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1.
Lancet Diabetes Endocrinol ; 8(9): 773-781, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32711725

RESUMO

BACKGROUND: Cross-sectional imaging regularly results in incidental discovery of adrenal tumours, requiring exclusion of adrenocortical carcinoma (ACC). However, differentiation is hampered by poor specificity of imaging characteristics. We aimed to validate a urine steroid metabolomics approach, using steroid profiling as the diagnostic basis for ACC. METHODS: We did a prospective multicentre study in adult participants (age ≥18 years) with newly diagnosed adrenal masses. We assessed the accuracy of diagnostic imaging strategies based on maximum tumour diameter (≥4 cm vs <4 cm), imaging characteristics (positive vs negative), and urine steroid metabolomics (low, medium, or high risk of ACC), separately and in combination, using a reference standard of histopathology and follow-up investigations. With respect to imaging characteristics, we also assessed the diagnostic utility of increasing the unenhanced CT tumour attenuation threshold from the recommended 10 Hounsfield units (HU) to 20 HU. FINDINGS: Of 2169 participants recruited between Jan 17, 2011, and July 15, 2016, we included 2017 from 14 specialist centres in 11 countries in the final analysis. 98 (4·9%) had histopathologically or clinically and biochemically confirmed ACC. Tumours with diameters of 4 cm or larger were identified in 488 participants (24·2%), including 96 of the 98 with ACC (positive predictive value [PPV] 19·7%, 95% CI 16·2-23·5). For imaging characteristics, increasing the unenhanced CT tumour attenuation threshold to 20 HU from the recommended 10 HU increased specificity for ACC (80·0% [95% CI 77·9-82·0] vs 64·0% [61·4-66.4]) while maintaining sensitivity (99·0% [94·4-100·0] vs 100·0% [96·3-100·0]; PPV 19·7%, 16·3-23·5). A urine steroid metabolomics result indicating high risk of ACC had a PPV of 34·6% (95% CI 28·6-41·0). When the three tests were combined, in the order of tumour diameter, positive imaging characteristics, and urine steroid metabolomics, 106 (5·3%) participants had the result maximum tumour diameter of 4 cm or larger, positive imaging characteristics (with the 20 HU cutoff), and urine steroid metabolomics indicating high risk of ACC, for which the PPV was 76·4% (95% CI 67·2-84·1). 70 (3·5%) were classified as being at moderate risk of ACC and 1841 (91·3%) at low risk (negative predictive value 99·7%, 99·4-100·0). INTERPRETATION: An unenhanced CT tumour attenuation cutoff of 20 HU should replace that of 10 HU for exclusion of ACC. A triple test strategy of tumour diameter, imaging characteristics, and urine steroid metabolomics improves detection of ACC, which could shorten time to surgery for patients with ACC and help to avoid unnecessary surgery in patients with benign tumours. FUNDING: European Commission, UK Medical Research Council, Wellcome Trust, and UK National Institute for Health Research, US National Institutes of Health, the Claire Khan Trust Fund at University Hospitals Birmingham Charities, and the Mayo Clinic Foundation for Medical Education and Research.


Assuntos
Neoplasias das Glândulas Suprarrenais/epidemiologia , Neoplasias das Glândulas Suprarrenais/urina , Metabolômica/métodos , Esteroides/urina , Neoplasias das Glândulas Suprarrenais/diagnóstico , Adulto , Idoso , Diagnóstico Diferencial , Europa (Continente)/epidemiologia , Feminino , Seguimentos , Humanos , Achados Incidentais , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos
2.
Mol Endocrinol ; 21(8): 1958-68, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17505056

