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Wiebke Arlt - One of the best experts on this subject based on the ideXlab platform.
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reply to fluck et al alternative androgen pathway biosynthesis drives fetal female virilization in P450 Oxidoreductase Deficiency
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Nicole Reisch, Cedric H.l. Shackleton, Richard J Auchus, Neil A Hanley, Wiebke ArltAbstract:Newborn girls with P450 Oxidoreductase (POR) Deficiency regularly present with virilized external genitalia despite low circulating androgens (1). In PNAS, we (2) explain this conundrum by enhanced prenatal activity of an alternative androgen pathway (Fig. 1) while classic androgen biosynthesis is disrupted. Fig. 1. Schematic representation of steroidogenesis including the classic androgen pathway (dark blue) and the alternative androgen biosynthesis pathway (light blue), both resulting in the synthesis of potent 5α-dihydrotestosterone (DHT). Alternative pathway steroids are 5α-reduced upon pathway entry and therefore cannot be aromatized. The electron donor enzyme P450 Oxidoreductase (POR) supports the activities of CYP21A2 21-hydroxylase (yellow), CYP17A1 17α-hydroxylase (white), CYP17A1 17,20-lyase (blue), and CYP19A1 aromatase (pink) in a mutation-dependent, differential manner. Mutations in the electron donor enzyme POR invariably disrupt … [↵][1]1To whom correspondence may be addressed. Email: w.arlt{at}bham.ac.uk. [1]: #xref-corresp-1-1
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impaired hepatic drug and steroid metabolism in congenital adrenal hyperplasia due to P450 Oxidoreductase Deficiency
European Journal of Endocrinology, 2010Co-Authors: Dorota Tomalikscharte, Hannah E Ivison, Dominique Maiter, Julia Kirchheiner, Uwe Fuhr, Wiebke ArltAbstract:Objective: Patients with congenital adrenal hyperplasia due to P450 Oxidoreductase (POR) Deficiency (ORD) present with disordered sex development and glucocorticoid Deficiency. This is due to disruption of electron transfer from mutant POR to microsomal cytochrome P450 (CYP) enzymes that play a key role in glucocorticoid and sex steroid synthesis. POR also transfers electrons to all major drugmetabolizing CYP enzymes, including CYP3A4 that inactivates glucocorticoid and oestrogens. However, whether ORD results in impairment of in vivo drug metabolism has never been studied. Design: We studied an adult patient with ORD due to homozygous POR A287P, the most frequent POR mutation in Caucasians, and her clinically unaffected, heterozygous mother. The patient had received standard dose oestrogen replacement from 17 until 37 years of age when it was stopped after she developed breast cancer. Methods: Both subjects underwent in vivo cocktail phenotyping comprising the oral administration of caffeine, tolbutamide, omeprazole, dextromethorphan hydrobromide and midazolam to assess the five major drug-metabolizing CYP enzymes. We also performed genotyping for variant CYP alleles known to affect drug metabolism. Results: Though CYP enzyme genotyping predicted normal or high enzymatic activities in both subjects, in vivo assessment showed subnormal activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in the patient and of CYP1A2 and CYP2C9 in her mother. Conclusions: Our results provide in vivo evidence for an important role of POR in regulating drug metabolism and detoxification. In patients with ORD, in vivo assessment of drug-metabolizing activities with subsequent tailoring of drug therapy and steroid replacement should be considered.
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gas chromatography mass spectrometry gc ms remains a pre eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry lc ms ms
The Journal of Steroid Biochemistry and Molecular Biology, 2010Co-Authors: Nils Krone, Wiebke Arlt, Beverly A Hughes, Gareth G Lavery, Paul M Stewart, Cedric H.l. ShackletonAbstract:Liquid chromatography tandem mass spectrometry (LC/MS/MS) is replacing classical methods for steroid hormone analysis. It requires small sample volumes and has given rise to improved specificity and short analysis times. Its growth has been fueled by criticism of the validity of steroid analysis by older techniques, testosterone measurements being a prime example. While this approach is the gold-standard for measurement of individual steroids, and panels of such compounds, LC/MS/MS is of limited use in defining novel metabolomes. GC/MS, in contrast, is unsuited to rapid high-sensitivity analysis of specific compounds, but remains the most powerful discovery tool for defining steroid disorder metabolomes. Since the 1930s almost all inborn errors in steroidogenesis have been first defined through their urinary steroid excretion. In the last 30 years, this has been exclusively carried out by GC/MS and has defined conditions such as AME syndrome, glucocorticoid remediable aldosteronism (GRA) and Smith–Lemli–Opitz syndrome. Our recent foci have been on P450 Oxidoreductase Deficiency (ORD) and apparent cortisone reductase Deficiency (ACRD). In contrast to LC/MS/MS methodology, a particular benefit of GC/MS is its non-selective nature; a scanned run will contain every steroid excreted, providing an integrated picture of an individual's metabolome. The “Achilles heel” of clinical GC/MS profiling may be data presentation. There is lack of familiarity with the multiple hormone metabolites excreted and diagnostic data are difficult for endocrinologists to comprehend. While several conditions are defined by the absolute concentration of steroid metabolites, many are readily diagnosed by ratios between steroid metabolites (precursor metabolite/product metabolite). Our work has led us to develop a simplified graphical representation of quantitative urinary steroid hormone profiles and diagnostic ratios.
