P450 Oxidoreductase Mutations Research Articles

Exploring Novel Variants of the Cytochrome P450 Reductase Gene (POR) from the Genome Aggregation Database by Integrating Bioinformatic Tools and Functional Assays.

Cytochrome P450 oxidoreductase (POR) is an essential redox partner for steroid and drug-metabolizing cytochromes P450 located in the endoplasmic reticulum. Mutations in POR lead to metabolic disorders, including congenital adrenal hyperplasia, and affect the metabolism of steroids, drugs, and xenobiotics. In this study, we examined approximately 450 missense variants of the POR gene listed in the Genome Aggregation Database (gnomAD) using eleven different in silico prediction tools. We found that 64 novel variants were consistently predicted to be disease-causing by most tools. To validate our findings, we conducted a population analysis and selected two variations in POR for further investigation. The human POR wild type and the R268W and L577P variants were expressed in bacteria and subjected to enzyme kinetic assays using a model substrate. We also examined the activities of several cytochrome P450 proteins in the presence of POR (WT or variants) by combining P450 and reductase proteins in liposomes. We observed a decrease in enzymatic activities (ranging from 35% to 85%) of key drug-metabolizing enzymes, supported by POR variants R288W and L577P compared to WT-POR. These results validate our approach of curating a vast amount of data from genome projects and provide an updated and reliable reference for diagnosing POR deficiency.

Diagnostic challenges and management advances in cytochrome P450 oxidoreductase deficiency, a rare form of congenital adrenal hyperplasia, with 46, XX karyotype.

Cytochrome P450 oxidoreductase deficiency (PORD) is a rare form of congenital adrenal hyperplasia that can manifest with skeletal malformations, ambiguous genitalia, and menstrual disorders caused by cytochrome P450 oxidoreductase (POR) mutations affecting electron transfer to all microsomal cytochrome P450 and some non-P450 enzymes involved in cholesterol, sterol, and drug metabolism. With the advancement of molecular biology and medical genetics, increasing numbers of PORD cases were reported, and the clinical spectrum of PORD was extended with studies on underlying mechanisms of phenotype-genotype correlations and optimum treatment. However, diagnostic challenges and management dilemma still exists because of unawareness of the condition, the overlapping manifestations with other disorders, and no clear guidelines for treatment. Delayed diagnosis and management may result in improper sex assignment, loss of reproductive capacity because of surgical removal of ruptured ovarian macro-cysts, and life-threatening conditions such as airway obstruction and adrenal crisis. The clinical outcomes and prognosis, which are influenced by specific POR mutations, the presence of additional genetic or environmental factors, and management, include early death due to developmental malformations or adrenal crisis, bilateral oophorectomies after spontaneous rupture of ovarian macro-cysts, genital ambiguity, abnormal pubertal development, and nearly normal phenotype with successful pregnancy outcomes by assisted reproduction. Thus, timely diagnosis including prenatal diagnosis with invasive and non-invasive techniques and appropriate management is essential to improve patients' outcomes. However, even in cases with conclusive diagnosis, comprehensive assessment is needed to avoid severe complications, such as chromosomal test to help sex assignment and evaluation of adrenal function to detect partial adrenal insufficiency. In recent years, it has been noted that proper hormone replacement therapy can lead to decrease or resolve of ovarian macro-cysts, and healthy babies can be delivered by in vitro fertilization and frozen embryo transfer following adequate control of multiple hormonal imbalances. Treatment may be complicated with adverse effects on drug metabolism caused by POR mutations. Unique challenges occur in female PORD patients such as ovarian macro-cysts prone to spontaneous rupture, masculinized genitalia without progression after birth, more frequently affected pubertal development, and impaired fertility. Thus, this review focuses only on 46, XX PORD patients to summarize the potential molecular pathogenesis, differential diagnosis of classic and non-classic PORD, and tailoring therapy to maintain health, avoid severe complications, and promote fertility.

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels.

