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Michel Kranendonk - One of the best experts on this subject based on the ideXlab platform.
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DMD #42820 1 Title page
2016Co-Authors: Daniela Moutinho, Christopher C Marohnic, Jose Rueff, Satya P. P, Bettie Sue Masters, Michel KranendonkAbstract:Altered human CYP3A4 activity caused by Antley-Bixler Syndrome-related variants of NADPH-cytochrome P450 oxidoreductase measured in a robust in vitro syste
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Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype.
The Journal of biological chemistry, 2016Co-Authors: Karen Mccammon, Michel Kranendonk, Satya Prakash Panda, Daniela Moutinho, Chuanwu Xia, Jung-ja P. Kim, Richard J. Auchus, Eileen M. Lafer, Debashis Ghosh, Pavel MartásekAbstract:Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo. Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the Syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.
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altered human cyp3a4 activity caused by antley bixler Syndrome related variants of nadph cytochrome p450 oxidoreductase measured in a robust in vitro system
Drug Metabolism and Disposition, 2012Co-Authors: Daniela Moutinho, Christopher C Marohnic, Satya Prakash Panda, Bettie Sue Siler Masters, Jose Rueff, Michel KranendonkAbstract:NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler Syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375–384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (kcat and Km) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained ∼10 and 30% activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (kcat/Km), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (kcat/Km) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.
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Altered human CYP3A4 activity caused by Antley−Bixler Syndrome-related variants of NADPHcytochrome P450 oxidoreductase measured in a robust in vitro system
2012Co-Authors: Daniela Moutinho, Christopher C Marohnic, Satya P. P, Jose ́ Rueff, Bettie Sue Masters, Michel KranendonkAbstract:been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler Syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR vari-ants were coexpressed with CYP3A4, resulting in relative expres-sion levels comparable to those found in hepatic tissue. Dibenzyl-fluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375–384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver mi-crosomes. CYP3A4, combined with WT CYPOR, demonstrated ki-netic parameters (kcat and Km) equal to those for pooled huma
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impairment of human cyp1a2 mediated xenobiotic metabolism by antley bixler Syndrome variants of cytochrome p450 oxidoreductase
Archives of Biochemistry and Biophysics, 2008Co-Authors: Michel Kranendonk, Christopher C Marohnic, Satya Prakash Panda, Maria Paula Duarte, Jose Santos Oliveira, Bettie Sue Siler Masters, Jose RueffAbstract:Abstract Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley–Bixler Syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: POR null , POR wt , POR YH , and POR VE , for which equivalent CYP1A2 and CYPOR levels were confirmed, except for POR null , not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5- f )quinoline, were significantly less mutagenic in POR YH and POR VE models than in POR wt , indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.
Walter L. Miller - One of the best experts on this subject based on the ideXlab platform.
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COMMENTS AND RESPONSES Did Akhenaten Have the Antley–Bixler Syndrome? TO THE EDITOR: Braverman and colleagues (1) hypothesize that
2015Co-Authors: Walter L. MillerAbstract:Akhenaten (Amenophis IV) had gynecomastia and brachycephaly attributable to the form of the Antley–Bixler Syndrome (ABS) caused by mutations in P450 oxidoreductase (POR). Having reviewed the clinical findings and identified the genetic defects in most reported cases of POR deficiency (2–4), I find this unlikely. First described in 1975, ABS is a rare skeletal dysplasia Syndrome characterized by craniosynostosis, radioulnar or radiohumeral synostosis, brachyceph-aly, femoral bowing, femoral fractures, midface hypoplasia, propto-sis, a “pear-shaped ” nose, choanal atresia, and other bony findings (5, 6). The ABS phenotype can result from either of 2 distinct genetic disorders: autosomal dominant gain-of-function mutations of FGFR2 or autosomal recessive loss-of-function mutations of POR (3). The multigenerational history of brachycephaly in Akhenaten’s family could be consistent with an autosomal dominant disorder, possibly in FGFR2. However, nothing in Akhenaten’s kindred sug
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did akhenaten have the antley bixler Syndrome
