RESUMEN
Differences of Sex Development (DSD) comprise a variety of congenital conditions characterized by atypical chromosomal, gonadal, or anatomical sex. Diagnosis and monitoring of treatment of patients suspected of DSD conditions include clinical examination, measurement of peptide and steroid hormones, and genetic analysis. This position paper on peptide hormone analyses in the diagnosis and control of patients with DSD was jointly prepared by specialists in the field of DSD and/or peptide hormone analysis from the European Cooperation in Science and Technology (COST) Action DSDnet (BM1303) and the European Reference Network on rare Endocrine Conditions (Endo-ERN). The goal of this position paper on peptide hormone analysis was to establish laboratory guidelines that may contribute to improve optimal diagnosis and treatment control of DSD. The essential peptide hormones used in the management of patients with DSD conditions are follicle-stimulating hormone, luteinising hormone, anti-Müllerian hormone, and Inhibin B. In this context, the following position statements have been proposed: serum and plasma are the preferred matrices; the peptide hormones can all be measured by immunoassay, while use of LC-MS/MS technology has yet to be implemented in a diagnostic setting; sex- and age-related reference values are mandatory in the evaluation of these hormones; and except for Inhibin B, external quality assurance programs are widely available.
Asunto(s)
Trastornos del Desarrollo Sexual/diagnóstico , Trastornos del Desarrollo Sexual/terapia , Inmunoensayo/normas , Hormonas Peptídicas/sangre , Hormona Antimülleriana/sangre , Cromatografía Liquida/normas , Manejo de la Enfermedad , Europa (Continente) , Femenino , Hormona Folículo Estimulante/sangre , Humanos , Inhibinas/sangre , Hormona Luteinizante/sangre , Masculino , Guías de Práctica Clínica como Asunto , Enfermedades Raras , Estándares de Referencia , Espectrometría de Masas en Tándem/normasRESUMEN
The differential diagnosis of differences or disorders of sex development (DSD) belongs to the most complex fields in medicine. It requires a multidisciplinary team conducting a synoptic and complementary approach consisting of thorough clinical, hormonal and genetic workups. This position paper of EU COST (European Cooperation in Science and Technology) Action BM1303 'DSDnet' was written by leading experts in the field and focuses on current best practice in genetic diagnosis in DSD patients. Ascertainment of the karyotpye defines one of the three major diagnostic DSD subclasses and is therefore the mandatory initial step. Subsequently, further analyses comprise molecular studies of monogenic DSD causes or analysis of copy number variations (CNV) or both. Panels of candidate genes provide rapid and reliable results. Whole exome and genome sequencing (WES and WGS) represent valuable methodological developments that are currently in the transition from basic science to clinical routine service in the field of DSD. However, in addition to covering known DSD candidate genes, WES and WGS help to identify novel genetic causes for DSD. Diagnostic interpretation must be performed with utmost caution and needs careful scientific validation in each DSD case.
Asunto(s)
Trastornos del Desarrollo Sexual/diagnóstico , Secuenciación del Exoma , Cariotipo , Secuenciación Completa del Genoma , Hiperplasia Suprarrenal Congénita/diagnóstico , Hiperplasia Suprarrenal Congénita/genética , Variaciones en el Número de Copia de ADN , Trastornos del Desarrollo Sexual/genética , Unión Europea , Disgenesia Gonadal/diagnóstico , Disgenesia Gonadal/genética , Humanos , Biología Molecular , Técnicas de Diagnóstico Molecular , Guías de Práctica Clínica como Asunto , Análisis de Secuencia de ADNRESUMEN
Disorders or differences in sex development (DSD) comprise a heterogeneous group of conditions with an atypical sex development. For optimal diagnosis, highly specialised laboratory analyses are required across European countries. Working group 3 of EU COST (European Cooperation in Science and Technology) Action BM 1303 'DSDnet' 'Harmonisation of Laboratory Assessment' has developed recommendations on laboratory assessment for DSD regarding the use of technologies and analytes to be investigated. This position paper on steroid hormone analysis in diagnosis and treatment of DSD was compiled by a group of specialists in DSD and/or hormonal analysis, either from participating European countries or international partner countries. The topics discussed comprised analytical methods (immunoassay/mass spectrometry-based methods), matrices (urine/serum/saliva) and harmonisation of laboratory tests. The following positions were agreed upon: support of the appropriate use of immunoassay- and mass spectrometry-based methods for diagnosis and monitoring of DSD. Serum/plasma and urine are established matrices for analysis. Laboratories performing analyses for DSD need to operate within a quality framework and actively engage in harmonisation processes so that results and their interpretation are the same irrespective of the laboratory they are performed in. Participation in activities of peer comparison such as sample exchange or when available subscribing to a relevant external quality assurance program should be achieved. The ultimate aim of the guidelines is the implementation of clinical standards for diagnosis and appropriate treatment of DSD to achieve the best outcome for patients, no matter where patients are investigated or managed.