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cytochrome P450 Oxidoreductase Deficiency
2009Co-Authors: Jan Idkowiak, Deborah Cragun, Robert J Hopkin, Wiebke ArltAbstract:Clinical characteristics Cytochrome P450 Oxidoreductase Deficiency (PORD) is a disorder of steroidogenesis with a broad phenotypic spectrum including cortisol Deficiency, altered sex steroid synthesis, disorders of sex development (DSD), and skeletal malformations of the Antley-Bixler syndrome (ABS) phenotype. Cortisol Deficiency is usually partial, with some baseline cortisol production but failure to mount an adequate cortisol response in stress. Mild mineralocorticoid excess can be present and causes arterial hypertension, usually presenting in young adulthood. Manifestations of altered sex steroid synthesis include ambiguous genitalia/DSD in both males and females, large ovarian cysts in females, poor masculinization and delayed puberty in males, and maternal virilization during pregnancy with an affected fetus. Skeletal malformations can manifest as craniosynostosis, mid-face retrusion with proptosis and choanal stenosis or atresia, low-set dysplastic ears with stenotic external auditory canals, hydrocephalus, radiohumeral synostosis, neonatal fractures, congenital bowing of the long bones, joint contractures, arachnodactyly, and clubfeet; other anomalies observed include urinary tract anomalies (renal pelvic dilatation, vesicoureteral reflux). Cognitive impairment is of minor concern and likely associated with the severity of malformations; studies of developmental outcomes are lacking. Diagnosis/testing The diagnosis of PORD can be established by urinary steroid profiling using gas chromatography / mass spectrometry (GC/MS), which documents combined impairment of 17α-hydroxylase (CYP17A1) and 21-hydroxylase (CYP21A2) enzymatic activity located at key branch points of cortisol, aldosterone, and sex steroid synthesis. Identification of biallelic POR pathogenic variants on molecular genetic testing confirms the diagnosis. Molecular genetic testing is desirable for all individuals affected by PORD to confirm the diagnosis, but is mandatory if clinical and laboratory features are inconclusive. Management Treatment of manifestations: Glucocorticoid replacement therapy for cortisol Deficiency including stress-dose cover in intercurrent illness; surgery as needed for craniosynostosis, hypospadias, and cryptorchidism in males and clitoromegaly and vaginal hypoplasia in females; dihydrotestosterone treatment has been successful in some males with micropenis; testosterone replacement in males in whom testosterone levels remain relatively low after onset of puberty; females with absent pubertal development may require estrogen replacement therapy; treatment with estradiol to reduce the size of ovarian cysts; endotracheal intubation, nasal stints or tracheotomy, and tracheostomy as needed; physical and occupational therapy for joint contractures and help with fine and gross motor skills. Prevention of secondary complications: Supplementation with appropriate steroid hormones in individuals who are deficient has helped alleviate adrenal crisis, lack of or poor pubertal development in males and females, sleepiness, and fatigue. Early intervention services may improve the outcome for individuals at risk for developmental delays and learning difficulties. Surveillance: Evaluations with a specialist tertiary pediatric endocrine service throughout childhood to closely monitor development and adjust steroid supplementation. Periodic formal developmental assessments in centers with expertise and experience in developmental testing. Evaluation of relatives at risk: It is appropriate to evaluate apparently asymptomatic older and younger sibs of a proband in order to identify as early as possible those who would benefit from initiation of treatment and preventive measures. Genetic counseling PORD is inherited in an autosomal recessive manner. At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Carrier testing for at-risk family members and prenatal genetic testing for pregnancies at increased risk are possible if the POR pathogenic variants have been identified in the family. In addition, noninvasive testing of maternal urine steroid excretion by GC/MS can indicate whether the unborn child is affected by PORD from gestational week 12 onwards.