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

Loss of protein stability and function caused by a single point mutation (P228L) in the Cytochrome P450 Oxidoreductase

Cytochrome P450 oxidoreductase (POR) is the obligatory redox partner of steroid and drug metabolizing cytochrome P450s located in the endoplasmic reticulum. Mutations in POR cause a broad range of disorders like congenital adrenal hyperplasia. Genome sequencing studies have revealed the existence of a POR missense variant P228L which was linked with reduced function of some P450 enzymes. We aimed to expand the enzymatic studies of POR variant P228L for its role in human metabolism.We expressed human wild type and the P228L variant POR in bacteria and purified the proteins by Affinity Chromatography. We tested the stability of the proteins using fast proteolysis and performed kinetics assays of POR activities using small molecules substrates. POR (WT or P228L) were mixed with purified cytochrome P450 proteins and activities of cytochrome P450 proteins were assayed.The single point mutation P228L had reduced thermal stability indicated by a lower melting point comparing to the WT. In the kinetics studies, the rates of the reactions with P228L were considerably lower than the WT but the Km did not show substantial changes. We observed a decrease in the enzymatic activities of CYP3A5 and CYP2C9 of more than 40% with P228L form of POR comparing to WT.A single change in the amino acid sequence can affect the protein stability and cause a severe reduction in POR activity. Molecular characterization of POR mutations is crucial to have a better understanding of the impact on the functionality of its redox Partners.

Successful live birth in a Chinese woman with P450 oxidoreductase deficiency through frozen-thawed embryo transfer: a case report with review of the literature

BackgroundCongenital adrenal hyperplasia (CAH) caused by P450 oxidoreductase deficiency (PORD) in 46, XX patients is characterized by genital ambiguity, primary amenorrhea, absent or incomplete sexual maturation, infertility, skeletal malformations and so on. But few pregnancies have been reported from these female patients with PORD.Case descriptionA 29-year-old Chinese woman with PORD due to the compound heterozygous mutation (c.1370G > A/c.1196_1204del) in the P450 oxidoreductase (POR) gene had suffered from primary amenorrhea and infertility. She had one cancelled cycle of ovulation induction due to low serum estradiol(E2), high progesterone(P) levels and thin endometrium, then in vitro fertilization (IVF) was recommended. At the first IVF cycle, 4 oocytes were retrieved and 4 viable embryos were cryopreserved due to thin endometrium associated with low E2 and prematurely elevated P after ovarian stimulation, even though oral dexamethasone were used to control adrenal P overproduction at the same time. When basal P fell to < 1.5 ng/ml after the therapy of oral dexamethasone, artificial endometrial preparation and frozen embryo transfer were performed, resulting in a twin pregnancy. She delivered a healthy boy and a healthy girl by caesarean section at 37 weeks and 2 days of gestation. After the literature search in PORD women, no spontaneous pregnancy has been reported and only two previous case reports of 3 successful pregnancies through IVF were summarized.ConclusionsIt is the third report that successful pregnancy was achieved in a CAH woman caused by a compound heterozygous POR mutation, with primary amenorrhea and disorders of steroidogenesis. It seemed that disorders of steroidogenesis caused by PORD didn’t impair the developmental potential of oocytes. IVF and frozen embryo transfer after adequate hormonal control and endometrial preparation should be an effective infertility treatment for PORD women.

Generation of iPSC lines from two (BGUi002-A and BGUi003-A) homozygous p450 oxidoreductase-deficient patients and from one (BGUi001-A) heterozygous healthy family relative

p450 oxidoreductase (POR) cytochromes are enzymes involved in the metabolism of steroids and sex hormones, in which POR acts as an electron donor. Inactivating mutations in the POR gene cause diverse deficiencies. Access to patient samples carrying these POR mutations can contribute to the understanding of metabolic and developmental processes. We report the generation of three iPSC lines from two POR-deficient patients carrying a rare G539R homozygous mutation, and one healthy heterozygous family relative. All generated lines highly expressed pluripotency markers, spontaneously differentiated into three germ layers, retained the deficiency causing mutation and displayed normal karyotypes.

Clinical, endocrinological, and molecular features of four Korean cases of cytochrome P450 oxidoreductase deficiency