Asunto(s)
Trastornos del Desarrollo Sexual/diagnóstico , Hormonas/análisis , Hormonas/genética , Esteroides/análisis , Trastornos del Desarrollo Sexual 46, XX/diagnóstico , Trastornos del Desarrollo Sexual 46, XX/genética , Trastornos Testiculares del Desarrollo Sexual 46, XX/diagnóstico , Trastornos Testiculares del Desarrollo Sexual 46, XX/genética , Trastornos del Desarrollo Sexual/genética , Europa (Continente) , Femenino , Humanos , MasculinoRESUMEN
CONTEXT: Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene. OBJECTIVE: The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls. DESIGN: Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus. SETTING: The study was conducted at a university hospital endocrine research laboratory. PATIENTS: GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46). INTERVENTION(S): There were no interventions. MAIN OUTCOME MEASURE(S): DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured. RESULTS: The APOD assay clearly separated control individuals (healthy males and molecular defined DSD patients other than AIS) from genetically proven AIS (cutoff < 2.3-fold APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling. CONCLUSIONS: AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high clinical relevance.
Asunto(s)
Síndrome de Resistencia Androgénica/diagnóstico , Apolipoproteínas D , Bioensayo/normas , Trastornos del Desarrollo Sexual/diagnóstico , Receptores Androgénicos/metabolismo , Testosterona/análogos & derivados , Adulto , Síndrome de Resistencia Androgénica/genética , Síndrome de Resistencia Androgénica/metabolismo , Células Cultivadas , Trastornos del Desarrollo Sexual/genética , Trastornos del Desarrollo Sexual/metabolismo , Fibroblastos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Mutación , Receptores Androgénicos/genética , Sensibilidad y Especificidad , Testosterona/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: In boys with suspected partial androgen insensitivity syndrome (PAIS), systematic evidence that supports the long-term prognostic value of identifying a mutation in the androgen receptor gene (AR) is lacking. OBJECTIVE: To assess the clinical characteristics and long-term outcomes in young men with suspected PAIS in relation to the results of AR analysis. METHODS: Through the International Disorders of Sex Development Registry, clinical information was gathered on young men suspected of having PAIS (n = 52) who presented before the age of 16 years and had genetic analysis of AR. RESULTS: The median ages at presentation and at the time of the study were 1 month (range, 1 day to 16 years) and 22 years (range, 16 to 52 years), respectively. Of the cohort, 29 men (56%) had 20 different AR mutations reported. At diagnosis, the median external masculinization scores were 7 and 6 in cases with and without AR mutation, respectively (P = .9), and median current external masculinization scores were 9 and 10, respectively (P = .28). Thirty-five men (67%) required at least one surgical procedure, and those with a mutation were more likely to require multiple surgeries for hypospadias (P = .004). All cases with an AR mutation had gynecomastia, compared to 9% of those without an AR mutation. Of the six men who had a mastectomy, five (83%) had an AR mutation. CONCLUSIONS: Boys with genetically confirmed PAIS are likely to have a poorer clinical outcome than those with XY DSD, with normal T synthesis, and without an identifiable AR mutation. Routine genetic analysis of AR to confirm PAIS informs long-term prognosis and management.
