title: Functional characterization of three CYP21A2 sequence variants (p.A265V, p.W302S, p.D322G) employing a yeast co-expression system.
abstract: Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase (CYP21A2) deficiency is the commonest inborn error in steroid hormone biosynthesis. Functional in vitro assessment of mutant activity generally correlates well with clinical phenotype and therefore has contributed greatly to phenotype prediction in this CAH variant. Three CYP21A2 sequence variants (g.1641C>T, p.A265V; g.1752G>C, p.W302S; and g.2012A>G, p.D322G) identified in patients with non-classic and simple virilizing CAH were characterized using a yeast co-expression system and a computational three-dimensional CYP21A2 model. Computational analysis of the mutants in the three-dimensional structural model predicted no relevant effect of p.A265V, while p.W302S and p.D322G were predicted to impact significantly on enzyme function. Consistent with these findings, in vitro mutant analysis revealed enzyme activity similar to wild-type for p.A265V, whereas p.W302S and p.D322G exerted activities compatible with simple virilizing and non-classical CAH, respectively. The results indicate that p.A265V is an allelic variant rather than a disease-causing amino acid change, whilst p.W302S and p.D322G could be confirmed as functionally relevant mutations. These findings emphasize the value of in vitro functional analysis of sequence variations in predicting genotype-phenotype correlations and disease severity.
authors.affiliation: Institute of Biomedical Research, Division of Medical Sciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Caroline
authors.initials: C
authors.lastname: Bleicken
authors.forename: Lourdes
authors.initials: L
authors.lastname: Loidi
authors.forename: Vivek
authors.initials: V
authors.lastname: Dhir
authors.forename: Silvia
authors.initials: S
authors.lastname: Parajes
authors.forename: Celsa
authors.initials: C
authors.lastname: Quinteiro
authors.forename: Fernando
authors.initials: F
authors.lastname: Dominguez
authors.forename: Joachim
authors.initials: J
authors.lastname: Grötzinger
authors.forename: Wolfgang G
authors.initials: WG
authors.lastname: Sippell
authors.forename: Felix G
authors.initials: FG
authors.lastname: Riepe
authors.forename: Wiebke
authors.initials: W
authors.lastname: Arlt
authors.forename: Nils
authors.initials: N
authors.lastname: Krone
mesh_terms: D000293:Adolescent; D002648:Child; D005260:Female; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013255:Steroid 21-Hydroxylase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D050505:Mutant Proteins; C531510:CYP21A2 protein, human; D013255:Steroid 21-Hydroxylase