RESUMO

P450 oxidoreductase (POR) has a pivotal role in facilitating electron transfer from nicotinamide adenine dinucleotide phosphate to microsomal cytochrome P450 (CYP) enzymes, including the steroidogenic enzymes CYP17A1 and CYP21A2. Mutations in POR have been shown recently to cause congenital adrenal hyperplasia with apparent combined CYP17A1 and CYP21A2 deficiency that comprises a variable clinical phenotype, including glucocorticoid deficiency, ambiguous genitalia, and craniofacial malformations. To dissect structure-function relationships potentially explaining this phenotypic diversity, we investigated whether specific POR mutations have differential effects on CYP17A1 and CYP21A2. We compared the impact of missense mutations encoding for single amino acid changes in three distinct regions of the POR molecule: 1), Y181D and H628P close to the central electron transfer area, 2) S244C located within the hinge close to the flavin adenine dinucleotide and flavin mononucleotide domains of POR, and 3) A287P that is clearly distant from the two other regions. Functional analysis using a yeast microsomal assay with coexpression of human CYP17A1 or CYP21A2 with wild-type or mutant human POR revealed equivalent decreases in CYP17A1 and CYP21A2 activities by Y181D, H628P, and S244C. In contrast, A287P had a differential inhibitory effect, with decreased catalytic efficiency (Vmax/Km) for CYP17A1, whereas CYP21A2 retained near normal activity. In vivo analysis of urinary steroid excretion by gas chromatography/mass spectrometry in 11 patients with POR mutations showed that A287P homozygous patients had the highest corticosterone/cortisol metabolite ratios, further indicative of preferential inhibition of CYP17A1. These findings provide novel mechanistic insights into the redox regulation of human steroidogenesis. Differential interaction of POR with electron-accepting CYP enzymes may explain the phenotypic variability in POR deficiency, with additional implications for hepatic drug metabolism by POR-dependant CYP enzymes.


Assuntos
Substituição de Aminoácidos/genética , NADPH-Ferri-Hemoproteína Redutase/fisiologia , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Esteroide 21-Hidroxilase/antagonistas & inibidores , Alanina/genética , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , NADPH-Ferri-Hemoproteína Redutase/genética , Prolina/genética , Coelhos , Ratos , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroide 21-Hidroxilase/metabolismo
3.
Lancet ; 363(9427): 2128-35, 2004 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-15220035

RESUMO

BACKGROUND: Congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency is associated with accumulation of steroid metabolites, indicating impaired activity of 17alpha-hydroxylase and 21-hydroxylase. However, no mutations have been reported in the CYP17 and CYP21 genes, which encode these P450 enzymes. Affected girls are born with ambiguous genitalia, but their circulating androgens are low, and virilisation does not progress. We aimed to investigate the underlying molecular basis of congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency in affected children. METHODS: We did sequence analysis of the human gene encoding P450 oxidoreductase, an enzyme that is important in electron transfer from NADPH to P450C17 and P450C21. We studied two unrelated families with a total of three affected children and 100 healthy controls. Wild-type and mutant P450 oxidoreductase proteins were bacterially expressed, purified, and assayed for cytochrome c reductase activity. FINDINGS: We identified four mutations encoding single aminoacid changes in P450 oxidoreductase. All patients were compound heterozygotes, whereas their parents and an unaffected sibling harboured a mutation in only one allele. By contrast, no mutations were noted in the controls. Bacterial expression of recombinant mutant proteins revealed deficient or reduced enzyme activity. INTERPRETATION: Molecular pathogenesis of this form of congenital adrenal hyperplasia is caused by mutations in the gene encoding P450 oxidoreductase. Deficiency of this enzyme could suggest an alternative pathway in human androgen synthesis, present only in fetal life, which explains the combination of antenatal androgen excess and postnatal androgen deficiency.


Assuntos
Hiperplasia Suprarrenal Congênita/genética , Androgênios/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Mutação , Adolescente , Hiperplasia Suprarrenal Congênita/metabolismo , Substituição de Aminoácidos , Pré-Escolar , Sistema Enzimático do Citocromo P-450/química , Feminino , Humanos , Masculino
4.
J Clin Endocrinol Metab ; 98(3): E528-36, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23365120