Asunto(s)
Envejecimiento , Síndrome de Resistencia Androgénica/diagnóstico , Síndrome de Resistencia Androgénica/genética , Mutación , Receptores Androgénicos/genética , Adolescente , Adulto , Síndrome de Resistencia Androgénica/fisiopatología , Niño , Preescolar , Estudios de Cohortes , Progresión de la Enfermedad , Trastorno del Desarrollo Sexual 46,XY/diagnóstico , Trastorno del Desarrollo Sexual 46,XY/genética , Trastorno del Desarrollo Sexual 46,XY/fisiopatología , Ginecomastia/etiología , Ginecomastia/cirugía , Humanos , Hipospadias/etiología , Hipospadias/cirugía , Lactante , Recién Nacido , Agencias Internacionales , Masculino , Mastectomía , Persona de Mediana Edad , Pronóstico , Pubertad Tardía , Receptores Androgénicos/metabolismo , Sistema de Registros , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
BACKGROUND: Data on regional differences in the quality of medical care in Germany are scarce. This study aimed to compare outcome quality and medical treatment of pediatric patients with type 1 diabetes between the federal states of Germany. METHODS: 24,928 patients (< 18 years of age) with type 1 diabetes and German residence were selected from the Diabetes-Patienten-Verlaufsdokumentation database. Indicators of outcome quality were HbA1C, overweight prevalence, and rate of severe hypoglycemia. To reflect medical treatment, use of insulin pumps and use of rapid-acting or long-acting insulin analogues were analyzed. Logistic regression models were created for binary variables with federal state as independent predictor. Linear regression was applied for HbA1C and Poisson regression for rate of severe hypoglycemia. Confounders: Sex, age, diabetes duration, migratory background. RESULTS: Disparity was observed for indicators of outcome quality between the 16 federal states of Germany (all p<0.05). After adjustment, HbA1C varied between 55.8 mmol/mol and 67.3 mmol/mol, overweight prevalence between 10.0 and 15.3%, severe hypoglycemia ranged from 0.06 events/PY to 0.21 events/PY. Overall, the best outcome quality appeared to be present in Saxony. Medical treatment also differed. The percentage of pediatrics on insulin pumps varied between 26.3 and 51.8%. The use of rapid-acting analogues ranged from 56.6 to 96.2% and the use of long-acting analogues varied between 41.9 and 96.9% (all p<0.0001). CONCLUSIONS: Medical treatment and outcome quality in pediatrics with type 1 diabetes differed within Germany. Disparities in individual socioeconomic status, regional deprivation, or differences in medical reimbursement decisions might have contributed to the patterns observed.
Asunto(s)
Atención a la Salud , Diabetes Mellitus Tipo 1/terapia , Modelos Teóricos , Calidad de la Atención de Salud , Adolescente , Niño , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/epidemiología , Alemania , Hemoglobina Glucada/metabolismo , Humanos , Sistema de RegistrosRESUMEN
OBJECTIVE: Heterozygosity in 21-hydroxylase deficiency (21OHD) has been associated with hyperandrogenemic symptoms in children and adults. Moreover, the carrier status is mandatory for genetic counseling. We aimed at defining a hormonal parameter for carrier detection by mass spectrometry. DESIGN: Eleven basal and ACTH-stimulated steroid hormones of heterozygous carriers of CYP21A2 mutations and control individuals were compared. METHOD: Hormones were determined in plasma samples by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 58 carriers (35 males, 23 females, age range 6-78 years) and 44 random controls (25 males, 19 females, age range 8-58 years). RESULTS: Heterozygotes could be identified best applying the 17-hydroxyprogesterone+21-deoxycortisol/cortisol×1000 ((17OHP+21S)/F×1000) equation 30â min after ACTH injection. An optimal cut-off value of 8.4 provided 89% sensitivity and specificity. Considering this data and a published frequency of heterozygotes of 1/50 to 1/61, the positive predictive value (PPV) of this cut-off is 12%. Of note, the negative predictive value (NPV) excluding heterozygosity in a given patient is 99.8%. CONCLUSION: Considering only marginal biochemical effects anticipated from heterozygosity, the stimulated ((17OHP+21S)/F×1000) identifies and excludes heterozygotes remarkably well. Nevertheless, LC-MS/MS cannot replace genetic testing, since sensitivity and specificity did not reach 100%. However, due to the considerably high NPV of the optimal cut-off and to a specificity of even 100% applying a cut-off higher than 14.7, hormonal assessment of heterozygosity can be of significant aid in conditions with limited access to genetic testing, as in some health care systems. The ((17OHP+21S)/F×1000) equation can guide diagnostic considerations in the differential diagnosis of hyperandrogenism.
