Ningwu Huang

Genetics of P450 oxidoreductase: Sequence variation in 842 individuals of four ethnicities and activities of 15 missense mutations

P450 oxidoreductase (POR) is an electron-donating flavoprotein required for the activity of all microsomal cytochrome P450 enzymes. We sequenced 5,655 bp of the POR gene in a representative population of 842 healthy unrelated individuals in four ethnic groups: 218 African Americans, 260 Caucasian Americans, 179 Chinese Americans, and 185 Mexican Americans. One hundred forty SNPs were detected, of which 43 were found in ≥1% of alleles. Twelve SNPs were in the POR promoter region. Fifteen of 32 exonic variations altered the POR amino acid sequence; 13 of these 15 are previously undescribed missense variations. We found eight indels, only one of which was in the coding region. A previously described variant, A503V, was found on 27.9% of all alleles with some ethnic predilection (19.1% in African Americans, 26.4% in Caucasian Americans, 36.7% Chinese Americans, and 31.0% in Mexican Americans). We built cDNA expression vectors for the 13 previously undescribed missense variants, expressed each protein lacking 27 N-terminal residues in Escherichia coli, and assayed the apparent Km and Vmax of each in four assays: reduction of cytochrome c, oxidation of NADPH, 17α-hydroxylase activity of P450c17, and 17,20 lyase activity of P450c17. The catalytic activities of several missense mutants differed substantially in these assays, indicating that each POR mutant must be assayed separately with each potential target P450 enzyme. The activity of A503V was reduced to a modest but statistically significant degree in all four assays, suggesting that it may play an important role in interindividual variation in drug response.

Severe Combined Adrenal and Gonadal Deficiency Caused by Novel Mutations in the Cholesterol Side Chain Cleavage Enzyme, P450scc

CONTEXT Mitochondrial cytochrome P450scc converts cholesterol to pregnenolone in all steroidogenic tissues. Although progesterone production from the fetally-derived placenta is necessary to maintain pregnancy to term, four patients with mutations in the gene encoding P450scc (CYP11A1), have been described, one in a 46,XX female and three in underandrogenized 46,XY individuals, all with primary adrenal failure. OBJECTIVE Our aim was to determine whether P450scc mutations might be found in other children and to explore genotype/phenotype correlations. METHODS AND PATIENTS We performed mutational analysis of CYP11A1 in individuals with 46,XY disorders of sex development and primary adrenal failure, followed by functional studies of P450scc activity and of P450scc RNA splicing. RESULTS Among nine 46,XY infants with adrenal failure and disordered sexual differentiation, two infants had compound heterozygous mutations in CYP11A1. One patient harbored the novel P450scc missense mutations L141W and V415E, which retained 38 and 0% activity, respectively. The other carried a CYP11A1 frameshift mutation c835delA (0% activity) and a splice site mutation [IVS3+(2-3)insT] that prevented correct splicing of P450scc mRNA. CONCLUSIONS P450scc deficiency is a recently recognized disorder that may be more frequent than originally thought. The phenotypic spectrum ranges from severe loss-of-function mutations associated with prematurity, complete underandrogenization, and severe, early-onset adrenal failure, to partial deficiencies found in children born at term with clitoromegaly and later-onset adrenal failure. In contradistinction to congenital lipoid adrenal hyperplasia caused by steroidogenic acute regulatory protein mutations, adrenal hyperplasia has not been reported in any of the six patients with P450scc deficiency.

Pharmacogenetics of P450 oxidoreductase: effect of sequence variants on activities of CYP1A2 and CYP2C19.

Objectives All microsomal cytochrome P450s enzymes, including those that metabolize the majority of clinically used drugs, require electron transfer through P450 oxidoreductase (POR). Mutations in human POR cause altered steroidogenesis and congenital malformations, but the clinical effects on drug metabolism are unclear. We examined the effects of POR sequence variants on two drug-metabolizing P450 enzymes, CYP1A2 and CYP2C19. Methods Our previous sequencing of the human POR gene in POR-deficient patients and in 842 normal individuals identified 35 sequence variants. We expressed these 35 POR sequence variants in bacteria, reconstituted them with the CYP enzymes in vitro, and assayed their activities with human CYP1A2 and CYP2C19. Results POR variants affected the activities of these enzymes to different extents. Disease-causing POR mutations A287P and R457H diminished catalysis by CYP1A2 and CYP2C19 to barely detectable levels. POR A503V, a polymorphism found in 28% of alleles in the normal population, had 85% of wild-type activity with CYP1A2 and 113% of wild-type activity with CYP2C19. Q153R, a disease-causing mutation that severely impaired steroidogenic activity and cytochrome c reduction, increased the activity of CYP1A2 to 144% and CYP2C19 activity to 284% of control. Conclusion The activity of individual POR mutants may vary greatly depending on the electron recipient used to assay activity. Thus, the activity of a POR mutant to support catalysis by a particular P450 enzyme cannot be predicted by the activity of that POR mutant in an assay with a different P450 or with cytochrome c.

Cloning of factors related to HIV-inducible LBP proteins that regulate steroidogenic factor-1-independent human placental transcription of the cholesterol side-chain cleavage enzyme, P450scc.

