Urine Steroid Hormone Profile Analysis in Cytochrome P450 Oxidoreductase Deficiency: Implication for the Backdoor Pathway to Dihydrotestosterone

Homma, Keiko; Hasegawa, Tomonobu; Nagai, Toshiro; Adachi, Masanori; Horikawa, Reiko; Fujiwara, Ikuma; Tajima, Toshihiro; Takeda, Ryoujun; Fukami, Maki; Ogata, Tsutomu

doi:10.1210/jc.2005-2460

Cited by 144 publications

(121 citation statements)

References 16 publications

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“…Four POR variants that the authors found in the BioVentures Web data base (P228L, R316W, G413S, and G504R) were also analyzed. Most recently, Homma et al (10) described the POR mutations Q201X, A462_S463insIA, and E580Q.…”

Section: Antley-bixler Syndrome (Abs)mentioning

confidence: 99%

Diminished FAD Binding in the Y459H and V492E Antley-Bixler Syndrome Mutants of Human Cytochrome P450 Reductase

Marohnic

Panda

Martásek

et al. 2006

Journal of Biological Chemistry

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Numerous mutations/polymorphisms of the POR gene, encoding NADPH:cytochrome P450 oxidoreductase (CYPOR), have been described in patients with Antley-Bixler syndrome (ABS), presenting with craniofacial dysmorphogenesis, and/or disordered steroidogenesis, exhibiting ambiguous genitalia. CYPOR is the obligate electron donor to 51 microsomal cytochromes P450 that catalyze critical steroidogenic and xenobiotic reactions, and to two heme oxygenase isoforms, among other redox partners. To address the molecular basis of CYPOR dysfunction in ABS patients, the soluble catalytic domain of human CYPOR was bacterially expressed. WT enzyme was green, due to air-stable FMN semiquinone (blue) and oxidized FAD (yellow). The ABS mutant V492E was blue-gray. Flavin analysis indicated that WT had a protein:FAD:FMN ratio of ϳ1:1:1, whereas ϳ1:0.1:0.9 was observed for V492E, which retained 9% of the WT k cat /K m in NADPH:cytochrome c reductase assays. V492E was reconstituted upon addition of FAD, post-purification, as shown by flavin analysis, activity assay, and near UV-visible CD. Both Y459H and V492E were expressed as membrane anchor-containing proteins, which also exhibited FAD deficiency. CYP4A4-catalyzed -hydroxylation of prostaglandin E 1 was supported by WT CYPOR but not by either of the ABS mutants. Hydroxylation activity was rescued for both Y459H and V492E upon addition of FAD to the reaction. Based on these findings, decreased FAD-binding affinity is proposed as the basis of the observed loss of CYPOR function in the Y459H and V492E POR mutations in ABS. Antley-Bixler syndrome (ABS)3 is a disorder characterized by severe midface hypoplasia, humeroradial synostosis, bowing and fracture of femora, and other severe developmental malformations (1). The disease was initially attributed to fibroblast growth factor receptor 2 (FGFR2) mutations (2).Miller's group discovered a correlation between six allelic variants of the POR gene (encoding CYPOR, EC 1.6.2.4) and disordered steroidogenesis observed in patients with and without ABS (3). The variants were A287P, R457H, V492E, C569Y, V608F, and an intron 6 splice variant resulting in a premature stop codon. The wild-type human CYPOR cDNA and mutants, created by site-directed mutagenesis, were expressed in Escherichia coli. The resultant proteins were assayed for their respective NADPH-cytochrome c and NADPH-cytochrome P450 reductase activities. The authors concluded that deleterious POR mutations correlated well with the decreased lanosterol demethylase (CYP51) activity that had previously been observed in ABS patients, and that severe POR mutations were sufficient to cause the ABS phenotype, even in the absence of FGFR2 mutations. Retardation of somite and limb bud formation, which was observed previously in embryonic lethal CYPOR knock-out mice (4, 5), seems to substantiate this assertion. The authors also hypothesized that POR mutations were more common than the relatively low incidence of ABS suggested and that milder mutations could result in disordered steroidogenesis or incr...