Asunto(s)
Hiperplasia Suprarrenal Congénita/diagnóstico , Hormona Adrenocorticotrópica , Tamización de Portadores Genéticos/métodos , Hormonas , Esteroide 21-Hidroxilasa/genética , 17-alfa-Hidroxiprogesterona/sangre , Adolescente , Hiperplasia Suprarrenal Congénita/sangre , Hiperplasia Suprarrenal Congénita/genética , Adulto , Anciano , Androstenodiona/sangre , Estudios de Casos y Controles , Niño , Cromatografía Liquida , Corticosterona/sangre , Cortisona/sangre , Cortodoxona/sangre , Desoxicorticosterona/sangre , Dihidrotestosterona/sangre , Femenino , Humanos , Hidrocortisona/sangre , Masculino , Persona de Mediana Edad , Progesterona/sangre , Espectrometría de Masas en Tándem , Testosterona/sangre , Adulto JovenAsunto(s)
Diabetes Mellitus/diagnóstico , Diabetes Mellitus/terapia , Adolescente , Niño , Preescolar , Continuidad de la Atención al Paciente/normas , Diabetes Mellitus/epidemiología , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/epidemiología , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/terapia , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/terapia , Dieta , Diagnóstico Precoz , Humanos , Insulina/análogos & derivados , Insulina/uso terapéutico , Sistemas de Infusión de Insulina , Educación del Paciente como Asunto/normas , Factores de Riesgo , Instituciones AcadémicasRESUMEN
Intersex is an inherited incongruence of chromosomal, gonadal, and genital sexual characteristics. A typical clinical situation of intersex is the ambiguous genitalia in the newborn. Diagnostics, counseling, and therapy should be offered by specialized multidisciplinary health-care teams. The focus is not only on medical issues but also on psychological, social, and ethical aspects. In the international literature, intersex is now termed "disorders of sex development" (DSD). Alternatively, some authors use "differences of sex development" to underline that patients do not necessarily feel they have a "disorder" but rather a "difference" of sex development compared with normal sex development.
Asunto(s)
Consejo/métodos , Trastornos del Desarrollo Sexual/diagnóstico , Trastornos del Desarrollo Sexual/terapia , Identidad de Género , Análisis para Determinación del Sexo/métodos , Trastornos del Desarrollo Sexual/psicología , Femenino , Alemania , Humanos , MasculinoRESUMEN
17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD 3) deficiency is a rare cause of 46,XY disorders of sex development (DSD). At puberty, these patients experience a surge of androstenedione and also testosterone, leading to substantial virilization. The origin of testosterone synthesis in these patients remains elusive. We investigated the expression of the isoenzyme AKR1C3 (17ß-HSD 5) in the testis and patient-derived genital skin fibroblasts (GSF) as well as the ability of GSF to synthesize testosterone. Supernatants of GSF cultures and serum samples of one patient before and after gonadectomy were analyzed by liquid and gas chromatography/mass spectrometry. The androgenic potential of GSF-derived supernatants was also assessed by androgen receptor-mediated transactivation of a reporter gene in transiently transfected Chinese hamster ovary cells. Although AKR1C3 is expressed both in the testes and in GSF, androstenedione is rapidly metabolized and is not synthesized to testosterone. The transactivation potential of GSF supernatants towards the androgen receptor is declining within 48 h. However, under testis-equivalent androstenedione concentration, testosterone can be synthesized in 17ß-HSD 3-negative GSF. After gonadectomy, both androstenedione and testosterone decline rapidly in vivo. In 17ß-HSD 3 deficiency, relevant amounts of testosterone are synthesized most probably through AKR1C3 in the testis and not peripherally in GSF.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/deficiencia , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Testosterona/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/genética , Adolescente , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Androstenodiona/metabolismo , Células Cultivadas , Niño , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Hidroxiprostaglandina Deshidrogenasas/genética , Inmunohistoquímica , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/metabolismoRESUMEN
Liquid-chromatography - tandem mass spectrometry (LC-MS/MS) is becoming the method of choice for clinical steroid analysis. In most instances, it has the advantage of higher sensitivity, better reproducibility and greater specificity than commercial immunoassay techniques. The method requires only minimal sample preparation and a small sample volume. Furthermore, it has the potential to analyze multiple steroids simultaneously. Modern instruments guarantee high throughput, allowing an affordable price for the individual assay. All this makes LC-MS/MS an attractive method for use in a clinical setting. Reliable reference ranges for the detected analytes are the pre-requisite for their clinical use. If these are available, LC-MS/MS can find application in congenital disorders of steroid metabolism, such as congenital adrenal hyperplasia, disorders of sex development and disorders of salt homeostasis, as well as in acquired disorders of steroid metabolism, such as primary aldosteronism, Cushing's disease, Addison's disease, and hyperandrogenemia, as well as in psychiatric disease states such as depression or anxiety disorders. The principles of LC-MS/MS for steroid measurement, the pros and cons of LC-MS/MS compared with conventional immunoassays and the possible applications in clinical routine, with a special focus on pediatric endocrinology needs, are discussed here.
