RESUMEN
NMR measurements for metabolic characterization of biological samples like cells, biopsies or plasma, may take several hours for advanced methods. Preanalytical issues, such as sample preparation and stability over the measurement time, may have a high impact on metabolite content, and potentially lead to misinterpretation. The aim of this study was therefore to investigate by 1H HR-MAS NMR the impact of different cell handling preparation protocols on the stability of the cell metabolite content over the measurement time. For this purpose, the metabolite content of fibroblasts and adrenal cells were measured at different time points after lysis and after additional heating. Interestingly the results showed similar metabolite concentrations between lysed and lysed-heated cells at the beginning of the measurement, but increasing differences after some hours of measurement. In lysed cells, metabolism was ongoing, producing metabolite changes over time, contrary to a stable metabolite content of the lysed-heated cells. These results were confirmed in both fibroblasts and adrenal cells. Therefore, in order to minimize metabolite content modifications over the measurement time, it is suggested to use cell lysis in combination with heat inactivation for extended HR-MAS NMR measurements.
Asunto(s)
Glándulas Suprarrenales/citología , Fibroblastos/citología , Metaboloma , Espectroscopía de Protones por Resonancia Magnética , Biopsia , Línea Celular , Calor , Humanos , Piel/citologíaRESUMEN
STUDY QUESTION: Is the steroid hormone profile in follicular fluid (FF) at the time of oocyte retrieval different in naturally matured follicles, as in natural cycle IVF (NC-IVF), compared with follicles stimulated with conventional gonadotrophin stimulated IVF (cIVF)? SUMMARY ANSWER: Anti-Mullerian hormone (AMH), testosterone (T) and estradiol (E2) concentrations are â¼3-fold higher, androstenedione (A2) is â¼1.5-fold higher and luteinizing hormone (LH) is â¼14-fold higher in NC-IVF than in cIVF follicles, suggesting an alteration of the follicular metabolism in conventional gonadotrophin stimulated IVF. WHAT IS KNOWN ALREADY: In conventional IVF, the implantation rate of unselected embryos appears to be lower than in NC-IVF, which is possibly due to negative effects of the stimulation regimen on follicular metabolism. In NC-IVF, the intrafollicular concentration of AMH has been shown to be positively correlated with the oocyte fertilization and implantation rates. Furthermore, androgen treatment seems to improve the ovarian response in low responders. STUDY DESIGN, SIZE, DURATION: This cross-sectional study involving 36 NC-IVF and 40 cIVF cycles was performed from 2011 to 2013. Within this population, 13 women each underwent 1 NC-IVF and 1 cIVF cycle. cIVF was performed by controlled ovarian stimulation with HMG and GnRH antagonists. PARTICIPANTS/MATERIALS, SETTING, METHODS: Follicular fluid was collected from the leading follicles. AMH, T, A2, dehydroepiandrosterone (DHEA), E2, FSH, LH and progesterone (P) were determined by immunoassays in 76 women. Aromatase activity in follicular fluid cells was analysed by a tritiated water release assay in 33 different women. For statistical analysis, the non-parametric Mann-Whitney U or Wilcoxon tests were used. MAIN RESULTS AND ROLE OF CHANCE: In follicular fluid from NC-IVF and from cIVF, median levels were 32.8 and 10.7 pmol/l for AMH (P < 0.0001), 47.2 and 18.8 µmol/l for T (P < 0.0001), 290 and 206 nmol/l for A2 (P = 0.0035), 6.7 and 5.6 pg/ml for DHEA (n.s.), 3292 and 1225 nmol/l for E2 (P < 0.0001), 4.9 and 7.2 mU/ml for FSH (P < 0.05), 14.4 and 0.9 mU/ml for LH (P < 0.0001) and 62 940 and 54 710 nmol/l for P (n.s.), respectively. Significant differences in follicular fluid concentrations for AMH, E2 and LH were also found in the 13 patients who underwent both NC-IVF and cIVF when they were analysed separately in pairs. Hormone analysis in serum excluded any relevant impact of AMH, T, A2, and E2 serum concentration on the follicular fluid hormone concentrations. Median serum concentrations were 29.4 and 0.9 mU/ml for LH (P < 0.0001) and 2.7 