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Section: Antley-bixler Syndrome (Abs)mentioning

confidence: 99%

Diminished FAD Binding in the Y459H and V492E Antley-Bixler Syndrome Mutants of Human Cytochrome P450 Reductase

Marohnic

Panda

Martásek

et al. 2006

Journal of Biological Chemistry

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“…Serum concentrations of AAS are of particular importance and often monitored for diagnosis, treatment or research on the relationship between their concentrations and diseases [8][9][10][11]. Urinary concentrations are also received great attention because of its noninvasive detection [12][13][14]. Furthermore, AAS are even misused by athletes to improve their sports performance [6,15].…”

Section: Introductionmentioning

confidence: 99%

Automated and sensitive determination of four anabolic androgenic steroids in urine by online turbulent flow solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry: A novel approach for clinical monitoring and doping control

Guo

Shao²,

Liu

et al. 2014

Talanta

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“…On the one hand, androgen metabolites showed a clear overlap that can be explained by androgen production in the backdoor pathway (Fig. 1, dashed arrow), which is proven in the tammar wallaby (12 ) and postulated in the human newborn (13 ). Androsterone can be derived not only from AD4 and dihydrotestosterone in the conventional pathway but also from 5␣-pregnane-3␣,17␣-diol-20-one in the backdoor pathway (12,13 ).…”

Section: Biochemical Diagnosis Of C21ohd and Pordmentioning

confidence: 85%

“…Androsterone can be derived not only from AD4 and dihydrotestosterone in the conventional pathway but also from 5␣-pregnane-3␣,17␣-diol-20-one in the backdoor pathway (12,13 ). In PORD, androsterone could increase during early infancy (13 ), resulting in overlap in androgen metabolites.…”

Section: Biochemical Diagnosis Of C21ohd and Pordmentioning

confidence: 99%

Two-Step Biochemical Differential Diagnosis of Classic 21-Hydroxylase Deficiency and Cytochrome P450 Oxidoreductase Deficiency in Japanese Infants by GC-MS Measurement of Urinary Pregnanetriolone/ Tetrahydroxycortisone Ratio and 11β-Hydroxyandrosterone

et al. 2012

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BACKGROUND The clinical differential diagnosis of classic 21-hydroxylase deficiency (C21OHD) and cytochrome P450 oxidoreductase deficiency (PORD) is sometimes difficult, because both deficiencies can have similar phenotypes and high blood concentrations of 17α-hydroxyprogesterone (17OHP). The objective of this study was to identify biochemical markers for the differential diagnosis of C21OHD, PORD, and transient hyper 17α-hydroxyprogesteronemia (TH17OHP) in Japanese newborns. We established a 2-step biochemical differential diagnosis of C21OHD and PORD. METHODS We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH17OHP, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11β-hydroxyandrostenedione (11OHAD4) by GC-MS. RESULTS At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH17OHP and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11β-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine. CONCLUSIONS A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH17OHP and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.

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Urine Steroid Hormone Profile Analysis in Cytochrome P450 Oxidoreductase Deficiency: Implication for the Backdoor Pathway to Dihydrotestosterone

Abstract: The increased androsterone excretion during early infancy, as compared with the etiocholanolone and 11-hydroxyandrosterone excretions in the same period, suggests the presence of the backdoor pathway in PORD.

Cited by 144 publications

References 16 publications

Diminished FAD Binding in the Y459H and V492E Antley-Bixler Syndrome Mutants of Human Cytochrome P450 Reductase

Diminished FAD Binding in the Y459H and V492E Antley-Bixler Syndrome Mutants of Human Cytochrome P450 Reductase

Automated and sensitive determination of four anabolic androgenic steroids in urine by online turbulent flow solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry: A novel approach for clinical monitoring and doping control

Two-Step Biochemical Differential Diagnosis of Classic 21-Hydroxylase Deficiency and Cytochrome P450 Oxidoreductase Deficiency in Japanese Infants by GC-MS Measurement of Urinary Pregnanetriolone/ Tetrahydroxycortisone Ratio and 11β-Hydroxyandrosterone

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