Molecular defects of the CYP21 gene in Spanish girls with isolated precocious pubarche.
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OBJECTIVE: To determine the frequency of mutant alleles in the CYP21 gene in Spanish girls presenting with precocious pubarche (PP) and to assess the relationships between genotype and endocrine-metabolic variables. DESIGN: Fifty-three unrelated girls with a history of PP (14 prepubertal, 8 pubertal and 31 postmenarcheal) and 35 controls were studied. METHODS: Genomic DNA was extracted from peripheral blood leukocytes. After selection against the pseudogen, an allele-specific PCR was used to identify 14 known mutations in the CYP21 gene. The mutations studied were Pro30Leu, splice intron 2, Ilel72Asn, Cluster E(6), Glyl92Ser, Ins T, GT-CT, Gln318-stop, Arg357Trp, Trp406-stop, Pro453Ser, Arg483Pro, Arg483 frameshift and Val281Leu. A standard 2-h oral glucose tolerance test was performed in all PP girls. Ovarian 17-hydroxyprogesterone (17-OHP) responses to gonadotrophin-releasing hormone-agonist stimulation was assessed in postmenarcheal PP girls. RESULTS: Thirteen PP girls and eight control girls were heterozygous for one of the mutations studied. The frequency of the carrier status was 25% and 23% in the PP and control groups respectively. Severe mutations were found in 33% of the carrier girls. Serum 17-OHP responses to ACTH stimulation were similar in carriers and non-carriers (351+/-65 vs 334+/-22 ng/dl). The presence of ovarian hyperandrogenism and/or hyperinsulinism was also not related to the carrier status. CONCLUSION: The incidence of molecular defects in the CYP21 gene in the present study was comparable in the PP and control groups. We found no relationship between the presence of carrier status and endocrine-metabolic abnormalities. Prospective studies of larger cohorts of PP girls are needed to ascertain the long-term clinical relevance of CYP21 heterozygosity. (+info)
Effects of G169R and P34S substitutions produced by mutations of CYP2D6*14 on the functional properties of CYP2D6 expressed in V79 cells.
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CYP2D6 is a polymorphic enzyme that catalyzes the oxidation of various drugs. At least 40-mutant alleles of CYP2D6 have been reported. CYP2D6*14, which is one of them found in Asian populations, causes deficient activity of CYP2D6. Four amino acid substitutions, P34S, G169R, R296C, and S486T, are present in the protein encoded by CYP2D6*14 (CYP2D6 14). Among them, G169R is thought to be a definitive substitution because it is unique to CYP2D6 14. However, a previous study showed that the activity of G169R-substituted CYP2D6 was about 40% of wild-type CYP2D6, suggesting that a combination of G169R and other substitutions may be required to abolish the activity of CYP2D6. In the present study, we examined the effects of combined substitutions of G169R and P34S on the functional properties of CYP2D6 and compared them with those of a single substitution of G169R or P34S using a cDNA expression system of V79 cells. The results showed that a combined substitution of G169R and P34S reduced the activities of CYP2D6 to less than the detection limit of our analytical method for bufuralol 1'-hydroxylation and dextromethorphan O-demethylation. However, these activities were not completely abolished by a single substitution of P34S or G169R. The findings suggest that simultaneous substitution of G169R and P34S is crucial for almost completely abolishing the activity of CYP2D6 at least in V79 cells, although whether the absence of metabolism is due to the absence of functional protein or catalytic incompetency remains unclear because the levels of CYP2D6 protein expressed in V79 cells were too low to be determined by difference CO-reduced spectra. (+info)
Explicit water near the catalytic I helix Thr in the predicted solution structure of CYP2A4.
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The solution structure of mouse cytochrome P450 2A4 (CYP2A4), a monooxygenase of deoxysteroids, was obtained using homology modeling and molecular dynamics. The solvent-equilibrated CYP2A4 preserves the essential features of CYP450s. A comparison of the models CYP2A4 and CYP2A4 with testosterone bound CYP2A4/T illustrates the changes induced by the binding of the substrate. Experimental evidence links four amino acid residues to the catalytic activity, substrate specificity, and regioselectivity of this enzyme. Three of the four amino acids are found within contact distance of the testosterone substrate, and therefore may control the binding of the substrate through direct interaction. Remarkably, a water complex previously observed in x-ray crystal structure forms near the bulge in the central I helix that contains a conserved Thr. The properties of the I helix are computed in the context of the presence or absence of ligand. (+info)
Serum leptin levels in patients with 21-hydroxylase deficiency before and after treatment.
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Increase in serum androgen levels results in suppression of serum leptin levels. In this study, the changes in serum leptin concentrations of children with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) with respect to their hyperandrogenism were investigated. Eleven children with 21-OHD and 25 healthy control children were included in the study. Before initiation of hydrocortisone, serum leptin levels in children with CAH were lower (1.7 +/- 1.3 ng/ml) than in the control group (5.3 +/- 4.01 ng/ml) (p<0.001). After three months of treatment, serum leptin levels increased to the normal range (7.1 +/- 2.9 ng/ml). Prior to and on hydrocortisone treatment in CAH, serum leptin levels were positively correlated with cortisol (r:0.78, p:0.004 and r:0.80, p:0.003) but negatively correlated with testosterone (r:-0.62, p:0.04 and r:-0.65, p:0.002). These results suggest that serum leptin measurements may be used as an additional parameter in the follow-up of children with CAH to evaluate the efficacy of hydrocortisone treatment with respect to androgenemia. (+info)
Selectivities of human cytochrome P450 inhibitors toward rat P450 isoforms: study with cDNA-expressed systems of the rat.