and 23.5 nmol/l for P (P < 0.0001) after NC-IVF and c-IVF, respectively. Positive correlations were seen for FF-AMH with FF-T (r = 0.35, P = 0.0002), FF-T with FF-LH (r = 0.48, P < 0.0001) and FF-E2 with FF-T (r = 0.75, P < 0.0001). The analysis of aromatase activity was not different in NC-IVF and cIVF follicular cells. LIMITATION, REASONS FOR CAUTION: Any association between the hormone concentrations and the implantation potential of the oocytes could not be investigated as the oocytes in cIVF were not treated individually in the IVF laboratory. Since both c-IVF and NC-IVF follicles were stimulated by hCG before retrieval, the endocrine milieu in the natural cycle does not represent the pure physiological situation. WIDER IMPLICATIONS OF THE FINDINGS: The endocrine follicular milieu and the concentration of putative markers of oocyte quality, such as AMH, are significantly different in gonadotrophin-stimulated conventional IVF compared with natural cycle IVF. This could be a cause for the suggested lower oocyte quality in cIVF compared with naturally matured oocytes. The reasons for the reduced AMH concentration might be low serum and follicular fluid LH concentrations due to LH suppression, leading initially to low follicular androgen concentrations and then to low follicular AMH production. STUDY FUNDING/COMPETING INTERESTS: Funding for this study was obtained from public universities (for salaries) and private industry (for consumables). Additionally, the study was supported by an unrestricted grant from MSD Merck Sharp & Dohme GmbH and IBSA Institut Biochimique SA. The authors are clinically involved in low-dose monofollicular stimulation and IVF therapies, using gonadotrophins from all gonadotrophin distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. Otherwise, the authors have no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.
Asunto(s)
Fertilización In Vitro/métodos , Líquido Folicular/química , Inducción de la Ovulación/métodos , Adulto , Androstenodiona/análisis , Hormona Antimülleriana/análisis , Estudios Transversales , Estradiol/análisis , Femenino , Humanos , Hormona Luteinizante/análisis , Testosterona/análisis , Adulto JovenRESUMEN
CONTEXT: Steroidogenic factor-1 (SF-1/NR5A1) is a nuclear receptor that regulates adrenal and reproductive development and function. NR5A1 mutations have been detected in 46,XY individuals with disorders of sexual development (DSD) but apparently normal adrenal function and in 46,XX women with normal sexual development yet primary ovarian insufficiency (POI). OBJECTIVE: A group of 100 46,XY DSD and two POI was studied for NR5A1 mutations and their impact. DESIGN: Clinical, biochemical, histological, genetic, and functional characteristics of the patients with NR5A1 mutations are reported. SETTING: Patients were referred from different centers in Spain, Switzerland, and Turkey. Histological and genetic studies were performed in Barcelona, Spain. In vitro studies were performed in Bern, Switzerland. PATIENTS: A total of 65 Spanish and 35 Turkish patients with 46,XY DSD and two Swiss 46,XX patients with POI were investigated. MAIN OUTCOME: Ten novel heterozygote NR5A1 mutations were detected and characterized (five missense, one nonsense, three frameshift mutations, and one duplication). RESULTS: The novel NR5A1 mutations were tested in vitro by promoter transactivation assays showing grossly reduced activity for mutations in the DNA binding domain and variably reduced activity for other mutations. Dominant negative effect of the mutations was excluded. We found high variability and thus no apparent genotype-structure-function-phenotype correlation. Histological studies of testes revealed vacuolization of Leydig cells due to fat accumulation. CONCLUSIONS: SF-1/NR5A1 mutations are frequently found in 46,XY DSD individuals (9%) and manifest with a broad phenotype. Testes histology is characteristic for fat accumulation and degeneration over time, similar to findings observed in patients with lipoid congenital adrenal hyperplasia (due to StAR mutations). Genotype-structure-function-phenotype correlation remains elusive.
