the functional impact of any enriched variant. Whole-genome data from the 100,000 Genomes Project (100k GP) were filtered to produce a case cohort of seven participants with recorded CAH diagnoses and a control cohort of 33,412 individuals. Bioinformatic screening of the 1 kb region upstream of CYP21A2 identified 3 promoter variants in the case cohort. Fisher’s exact testing showed significant enrichment of rs150223621 (NC_000006.12:g.32037495G > A) in confirmed cases versus controls (p = 2.6 × 10⁻⁵, odds ratio 30.4, 95% CI 6.95–105.5) and versus gnomAD v4.1 (p = 5.3 × 10⁻⁵, OR 25.2, 95% CI 5.77–87.7). No other variant reached significance. A 1 kb wild-type or rs150223621 promoter fragment was cloned upstream of firefly luciferase and transfected into zebrafish PAC2 cells (9 replicate wells per construct). Two-way repeated-measures ANOVA found no interaction between promoter genotype and luminescence over 30 time points (p = 0.654); Wilcoxon testing of total luminescence also suggested equivalence (p = 0.863). Thus, rs150223621 is statistically associated with CAH but did not measurably alter transcript output in this cell model, highlighting the need for additional context-specific assays.