Annals of Internal Medicine, 2009Co-Authors: Walter L. MillerAbstract:deficiency (2–4), I find this unlikely. First described in1975, ABS is a rare skeletal dysplasia Syndrome characterized bycraniosynostosis, radioulnar or radiohumeral synostosis, brachyceph-aly, femoral bowing, femoral fractures, midface hypoplasia, propto-sis, a “pear-shaped” nose, choanal atresia, and other bony findings (5,6). The ABS phenotype can result from either of 2 distinct geneticdisorders: autosomal dominant gain-of-function mutations of
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Apparent Manifesting Heterozygosity in P450 Oxidoreductase Deficiency and Its Effect on Coexisting 21-Hydroxylase Deficiency
The Journal of clinical endocrinology and metabolism, 2007Co-Authors: Rachel R. Scott, Ningwu Huang, Larissa G. Gomes, Guy Van Vliet, Walter L. MillerAbstract:Context: P450 oxidoreductase (POR) deficiency is a disorder of steroidogenesis affecting the microsomal P450 enzymes that use POR as an electron donor. The clinical presentation is variable; patients can be asymptomatic or can present with genital anomalies and the Antley-Bixler Syndrome, characterized by craniosynostosis and other bony anomalies. Obligately heterozygous parents are normal. Combined POR and 21-hydroxylase deficiencies have not been reported. Objective: The aim was to explore the manifestations of combined deficiencies of 21-hydroxylase and POR and to search for lesions in apparent manifesting POR heterozygotes. Patients and Methods: A newborn female had craniosynostosis, severe salt wasting, minimal virilization, grossly elevated 17OH-progesterone, and minimally elevated androgens. DNA encoding 21-hydroxylase, POR, and fibroblast growth factor receptor 2 was sequenced. For POR, the first untranslated exon (exon 1U), 5′ flanking DNA, and most introns were sequenced in five apparent manifes...
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P450 oxidoreductase deficiency: a new form of congenital adrenal hyperplasia.
Current opinion in pediatrics, 2006Co-Authors: Christa E Fluck, Walter L. MillerAbstract:Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler Syndrome (a craniosynostosis Syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate ‘backdoor’ pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.
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antley bixler Syndrome a reply to cragun and hopkin
American Journal of Human Genetics, 2005Co-Authors: Walter L. MillerAbstract:To the Editor: Cragun and Hopkin (2005 [in this issue]) raise a series of points concerning use of the term “Antley-Bixler Syndrome” (ABS), both in our recent paper in this journal (Huang et al. 2005) and elsewhere. We agree that it is useful to minimize confusion: the best way to do this may be to discard the term “ABS” and other eponymic terms once the molecular genetics and cell biology of a disease have been worked out. However, until such eponyms are discarded, as stated in our paper, “we propose that the term ‘ABS’ be reserved for those patients with the skeletal dysmorphologic findings initially reported by Antley and Bixler (1975) but who have normal genitalia and normal steroidogenesis” (Huang et al. 2005, p. 745). Cragun and Hopkin suggest that “patients with FGFR mutations should be grouped with those with other autosomal dominant FGFR-related craniosynostosis Syndromes.” We agree. Describing dysmorphologic disorders by eponyms was appropriate when the responsible genes were not known, but little purpose is served in preserving these terms once the molecular genetics and cell biology are delineated. Although one may wish to honor investigators with eponyms, such nosology inhibits understanding of mechanisms: “post-streptococcal glomerulonephritis” may be less euphonious than “Bright's disease” but is more specific and informative. Cragun and Hopkin raise questions about the patient initially described by Antley and Bixler and about several other reported cases. Clearly, it is difficult (if not impossible) to make or exclude a diagnosis retrospectively. Lacking the individual’s DNA, we cannot know the molecular lesion in the patient described by Antley and Bixler (but we’d be happy to study it!). Whereas Cragun and Hopkin lament the paucity of published photographs and clinical detail in many case reports, we lament the poor descriptions of the external genitalia and the incomplete hormonal evaluations in most patients with “ABS.” Careful steroidal evaluation is crucial: mass spectrometric analysis of urinary steroids is highly reliable but not widely available; appropriate evaluation of pre- and poststimulation plasma steroids is equally reliable. The data available to Reardon et al. (2000) were insufficient to establish or exclude a steroidogenic disorder for several of their patients; hence, their report does not provide evidence for the suggestion that POR (P450 oxidoreductase) mutations might cause a skeletal disorder associated with normal genitalia. Thus, we believe that our conclusion that “aside from the genital anomalies attributable to disordered steroidogenesis, no dysmorphological feature distinguished patients with POR mutations from those with FGFR mutations” (Huang et al. 2005, p. 736) is well supported by the data in table 1 