PurposeCytochrome P450 oxidoreductase (POR) deficiency is a rare autosomal recessive disorder caused by mutations in the POR gene encoding an electron donor for all microsomal P450 enzymes. It is characterized by adrenal insufficiency, ambiguous genitalia, maternal virilization during pregnancy, and skeletal dysplasia. In this study, we investigated the clinical, hormonal, and molecular characteristics of patients with POR deficiency in Korea.MethodsThis study included four patients with POR deficiency confirmed by biochemical and molecular analysis of POR. Clinical and biochemical findings were reviewed retrospectively. Mutation analysis of POR was performed by Sanger sequencing after polymerase chain reaction amplification of all coding exons and the exon-intron boundaries.ResultsAll patients presented with adrenal insufficiency and ambiguous genitalia regardless of their genetic sex. Two patients harbored homozygous p.R457H mutations in POR and presented with adrenal insufficiency and genital ambiguity without skeletal phenotypes. The other two patients with compound heterozygous mutations of c.[1329_1330insC];[1370G>A] (p.[I444Hfs*6];[R457H]) manifested skeletal abnormalities, such as craniosynostosis and radiohumeral synostosis, suggesting Antley-Bixler syndrome. They also had multiple congenital anomalies involving heart, kidney, and hearing ability. All patients were treated with physiologic doses of oral hydrocortisone.ConclusionsWe report the cases of 4 patients with POR deficiency identified by mutation analysis of POR. Although the study involved a small number of patients, the POR p.R457H mutation was the most common, suggesting founder effect in Korea. POR deficiency is rare and can be misdiagnosed as 21-hydroxylase or 17α-hydroxylase/17,20-lyase deficiency. Therefore, molecular analysis is critical for confirmatory diagnosis.

Changes in Steroids, Xenobiotics, and Drug‐Metabolism in Humans by Genetic Variations in NADPH Cytochrome P450 Oxidoreductase

A broad spectrum of human diseases, including disordered steroid metabolism, are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR) [1, 2]. Human POR is a diflavin reductase that transfers electrons from NADPH to small molecules, non‐P450 redox partners, and cytochrome P450 proteins in the endoplasmic reticulum [2]. Cytochrome P450 proteins perform an extensive range of reactions, including the metabolism of steroids, drugs, and other xenobiotics. Therefore, genetic variations in POR change several metabolic pathways by changing the activities cytochromes P450 [2]. Due to this unique role of POR in metabolism, it was believed for a long time that genetic defects in POR are unlikely, and indeed a POR knockout mouse is embryonically lethal. However, in 2004, the first human patients with defects in POR were reported, and since then, over 200 different variations in POR have been found [3, 4].Recently, we have characterized POR variations from non‐clinical samples [5]. By analyzing the POR sequences from the 1000 genome and other sequencing projects, we identified potentially disease‐causing variations and characterized these by functional studies using recombinant proteins produced in bacteria, yeast, and mammalian cells [6, 7].Identification of severe effects of POR mutations on both the drug and the steroid metabolizing cytochromes P450 indicates that likely pathogenic mutations may be found in non‐clinical population. Their combination as compound heterozygotes or homozygous may severely impact both steroid and drug metabolism by modifications of cytochrome P450 activities. Variations in POR need to be evaluated individually. Changes in drug and steroid metabolism due to genetic variations can be addressed using personalized metabolic profiling and supplementation using modified dosages of drugs and steroids.Support or Funding InformationSupported by grants from the Swiss National Science Foundation (31003A‐134926) &amp; Novartis Foundation for Medical‐Biological Research (18A053) to AVP.

Variability in Loss of Multiple Enzyme Activities Due to the Human Genetic Variation P284T Located in the Flexible Hinge Region of NADPH Cytochrome P450 Oxidoreductase.

Cytochromes P450 located in the endoplasmic reticulum require NADPH cytochrome P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause multiple disorders in humans related to the biosynthesis of steroid hormones and also affect drug-metabolizing cytochrome P450 activities. Electron transfer in POR occurs from NADH to FAD to FMN, and the flexible hinge region in POR is essential for domain movements to bring the FAD and FMN close together for electron transfer. We tested the effect of variations in the hinge region of POR to check if the effects would be similar across all redox partners or there will be differences in activities. Here we are reporting the effects of a POR genetic variant P284T located in the hinge region of POR that is necessary for the domain movements and internal electron transfer between co-factors. Human wild-type and P284T mutant of POR and cytochrome P450 proteins were expressed in bacteria, purified, and reconstituted for enzyme assays. We found that for the P284T variant of POR, the cytochrome c reduction activity was reduced to 47% of the WT and MTT reduction was reduced to only 15% of the WT. No impact on ferricyanide reduction activity was observed, indicating intact direct electron transfer from FAD to ferricyanide, but a severe loss of CYP19A1 (aromatase) activity was observed (9% of WT). In the assays of drug-metabolizing cytochrome P450 enzymes, the P284T variant of POR showed 26% activity for CYP2C9, 44% activity for CYP2C19, 23% activity for CYP3A4, and 44% activity in CYP3A5 assays compared to the WT POR. These results indicate a severe effect on several cytochrome P450 activities due to the P284T variation in POR, which suggests a negative impact on both the steroid as well as drug metabolism in the individuals carrying this variation. The negative impact of P284T mutation in the hinge region of POR seems to be due to disruption of FAD to FMN electron transfer. These results further emphasize the importance of hinge region in POR for protein flexibility and electron transfer within POR as well as the interaction of POR with different redox partners.