Asunto(s)
Trastornos del Desarrollo Sexual 46, XX/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Mutación Puntual , Insuficiencia Ovárica Primaria/genética , Factor Esteroidogénico 1/genética , Trastornos del Desarrollo Sexual 46, XX/complicaciones , Adolescente , Secuencia de Bases , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Datos de Secuencia Molecular , Fenotipo , Mutación Puntual/fisiología , Insuficiencia Ovárica Primaria/complicaciones , Adulto JovenRESUMEN
CONTEXT: Congenital isolated ACTH deficiency (IAD) is a rare disease characterized by low plasma ACTH and cortisol levels and preservation of all other pituitary hormones. This condition was poorly defined before we identified TPIT, a T-box transcription factor with a specific role in differentiation of the corticotroph lineage in mice and humans, as its principal molecular cause. OBJECTIVE: We have enlarged our series of IAD patients to better characterize the phenotype and the genotype of this rare disease. DESIGN: Each exon of the TPIT gene was amplified and sequenced in IAD patients without any identified cause. A functional analysis of each new TPIT mutation was performed. RESULTS: We described the largest series of 91 IAD patients and identified three distinct groups: neonatal onset complete or partial IAD or late onset IAD. We did not identify any TPIT mutation in patients with partial or late-onset IAD. However, we found a TPIT mutation in 65% of patients with neonatal-onset complete IAD. These patients are homozygous or compound heterozygous for TPIT mutations, and their parents are healthy heterozygous carriers. We identified nine new mutations: four missense, one one-nucleotide deletion, three splice-site mutations, and one large deletion. TPIT mutations lead to loss of function by different mechanisms, such as non-sense-mediated mRNA decay, abnormal mRNA splicing, loss of TPIT DNA binding or protein-protein interaction defects. CONCLUSION: TPIT mutations are responsible for two thirds of neonatal-onset complete IAD but can not be detected in partial or late-onset IAD.
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Hormona Adrenocorticotrópica/deficiencia , Enfermedades Genéticas Congénitas/genética , Proteínas de Homeodominio/genética , Enfermedades Hipotalámicas/genética , Proteínas de Dominio T Box/genética , Adolescente , Hormona Adrenocorticotrópica/genética , Adulto , Niño , Preescolar , Femenino , Genotipo , Humanos , Masculino , FenotipoRESUMEN
Glucocorticoids (GC) have important anti-inflammatory and pro-apoptotic activities. Initially thought to be exclusively produced by the adrenal glands, there is now increasing evidence for extra-adrenal sources of GCs. We have previously shown that the intestinal epithelium produces immunoregulatory GCs and that intestinal steroidogenesis is regulated by the nuclear receptor liver receptor homolog-1 (LRH-1). As LRH-1 has been implicated in the development of colon cancer, we here investigated whether LRH-1 regulates GC synthesis in colorectal tumors and whether tumor-produced GCs suppress T-cell activation. Colorectal cancer cell lines and primary tumors were found to express steroidogenic enzymes and regulatory factors required for the de novo synthesis of cortisol. Both cell lines and primary tumors constitutively produced readily detectable levels of cortisol, as measured by radioimmunoassay, thin-layer chromatography and bioassay. Whereas overexpression of LRH-1 significantly increased the expression of steroidogenic enzymes and the synthesis of cortisol, downregulation or inhibition of LRH-1 effectively suppressed these processes, indicating an important role of LRH-1 in colorectal tumor GC synthesis. An immunoregulatory role of tumor-derived GCs could be further confirmed by demonstrating a suppression of T-cell activation. This study describes for the first time cortisol synthesis in a non-endocrine tumor in humans, and suggests that the synthesis of bioactive GCs in colon cancer cells may account as a novel mechanism of tumor immune escape.