of Huang et al. (2005, p. 731). For convenience, we have compiled the clinical data from table 1 and the genetic data from table 4 (p. 737) in the table shown here. More-refined observations may identify other anatomic features distinguishing POR from FGFR mutations in infants with craniosynostosis; however, our data clearly show that dysmorphologists must pay very close attention to the external genitalia in these children and consider evaluation of steroid secretion before and after tropic stimulation with adrenocorticotropic hormone. Table 1 Genotype/Phenotype Correlations Collated from the Data in Tables 1 and 4 of Huang et al. (2005)[Note] Deficient POR activity can cause a broad spectrum of human disease (Fluck et al. 2004; Miller 2004; Huang et al. 2005), and clearly the patients described by us and others who have POR lesions without skeletal defects do not have ABS. Thus, we do not think it is reasonable to follow the suggestion of Cragun and Hopkin to use the term “ABS” for patients with the more severe form of POR deficiency associated with skeletal anomalies. If, as they propose (and we agree), all FGFR-based craniosynostosis disorders should be grouped together, then it is logical to group all POR disorders together. Regardless of whether the clinical genetics community agrees to discard eponyms in favor of a term such as “FGFR hyperactivity,” we believe that “POR deficiency,” which is a newly described monogenic disease, should be dissociated from the term “Antley-Bixler Syndrome.”
Christa E Fluck - One of the best experts on this subject based on the ideXlab platform.
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P450 Oxidoreductase Deficiency: Loss of Activity Caused by Protein Instability From a Novel L374H Mutation
The Journal of clinical endocrinology and metabolism, 2016Co-Authors: Shaheena Parween, Christa E Fluck, Florence Roucher-boulez, Anne Lienhardt-roussie, Delphine Mallet, Yves Morel, Amit V PandeyAbstract:Context: P450 oxidoreductase (POR) is required for the activities of steroid-metabolizing cytochrome P450 enzymes in the endoplasmic reticulum. POR deficiency (PORD) is a form of congenital adrenal hyperplasia. Objective and Aim: Enzymatic and structural analysis of a novel L374H POR mutation from a patient with 46,XX disorder of sexual development. Design, Setting, Patient, and Intervention: The patient was a 46,XX girl with nonconsanguineous Turkish parents. She had virilized external genitalia at birth, a uterus and ovaries, and no sign of Antley-Bixler Syndrome. The initial diagnosis was CYP21A2 deficiency with no mutations in CYP21A2, but POR mutations were found. Functional testing was done after producing recombinant POR proteins for analyzing enzymatic and structural properties. Main Outcome: Novel mutations were causing severe loss of POR activities for metabolism of steroids and small molecules. Results: The L374H mutation reduced activities by 80% in cytochrome c, 97% in thiazolyl blue tetrazol...
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reduction in hepatic drug metabolizing cyp3a4 activities caused by p450 oxidoreductase mutations identified in patients with disordered steroid metabolism
Biochemical and Biophysical Research Communications, 2010Co-Authors: Christa E Fluck, Primus E Mullis, Amit V PandeyAbstract:Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler Syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.
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Characterization of the first FGFRL1 mutation identified in a craniosynostosis patient
Biochimica et biophysica acta, 2008Co-Authors: Thorsten Rieckmann, Christa E Fluck, Lei Zhuang, Beat TruebAbstract:Fibroblast growth factor receptor-like 1 (FGFRL1) is a recently discovered transmembrane protein whose functions remain unclear. Since mutations in the related receptors FGFR1-3 cause skeletal malformations, DNA samples from 55 patients suffering from congenital skeletal malformations and 109 controls were searched for mutations in FGFRL1. One patient was identified harboring a frameshift mutation in the intracellular domain of this novel receptor. The patient showed craniosynostosis, radio-ulnar synostosis and genital abnormalities and had previously been diagnosed with Antley-Bixler Syndrome. The effect of the FGFRL1 mutation was studied in vitro. In a reporter gene assay, the wild-type as well as the mutant receptor inhibited FGF signaling. However, the mutant protein differed from the wild-type protein in its subcellular localization. Mutant FGFRL1 was mainly found at the plasma membrane where it interacted with FGF ligands, while the wild-type protein was preferentially located in vesicular structures and the Golgi complex. Two motifs from the intracellular domain of FGFRL1 appeared to be responsible for this differential distribution, a tandem tyrosine based motif and a histidine-rich sequence. Deletion of either one led to the preferential redistribution of FGFRL1 to the plasma membrane. It is therefore likely that mutant FGFRL1 contributes to the skeletal malformations of the patient.