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genetics of congenital adrenal hyperplasia
Best Practice & Research Clinical Endocrinology & Metabolism, 2009Co-Authors: Nils Krone, Wiebke ArltAbstract:Congenital adrenal hyperplasia (CAH) is one of the most common inherited metabolic disorders. It comprises a group of autosomal recessive disorders caused by the Deficiency of one of four steroidogenic enzymes involved in cortisol biosynthesis or in the electron donor enzyme P450 Oxidoreductase (POR) that serves as electron donor to steroidogenic cytochrome P450 (CYP) type II enzymes. The biochemical and clinical phenotype depends on the specific enzymatic defect and the impairment of specific enzyme activity. Defects of steroid 21-hydroxylase (CYP21A2) and 11β-hydroxylase (CYP11B1) only affect adrenal steroidogenesis, whereas 17α-hydroxylase (CYP17A1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) Deficiency also impact on gonadal steroid biosynthesis. Inactivating POR gene mutations are the cause of CAH manifesting with apparent combined CYP17A1–CYP21A2 Deficiency. P450 Oxidoreductase Deficiency (ORD) has a complex phenotype including two unique features not observed in any other CAH variant: skeletal malformations and severe genital ambiguity in both sexes.
Cedric H.l. Shackleton - One of the best experts on this subject based on the ideXlab platform.
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reply to fluck et al alternative androgen pathway biosynthesis drives fetal female virilization in P450 Oxidoreductase Deficiency
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Nicole Reisch, Cedric H.l. Shackleton, Richard J Auchus, Neil A Hanley, Wiebke ArltAbstract:Newborn girls with P450 Oxidoreductase (POR) Deficiency regularly present with virilized external genitalia despite low circulating androgens (1). In PNAS, we (2) explain this conundrum by enhanced prenatal activity of an alternative androgen pathway (Fig. 1) while classic androgen biosynthesis is disrupted. Fig. 1. Schematic representation of steroidogenesis including the classic androgen pathway (dark blue) and the alternative androgen biosynthesis pathway (light blue), both resulting in the synthesis of potent 5α-dihydrotestosterone (DHT). Alternative pathway steroids are 5α-reduced upon pathway entry and therefore cannot be aromatized. The electron donor enzyme P450 Oxidoreductase (POR) supports the activities of CYP21A2 21-hydroxylase (yellow), CYP17A1 17α-hydroxylase (white), CYP17A1 17,20-lyase (blue), and CYP19A1 aromatase (pink) in a mutation-dependent, differential manner. Mutations in the electron donor enzyme POR invariably disrupt … [↵][1]1To whom correspondence may be addressed. Email: w.arlt{at}bham.ac.uk. [1]: #xref-corresp-1-1
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gas chromatography mass spectrometry gc ms remains a pre eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry lc ms ms
The Journal of Steroid Biochemistry and Molecular Biology, 2010Co-Authors: Nils Krone, Wiebke Arlt, Beverly A Hughes, Gareth G Lavery, Paul M Stewart, Cedric H.l. ShackletonAbstract:Liquid chromatography tandem mass spectrometry (LC/MS/MS) is replacing classical methods for steroid hormone analysis. It requires small sample volumes and has given rise to improved specificity and short analysis times. Its growth has been fueled by criticism of the validity of steroid analysis by older techniques, testosterone measurements being a prime example. While this approach is the gold-standard for measurement of individual steroids, and panels of such compounds, LC/MS/MS is of limited use in defining novel metabolomes. GC/MS, in contrast, is unsuited to rapid high-sensitivity analysis of specific compounds, but remains the most powerful discovery tool for defining steroid disorder metabolomes. Since the 1930s almost all inborn errors in steroidogenesis have been first defined through their urinary steroid excretion. In the last 30 years, this has been exclusively carried out by GC/MS and has defined conditions such as AME syndrome, glucocorticoid remediable aldosteronism (GRA) and Smith–Lemli–Opitz syndrome. Our recent foci have been on P450 Oxidoreductase Deficiency (ORD) and apparent cortisone reductase Deficiency (ACRD). In contrast to LC/MS/MS methodology, a particular benefit of GC/MS is its non-selective nature; a scanned run will contain every steroid excreted, providing an integrated picture of an individual's metabolome. The “Achilles heel” of clinical GC/MS profiling may be data presentation. There is lack of familiarity with the multiple hormone metabolites excreted and diagnostic data are difficult for endocrinologists to comprehend. While several conditions are defined by the absolute concentration of steroid metabolites, many are readily diagnosed by ratios between steroid metabolites (precursor metabolite/product metabolite). Our work has led us to develop a simplified graphical representation of quantitative urinary steroid hormone profiles and diagnostic ratios.