Asunto(s)
Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Glucocorticoides/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Células CACO-2 , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Cromatografía en Capa Delgada , Neoplasias del Colon/patología , Medios de Cultivo Condicionados/farmacología , Regulación Neoplásica de la Expresión Génica , Glucocorticoides/farmacología , Células HEK293 , Células HT29 , Humanos , Hidrocortisona/metabolismo , Hidrocortisona/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fosfoproteínas/genética , Interferencia de ARN , Radioinmunoensayo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Esteroide 11-beta-Hidroxilasa/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Timo/citologíaRESUMEN
BACKGROUND/AIMS: Growth hormone insensitivity syndrome (GHIS) is a rare cause of growth retardation characterized by high serum GH levels, and low serum insulin-like growth factor I (IGF-I) levels associated with a genetic defect of the GH receptor (GHR) as well post-GHR signaling pathway. Based on clinical, as well as biochemical characteristics, GHIS can be genetically classified as classical/Laron's syndrome and nonclassical/atypical GHIS. Recombinant human IGF-I (rhIGF-I) treatment is effective in promoting growth in subjects who have GHIS. Further, pharmacological studies of a IGF-I compound containing a 1:1 molar complex of rhIGF-I and rhIGF-binding protein-3 (BP-3) demonstrated that the complex was effective in increasing levels of circulating total and free IGF-I and that the administration in patients with GHIS should be safe, well-tolerated and more effective than rhIGF-I on its own. PATIENT/METHODS: We describe the long-term effect of various IGF-I preparations (rhIGF; rhIGF-I/rhIGFBP-3) in a single subject treated for more than 14 years while focusing on height, height velocity as well as on additional auxological and laboratory data. RESULTS: This study confirms that rhIGF-I is effective in promoting growth in children with GHIS. However, on the combined rhIGF-I/rhIGFBP-3 treatment as well as off rhIGF-I therapy the height velocity decreased drastically (2 and 1.8 cm vs. overall 6.5 cm/year on rhIGF-I, respectively). On rhIGF-I treatment, serum IGF-I was found to be well within the normal range, whereas serum IGFBP-3 remained low. On the rhIGF-I/rhIGFBP-3 compound therapy, however, serum IGFBP-3 increased into the normal range, which was not the case for serum IGF-I. Importantly, the increase of the serum IGFBP-3 level excludes noncompliance. In addition, body mass index as well as dual-energy X-ray absorptiometry analysis underlined the positive effect of rhIGF-I treatment on body composition. CONCLUSIONS: The rhIGF-I/rhIGFBP-3 compound therapy seems to be not efficient in treating this individual patient with GHIS when compared with rhIGF-I alone.
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Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/uso terapéutico , Factor I del Crecimiento Similar a la Insulina/uso terapéutico , Síndrome de Laron/tratamiento farmacológico , Adolescente , Estatura/efectos de los fármacos , Índice de Masa Corporal , Niño , Preescolar , Hormona del Crecimiento/metabolismo , Humanos , Lactante , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Síndrome de Laron/fisiopatología , Masculino , Proteínas Recombinantes/uso terapéutico , Factores de TiempoRESUMEN
Neuropeptide Y (NPY) is abundantly expressed in the nervous system and acts on target cells through NPY receptors. The human adrenal cortex and adrenal tumors express NPY receptor subtype Y1, but its function is unknown. We studied Y1-mediated signaling, steroidogenesis and cell proliferation in human adrenal NCI-H295R cells. Radioactive ligand binding studies showed that H295R cells express Y1 receptor specifically. NPY treatment of H295R cells stimulated the MEK/ERK1/2 pathway, confirming that H295R cells express functional Y1 receptors. Studies of the effect of NPY and related peptide PYY on adrenal steroidogenesis revealed a decrease in 11-deoxycortisol production. RIA measurements of cortisol from cell culture medium confirmed this finding. Co-treatment with the Y1 antagonist BIBP2336 reversed the inhibitory effect of NPY on cortisol production proving specificity of this effect. At mRNA level, NPY decreased HSD3B2 and CYP21A2 expression. However NPY revealed no effect on cell proliferation. Our data show that NPY can directly regulate human adrenal cortisol production.