RESUMO

CONTEXT: Mutations in the electron donor enzyme P450 oxidoreductase (POR) result in congenital adrenal hyperplasia with apparent combined 17α-hydroxylase/17,20 lyase and 21-hydroxylase deficiencies, also termed P450 oxidoreductase deficiency (PORD). Major clinical features present in PORD are disordered sex development in affected individuals of both sexes, glucocorticoid deficiency, and multiple skeletal malformations. OBJECTIVE: The objective of the study was to establish a noninvasive approach to prenatal diagnosis of PORD including assessment of malformation severity to facilitate optimized prenatal diagnosis and timely treatment. DESIGN: We analyzed 20 pregnancies with children homozygous or compound heterozygous for disease-causing POR mutations and 1 pregnancy with a child carrying a heterozygous POR mutation by recording clinical and biochemical presentations and fetal ultrasound findings. In 4 of the pregnancies (3 homozygous and 1 heterozygous for disease-causing POR mutations), prenatal analysis of steroid metabolite excretion in maternal urine was carried out by gas chromatography/mass spectrometry during gestational weeks 11-23. RESULTS: Pregnancy complications in our cohort included maternal virilization (6 of 20) with onset in the second trimester. Seven pregnant women presented with low unconjugated estriol at prenatal screening (triple or quadruple antenatal screening test). Overt dysmorphic features were noted in 19 of the 20 babies at birth but observed in only 5 by prenatal ultrasound. These 5 had the most severe malformation phenotypes and poor outcome, whereas the other babies showed normal development. Steroid profiling of maternal urine revealed significantly increased steroids of fetal origin, namely the pregnenolone metabolite epiallopregnanediol and the androgen metabolite androsterone, with concomitant low values for estriol. Diagnostic steroid ratios conclusively indicated PORD as early as gestational week 12. In the heterozygous pregnancy, steroid ratios were only slightly elevated and estriol excretion was normal. CONCLUSION: Prenatal diagnosis in PORD is readily established via urinary steroid metabolite analysis of maternal urine. Visible malformations at prenatal ultrasound predict a severe malformation phenotype.


Assuntos
Anormalidades Múltiplas/diagnóstico por imagem , Hiperplasia Suprarrenal Congênita , Programas de Rastreamento/métodos , Diagnóstico Pré-Natal/métodos , Esteroide 17-alfa-Hidroxilase/urina , Esteroide 21-Hidroxilase/urina , Anormalidades Múltiplas/genética , Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/genética , Hiperplasia Suprarrenal Congênita/urina , Androsterona/urina , Estriol/urina , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Fenótipo , Valor Preditivo dos Testes , Gravidez , Segundo Trimestre da Gravidez/genética , Pregnanodiol/urina , Radiografia , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 21-Hidroxilase/genética , Ultrassonografia Pré-Natal , Virilismo/diagnóstico , Virilismo/genética
5.
J Clin Endocrinol Metab ; 97(2): E257-67, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22162478

RESUMO

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.


Assuntos
Hiperplasia Suprarrenal Congênita/genética , NADPH-Ferri-Hemoproteína Redutase/genética , Adolescente , Hiperplasia Suprarrenal Congênita/urina , Insuficiência Adrenal/genética , Insuficiência Adrenal/metabolismo , Insuficiência Adrenal/urina , Adulto , Criança , Estudos de Coortes , Análise Mutacional de DNA/métodos , Transtornos do Desenvolvimento Sexual , Feminino , Estudos de Associação Genética , Genitália/anormalidades , Hormônios Esteroides Gonadais/urina , Humanos , Masculino , Metaboloma , Modelos Biológicos , Modelos Moleculares , Reação em Cadeia da Polimerase Multiplex/métodos , NADPH-Ferri-Hemoproteína Redutase/deficiência , NADPH-Ferri-Hemoproteína Redutase/fisiologia , Adulto Jovem
6.
Eur J Endocrinol ; 163(6): 919-24, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20844025

RESUMO

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 drug-metabolizing 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.


Assuntos
Hiperplasia Suprarrenal Congênita/genética , Fenótipo de Síndrome de Antley-Bixler/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Transtornos do Desenvolvimento Sexual/genética , Fígado/metabolismo , NADPH-Ferri-Hemoproteína Redutase/deficiência , Esteroides/metabolismo , Hiperplasia Suprarrenal Congênita/metabolismo , Adulto , Idoso , Fenótipo de Síndrome de Antley-Bixler/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2C9 , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade
7.
Clin Endocrinol (Oxf) ; 66(2): 162-72, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17223983

RESUMO

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.


Assuntos
Hiperplasia Suprarrenal Congênita/enzimologia , Sistema Enzimático do Citocromo P-450/deficiência , Sistema Enzimático do Citocromo P-450/genética , Feminino , Genótipo , Humanos , Masculino , Mutação , Fenótipo , Virilismo
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