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Linking Antley-Bixler syndrome and congenital adrenal hyperplasia: A novel case of P450 Oxidoreductase Deficiency
American Journal of Medical Genetics Part A, 2006Co-Authors: L. Williamson, Richard I. Kelley, Wiebke Arlt, Cedric H.l. Shackleton, Stephen R. BraddockAbstract:The Antley-Bixler syndrome (ABS) is a multiple congenital malformation syndrome with craniosynostosis, radiohumeral synostosis, femoral bowing, choanal atresia or stenosis, joint contractures, urogenital abnormalities and, often, early death. Autosomal recessive and dominant inheritance have been postulated, as has fluconazole teratogenesis. Mutations in POR (P450 (cytochrome) Oxidoreductase, an essential electron donor to enzymes participating in cholesterol biosynthesis), have been identified in some patients with the ABS phenotype. Recent evidence suggests that these mutations cause attenuated steroid hydroxylation, which in turn, causes congenital adrenal hyperplasia (CAH) with ambiguous genitalia in both sexes and glucocorticoid Deficiency. Here, we report on a new patient with findings of both ABS and CAH that further illustrates how low maternal estriol at prenatal screening can serve as a marker steroid facilitating early diagnosis.
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prenatal diagnosis of P450 Oxidoreductase Deficiency ord a disorder causing low pregnancy estriol maternal and fetal virilization and the antley bixler syndrome phenotype
American Journal of Medical Genetics Part A, 2004Co-Authors: Cedric H.l. Shackleton, Wiebke Arlt, Josep Marcos, Berthold P. HauffaAbstract:We report studies on the second pregnancy of a woman who had previously given birth to a virilized female infant. The cause of the virilization had not been established, but common forms of congenital adrenal hyperplasia (CAH) were excluded. Longitudinal monitoring of the second pregnancy revealed that estriol excretion failed to increase normally, reaching a maximum 0.7 mg/24 hr at the end of pregnancy (normal mean 30 mg/24 hr). The mother showed signs of virilization by the 23rd week of gestation and aromatase Deficiency was suspected. However, predicted urinary metabolites for diagnosis of aromatase Deficiency (for example, 16alpha-hydroxyandrosterone) were not increased significantly during the pregnancy. Interestingly, excretion of the androgen metabolite androsterone increased rapidly at the beginning of pregnancy and peaked around the 20th week, suggesting increased production of testosterone and 5alphaDHT, probably the cause of maternal virilization. Urine steroid analysis by GC/MS showed gradually increasing excretion (9 mg/24 hr) of the normally minor metabolite 5alpha-pregnane-3beta,20alpha-diol (epiallopregnanediol), an epimer of the dominant progesterone metabolite pregnanediol (5beta-pregnane-3alpha,20alpha-diol). We believe epiallopregnanediol is largely the maternal urinary excretion product of fetal 5-pregnene-3beta,20alpha-diol, the principal metabolite of pregnenolone, implying a build-up of the latter steroid in the fetal adrenal. These findings suggested that the 'block' in the estriol biosynthetic pathway occurs at an early stage with 17-hydroxylation of pregnenolone being affected. The male baby born of this pregnancy had normal genitalia but showed a urinary steroid profile indicating partial deficiencies of P450c17 and P450c21. However, no mutations in the corresponding CYP17 and CYP21 genes were identified. Urinary steroid analysis carried out on his virilized older sibling showed the same pattern of metabolites. Recently, we determined that this disorder is caused by mutations in P450 Oxidoreductase (OR), the essential redox partner for CYP17 and CYP21 hydroxylases. The novel metabolic profile has now been seen in many patients, most diagnosed with the skeletal dysplasia Antley-Bixler syndrome. We propose that excessive excretion of epiallopregnanediol together with low estriol may be prenatally diagnostic for OR Deficiency (ORD).
Tsutomu Ogata - One of the best experts on this subject based on the ideXlab platform.
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Classic and non-classic 21-hydroxylase Deficiency can be discriminated from P450 Oxidoreductase Deficiency in Japanese infants by urinary steroid metabolites.
Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology, 2016Co-Authors: Yuhei Koyama, Keiko Homma, Maki Fukami, Masayuki Miwa, Kazushige Ikeda, Tsutomu Ogata, Mitsuru Murata, Tomonobu HasegawaAbstract:We previously reported a two-step biochemical diagnosis to discriminate classic 21-hydroxylase Deficiency (C21OHD) from P450 Oxidoreductase Deficiency (PORD) by using urinary steroid metabolites: the pregnanetriolone/tetrahydrocortisone ratio (Ptl / the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and 11β-hydroxyandrosterone (11OHAn). The objective of this study was to investigate whether both C21OHD and non-classic 21OHD (C+NC21OHD) could be biochemically differentiated from PORD. We recruited 55 infants with C21OHD, 8 with NC21OHD, 16 with PORD, 57 with transient hyper-17α-hydroxyprogesteronemia (TH17OHP), and 2,473 controls. All infants were Japanese with ages between 0-180 d. In addition to Ptl, THEs, and 11OHAn, we measured urinary tetrahydroaldosterone (THAldo) and pregnenediol (PD5). The first step: by Ptl with the age-specific cutoffs 0.06 mg/g creatinine (0-10 d of age) and 0.3 mg/g creatinine (11-180 d of age), we were able to differentiate C+NC21OHD and PORD from TH17OHP and controls (0-10 d of age: 0.065-31 vs. < 0.001-0.052, 11-180 d of age: 0.40-42 vs. < 0.001-0.086) with 100% sensitivity and specificity. The second step: by the 11OHAn/THAldo or 11OHAn/PD5 ratio with a cutoff of 0.80 or 1.0, we were able to discriminate between C+NC21OHD and PORD (1.0-720 vs. 0.021-0.61 or 1.8-160 vs. 0.005-0.32, respectively) with 100% sensitivity and specificity. Ptl, 11OHAn/THAldo, and 11OHAn/PD5 could differentiate between C+NC21OHD and PORD in Japanese infants.
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Cytochrome P450 Oxidoreductase Deficiency: Rare congenital disorder leading to skeletal malformations and steroidogenic defects
Pediatrics international : official journal of the Japan Pediatric Society, 2014Co-Authors: Maki Fukami, Tsutomu OgataAbstract: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.
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backdoor pathway for dihydrotestosterone biosynthesis implications for normal and abnormal human sex development
Developmental Dynamics, 2013Co-Authors: Maki Fukami, Keiko Homma, Tomonobu Hasegawa, Tsutomu OgataAbstract:We review the current knowledge about the “backdoor” pathway for the biosynthesis of dihydrotestosterone (DHT). While DHT is produced from cholesterol through the conventional “frontdoor” pathway via testosterone, recent studies have provided compelling evidence for the presence of an alternative “backdoor” pathway to DHT without testosterone intermediacy. This backdoor pathway is known to exist in the tammar wallaby pouch young testis and the immature mouse testis, and has been suggested to be present in the human as well. Indeed, molecular analysis has identified pathologic mutations of genes involved in the backdoor pathway in genetic male patients with undermasculinized external genitalia, and urine steroid profile analysis has argued for the relevance of the activated backdoor pathway to abnormal virilization in genetic females with cytochrome P450 Oxidoreductase Deficiency and 21-hydroxylase Deficiency. It is likely that the backdoor pathway is primarily operating in the fetal testis in a physiological condition to produce a sufficient amount of DHT for male sex development, and that the backdoor pathway is driven with a possible interaction between fetal and permanent adrenals in pathologic conditions with increased 17-hydroxyprogesterone levels. These findings provide novel insights into androgen biosynthesis in both physiological and pathological conditions. Developmental Dynamics 242:320–329, 2013. © 2012 Wiley Periodicals, Inc.
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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
Clinical Chemistry, 2012Co-Authors: Yuhei Koyama, Keiko Homma, Tomonobu Hasegawa, Maki Fukami, Masayuki Miwa, Kazushige Ikeda, Tsutomu Ogata, Mitsuru MurataAbstract: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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anorectal and urinary anomalies and aberrant retinoic acid metabolism in cytochrome P450 Oxidoreductase Deficiency
Molecular Genetics and Metabolism, 2010Co-Authors: Maki Fukami, Toshiro Nagai, Hiroshi Mochizuki, Koji Muroya, Gen Yamada, Kimitaka Takitani, Tsutomu OgataAbstract:Abstract Context: Cytochrome P450 Oxidoreductase (POR) is an electron donor for all microsomal P450 enzymes including CYP26 involved in inactivation of all-trans retinoic acid (atRA). Although previous studies in Por knockout mice suggest that atRA accumulation is relevant to various posterior organ abnormalities, a systematic analysis has not been performed for anorectal and urinary anomalies in patients with POR Deficiency (PORD). Objective: To report the frequencies of anorectal and urinary anomalies and plasma atRA values in PORD patients. Patients: We studied 37 Japanese patients with PORD, consisting of 15 homozygotes for R457H (group A), 15 compound heterozygotes for R457H and one apparently null mutation (group B), and seven patients with other combinations of mutations (group C). Since R457H is a severe hypomorphic mutation, the residual POR function is predicted to be higher in group A than in group B. Results: Imperforate anus was observed in four patients (10.8%) and vesicoureteral reflux was found in three patients (8.1%), with no significant difference in the frequencies of such anomalies between groups A and B. In addition, a complex urogenital malformation including penile agenesis was identified in one patient. Plasma atRA values were above the reference range in nine of 12 patients examined, and were similar between groups A and B and between patients with and without anomalies. Conclusions: The results imply that aberrant atRA metabolism due to CYP26 Deficiency underlies various anorectal and urinary anomalies in patients with PORD. Clinical phenotypes may be primarily determined by maternal oral retinol intake during pregnancy, and plasma atRA values may be largely influenced by the amount of postnatal oral retinol intake in such patients.
