Functional and Structural Consequences of NineCYP21A2Mutations Ranging from Very Mild to Severe Effects (original) (raw)
Related papers
PLoS ONE, 2014
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism and accounts for 90-95% of CAH cases. In the present work, we analyzed the functional consequence of four novel previously reported point CYP21A2 mutations -p.R132C, p.R149C, p.M283V, p.E431K-found in Argentinean 21-hydroxylase deficient patients. In addition, we report an acceptor splice site novel point mutation, c.652-2A.G, found in a classical patient in compound heterozygosity with the rare p.R483Q mutation. We performed bioinformatic and functional assays to evaluate the biological implication of the novel mutation. Our analyses revealed that the residual enzymatic activity of the isolated mutants coding for CYP21A2 aminoacidic substitutions was reduced to a lesser than 50% of the wild type with both progesterone and 17-OH progesterone as substrates. Accordingly, all the variants would predict mild non-classical alleles. In one non-classical patient, the p.E431K mutation was found in cis with the p.D322G one. The highest decrease in enzyme activity was obtained when both mutations were assayed in the same construction, with a residual activity most likely related to the simple virilizing form of the disease. For the c.652-2A.G mutation, bioinformatic tools predicted the putative use of two different cryptic splicing sites. Nevertheless, functional analyses revealed the use of only one cryptic splice acceptor site located within exon 6, leading to the appearance of an mRNA with a 16 nt deletion. A severe allele is strongly suggested due to the presence of a premature stop codon in the protein only 12 nt downstream.
Novel Mutations in CYP21 Detected in Individuals with Hyperandrogenism
The Journal of Clinical Endocrinology & Metabolism, 2002
We studied the functional and structural consequences of two novel missense mutations in CYP21 found in women with hyperandrogenism. The women were predicted to carry mutations by hormonal evaluation, but did not display any of the genotypes commonly associated with congenital adrenal hyperplasia. In one woman the novel mutation V304M was found in homozygous form. After expression in COS-1 cells the mutated enzyme was found to have a residual activity of 46% for conversion of 17-hydroxyprogesterone and 26% for conversion of progesterone compared with the normal enzyme. The V304M variant thus represents the sixth known missense mutation associated with nonclassical disease. A normal degradation pattern for this mutant enzyme indicates that the missense mutation is of functional, rather than structural, importance. The other mutation, G375S, was detected in a young woman with signs of hyperandrogenism, in heterozygous form together with P453S, a mutation known to cause nonclassical congenital adrenal hyperplasia (her genotype was G375S؉P453S/wild type). This novel variant almost completely abolished enzyme activity; conversion was 1.6% and 0.7% of normal for 17-hydroxyprogesterone and progesterone, respectively. These results underline the importance of genetic evaluation and counseling in hyperandrogenic women who are predicted to carry congenital adrenal hyperplasiacausing mutations by biochemical tests. It also supports the idea that the heterozygous carrier state for CYP21 mutations can be associated with symptoms of androgen excess in certain susceptible individuals.
Characterization of Mutations Causing CYP21A2 Deficiency in Brazilian and Portuguese Populations
Deficiency of Cytochrome P450 Steroid 21-hydroxylase (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction along with functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in the Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH act...
Scientific Reports, 2016
Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90-95% of CAH cases. In this work we performed an extensive survey of mutations and SNPs modifying the coding sequence of the CYP21A2 gene. Using bioinformatic tools and two plausible CYP21A2 structures as templates, we initially classified all known mutants (n = 343) according to their putative functional impacts, which were either reported in the literature or inferred from structural models. We then performed a detailed analysis on the subset of mutations believed to exclusively impact protein stability. For those mutants, the predicted stability was calculated and correlated with the variant's expected activity. A high concordance was obtained when comparing our predictions with available in vitro residual activities and/or the patient's phenotype. The predicted stability and derived activity of all reported mutations and SNPs lacking functional assays (n = 108) were assessed. As expected, most of the SNPs (52/76) showed no biological implications. Moreover, this approach was applied to evaluate the putative synergy that could emerge when two mutations occurred in cis. In addition, we propose a putative pathogenic effect of five novel mutations, p.L107Q, p.L122R, p.R132H, p.P335L and p.H466fs, found in 21-hydroxylase deficient patients of our cohort. Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (OMIM 201910) represents 90-95% of all CAH cases 1-3. This autosomal recessive disorder, which is the most frequent inborn error of metabolism, has a broad spectrum of clinical forms, ranging from severe or classical, to mild late onset or non-classical (NC). The classical form includes the salt-wasting (SW) and simple virilizing (SV) of early onset. Girls with classical CAH are typically born with ambiguous genitalia. Patients with NC CAH exhibit clinical manifestations of hyperandrogenism. The gene encoding 21-hydroxylase, CYP21A2 is mapped to the short arm of chromosome 6 (6p21.3), within the human leukocyte antigen (HLA) complex, in the so-called RCCX module. Approximately two thirds of the chromosomes analyzed have a duplicated RCCX module that includes a genomic DNA segment composed of the pseudogenes RP2, CYP21A1P, TNXA, and a second active copy of the C4 (long or short) gene 4,5. CYP21A1P shares 98% sequence identity with CYP21A2. Due to the high degree of sequence identity between the gene and its pseudogene, most of the disease-causing mutations described in 21-hydroxylase deficiency are likely to be the consequence of non-homologous recombination or gene conversion events 6,7. Although most of the patients carry CYP21A1P-derived mutations, an increasing number of naturally occurring mutations have been found in disease-causing alleles (see: http://www.hgmd.cf.ac.uk for details). Mutations in the CYP21A2 gene cause varying degrees of 21-hydroxylase activity loss. In vitro studies revealed that mutations leading to a complete inactivation of 21-hydroxylase are usually associated with the SW phenotype. Mutations that reduce enzyme activity close to 2% cause the SV phenotype, whereas those with a residual enzymatic activity in the range of 10% to 60% result in the mild NC CAH phenotype. In addition, a great number
