Cloning and Expression of cDNA Encoding a Bovine Adrenal Cytochrome P-450 Specific for Steroid 21-Hydroxylation (P-450 C21 ) (original) (raw)
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Proceedings of the National Academy of Sciences, 1986
P-450c21, a cytochrome P.450 enzyme [steroid 21-monooxygenase (steroid 21-hydroxylase), EC 1.14.99.10], mediates the 21-hydroxylation of glucocorticoid and mineralocorticoid hormones in the adrenal gland. The complete sequence of a bovine P-450c21 gene shows it is 3447 base pairs long and contains 10 exons. The intron/exon organization and encoded amino acid sequence indicate that P-450c21 represents a unique family of genes in the P450 gene superfamily. Primer extension and S1 nuclease protection experiments identified several cap sites for initiation of transcription; the principal cap site produces mRNA with a 5' untranslated region only 11 bases long. S1 nuclease protection experiments confirm that P-450c21 is actively expressed in the adrenal and the testis, an organ not known to secrete 21hydroxylated steroids.
The Journal of biological chemistry, 2017
Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxy(OH)progesterone to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of wild-type (WT) human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH progesterone). We found that the 17α-OH progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting (SW) or nonclassical (NC) forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2, and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37-13,000 fold higher than for WT P450 21A2. Cytochro...
Proceedings of the National Academy of Sciences, 1987
Congenital adrenal hyperplasia (CAH) is a common genetic disorder due to defective 21-hydroxylation of steroid hormones. The human P45OXXIA2 gene encodes cytochrome P450c2l [steroid 21-monooxygenase (steroid 21hydroxylase), EC 1.14.99.10], which mediates 21-hydroxylation. The P45OXXIA2 gene may be distinguished from the duplicated P45OXXIA1 pseudogene by cleavage with the restriction endonuclease Taq I, with the XXIA2 gene characterized by a 3.7-kilobase (kb) fragment and the XXIA1 pseudogene characterized by a 3.2-kb fragment. Restriction endonuclease mapping by several laboratories has suggested that deletion of the P45OXXIA2 gene occurs in about 25% of patients with CAH, as their genomic DNA lacks detectable 3.7-kb Taq I fragments. We have cloned human P450c21 cDNA and used it to study genomic DNA prepared from 51 persons in 10 families, each of which includes 2 or more persons with CAM. After Taq I digestion, apparent deletions are seen in 7 of the 20 alleles of the probands; using EcoRI, apparent deletions are seen in 9 of the 20 alleles. However, the apparently deleted alleles seen with Taq I do not coincide with those seen with EcoRI. Furthermore, studies with Bgl II, EcoRI, Kpn I, and Xba I yield normal patterns with at least two enzymes in all cases. Since all probands yielded normal patterns with at least two of the five enzymes used, we conclude that the P45OXXIA2 gene "deletions" widely reported in CAH patients probably represent gene conversions, unequal crossovers, or polymorphisms rather than simple gene deletions.
Journal of Clinical Investigation, 1992
Genotyping for 10 mutations in the CYP21 gene was performed in 88 families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Southern blot analysis was used to detect CYP21 deletions or large gene conversions, and allele-specific hybridizations were performed with DNA amplified by the polymerase chain reaction to detect smaller mutations. Mutations were detected on 95% of chromosomes examined. The most common mutations were an A G change in the second intron affecting pre-mRNA splicing (26%), large deletions (21%), Ile-172 --Asn (16%), and Val-281 --Leu (11%). Patients were classified into three mutation groups based on degree of predicted enzymatic compromise. Mutation groups were correlated with clinical diagnosis and specific measures of in vivo 21-hydroxylase activity, such as 17-hydroxyprogesterone, aldosterone, and sodium balance. Mutation group A (no enzymatic activity) consisted principally of salt-wasting (severely affected) patients, group B (2% activity) of simple virilizing patients, and group C (10-20% activity) of nonclassic (mildly affected) patients, but each group contained patients with phenotypes either more or less severe than predicted. These data suggest that most but not all of the phenotypic variability in 21-hydroxylase deficiency results from allelic variation in CYP21. Accurate prenatal diagnosis should be possible in most cases using the described strategy. (J. Clin. Invest. 1992. 90:584-595.) Key words: steroid 21-hydroxylase deficiency-CYP21 * allelic variation and in revisedform 21 Methods Patients. A total of 88 families of diverse ethnic backgrounds were studied. In 90 patients from 79 of these families, CYP21 mutations were identified on each of the two chromosomes. Clinical data are 584 Speiser, Dupont, Zhu, Serrat, Buegeleisen, Tusie-Luna, Lesser, New, and White J. Clin. Invest.