Nils Krone - One of the best experts on this subject based on the ideXlab platform.
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genotype phenotype analysis in congenital adrenal hyperplasia due to P450 Oxidoreductase Deficiency
The Journal of Clinical Endocrinology and Metabolism, 2012Co-Authors: Nils Krone, Beverly A Hughes, Vivek Dhir, Hannah E Ivison, Nicole Reisch, Jan Idkowiak, Ian T Rose, Donna M Oneil, Raymon Vijzelaar, Matthew J SmithAbstract:Context: P450 Oxidoreductase Deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid Deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available. Objective: The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort. Design: The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries. Results: We identified 23 P450 Oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles. Conclusions: We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.
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gas chromatography mass spectrometry gc ms remains a pre eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry lc ms ms
The Journal of Steroid Biochemistry and Molecular Biology, 2010Co-Authors: Nils Krone, Wiebke Arlt, Beverly A Hughes, Gareth G Lavery, Paul M Stewart, Cedric H.l. ShackletonAbstract:Liquid chromatography tandem mass spectrometry (LC/MS/MS) is replacing classical methods for steroid hormone analysis. It requires small sample volumes and has given rise to improved specificity and short analysis times. Its growth has been fueled by criticism of the validity of steroid analysis by older techniques, testosterone measurements being a prime example. While this approach is the gold-standard for measurement of individual steroids, and panels of such compounds, LC/MS/MS is of limited use in defining novel metabolomes. GC/MS, in contrast, is unsuited to rapid high-sensitivity analysis of specific compounds, but remains the most powerful discovery tool for defining steroid disorder metabolomes. Since the 1930s almost all inborn errors in steroidogenesis have been first defined through their urinary steroid excretion. In the last 30 years, this has been exclusively carried out by GC/MS and has defined conditions such as AME syndrome, glucocorticoid remediable aldosteronism (GRA) and Smith–Lemli–Opitz syndrome. Our recent foci have been on P450 Oxidoreductase Deficiency (ORD) and apparent cortisone reductase Deficiency (ACRD). In contrast to LC/MS/MS methodology, a particular benefit of GC/MS is its non-selective nature; a scanned run will contain every steroid excreted, providing an integrated picture of an individual's metabolome. The “Achilles heel” of clinical GC/MS profiling may be data presentation. There is lack of familiarity with the multiple hormone metabolites excreted and diagnostic data are difficult for endocrinologists to comprehend. While several conditions are defined by the absolute concentration of steroid metabolites, many are readily diagnosed by ratios between steroid metabolites (precursor metabolite/product metabolite). Our work has led us to develop a simplified graphical representation of quantitative urinary steroid hormone profiles and diagnostic ratios.
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genetics of congenital adrenal hyperplasia
Best Practice & Research Clinical Endocrinology & Metabolism, 2009Co-Authors: Nils Krone, Wiebke ArltAbstract:Congenital adrenal hyperplasia (CAH) is one of the most common inherited metabolic disorders. It comprises a group of autosomal recessive disorders caused by the Deficiency of one of four steroidogenic enzymes involved in cortisol biosynthesis or in the electron donor enzyme P450 Oxidoreductase (POR) that serves as electron donor to steroidogenic cytochrome P450 (CYP) type II enzymes. The biochemical and clinical phenotype depends on the specific enzymatic defect and the impairment of specific enzyme activity. Defects of steroid 21-hydroxylase (CYP21A2) and 11β-hydroxylase (CYP11B1) only affect adrenal steroidogenesis, whereas 17α-hydroxylase (CYP17A1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) Deficiency also impact on gonadal steroid biosynthesis. Inactivating POR gene mutations are the cause of CAH manifesting with apparent combined CYP17A1–CYP21A2 Deficiency. P450 Oxidoreductase Deficiency (ORD) has a complex phenotype including two unique features not observed in any other CAH variant: skeletal malformations and severe genital ambiguity in both sexes.
