Analysis of single nucleotide substitution in CYP21A2 gene using Alphagenome

Abstract

Context: CYP21A2 encodes steroid 21-hydroxylase, which supplies precursors for adrenal glucocorticoid and mineralocorticoid synthesis. Interpretation of its variants is complicated by a homologous pseudogene, complex alleles, heterogeneous functional assays and uncertain noncoding effects. We integrated observed variation with a reference-locus saturation analysis to distinguish transcriptional, splicing and protein-level mechanisms. Methods: The NM_000500.9 MANE transcript was reconstructed from GRCh38 genomic sequence and verified against RefSeq and UniProt P08686. ClinVar, gnomAD and UniProt records were retrieved with provenance, and a cutoff-limited PubMed search was reconciled against Entrez. AlphaGenome Atlas scores were obtained for every possible single nucleotide substitution across the 3,230-bp transcript span. Reported non-SNV alleles were analyzed by direct AlphaGenome inference. Adrenal, testis, ovary, placenta, liver and kidney expression predictions were calculated. Results: The results include 483 ClinVar records, 3,208 gnomAD records and 82 UniProt natural-variants. All 9,690 locus-wide substitutions were scored, comprising 6,018 exonic and 3,672 intronic substitutions. The coding set contained 3,201 missense, 1,098 synonymous, 147 stop-gained, nine start-lost, eight stop-lost and one stop-retained substitution. Direct inference was completed for 413 additional reported alleles and four context controls. Established splice defects, including c.293-13C>G and c.738+75C>T, showed appreciable splicing scores without corresponding large reductions in predicted total RNA. Clinical classification groups differed in AVI score distributions. Conclusions: A sequence-verified CYP21A2 catalogue connects clinical annotations, population observations, splicing predictions and measured enzyme function. Complete single-base coverage of the defined CYP21A2 transcript was achievable. Regulatory predictions should guide mechanistic experiments and remain separate from enzyme activity, tissue function and clinical classification.