Human Genetics, 1994
We have defined the mutations causing congenital adrenal hyperplasia in three Swedish patients carrying a rare haplotype containing two mutated steroid 21hydroxylase genes (CYP21) in addition to one pseudogene (CYP21P). The presence of such haplotypes complicates genetic diagnosis and screening of mutations in 21hydroxylase deficiency, and we show how these genotypes can be resolved by amplification and analysis of each gene separately. In all cases, the rare haplotype carfled the same combination of disease-causing mutations; one of the genes had the splice mutation at base 659 in intron 2, and the other had the nonsense mutation at base 1999 in exon 8 (CAG to TAG). We have thus characterized the most common haplotype containing duplicated CYP21 genes. The frequency of this haplotype is low, and if additional such haplotypes are present, they are rare in this population.
The Journal of Steroid Biochemistry and Molecular Biology, 2011
The study subject was a 13 day-old boy admitted to hospital, with weight loss since birth. He presented with the vomiting and hypotension that are classical features of congenital adrenal hyperplasia (CAH). The most common type of CAH is an autosomal recessive disorder caused by mutations in the 21hydroxylase (CYP21A2) gene. To examine the CYP21A2 gene, gene-specific PCR was carried out, followed by sequencing. The baby was shown to be a compound heterozygote H365Y/R356W for two CYP21A2 gene mutations each inherited from a different parent. One of the mutations has not previously been functionally characterised. The mutations were reconstructed in an expression plasmid and characterised in vitro after transient transfection into human embryonic kidney (HEK293T) and hepatoblastoma (C3A) cell lines followed by measurement of enzyme activity. The CYP21A2 H365Y mutant exhibited minimal 21-hydroxylase activity to convert 17-hydroxyprogesterone to 11-deoxycortisol or progesterone to 11deoxycorticosterone. Western immunoblotting indicated that the H365Y enzyme was produced in more variable amounts than wild type; in particular, the H365Y mutant protein may be unstable and/or subject to a more rapid degradation by the human proteosome as well as catalytically inefficient. The double mutant genotype with a severe mutation on each allele is compatible with the clinical presentation.
Journal of Biological Chemistry, 1998
Three mutants (deletion of E196, G291S, and R483P) of steroid 21-hydroxylase (P450c21) from patients with inherited congenital adrenal hyperplasia had reduced activity toward progesterone and 17-hydroxyprogesterone after transient expression in cultured mammalian cells. In addition, both the E196 deletion and the R483P mutant had shorter half-lives than the wild-type enzyme, whereas the half-life of the G291S mutant was comparable with that of the normal protein. These results directly link the clinical situation with the three mutations and suggest that G291 is important for the catalytic activity of P450c21.
HETEROGENEITY OF STEROID 21-HYDROXYLASE GENES IN CLASSICAL CONGENITAL ADRENAL HYPERPLASIA
European Journal of Immunogenetics, 1987
Careful genotyping of three families, each having a member with classical saltlosing steroid 21-hydroxylase deficiency, has allowed identification of carrier haplotypes. Digestion with TaqI or EcoRI and probing with a cDNA probe for the 21-hydroxylase genes (pC21/3c) revealed that all six affected haplotypes are abnormal with at least EcoRI. The data suggest that there is extreme polymorphism of the 21-hydroxylase genes and that dysfunction may result from several different abnormalities.
The Genetics of Steroid 21-Hydroxylase Deficiency
The Endocrinologist, 2005
Congenital adrenal hyperplasia (CAH) is a prevalent cause of newborn genital ambiguity and adrenal insufficiency. The most common form of CAH, steroid 21-hydroxylase deficiency, accounts for about 95% of affected individuals. The main forms of this disease are classic salt-wasting, simple virilizing, and nonclassic. Phenotypic variability is attributable to allelic variation in the gene encoding active steroid 21-hydroxylase, CYP21A2. The disease is inherited as an autosomal recessive trait. There are more than 80 known disease-causing mutations, but about 10 mutations comprise 80% to 90% of alleles in most populations. It has become clear that there is a spectrum of disease, ranging from severe to mild, depending on which CYP21A2 mutations a patient carries. Genotyping can be useful in verifying the diagnosis of CAH in newborn screening programs primarily based on hormone assays and is also valuable in prenatal diagnosis.