Asunto(s)
Glándulas Suprarrenales/química , Cromatografía Liquida/métodos , Hormonas Esteroides Gonadales/análisis , Esteroides/análisis , Espectrometría de Masas en Tándem/métodos , Enfermedades de las Glándulas Suprarrenales/diagnóstico , Cromatografía Liquida/economía , Cromatografía Liquida/instrumentación , Endocrinología/economía , Endocrinología/métodos , Humanos , Inmunoensayo/economía , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Estructura Molecular , Valores de Referencia , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem/economía , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
Male external genital differentiation is accompanied by implementation of a long-term, male-specific gene expression pattern indicating androgen programming in cultured genital fibroblasts. We hypothesized the existence of an epigenetic background contributing to this phenomenon. DNA methylation levels in 2 normal scrotal fibroblast strains from 46,XY males compared to 2 labia majora fibroblast strains from 46,XY females with complete androgen insensitivity syndrome (AIS) due to androgen receptor (AR) mutations were analyzed by Illumina GoldenGate methylation arrays®. Results were validated with pyrosequencing in labia majora fibroblast strains from fifteen 46,XY patients and compared to nine normal male scrotal fibroblast strains. HOXA5 showed a significantly higher methylation level in complete AIS. This finding was confirmed by bisulfite pyrosequencing of 14 CpG positions within the HOXA5 promoter in the same strains. Extension of the 2 groups revealed a constant low HOXA5 methylation pattern in the controls in contrast to a highly variable methylation pattern in the AIS patients. HOXA5 represents a candidate gene of androgen-mediated promoter methylation. The constantly low HOXA5 DNA methylation level of normal male scrotal fibroblast strains and the frequently high methylation levels in labia majora fibroblast strains in AIS indicate for the first time that androgen programming in sexual differentiation is not restricted to global gene transcription but also occurs at the epigenetic level.
Asunto(s)
Epigénesis Genética/genética , Proteínas de Homeodominio/genética , Receptores Androgénicos/genética , Metilación de ADN/genética , Femenino , Humanos , Masculino , Mutación , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
CONTEXT: In 21-hydroxylase (CYP21A2) deficiency (21OHD), the level of in vitro enzymatic function allows for classification of mutation groups (null, A, B, C) and prediction of disease severity. However, genital virilization in affected females correlates only weakly with CYP21A2 mutation groups, suggesting the influence of genetic modifiers. OBJECTIVE: The objective of the study was to investigate the influence of the polymorphic CAG and GGn repeats of the androgen receptor (AR) gene on the degree of genital virilization in 21OHD females. DESIGN AND PATIENTS: Design of the study was the determination of CYP21A2 genotype, degree of genital virilization (Prader stage), and X-weighted biallelic mean of AR CAG and GGn repeat length in 205 females with 21OHD. OUTCOME MEASUREMENTS: Correlation of AR CAG and GGn repeat lengths with Prader stages using nested stepwise logistic regression analysis was measured. RESULTS: CYP21A2 mutation groups null and A showed significantly higher levels of genital virilization than groups B and C (P < 0.01). However, Prader stages varied considerably within mutation groups: null, Prader I-V (median IV); A, Prader I-V (median IV); B, Prader I-V (median III); C, 0-III (median I). Mean GGn repeat length of patients was not significantly associated with Prader stages, classified as low (0-I), intermediate (II-III), or severe (IV-V) (odds ratio per repeat: 0.98, 95% confidence interval 0.71-1.35). In contrast, patients with Prader 0-I showed a trend toward longer CAG repeats without reaching statistical significance (P = 0.07, odds ratio per repeat: 0.82, 95% confidence interval 0.65-1.02). CONCLUSION: Neither CAG nor GGn repeat lengths are statistically significant modifiers of genital virilization in females with 21OHD.