The cholesterol side-chain cleavage enzyme, cytochrome P450scc, initiates the biosynthesis of all steroid hormones. Adrenal and gonadal strategies for P450scc gene transcription are essentially identical and depend on the orphan nuclear receptor steroidogenic factor-1, but the placental strategy for transcription of P450scc employs cis-acting elements different from those used in the adrenal strategy and is independent of steroidogenic factor-1. Because placental expression of P450scc is required for human pregnancy, we sought factors that bind to the −155/−131 region of the human P450scc promoter, which participates in its placental but not adrenal or gonadal transcription. A yeast one-hybrid screen of 2.4 × 106 cDNA clones from human placental JEG-3 cells yielded two unique clones; one is the previously described transcription factor LBP-1b, which is induced by HIV, type I infection of lymphocytes, and the other is a new factor, termed LBP-9, that shares 83% amino acid sequence identity with LBP-1b. When expressed in transfected yeast, both factors bound specifically to the −155/−131 DNA; antisera to LBP proteins supershifted the LBP-9·DNA complex and inhibited formation of the LBP-1b·DNA complex. Reverse transcriptase-polymerase chain reaction detected LBP-1b in human placental JEG-3, adrenal NCI-H295A, liver HepG2, cervical HeLa, and monkey kidney COS-1 cells, but LBP-9 was detected only in JEG-3 cells. When the −155/−131 fragment was linked to a minimal promoter, co-expression of LBP-1b increased transcription 21-fold in a dose-dependent fashion, but addition of LBP-9 suppressed the stimulatory effect of LBP-1b. The roles of LBP transcription factors in normal human physiology have been unclear. Their modulation of placental but not adrenal P450scc transcription underscores the distinctiveness of placental strategies for steroidogenic enzyme gene transcription.

The Mechanism of 1,25-Dihydroxyvitamin D3Autoregulation in Keratinocytes

The synthesis of 1,25-dihydroxyvitamin D3(1,25(OH)2D3) from its precursor, 25-dihydroxyvitamin D3 (25(OH)D3), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase). It has been generally assumed that 1,25(OH)2D3inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)2D3 reduced the apparent conversion of 25(OH)D3 to 1,25(OH)2D3 while stimulating the conversion of 1,25(OH)2D3 and 25(OH)D3 to 1,24,25(OH)3D3 and 24,25(OH)2D3, respectively. However, 1,25(OH)2D3 failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)2D3 was blocked with a specific inhibitor of the 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1α-hydroxylase activity by 1,25(OH)2D3 was reversed. Thus, the apparent reduction in 1α-hydroxylase activity induced by 1,25(OH)2D3 is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.

Extraadrenal 21-Hydroxylation by CYP2C19 and CYP3A4: Effect on 21-Hydroxylase Deficiency

CONTEXT 21-Hydroxylase deficiency (21OHD) is caused by CYP21A2 gene mutations disrupting the adrenal 21-hydroxylase, P450c21. CYP21A2 mutations generally correlate well with the 21OHD phenotype, but some children with severe CYP21A2 mutations have residual 21-hydroxylase activity. Some hepatic P450 enzymes can 21-hydroxylate progesterone, but their physiological relevance in modifying 21OHD is not known. OBJECTIVE We determined the ability of CYP2C19 and CYP3A4 to 21-hydroxylate progesterone and 17-hydroxyprogesterone (17OHP), determined the impact of the common P450 oxidoreductase (POR) variant A503V on these activities, and examined correlations between CYP2C19 variants and phenotype in patients with 21OHD. METHODS Bacterially expressed, N-terminally modified, C-His-tagged human P450c21, CYP2C19, and CYP3A4 were combined with bacterially expressed wild-type and A503V POR. The 21-hydroxylation of radiolabeled progesterone and 17OHP was assessed, and the Michaelis constant (Km) and maximum velocity (Vmax) of the reactions were measured. CYP2C19 was genotyped in 21OHD patients with genotypes predicting severe congenital adrenal hyperplasia. RESULTS Compared to P450c21, the Vmax/Km for 21-hydroxylation of progesterone by CYP2C19 and CYP3A4 were 17 and 10%, respectively. With both forms of POR, the Km for P450c21 was approximately 2.6 microm, the Km for CYP2C19 was approximately 11 microm, and the Km for CYP3A4 was approximately 110 microm. Neither CYP2C19 nor CYP3A4 could 21-hydroxylate 17OHP. The CYP2C19 ultrametabolizer allele CYP2C19 17 was homozygous in one of five patients with a 21OHD phenotype that was milder than predicted by the CYP21A2 genotype. CONCLUSIONS CYP2C19 and CYP3A4 can 21-hydroxylate progesterone but not 17OHP, possibly ameliorating mineralocorticoid deficiency, but not glucocorticoid deficiency. Multiple enzymes probably contribute to extraadrenal 21-hydroxylation.

Consequences of POR mutations and polymorphisms

P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ∼40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ∼60%, is found on ∼28% of human alleles. A promoter polymorphism (∼8% of Asians and ∼13% of Caucasians) at -152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ∼30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism.