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age specific changes in sex steroid biosynthesis and sex development
Best Practice & Research Clinical Endocrinology & Metabolism, 2007Co-Authors: Nils Krone, Neil A Hanley, Wiebke ArltAbstract:Normal male sex development requires the SRY gene on the Y chromosome, the regression of Mullerian structures via anti-Mullerian hormone (AMH) signalling, the development of the Wolffian duct system into normal male internal genital structures consequent to testosterone secretion by the testicular Leydig cells, and finally, sufficient activation of testosterone to dihydrotestosterone by 5α-reductase. All these events take place during weeks 8–12 of gestation, a narrow window of sexual differentiation. Recent studies in human fetal development have demonstrated the early fetal expression of the adrenocorticotrophic hormone (ACTH) receptor and all steroidogenic components necessary for the biosynthesis of cortisol. These findings provide compelling evidence for the assumed pathogenesis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase Deficiency, diminished feedback to the pituitary due to glucocorticoid Deficiency, subsequent ACTH excess, and up-regulation of adrenal androgen production with subsequent virilization. Another CAH variant, P450 Oxidoreductase Deficiency, manifests with 46,XX disorder of sex development (DSD), i.e., virilized female genitalia, despite concurrently low circulating androgens. This CAH variant illustrates the existence of an alternative pathway toward the biosynthesis of active androgens in humans which is active in human fetal life only. Thus CAH teaches important lessons from nature, providing privileged insights into the window of human sexual differentiation, and particularly highlighting the importance of steroidogenesis in the process of human sexual differentiation.
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congenital adrenal hyperplasia and P450 Oxidoreductase Deficiency
Clinical Endocrinology, 2007Co-Authors: Nils Krone, Vivek Dhir, Hannah E Ivison, Wiebke ArltAbstract:Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders, which are usually due to inactivating mutations in single enzymes involved in adrenal steroid biosynthesis. The characteristics of the biochemical and clinical phenotype depend on the specific enzymatic defect. In 21-hydroxylase and 11beta-hydroxylase Deficiency only adrenal steroidogenesis is affected, whereas a defect in 3beta-hydroxysteroid dehydrogenase or 17alpha-hydroxylase also involves gonadal steroid biosynthesis. Recently, mutations in the electron donor enzyme P450 Oxidoreductase were identified as the cause of CAH with apparent combined 17alpha-hydroxylase and 21-hydroxylase Deficiency, thereby illustrating the impact of redox regulation enzymes on steroidogenesis. P450 Oxidoreductase Deficiency (ORD) has a complex phenotype including two unique features not observed in any other CAH variant, skeletal malformations and severe genital ambiguity in both sexes. Despite invariably low circulating androgens, females with ORD may present with virilized genitalia and mothers may suffer from virilization during pregnancy. This apparently contradictory finding may be explained by the existence of an alternative pathway in human androgen biosynthesis, with important implications for physiology and pathophysiology. This review discusses the biochemical and clinical presentation and the genetic and functional basis of the currently known CAH variants, with a specific focus on ORD.
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Classic and non-classic 21-hydroxylase Deficiency can be discriminated from P450 Oxidoreductase Deficiency in Japanese infants by urinary steroid metabolites.
Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology, 2016Co-Authors: Yuhei Koyama, Keiko Homma, Maki Fukami, Masayuki Miwa, Kazushige Ikeda, Tsutomu Ogata, Mitsuru Murata, Tomonobu HasegawaAbstract:We previously reported a two-step biochemical diagnosis to discriminate classic 21-hydroxylase Deficiency (C21OHD) from P450 Oxidoreductase Deficiency (PORD) by using urinary steroid metabolites: the pregnanetriolone/tetrahydrocortisone ratio (Ptl / the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and 11β-hydroxyandrosterone (11OHAn). The objective of this study was to investigate whether both C21OHD and non-classic 21OHD (C+NC21OHD) could be biochemically differentiated from PORD. We recruited 55 infants with C21OHD, 8 with NC21OHD, 16 with PORD, 57 with transient hyper-17α-hydroxyprogesteronemia (TH17OHP), and 2,473 controls. All infants were Japanese with ages between 0-180 d. In addition to Ptl, THEs, and 11OHAn, we measured urinary tetrahydroaldosterone (THAldo) and pregnenediol (PD5). The first step: by Ptl with the age-specific cutoffs 0.06 mg/g creatinine (0-10 d of age) and 0.3 mg/g creatinine (11-180 d of age), we were able to differentiate C+NC21OHD and PORD from TH17OHP and controls (0-10 d of age: 0.065-31 vs. < 0.001-0.052, 11-180 d of age: 0.40-42 vs. < 0.001-0.086) with 100% sensitivity and specificity. The second step: by the 11OHAn/THAldo or 11OHAn/PD5 ratio with a cutoff of 0.80 or 1.0, we were able to discriminate between C+NC21OHD and PORD (1.0-720 vs. 0.021-0.61 or 1.8-160 vs. 0.005-0.32, respectively) with 100% sensitivity and specificity. Ptl, 11OHAn/THAldo, and 11OHAn/PD5 could differentiate between C+NC21OHD and PORD in Japanese infants.