Modulation of human CYP19A1 activity by mutant NADPH P450 oxidoreductase
2007
Mutations in NADPH P450 oxidoreductase (POR) cause a broad spectrum of human disease with abnormalities in steroidogenesis. We have studied the impact of P450 reductase mutations on the activity of CYP19A1. POR supported CYP19A1 activity with a calculated K m of 126 nM for androstenedione and a V max of 1.7 pmol/min. Mutations R457H and V492E located in the FAD domain of POR that disrupt electron transfer caused a complete loss of CYP19A1 activity. The A287P mutation of POR decreased the activities of CYP17A1 by 60-80% but had normal CYP19A1 activity. Molecular modeling and protein docking studies suggested that A287P is involved in the interaction of POR:CYP17A1 but not in the POR:CYP19A1 interaction. Mutations C569Y and V608F in the NADPH binding domain of POR had 49 and 28% of activity of CYP19A1 compared with normal reductase and were more sensitive to the amount of NADPH available for supporting CYP19A1 activity. Substitution of NADH for NADPH had a higher impact on C569Y and V608F mutants of POR. Similar effects were obtained at low/high (5.5/8.5) pH, but using octanol to limit the flux of electrons from POR to CYP19A1 inhibited activity supported by all variants. High molar ratios of KCl also reduced the CYP19A1 supporting activities of C569Y and V608F mutants of POR to a greater extent compared to normal POR and A287P mutant. Because POR supports many P450s involved in steroidogenesis, bone formation, and drug metabolism, variations in the effects of POR mutations on specific enzyme activities may explain the broad clinical spectrum of POR deficiency.
Genes
Polycystic ovary syndrome (PCOS) is a reproductive disorder with multiple etiologies, mainly characterized by the excess production of androgens. It is equally contributed to by genes and environment. The CYP11A1 gene is imperative for steroidogenesis, so any dysregulation or mutation in this gene can lead to PCOS pathogenesis. Therefore, nucleotide diversity in this gene can be helpful in spotting the likelihood of developing PCOS. The present study was initiated to investigate the effect of single nucleotide polymorphisms in human CYP11A1 gene on different attributes of encoded mutated proteins, i.e., sub-cellular localization, ontology, half-life, isoelectric point, instability index, aliphatic index, extinction coefficient, 3-D and 2-D structures, and transmembrane topology. For this purpose, initially coding sequence (CDS) and single nucleotide polymorphisms (SNPs) were retrieved for the desired gene from Ensembl followed by translation of CDS using EXPASY tool. The protein seq...
The Journal of Steroid Biochemistry and Molecular Biology, 2010
In adrenal steroidogenesis, CYP17 catalyses the 17␣-hydroxylation of pregnenolone and progesterone and the subsequent 17,20-lyase reaction, yielding adrenal androgens. The enzyme exhibits distinctly different selectivities towards these substrates in various species. CYP17 has also been shown to exhibit 16␣-hydroxylase activity towards progesterone in some species, with only human and chimp CYP17 catalysing the biosynthesis of substantial amounts of 16-OHprogesterone. The 16␣-hydroxylase activity was investigated by introducing an Ala105Leu substitution into human CYP17. The converse mutation, Leu105Ala was introduced into the baboon, goat and pig enzymes. Wt human CYP17 converted ∼30% progesterone to 16-OHprogesterone while the Ala105Leu mutant converted negligible amounts to 16-OHprogesterone (∼9%), comparable to wt CYP17 of the other three species when expressed in COS-1 cells. The ratio of 17-hydroxylated products to 16-OHprogesterone of human CYP17 was 2.7 and that of the mutant human construct 10.5. Similar ratios were observed for human and goat CYP17 with the corresponding Ala or Leu residues. Although the Leu105Ala mutation of both baboon and pig CYP17 exhibited the same trend regarding the ratios, the rate of progesterone conversion was reduced. Coexpression with cytochrome b 5 significantly decreased the ratio of 17-hydroxylated products to 16-OHprogesterone in the Leu105 constructs, while effects were negligible with Ala at this position. Homology models show that Ala105 faces towards the active pocket in the predicted B -C domain of CYP17. The smaller residue allows more flexibility of movement in the active pocket than Leu, presenting both the C16 and C17 of progesterone to the iron-oxy complex.
Endocrine journal, 2017
Human cytochrome P450 11B2 (CYP11B2) is an essential enzyme in the steroid hormone biosynthesis, which catalyzes the last three reaction steps of the aldosterone synthesis. These reactions comprise a hydroxylation at position C11 of the steroid intermediate deoxycorticosterone yielding corticosterone, followed by a hydroxylation at position C18 yielding 18-hydroxy-corticosterone and a subsequent oxidation of the hydroxyl group at C18, which results in the formation of aldosterone. Alterations in the amino acid sequence of CYP11B2 often cause severe disease patterns. We previously described a procedure for expression and purification of human CYP11B2 employing recombinant E. coli, which allows the rapid characterization of CYP11B2 mutants on a molecular level. This system was now utilized for the examination of the influence of the polymorphism at position 173 in combination with the mutation V386A on the activity of CYP11B2. Our in vitro findings show that the combination of the V38...