Mutations of P450c21 (steroid 21-hydroxylase) at Cys428, Val281, and Ser268 result in complete, partial, or no loss of enzymatic activity, respectively.

Abstract

Steroid 21-hydroxylase deficiency is the major cause ofcongenital adrenal hyperplasia (CAH), a common genetic disease. To define the relationship between gene mutations and enzyme deficiency, we generated missense mutations of the 21-hydroxylase cDNA at three different sites and characterized the mutant proteins after expressing them in cultured mammain and yeast cells. Among them, Serl and Val"' have been found to be mutated in CAH patients, whereas Cys' has been implicated as the heme ligand. Our results show mutations at these sites result in complete, partial, or no loss of the enzymatic activity. All the Cys4' mutants had neither enzymatic activity nor P450 absorption, thus supporting the notion that Cys4' is the heme ligand. All the 268-mutants exhibited the same activity as normal 21-hydroxylase, demonstrating that the clinically observed Ser2" --Thr change represents a polymorphism rather than the cause of the enzyme deficiency. The 281-mutants had normal K., but greatly reduced V.. values that also paralleled the reduction in the heme content, in the order Val" (normal, 100%) > Ile2' (50%) > Leul' (20%) > Thr"' (10%).Our findings suggest that the methyl group at the l-carbon of Val"' is required for heme incorporation and consequently enzymatic activity. (J. Clin. Invest. 1991. 88:519-523.)

Mutational spectrum of steroid 21-hydroxylase and the genotype-phenotype association in Middle European patients with congenital adrenal hyperplasia

“…The presence of elevated basal and ACTH-stimulated 17OHP, premature pubarche, advanced bone age in all patients and also clitoral hypertrophy in one girl implicates a direct role of this substitution in the disease manifestation of NC and even SV-CAH. Lys102Arg and Ser268Thr mutations were also reported to be normal polymorphisms, however, a synergistic effect resulting in a decreased enzymatic activity was observed when both mutations were transmitted on the same haplotype (30,31). This could explain the finding of clitoral hypertrophy and elevated basal 17OHP levels in one NC patient hemizygous for Lys102Arg Ser268Thr substitutions.…”

“…In Mexican CAH patients a higher proportion of homozygosity for the Asn493Ser substitution was observed than in a healthy population (29). It was proposed that a synergistic effect between two mutations could lead to decreased enzymatic activity in CAH patients homozygous for Ser268Thr and Asn493Ser (30,31), but, except in one patient with a T-107C substitution and one patient with concomitant Lys102Arg on one haplotype, the sequencing of all the exons, introns and 340 nucleotides of the proximal promoter region revealed no other sequence alteration on alleles characterised by Asn493Ser. The presence of elevated basal and ACTH-stimulated 17OHP, premature pubarche, advanced bone age in all patients and also clitoral hypertrophy in one girl implicates a direct role of this substitution in the disease manifestation of NC and even SV-CAH.…”

The Common I172N Mutation Causes Conformational Change of Cytochrome P450c21 Revealed by Systematic Mutation, Kinetic, and Structural Studies

“…Plasmid Construction-Site-directed mutagenesis of the P450c21 cDNA cloned in M13 using a kit was performed as described previously (20). The mutated cDNA was subcloned into p5Јc21 and pYE8 vectors for in vitro transcription/translation and yeast expression, respectively, as described before (7,25).…”

“…The loss of enzymatic activity as a result of mutations of the c21B gene is shown by the expression of mutant proteins corresponding to each mutation in various cell types in mammalian (18), vaccinia (19), or yeast vectors (20). Some mutations cause the complete loss of enzymatic activity, resulting in the severe salt-wasting type of the disease.…”

Molecular analysis of Japanese patients with steroid 21-hydroxylase deficiency

“…Many mutations responsible for the disease have been described (Rodrigues et al 1987;Higashi et al 1988bHigashi et al , 1991Wu and Chung 1991). Most of these mutations are results of gene conversion events between the functional CYP21 gene and the pseudogene (CYP21P) acting as a reservoir of mutations (Higashi et al 1988a).…”