Alternative pathway androgen biosynthesis and human fetal female virilization

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenals, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography-tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography-mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

Variability in human drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase

A broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20–50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed.

Changes in Drug Metabolizing Enzyme Cytochrome P450 CYP3A4 Activities Due to Polymorphisms in Human Cytochrome P450 Oxidoreductase

BackgroundCytochromes P450 proteins are responsible for the metabolism of drugs and xenobiotics. All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Previously it has been shown that mutations in POR cause metabolic disorders of steroid hormone biosynthesis but less attention has been paid to drug metabolizing P450 activities. Considering the critical role of POR in activities of drug metabolizing P450 enzymes it is likely that polymorphisms and mutations in POR also affect drug metabolism. We aimed to characterize the variations identified in nonclinical individuals to access their effects on drug metabolizing cytochrome P450 CYP3A4. The variant P284T of POR (rs72557937, NM_000941.2:c.850C&gt;A) is exclusively present in the African population, while A503V allele of POR is found in about 25% of alleles across different populations.MethodsWT and mutant POR proteins, as well as cytochrome P450s, were expressed in recombinant form in bacteria and purified. We used liposomes to embed the P450 and POR proteins and created a functional P450 metabolic system. Metabolism of Testosterone, small molecule dyes, and tracer compounds was evaluated by radioactive ligand metabolism, fluorescent substrate metabolism and colorimetric assays using chromogenic substrates. Enzyme kinetic analysis was performed using Prizm.ResultsTo study the effect of the P284T variant on POR activity, we first measured the ability of WT and P284T variant to transfer electrons from NADPH to cytochrome c, MTT or ferricyanide. Compared to WT POR, the P284T variant had ~50% activity to reduce cytochrome c and 15 % activity to reduce MTT. However, the ferricyanide reduction activity was not affected by the P284T mutation. As compared to WT POR, both FMN and FAD binding was affected due to the P284T mutation. The FMN content of POR‐P284T was 64% as compared to WT while the FAD content was reduced by 35% suggesting that these mutations do affect FMN and FAD binding to POR. The PORvariant P284T had severe loss of CYP3A4 activity with only 22.8% residual activity as compared to WT indicating a potential impact on drug metabolism. The POR common polymorphism A503V which is found in around 25% of all alleles across different genetic populations showed increased activities of drug metabolizing enzyme CYP3A4.ConclusionsThese results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to determine the genotype‐phenotype correlations of POR variants accurately. These results further affirm the critical role of POR in drug metabolism and suggest that variations in POR should also be considered during the evaluation of abnormal drug‐metabolizing cytochrome P450 activities.Support or Funding InformationThis research was supported by grants to Amit V Pandey from the Swiss National Science Foundation, Novartis Foundation for Medical Biological Research and BurgerGemeinde Bern, Switzerland.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Isoform‐Dependent Effects of Cytochrome P450 Oxidoreductase Polymorphisms on Drug Metabolism by Cytochrome P450 Enzymes in Dogs

Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 (CYP) enzymes. We have recently discovered three polymorphisms in the dog POR coding region resulting in one (POR‐H2 and POR‐H4) or two (POR‐H3) amino acid substitutions. In humans, POR mutations can have CYP isoform‐dependent effects on CYP enzyme activity depending on the POR amino acid residue that is substituted.1 Genotyping of 2,040 DNA samples collected from 65 different dog breeds revealed a heterogeneous distribution of POR alleles across breeds. The POR‐H2 allele was found in 19 of 65 breeds and was most common in Rottweilers and Doberman Pinschers (51% and 19% allele frequencies, respectively). The POR‐H3 allele was found in 8 of 65 breeds and was most common in Scottish Deerhounds and Greyhounds (36% and 35% allele frequencies, respectively). The POR‐H4 allele was rare, only being found in 5 of 2,040 dogs. Greyhounds have been shown to metabolize substrates of CYP2B11 (but not other CYPs including CYP2D15) much more slowly compared with mixed‐breed dogs.2 Preliminary studies identified polymorphisms in the CYP2B11 gene that only partially explained reduced CYP2B11 metabolism in Greyhounds. Consequently, we hypothesized that the newly discovered canine POR variants found in Greyhounds would reduce CYP metabolism in a CYP isoform‐dependent manner. The aim of this study was to determine the functional impact of the canine POR variants on drug metabolism by CYP2B11 and CYP2D15 in vitro. In silico molecular structure modelling predicted that POR‐H3 and POR‐H4 but not POR‐H2 would have altered function. The three POR variants and wild‐type were expressed alone or co‐expressed with CYP2B11 or CYP2D15 using an insect cell system. Enzyme kinetic experiments were conducted to determine the impact of the mutations on POR function and CYP‐catalyzed reactions. The POR variants were not significantly different from wild‐type POR in protein expression or function (cytochrome c reduction) when expressed alone. POR variants also did not significantly alter CYP2D15 protein level or catalytic activities or CYP2B11 protein level when co‐expressed. However, POR‐H3 and POR‐H4 (but not POR‐H2) significantly reduced CYP2B11 catalytic efficiency (CLint) by 37% and 69%, respectively, compared to wild‐type POR. This was due to reduced maximal velocities (Vmax) but not substrate affinities (Km). POR‐H3 and POR‐H4 appear to reduce CYP activities in a CYP isoform‐dependent manner. Further investigations are needed to determine POR variant effects on other CYP isoforms and redox partners. The POR‐H3 and POR‐H4 variants are likely to contribute to slower CYP2B11 drug metabolism in Greyhounds and possibly related sighthound breeds (such as Scottish Deerhounds), but results must be confirmed in vivo.Support or Funding InformationAmerican Kennel Club Canine Health Foundation (Grants 2242 and 2529) and the William R. Jones Endowment at Washington State University College of Veterinary Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of single nucleotide polymorphisms on P450 oxidoreductase and peroxisome proliferator-activated receptor alpha on tacrolimus pharmacokinetics in renal transplant recipients.

The P450 oxidoreductase (POR) and peroxisome proliferator-activated receptor alpha (PPARA) genes are associated with the activity of cytochrome P450 enzymes in vivo. We aimed to investigate the impact of single nucleotide polymorphisms (SNPs) in the POR and PPARA genes on the pharmacokinetics of tacrolimus (TAC) in renal transplant recipients. A total of 220 recipients were assessed and 105 recipients were included for final quantitative analysis. Blood samples were collected and DNA was extracted. Targeting sequencing based on next-generation sequencing was applied to detect the SNPs in the POR and PPARA genes. In addition, a systematic review and meta-analysis was performed to comprehensively evaluate the influence of POR and PPARA mutations on the TAC concentrations. A total of 81 SNPs were obtained. Three SNPs (POR*28, Chr7:75619677 and Chr7:75614288) were found to be significantly associated with the TAC pharmacokinetics at 3 months, 6 months, and more than 12 months. No significant association was observed in the combined effect analysis of CYP3A4*1G and CYP3A5*3 with three significant SNPs in the POR gene. Age, post-transplant duration, and the use of sirolimus were identified as the most important factors that influenced the TAC concentrations. A meta-analysis of four studies results and our cohort indicated that compared with recipients carrying the CT or TT genotypes, recipients carrying the CC genotypes of POR*28 showed significantly higher TAC concentrations. Our study suggested the positive influence of mutations in the POR gene on TAC exposure at 3 months after kidney transplantation.

Effect of P450 Oxidoreductase Polymorphisms on the Metabolic Activities of Ten Cytochrome P450s Varied by Polymorphic CYP Genotypes in Human Liver Microsomes

Background/ Aims: Little is known about the effect of P450 oxidoreductase (POR) gene polymorphisms on the activities of CYPs with multiple genotypes. Methods: We genotyped 102 human livers for 18 known POR single nucleotide polymorphisms (SNPs) with allelic frequencies greater than 1% as well as for 27 known SNPs in 10 CYPs. CYP enzyme activities in microsomes prepared from these livers were determined by measuring probe substrate metabolism by high performance liquid chromatograph. Results: We found that the effects of the 18 POR SNPs on 10 CYP activities were CYP genotype-dependent. The POR mutations were significantly associated with decreased overall K_m for CYP2B6 and 2E1, and specific genotypes within CYP1A2, 2A6, 2B6, 2C8, 2D6 and 2E1 were identified as being affected by these POR SNPs. Notably, the effect of a specific POR mutation on the activity of a CYP genotype could not be predicted from other CYP genotypes of even the same CYP. When combining one POR SNP with other POR SNPs, a hitherto unrecognized effect of multiple-site POR gene polymorphisms (MSGP) on CYP activity was uncovered, which was not necessarily consistent with the effect of either single POR SNP. Conclusions: The effects of POR SNPs on CYP activities were not only CYP-dependent, but more importantly, CYP genotype-dependent. Moreover, the effect of a POR SNP alone and in combination with other POR SNPs (MSGP) was not always consistent, nor predictable. Understanding the impact of POR gene polymorphisms on drug metabolism necessitates knowing the complete SNP complement of POR and the genotype of the relevant CYPs.