The Common P450 Oxidoreductase Variant A503V Is Not a Modifier Gene for 21-Hydroxylase Deficiency

CONTEXT 21-hydroxylase deficiency (21OHD) is a common genetic disorder caused by mutations in the CYP21A2 gene, which encodes the adrenal 21-hydroxylase, microsomal P450c21. CYP21A2 gene mutations generally correlate well with impaired P450c21 enzymatic activity and the clinical findings in 21OHD, but occasional discrepancies between genotype and phenotype suggest the effects of modifier genes. Mutations in P450 oxidoreductase (POR), the protein that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate to all microsomal P450s, can ameliorate the 21OHD phenotype and, therefore, could be a modifier gene. OBJECTIVES We sought to identify POR variants in patients with 21OHD having discordant phenotype and genotype, and to evaluate their effect on 21-hydroxylase activity. PATIENTS AND METHODS We determined the CYP21A2 genotypes of 313 Brazilian patients with 21OHD and correlated the genotype and phenotype. The POR gene was sequenced in 17 patients with discordant genotype and phenotype. Wild-type and A503V POR, and P450c21 were expressed in bacteria and reconstituted in vitro. Activities were assayed by conversion of [(14)C]progesterone to deoxycorticosterone and [(3)H]17-hydroxyprogesterone to 11-deoxycortisol, and assessed by thin layer chromatography and phosphorimaging. RESULTS The A503V POR variant was found in 10 of 30 alleles, the same ratio as in the normal population. There were no significant differences in Michaelis constant, maximum velocity and maximum velocity/Michaelis constant of 21-hydroxylase activity supported by wild-type and A503V POR. CONCLUSION The only POR missense polymorphism found in atypical 21OHD patients was A503V. Although A503V reduces P450c17 enzymatic activity, it does not influence P450c21 activity, indicating that POR A503V does not modify the 21OHD phenotype.

Genetic variation in human P450 oxidoreductase

Catalysis by all 50 Type II (microsomal) P450 enzymes, including steroidogenic P450c17, P450c21, and P450aro and hepatic drug-metabolizing enzymes requires electron donation from P450 oxidoreductase (POR). POR knockout mice are embryonic lethal, but human POR mutations cause a complex disorder of steroidogenesis. Disorders of hepatic drug metabolism in human POR deficiency have not yet been described. To understand the potential contribution of POR to pharmacogenetics, we sequenced the POR gene in 842 normal persons from 4 ethnic groups. We detected 140 single nucleotide sequence variants of which 43 were in >1% of alleles, including 15 missense mutants; this brings the total of known POR missense mutants to 35. A503V was found on 28% of alleles, varying from 19% in African Americans to 37% in Chinese Americans. We expressed all 35 missense mutants in E. coli and assayed their activities to: oxidize NADPH, reduce cytochrome c, support the 17alpha-hydroxylase and 17,20 lyase activities of bacterially expressed human P450c17, and support the metabolism of fluorogenic EOMCC by bacterially expressed human CYP1A2 and CYP2C19. These data show that there are great differences in the activities of some POR mutants depending on the electron recipient assayed; for example, Q153R causes severely impaired steroid biosynthesis in human patients and in vitro, but is a gain-of-function mutant with CYP1A2 and 2C19. A503V reduces both activities of P450c17 in half, but had no effect on CYP1A2 or 2C19. POR variants are a previously unappreciated source of genetic variation in patterns of steroid synthesis and drug metabolism.

Transcriptional Regulation of the Human P450 Oxidoreductase Gene: Hormonal Regulation and Influence of Promoter Polymorphisms

P450 oxidoreductase (POR) is the flavoprotein that acts as the obligatory electron donor to all microsomal P450 enzymes, including those involved in hepatic drug metabolism as well as three steroidogenic P450 enzymes. The untranslated first exon of human POR was located recently, permitting analysis of human POR transcription. Expression of deletional mutants containing up to 3193 bp of the human POR promoter in human adrenal NCI-H295A and liver Hep-G2 cells located the proximal promoter at -325/-1 bp from the untranslated exon. Common human POR polymorphisms at -208 and -173 had little influence on transcription, but the polymorphism at -152 reduced transcription significantly in both cell lines. EMSA and supershift assays identified binding of Smad3/Smad4 between -249 and -261 and binding of thyroid hormone receptor-β (TRβ) at -240/-245. Chromatin immunoprecipitation showed that Smad3, Smad4, TRα, TRβ, and estrogen receptor-α were bound between -374 and -149. Cotransfection of vectors for these transcription factors and POR promoter-reporter constructs into both cell types followed by hormonal treatment showed that T(3) exerts major tropic effects via TRβ, with TRα, estrogen receptor-α, Smad3, and Smad4 exerting lesser, modulatory effects. T(3) also increased POR mRNA in both cell lines. Thyroid hormone also is essential for rat liver POR expression but acts via different transcription factor complexes. These are the first data on human POR gene transcription, establishing roles for TRβ and Smad3/4 in its expression and indicating that the common polymorphism at -152 may play a role in genetic variation in steroid biosynthesis and drug metabolism.