Asunto(s)
Hiperplasia Suprarrenal Congénita/genética , Receptores Androgénicos/genética , Esteroide 21-Hidroxilasa/genética , Repeticiones de Trinucleótidos/genética , Virilismo/genética , Hiperplasia Suprarrenal Congénita/clasificación , Hiperplasia Suprarrenal Congénita/patología , Alelos , Cartilla de ADN , Femenino , Amplificación de Genes , Genotipo , Humanos , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia , Virilismo/clasificación , Virilismo/patologíaRESUMEN
CONTEXT: Current immunoassays for analysis of plasma androgens in children have several limitations due to antibody-specific variations of data and normal ranges. Mass spectrometry-based methods are available for individual steroids but need complex sample preparation and report only fragmentary reference data for the pediatric population. OBJECTIVE: Our objective was to develop a state of the art sensitive and specific tandem mass spectrometry method for high-throughput simultaneous determination of plasma concentrations of androstenedione (A), testosterone (T), and dihydrotestosterone (DHT) and to report age-, sex-, and pubertal stage-specific reference levels for these steroids in children aged 0-18 yr. SUBJECTS AND METHODS: Plasma (100 microl) was mixed with internal standard and extracted by solid-phase extraction. Androgens were measured by ultrapressure liquid chromatography tandem mass spectrometry. Samples of 138 boys and 131 girls with neither signs of endocrine nor systemic disease were considered for the generation of reference data. The following age groups were used: less than 1 wk, 2 wk to 2 months, 3-5 months, 6-11 months, 1-3 yr, 4-6 yr, 7-9 yr, 10-12 yr, 13-15 yr, and over 16 yr. RESULTS: Lower quantification limit was 2.9 ng/dl (0.1 nmol/liter) for A, T, and DHT. No relevant interference with other steroids was detected. Reference data for A, T, and DHT are reported as functions of age, sex, pubertal maturation, and testicular volume. CONCLUSION: Simplicity, velocity, sensitivity, specificity, and the availability of pediatric reference data allow application of our new method in clinical routine as well as in research settings.
Asunto(s)
Androstenodiona/sangre , Dihidrotestosterona/sangre , Testosterona/sangre , Adolescente , Factores de Edad , Andrógenos/sangre , Niño , Preescolar , Cromatografía Liquida/métodos , Femenino , Humanos , Lactante , Masculino , Espectrometría de Masas/métodos , Reproducibilidad de los Resultados , Caracteres Sexuales , Testículo/anatomía & histologíaRESUMEN
Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by mutations in SLC34A3, the gene encoding the renal sodium-phosphate co-transporter NaPi-IIc. Despite increased urinary calcium excretion, HHRH is typically not associated with kidney stones prior to treatment. However, here we describe two sisters, who displayed nephrolithiasis or nephrocalcinosis upon presentation. The index patient, II-4, presented with short stature, bone pain, and knee X-rays suggestive of mild rickets at age 8.5 years. Laboratory evaluation showed hypophosphatemia, elevated 1,25(OH) (2) vitamin D levels, and hypercalciuria, later also developing vitamin D deficiency. Her sister, II-6, had a low normal serum phosphorous level, biochemically vitamin D deficiency and no evidence for osteomalacia, but had undergone left nephro-ureterectomy at age 17 because of ureteral stricture secondary to renal calculi. Nucleotide sequence analysis of DNA from II-4 and II-6 revealed a homozygous missense mutation c.586G>A (p.G196R) in SLC34A3/NaPi-IIc. Ultrasonographic examinations prior to treatment showed grade I nephrocalcinosis for II-4, while II-6 had grade I-II nephrocalcinosis in her remaining kidney. Four siblings and the mother were heterozygous carriers of the mutation, but showed no biochemical abnormalities. With oral phosphate supplements, hypophosphatemia and hypercalciuria improved in both homozygous individuals. Renal calcifications that are presumably due to increased urinary calcium excretion can be the presenting finding in homozygous carriers of G196R in SLC34A3/NaPi-IIc, and some or all laboratory features of HHRH may be masked by vitamin D deficiency.