Polymorphisms in cytochrome P450 oxidoreductase and its effect on drug metabolism and efficacy.

Cytochrome P450 oxidoreductase (POR) has played a potential role in the metabolism of drugs and steroids by supplying electrons to microsomal cytochrome P450 (CYP) enzymes. More than 200 different POR mutations and polymorphisms causing more than 130 amino acid changes in the POR protein have been reported since 2004. A503V is a common amino acid sequence variant encoded by POR*28, whereas A287P and R457H are the most common disease-causing mutations in Europeans and Asians, respectively. Polymorphisms in the POR gene can affect POR activity, CYP-mediated drug metabolism activities, and the efficacy of several clinically used drugs. The effects of POR variants on CYP activities are substrate dependent. In this review, recent research on the effects of POR genetic polymorphisms on drug metabolism and therapy has been summarized and discussed, which can contribute to the rational use of drugs in clinic and the development of personalized medicine.

Altered CYP19A1 and CYP3A4 Activities Due to Mutations A115V, T142A, Q153R and P284L in the Human P450 Oxidoreductase.

All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT) and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein–protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants.

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

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.

Impact on CYP19A1 activity by mutations in NADPH cytochrome P450 oxidoreductase

Cytochrome P450 aromatase (CYP19A1), in human placenta metabolizes androgens to estrogens and uses reduced nicotinamide adenine dinucleotide phosphate through cytochrome P450 oxidoreductase (POR) for the energy requirements of its metabolic activities. Mutations in the human POR lead to congenital adrenal hyperplasia due to loss of activities of several steroid metabolizing enzymatic reactions conducted by the cytochrome P450 proteins located in the endoplasmic reticulum. Effect of POR mutations on different P450 activities depend on individual partner proteins. In this report we have studied the impact of mutations found in the POR on the enzymatic activity of CYP19A1. We expressed wild type as well mutant human POR proteins in bacteria and purified the recombinant proteins, which were then used in an in vitro reconstitution system in combination with CYP19A1 and lipids for enzymatic analysis. We found that several mutations as well as polymorphisms in human POR can cause reduction of CYP19A1 activity. This would affect metabolism of estrogens in people with variations of POR allele. The POR mutants Y181D and R616X were found to have no activity in supporting CYP19A1 reactions. The POR mutations Y607C and delF646 showed a loss of 60–90% activity and two polymorphic forms of POR, R316W and G413S showed similar to WT activity. One POR variant, Q153R had almost double the activity of WT. Loss of CYP19A1 activity may contribute to disordered steroidogenesis in female patients with POR mutations as well as in mothers with POR variants carrying a male child.

Cytochrome P450 oxidoreductase deficiency: rare congenital disorder leading to skeletal malformations and steroidogenic defects.

Cytochrome P450 oxidoreductase (POR) deficiency (PORD) is a newly characterized disorder. PORD is caused by homozygous or compound heterozygous mutations in POR encoding an electron donor for several microsomal enzymes such as CYP21A2, CYP17A1, CYP19A1, CYP51A1, and CYP26A1-C1. Molecular defects of PORD include a Japanese founder mutation p.R457H, as well as various missense, nonsense, frameshift, and splice-site mutations and exonic deletions. PORD leads to unique skeletal malformations referred to as Antley-Bixler syndrome, in addition to 46,XX and 46,XY disorders of sex development, pubertal failure, adrenal dysfunction, and maternal virilization during pregnancy. Such clinical features are ascribable to impaired activities of the POR-dependent microsomal enzymes. PORD represents one form of congenital adrenal hyperplasia, although it can occur as a congenital malformation syndrome and a disorder of sex development. Phenotypic severity of PORD is highly variable and only partly depends on the residual activity of the mutant proteins. It is possible that PORD remains undiagnosed in several patients. Detailed hormonal assessment and molecular analysis are useful for diagnosis of PORD.