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The aim of this study was to determine the selectivities of chemical inhibitors for human cytochrome P450 (P450) isoforms toward the corresponding rat P450 isoforms by using cDNA-expressed rat P450s (CYP1A2, CYP2A1, CYP2C6, CYP2C11, CYP2D2, CYP2E1, CYP3A1, and CYP3A2). Among the inhibitor probes for human P450s used in this study, only sulfaphenazole showed a selective inhibitory effect on the activity of the corresponding rat P450 isoform (CYP2C6). Furafylline also preferentially inhibited the activity of rat CYP1A2. However, methoxalen and ketoconazole more strongly inhibited the activities of other P450 isoforms than those of the corresponding rat P450 isoforms, CYP2A1 and CYP3A1/2, respectively. On the other hand, quinidine and aniline had little effect on the activities of the corresponding rat P450 isoforms, CYP2D2, and rat CYP2E1, respectively. These results suggest that chemical probes that have been used for human P450 isoforms do not always exhibit the same selectivity for the corresponding rat P450 isoforms. However, it appears that sulfaphenazole can be used as a selective inhibitor for rat CYP2C6. In addition, furafylline may also be a relatively selective inhibitor for rat CYP1A2. (+info)
Activin signal transduction in the fetal rat adrenal gland and in human H295R cells.
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The presence of activin A and its effects have previously been documented in the adrenal gland, particularly in the human fetal adrenal gland and the rat adrenal gland. The primary signaling pathway of activin involves interactions between receptor and intracellular (Smad) proteins that have not been completely described in the adrenal gland. In this study, we demonstrate that the components of the activin signaling cascade are present in two complementary models, the fetal rat adrenal gland and the human adrenocortical cell line, H295R, by means of RT-PCR, western analysis, and immunoprecipitation techniques. Using the cell line, activin signaling was analyzed using an activin-responsive reporter gene, p3TP-luc, and luciferase assays to assess transcriptional activity with co-expression of the different activin receptors and Smads to demonstrate the functionality of the signaling cascade. In the fetal rat adrenal gland, the relative amounts of mRNA of the type II receptors, RII and RIIB, were regulated by gestational age, such that the RIIB levels increased after birth while RII levels fell. Using immunodetection techniques, the activin receptors and the different Smad proteins were detected in the rat fetal adrenal glands. Notably, the presence of Smad4 protein is significantly increased after birth in the rat adrenal gland. RT-PCR established a similar profile in the H295R cells. Using p3TP-luc, the H295R cells show transcriptional activation of this activin-responsive reporter in the presence of activin A. Co-expression of type I and type II receptors as well as Smads, results in ligand-independent transcriptional activity in addition to an activin-stimulated response. In determining activin's effects on adrenal function, adrenal steroid production was evaluated by incubation of the H295R cells with increasing doses of activin A and inhibin A, resulting in a detectable increase in P450c17 expression. Co-incubation of activin A with follistatin diminishes this response. These results are consistent with a role for activin A in the adrenal gland by demonstrating that the elements of the activin signaling pathway are present, intact, and functional. This suggests that in the adrenal gland the components of the activin receptor/Smad pathway are dynamically changing in the transition from fetal to neonatal life, and are important to the function of this organ. (+info)
Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: a novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease.
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OBJECTIVE: To analyse the mutational spectrum, the associated haplotypes and the genotype-phenotype correlation, and to design a reliable and rational approach for CYP21 mutation detection in Slovenian congenital adrenal hyperplasia (CAH) patients. DESIGN: Molecular analysis of the CYP21 gene was performed in 36 CAH patients and 79 family members. METHODS: Southern blotting, sequence-specific PCR amplification (PCR-SSP), sequence-specific oligonucleotide hybridisation (PCR-SSO) and sequencing were used to detect CYP21 gene deletions, conversions and point mutations. RESULTS: CYP21 gene deletion was the most frequent mutation (36.4%). Large gene conversions detectable only by Southern blotting represented 12.1%, and gene conversions involving the promoter region represented 7.6% of the mutated alleles. The most frequent point mutations were: intron 2 splice mutation 16.7%, Ile172Asn mutation 7.6%, Gln318Stop 7.5% and Pro30Leu 12.2% of alleles. A correlation between the genotype and the clinical phenotype similar to those described for large populations was observed. The finding of Pro30Leu mutation linked to a gene conversion could explain the simple virilising (SV) phenotype in compound heterozygotes for the Pro30Leu and a severe mutation. In two siblings with a salt wasting form of CAH (SW-CAH), a novel mutation Ala15Thr was found on the allele characterised by Pro30Leu mutation and gene conversion involving the promoter region. CONCLUSIONS: Our genotyping approach allowed reliable diagnosis of CAH in the Slovenian population. The high frequency of CYP21 gene aberrations on Pro30Leu positive alleles justified systematic searching for a gene conversion in the promoter region using the PCR-SSP reaction. (+info)
Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots.
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Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hybridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, beta 2-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFMPs were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMPs. RFMPs can be used as genetic markers, because their alleles segregate in a Mendelian manner. Unlike most other methods for detecting DNA sequence polymorphisms, a genomic DNA blot made from one gel can be hybridized consecutively with many (30 or more) different probes. (+info)