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P450 oxidoreductase deficiency: a new form of congenital adrenal hyperplasia.
Current opinion in pediatrics, 2006Co-Authors: Christa E Fluck, Walter L. MillerAbstract:Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler Syndrome (a craniosynostosis Syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate ‘backdoor’ pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.
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diversity and function of mutations in p450 oxidoreductase in patients with antley bixler Syndrome and disordered steroidogenesis
American Journal of Human Genetics, 2005Co-Authors: Ningwu Huang, Ethylin Wang Jabs, William Reardon, Amit V Pandey, Vishal Agrawal, Pablo Lapunzina, David Mowat, Guy Van Vliet, Joseph Sack, Christa E FluckAbstract:P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler Syndrome (ABS), which has also been attributed to fibroblast growth factor receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and/or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); 17 of the 34 alleles carried 16 “private” mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of 17α-hydroxylase activity, and support of 17,20 lyase using human P450c17. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR’s support of catalysis by P450c17—the enzyme most closely associated with the hormonal phenotype—provided an excellent genotype/phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency.
Amit V Pandey - One of the best experts on this subject based on the ideXlab platform.
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P450 Oxidoreductase Deficiency: Loss of Activity Caused by Protein Instability From a Novel L374H Mutation
The Journal of clinical endocrinology and metabolism, 2016Co-Authors: Shaheena Parween, Christa E Fluck, Florence Roucher-boulez, Anne Lienhardt-roussie, Delphine Mallet, Yves Morel, Amit V PandeyAbstract:Context: P450 oxidoreductase (POR) is required for the activities of steroid-metabolizing cytochrome P450 enzymes in the endoplasmic reticulum. POR deficiency (PORD) is a form of congenital adrenal hyperplasia. Objective and Aim: Enzymatic and structural analysis of a novel L374H POR mutation from a patient with 46,XX disorder of sexual development. Design, Setting, Patient, and Intervention: The patient was a 46,XX girl with nonconsanguineous Turkish parents. She had virilized external genitalia at birth, a uterus and ovaries, and no sign of Antley-Bixler Syndrome. The initial diagnosis was CYP21A2 deficiency with no mutations in CYP21A2, but POR mutations were found. Functional testing was done after producing recombinant POR proteins for analyzing enzymatic and structural properties. Main Outcome: Novel mutations were causing severe loss of POR activities for metabolism of steroids and small molecules. Results: The L374H mutation reduced activities by 80% in cytochrome c, 97% in thiazolyl blue tetrazol...
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Pharmacogenomics of human P450 oxidoreductase
Frontiers in pharmacology, 2014Co-Authors: Amit V Pandey, Patrick SprollAbstract:Cytochrome P450 oxidoreductase (POR) supports reactions of microsomal cytochrome P450 which metabolize drugs and steroid hormones. Mutations in POR cause disorders of sexual development. P450 oxidoreductase deficiency (PORD) was initially identified in patients with Antley-Bixler Syndrome but now it has been established as a separate disorder of sexual development (DSD). Here we are summarizing the work on variations in POR related to metabolism of drugs and xenobiotics. We have compiled mutation data on reported cases of PORD from clinical studies. Mutations found in patients with defective steroid profiles impact metabolism of steroid hormones as well as drugs. Some trends are emerging that establish certain founder mutations in distinct populations with Japanese (R457H), Caucasian (A287P) and Turkish (399-401) populations showing repeated findings of similar mutations. Most other mutations are found as single occurrences. A large number of different variants in POR gene with more than 130 amino acid changes are now listed in databases. Among the polymorphisms, the A503V allele is found in about 30% of all alleles but there are some differences across different population groups.