Asunto(s)
Calcinosis/metabolismo , Hipercalciuria/metabolismo , Hipofosfatemia/metabolismo , Enfermedades Renales/metabolismo , Raquitismo/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/metabolismo , Adolescente , Adulto , Calcinosis/complicaciones , Calcinosis/diagnóstico por imagen , Calcinosis/genética , Niño , Susceptibilidad a Enfermedades , Femenino , Humanos , Hipercalciuria/complicaciones , Hipercalciuria/diagnóstico por imagen , Hipercalciuria/genética , Hipofosfatemia/complicaciones , Hipofosfatemia/diagnóstico por imagen , Hipofosfatemia/genética , Enfermedades Renales/complicaciones , Enfermedades Renales/diagnóstico por imagen , Enfermedades Renales/genética , Masculino , Persona de Mediana Edad , Mutación/genética , Linaje , Raquitismo/complicaciones , Raquitismo/diagnóstico por imagen , Raquitismo/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/genética , UltrasonografíaRESUMEN
AIMS: To conduct a multicentre, matched-pair cohort analysis comparing glycaemic control and adverse events of continuous subcutaneous insulin infusion (CSII) with multiple daily injections (MDI) in paediatric patients. METHODS: Using standardized computer-based prospective documentation, HbA(1c), insulin dose, body mass index-standard deviation score (BMI-SDS), rate of hypoglycaemia, rate of diabetic ketoacidosis (DKA) and intensity of care were analysed in 434 matched pairs during a follow-up period of 3 years after initiation of MDI or CSII. RESULTS: HbA(1c) was significantly lower in the CSII group during the first year of new regimen (CSII 7.5 +/- 0.05 vs. MDI 7.7 +/- 0.06; P < 0.05), but rose to the same level as in the MDI group during year 3. Insulin requirement remained significantly lower in the CSII group. The BMI-SDS increased in both study groups, with no significant difference. The rate of severe hypoglycaemia decreased significantly after the change of regimen (CSII 17.87 +/- 2.85 vs. MDI 25.14 +/- 3.79; P < 0.05) and during year 3 of the regimen, particularly when compared with baseline (-21% vs. -16%). The rate of DKA was lower at baseline in the CSII group and remained significantly lower over all 3 years. Intensity of care was the same in both subsets. CONCLUSIONS: Employing a large cohort, this matched-pair analysis has demonstrated over a 3-year study period that CSII is a safe form of intensive insulin therapy with similar glycaemic effects, but with significantly reduced rates of hypoglycaemia and DKA and a lower insulin requirement when compared with MDI.
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Glucemia/metabolismo , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Adolescente , Niño , Preescolar , Estudios de Cohortes , Diabetes Mellitus Tipo 1/sangre , Cetoacidosis Diabética/inducido químicamente , Esquema de Medicación , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemiantes/efectos adversos , Lactante , Recién Nacido , Inyecciones , Insulina/efectos adversos , Sistemas de Infusión de Insulina , Masculino , Resultado del TratamientoRESUMEN
In contrast to disorders of sexual differentiation caused by lack of androgen production or inhibited androgen action, defects affecting development of the bipotent genital anlagen have rarely been investigated in humans. We have previously documented that the transcription factor FOXF2 is highly expressed in human foreskin. Moreover, Foxf2 knockout mice present with cleft palate in combination with hypoplasia of the genital tubercle. We hypothesized that humans with disorders of sex development (DSD) in combination with cleft palate could have mutations in the FOXF2 gene. Eighteen children with DSD and cleft palate were identified in the Lübeck DSD database (about 1,500 entries). Genomic DNA sequence analysis of the FOXF2 gene was performed and compared with 10 normal female and 10 normal male controls, respectively. Two heterozygous DNA sequence variations were solely present in one single patient each but in none of the 20 normal controls: a duplication of GCC (c.97GCC[9]+[10]) resulting in an extra alanine within exon 1 and a 25*G>A substitution in the 3'-untranslated region. Two patients carried a c.262G>A sequence variation predicting for an Ala88Thr exchange which was also detected in 2 normal controls. Two silent mutations, c.1272C>T (Ser424Ser) and c.1284T>C (Tyr428Tyr), respectively, occurred in the coding region of exon 2, again in both patients and normal controls. In conclusion, the majority of the detected sequence alterations were polymorphisms without obvious functional relevance. However, it cannot be excluded that the 2 unique DNA sequence alterations could have affected FOXF2 on the mRNA or protein level thus contributing to the observed disturbances in genital and palate development.
