Functional Characterization Of Novel CYP21 Mutations P459H And R483W Detected In Two Chinese Patients With Simple-virilizing Form Of Congenital Adrenal Hyperplasia

1.Site-directed mutagenesis of CYP21Objectives:21-hydroxylase deficiency(21-OHD)is the most frequent cause of congenital adrenal hyperplasia,a group of inherited metabolic disorders.Clinically,21-OHD is classified into three types:salt-wasting,simple-virilizing and non-classic.Deletion or mutations of CYP21(gene encoding 21-hydroxylsae)account for the disease. Generally there is good correlation between phenotype and genotype of 21-OHD,but exceptions also happen.Recently,by direct sequencing of the whole CYP21 gene of two simple-virilizing patients,a novel mutation P459H and a rare mutation R483W were detected.In order to determine how much enzyme activity are damaged respectively by each of the two mutations and then make genotype-phenotype correlation analysis,we did in vitro expression and assays of enzyme activity.First,we must introduce P459H and R483W mutation respectively into wild-type CYP21 and reconstruct plasmids containing mutant CYP21.Methods:To construct mutated CYP21,PCR reactions were done with template wide-type CYP21 and specially designed primers.Pfu DNA polymerase was used to avoid additional mutations.This is site-directed mutagenesis method.Finally the constructs was sequenced to confirm target mutations.Results:(1)Mutated pCMV4-CYP21 was digested by two restriction enzymes BglII and KpnI,which produced two fragments of approximately 1800bp and 4800bp.(2)Sequencing of mutated constructs verified target mutations but no additional mutaions. Conclusions:Plasmids with P459H or R483W mutated CYP21 were constructed by site-directed mutagenesis and can be used to express mutated CYP21 enzyme proteins.2.Functional analysis of mutated CYP21Objectives:To determine enzyme activity of P459H and R483 W mutated CYP21 and make genotype-phenotype correlation analysis.Methods:1.Transient expression of plasmids containing CYP21 in COS-7 cells.2.~3H-labeled progesterone was added to each well as the substrate to test enzyme activities.Results:1.As the result of three independent experiments,the two missense mutations P459H and R483W both show harm to 21-hydroxylase activity,as shown in Fig.2.With the WT CYP21 activity is defined as 100%,the residual activity toward progesterone of P459H mutated protein is reduced to 6.8%(SD=2.1%)of WT 21-hydroxylase,while R483W mutant was reduced to 2.9%(SD=1.5%)(P＜0,05,Student's t test)2.P459 is highly conservative in all orthologs known today,and is deeply buried in the inner core of the protein.When praline is substituted by histidine,interreactions between P459 and L464 or Q477 are lost,which may influence function of the protein.R483 is also highly conserved.Substitution of arginine by tryptophane destroyes interreactions between R483 and D322 of helix J as well as L445 and L446.Conclusions:The results revealed that both mutanted CYP21 significantly reduced enzyme activity towards progesterone.Thus the phenotype -genotype correlation of the two patients was well established.We also tried to explain their partial 21-hydroxylase activity loss by constructing three-dimensional model of mutated CYP21.The study had evident significance to predicting severity of the disease and to family genetic consultation.