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reduction in hepatic drug metabolizing cyp3a4 activities caused by p450 oxidoreductase mutations identified in patients with disordered steroid metabolism
Biochemical and Biophysical Research Communications, 2010Co-Authors: Christa E Fluck, Primus E Mullis, Amit V PandeyAbstract:Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler Syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.
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diversity and function of mutations in p450 oxidoreductase in patients with antley bixler Syndrome and disordered steroidogenesis
American Journal of Human Genetics, 2005Co-Authors: Ningwu Huang, Ethylin Wang Jabs, William Reardon, Amit V Pandey, Vishal Agrawal, Pablo Lapunzina, David Mowat, Guy Van Vliet, Joseph Sack, Christa E FluckAbstract:P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler Syndrome (ABS), which has also been attributed to fibroblast growth factor receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and/or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); 17 of the 34 alleles carried 16 “private” mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of 17α-hydroxylase activity, and support of 17,20 lyase using human P450c17. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR’s support of catalysis by P450c17—the enzyme most closely associated with the hormonal phenotype—provided an excellent genotype/phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency.
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mutant p450 oxidoreductase causes disordered steroidogenesis with and without antley bixler Syndrome
Nature Genetics, 2004Co-Authors: Christa E Fluck, Ethylin Wang Jabs, Berenice B. Mendonca, Toshihro Tajima, Amit V Pandey, Kouji Okuhara, Charles F Verge, Kenji Fujieda, Walter L. MillerAbstract:Deficient activities of multiple steroidogenic enzymes have been reported without and with Antley-Bixler Syndrome (ABS), but mutations of corresponding cytochrome P450 enzymes have not been found. We identified mutations in POR, encoding P450 oxidoreductase, the obligate electron donor for these enzymes, in a woman with amenorrhea and three children with ABS, even though knock-out of POR is embryonically lethal in mice. Mutations of POR also affect drug-metabolizing P450 enzymes, explaining the association of ABS with maternal fluconazole ingestion.
Christopher C Marohnic - One of the best experts on this subject based on the ideXlab platform.
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DMD #42820 1 Title page
2016Co-Authors: Daniela Moutinho, Christopher C Marohnic, Jose Rueff, Satya P. P, Bettie Sue Masters, Michel KranendonkAbstract:Altered human CYP3A4 activity caused by Antley-Bixler Syndrome-related variants of NADPH-cytochrome P450 oxidoreductase measured in a robust in vitro syste
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altered human cyp3a4 activity caused by antley bixler Syndrome related variants of nadph cytochrome p450 oxidoreductase measured in a robust in vitro system
Drug Metabolism and Disposition, 2012Co-Authors: Daniela Moutinho, Christopher C Marohnic, Satya Prakash Panda, Bettie Sue Siler Masters, Jose Rueff, Michel KranendonkAbstract:NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler Syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375–384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (kcat and Km) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained ∼10 and 30% activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (kcat/Km), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (kcat/Km) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.
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Altered human CYP3A4 activity caused by Antley−Bixler Syndrome-related variants of NADPHcytochrome P450 oxidoreductase measured in a robust in vitro system
2012Co-Authors: Daniela Moutinho, Christopher C Marohnic, Satya P. P, Jose ́ Rueff, Bettie Sue Masters, Michel KranendonkAbstract:been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler Syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR vari-ants were coexpressed with CYP3A4, resulting in relative expres-sion levels comparable to those found in hepatic tissue. Dibenzyl-fluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375–384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver mi-crosomes. CYP3A4, combined with WT CYPOR, demonstrated ki-netic parameters (kcat and Km) equal to those for pooled huma
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impairment of human cyp1a2 mediated xenobiotic metabolism by antley bixler Syndrome variants of cytochrome p450 oxidoreductase
Archives of Biochemistry and Biophysics, 2008Co-Authors: Michel Kranendonk, Christopher C Marohnic, Satya Prakash Panda, Maria Paula Duarte, Jose Santos Oliveira, Bettie Sue Siler Masters, Jose RueffAbstract:Abstract Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley–Bixler Syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: POR null , POR wt , POR YH , and POR VE , for which equivalent CYP1A2 and CYPOR levels were confirmed, except for POR null , not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5- f )quinoline, were significantly less mutagenic in POR YH and POR VE models than in POR wt , indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.