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Fisura del Paladar/genética , Discapacidades del Desarrollo/genética , Factores de Transcripción Forkhead/genética , Desarrollo Sexual/genética , Niño , Análisis Mutacional de ADN , Cartilla de ADN , Exones/genética , Femenino , Duplicación de Gen , Genitales/crecimiento & desarrollo , Humanos , Masculino , Polimorfismo de Nucleótido Simple , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
AIMS: To analyse current therapeutic strategies for prandial insulin substitution in a large number of children and adolescents with Type 1 diabetes in Germany and Austria, along with changes in therapeutic habits and outcome. METHODS: We classified the data of 26 687 patients, treated from 1995 to 2005 in 152 paediatric clinics, using a database established for quality control and scientific surveys in paediatric diabetology (DPV). RESULTS: Seventy-three per cent of all patients (mean age 13.6 years., mean duration of diabetes 5.4 years.) were treated with > or = 4 daily injections (intensified conventional treatment; ICT), 14% with continuous subcutaneous insulin infusion (CSII), 13% with 1-3 injections per day (conventional treatment). Frequency of daily injections increased with age, duration of diabetes and insulin dose. The insulin dose at breakfast was higher than for the evening meal or lunch, from diagnosis onwards. Individuals using insulin analogues received up to 11% higher insulin doses per day compared with patients treated with human insulin. The time of day, age, duration of diabetes, female gender, insulin analogues and ICT all had a significant influence on prandial insulin doses. Although the number of patients treated with ICT or CSII increased over the period of observation, mean glycated haemoglobin (HbA(1c)) was approximately 8.0% each year, and decreased by only 0.01%. CONCLUSIONS: Eighty-seven per cent of patients were treated with ICT or CSII. However, while this percentage increased over the observation period, mean HbA(1c) was almost constant. Longer duration of diabetes, increasing age, female gender, insulin analogues and ICT were associated with higher prandial insulin doses.
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Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Insulina/análogos & derivados , Insulina/administración & dosificación , Adolescente , Adulto , Austria , Glucemia/análisis , Niño , Preescolar , Estudios de Cohortes , Esquema de Medicación , Alemania , Humanos , Lactante , Insulina Detemir , Insulina Glargina , Insulina Lispro , Insulina de Acción Prolongada , MasculinoRESUMEN
Normal synthesis and action of androgens is essential for normal male sex differentiation. 17Beta-hydroxysteroid dehydrogenase (17beta-HSD) and 5alpha-reductase isoenzymes play essential roles in normal androgen biosynthesis. We hypothesized that differences in expression of these enzymes in genital skin could contribute to the pathogenesis of 46,XY disorders of sex development (DSD). We investigated the mRNA transcription patterns of 17beta-hydroxysteroid dehydrogenase-isoenzymes type 1, 2, 3, 4, 5, 7, and 10, 5alpha-reductase type 1 and 2 and the androgen receptor in genital skin fibroblasts from foreskin and scrotal skin obtained from healthy males and patients with unclassified 46,XY DSD. mRNA expression was semi-quantified by real-time PCR. Although no systematic differences of gene expression of any enzyme between normal controls and hypospadias patients could be detected, we found in nearly half of all investigated patients' samples noticeable differences in the transcription profiles of 17beta-hydroxysteroid dehydrogenase type 5. In scrotal skin samples of patients a significantly higher transcription of the androgen receptor was detected. A role for an altered expression pattern of different enzymes of steroidogenesis in the etiology of genital malformations in some patients may be postulated.
Asunto(s)
Andrógenos/biosíntesis , Trastornos del Desarrollo Sexual/genética , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Disgenesia Gonadal 46 XY/genética , 17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Niño , Preescolar , Trastornos del Desarrollo Sexual/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/enzimología , Genitales/citología , Genitales/enzimología , Genitales/metabolismo , Disgenesia Gonadal 46 XY/metabolismo , Humanos , Lactante , Masculino , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citologíaRESUMEN
17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 5alpha-reductase isoenzymes play a crucial role in the formation and metabolism of sex steroids. Not only the key androgens testosterone and dihydrotestosterone but also their precursors are potent activators of the androgen receptor and are, therefore, likely to act as determinants of male sexual differentiation and maturation in a differentially regulated way. The aim of the present study was to relatively quantify the expression of the mRNA of 17beta-HSD isoenzymes, namely, type 1, 2, 3, 4, 5, 7, and 10, together with the 5alpha-reductase type 1 and 2, and the androgen receptor in normal human males and females. RNA was isolated from peripheral blood cells of both sexes and from genital skin fibroblasts (GSFs) of two different localizations (foreskin and scrotal skin) obtained from phenotypically normal males. mRNA expression was semi-quantified by quantitative reverse-transcriptase polymerase chain reaction with the LightCycler Instrument (Roche). The examined enzymes show statistically significant differences in their transcription pattern between the blood and the GSF RNA samples. Within the GSF samples, there are also significant variations between the two examined localizations in the transcription of 17beta-HSD type 1, 2, 4, and 5 as well as for the androgen receptor. We found large interindividual variation of enzyme transcription patterns in all investigated tissues. In peripheral blood cells, no sex-specific differences were seen. We conclude that sex steroid enzymes are expressed not only in genital primary target tissues but also in peripheral blood. The expression in different target tissues may contribute to both the individual sexual and tissue-specific phenotype in humans.