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 17alpha-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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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 K m and Vmax of each in four assays: reduction of cytochrome c, oxidation of NADPH, 17␣-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.cytochrome P450 ͉ drug metabolism ͉ electron transfer ͉ pharmacogenomics ͉ steroidogenesis P 450 oxidoreductase (POR) is a single protein containing both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) moieties that transfers electrons from NADPH to microsomal (type II) cytochrome P450 enzymes (for review, see ref. 1) (Fig. 1). Cytochrome P450 enzymes (http://drnelson.utmem.edu/ CytochromeP450.html) are heme-containing proteins that catalyze a broad range of oxidative reactions. The human genome contains 57 genes for cytochrome P450 enzymes; 7 encode Type I (mitochondrial) P450s and 50 encode Type II P450s, found in the endoplasmic reticulum (2). Among the 50 human microsomal P450 enzymes, approximately 20 are involved in the biosynthesis of steroids, sterols, fatty acids, and eicosanoids, approximately 15 participate in hepatic drug metabolism, and approximately 15 are ''orphan'' P450 enzymes whose catalytic roles are unclear (3).POR transfers electrons to three steroidogenic enzymes: P450c17 (17␣-hydroxylase/17,20 lyase), P450c21 (21-hydroxylase), and P450aro (aromatase) (4). Individuals with POR deficiency have a broad range of steroidogenic disorders, extending from infants with ambiguous genitalia and skeletal malformations to women with the polycystic ovary syndrome (5-8). Finding severe POR mutations in human patients was unexpected, because POR knockout mice die during embryonic devel...

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.