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Cytochrome P450 Oxidoreductase Deficiency: Rare congenital disorder leading to skeletal malformations and steroidogenic defects
Pediatrics international : official journal of the Japan Pediatric Society, 2014Co-Authors: Maki Fukami, Tsutomu OgataAbstract: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.
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backdoor pathway for dihydrotestosterone biosynthesis implications for normal and abnormal human sex development
Developmental Dynamics, 2013Co-Authors: Maki Fukami, Keiko Homma, Tomonobu Hasegawa, Tsutomu OgataAbstract:We review the current knowledge about the “backdoor” pathway for the biosynthesis of dihydrotestosterone (DHT). While DHT is produced from cholesterol through the conventional “frontdoor” pathway via testosterone, recent studies have provided compelling evidence for the presence of an alternative “backdoor” pathway to DHT without testosterone intermediacy. This backdoor pathway is known to exist in the tammar wallaby pouch young testis and the immature mouse testis, and has been suggested to be present in the human as well. Indeed, molecular analysis has identified pathologic mutations of genes involved in the backdoor pathway in genetic male patients with undermasculinized external genitalia, and urine steroid profile analysis has argued for the relevance of the activated backdoor pathway to abnormal virilization in genetic females with cytochrome P450 Oxidoreductase Deficiency and 21-hydroxylase Deficiency. It is likely that the backdoor pathway is primarily operating in the fetal testis in a physiological condition to produce a sufficient amount of DHT for male sex development, and that the backdoor pathway is driven with a possible interaction between fetal and permanent adrenals in pathologic conditions with increased 17-hydroxyprogesterone levels. These findings provide novel insights into androgen biosynthesis in both physiological and pathological conditions. Developmental Dynamics 242:320–329, 2013. © 2012 Wiley Periodicals, Inc.
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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
Clinical Chemistry, 2012Co-Authors: Yuhei Koyama, Keiko Homma, Tomonobu Hasegawa, Maki Fukami, Masayuki Miwa, Kazushige Ikeda, Tsutomu Ogata, Mitsuru MurataAbstract: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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anorectal and urinary anomalies and aberrant retinoic acid metabolism in cytochrome P450 Oxidoreductase Deficiency
Molecular Genetics and Metabolism, 2010Co-Authors: Maki Fukami, Toshiro Nagai, Hiroshi Mochizuki, Koji Muroya, Gen Yamada, Kimitaka Takitani, Tsutomu OgataAbstract:Abstract Context: Cytochrome P450 Oxidoreductase (POR) is an electron donor for all microsomal P450 enzymes including CYP26 involved in inactivation of all-trans retinoic acid (atRA). Although previous studies in Por knockout mice suggest that atRA accumulation is relevant to various posterior organ abnormalities, a systematic analysis has not been performed for anorectal and urinary anomalies in patients with POR Deficiency (PORD). Objective: To report the frequencies of anorectal and urinary anomalies and plasma atRA values in PORD patients. Patients: We studied 37 Japanese patients with PORD, consisting of 15 homozygotes for R457H (group A), 15 compound heterozygotes for R457H and one apparently null mutation (group B), and seven patients with other combinations of mutations (group C). Since R457H is a severe hypomorphic mutation, the residual POR function is predicted to be higher in group A than in group B. Results: Imperforate anus was observed in four patients (10.8%) and vesicoureteral reflux was found in three patients (8.1%), with no significant difference in the frequencies of such anomalies between groups A and B. In addition, a complex urogenital malformation including penile agenesis was identified in one patient. Plasma atRA values were above the reference range in nine of 12 patients examined, and were similar between groups A and B and between patients with and without anomalies. Conclusions: The results imply that aberrant atRA metabolism due to CYP26 Deficiency underlies various anorectal and urinary anomalies in patients with PORD. Clinical phenotypes may be primarily determined by maternal oral retinol intake during pregnancy, and plasma atRA values may be largely influenced by the amount of postnatal oral retinol intake in such patients.