Asunto(s)
Glándulas Suprarrenales/citología , Neuropéptido Y/metabolismo , Receptores de Neuropéptido Y/metabolismo , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/metabolismo , Aldosterona/biosíntesis , Línea Celular , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Hidrocortisona/biosíntesis , Hidroxiesteroide Deshidrogenasas/genética , Hidroxiesteroide Deshidrogenasas/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Neuropéptido Y/genética , Neuropéptido Y/farmacología , Péptido YY/metabolismo , Radioinmunoensayo , Receptores de Neuropéptido Y/genética , Transducción de Señal/fisiología , Testosterona/biosíntesisRESUMEN
Insulin replacement is the only effective treatment of type 1 Diabetes mellitus (T1DM). Nevertheless, many complementary treatments are in use for T1DM. In this study we assessed by questionnaire that out of 342 patients with T1DM, 48 (14%; 13.4% adult, 18.5% paediatric; 20 male, 28 female) used complementary medicine (CM) in addition to their insulin therapy. The purpose of the use of CM was to improve general well-being, ameliorate glucose homeostasis, reduce blood glucose levels as well as insulin doses, improve physical fitness, reduce the frequency of hypoglycaemia, and control appetite. The modalities most frequently used are cinnamon, homeopathy, magnesium and special beverages (mainly teas). Thus, good collaboration between health care professionals will allow optimal patient care.
Asunto(s)
Terapias Complementarias/estadística & datos numéricos , Diabetes Mellitus Tipo 1/terapia , Insulina/uso terapéutico , Adolescente , Adulto , Anciano , Niño , Preescolar , Terapia Combinada , Femenino , Humanos , Masculino , Persona de Mediana Edad , Encuestas y Cuestionarios , Suiza , Resultado del Tratamiento , Revisión de Utilización de Recursos/estadística & datos numéricos , Adulto JovenRESUMEN
A six month old boy is admitted to the children's hospital for sudden loss of consciousness. Hypoglycemia is diagnosed and corrected. Further investigations reveal the diagnosis of hyperinsulinism as underlying cause for hypoglycaemic episodes. Differential diagnosis and therapy of hypoglycemia in infancy are discussed.
Asunto(s)
Hiperinsulinismo Congénito , Hiperinsulinismo/diagnóstico , Hipoglucemia/diagnóstico , Factores de Edad , Hiperinsulinismo Congénito/diagnóstico , Diagnóstico Diferencial , Urgencias Médicas , Humanos , Lactante , Masculino , Pronóstico , Inconsciencia/etiologíaRESUMEN
Overweight and obesity in children and adolescents have become a major public health problem in recent years throughout the world. The medical consequences of obesity may manifest as an increase in the prevalence of the metabolic syndrome in children and adolescents putting them at increased risk for future cardiovascular diseases. Obesity can cause insulin resistance and might disturb glucose homeostasis eventually leading to type 2 diabetes in susceptible patients. Insulin resistance is also involved in the pathogenesis of dyslipidemia in obese children characteristically presenting as hypertriglyceridemia and low HDL cholesterol. Even elevated blood pressure might be present in obese kids. Here we present a 12-year-old boy diagnosed with the metabolic syndrome. The diagnostic criteria of the metabolic syndrome in children and adolescents are discussed. Thoughts about pathophysiology and therapeutic options are offered.
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Síndrome Metabólico , Adolescente , Adulto , Factores de Edad , Algoritmos , Índice de Masa Corporal , Niño , Preescolar , Diagnóstico Diferencial , Dislipidemias/diagnóstico , Ejercicio Físico , Humanos , Hipertensión/diagnóstico , Lactante , Resistencia a la Insulina , Masculino , Síndrome Metabólico/diagnóstico , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/epidemiología , Síndrome Metabólico/etiología , Síndrome Metabólico/terapia , Obesidad/diagnóstico , Pronóstico , Factores de RiesgoAsunto(s)
3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Anticonvulsivantes/administración & dosificación , Inhibidores Enzimáticos/administración & dosificación , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Anticonvulsivantes/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidoresRESUMEN
Diabetic nephropathy and end-stage renal failure are still a major cause of mortality amongst patients with diabetes mellitus (DM). In this study, we evaluated the Clinitek-Microalbumin (CM) screening test strip for the detection of microalbuminuria (MA) in a random morning spot urine in comparison with the quantitative assessment of albuminuria in the timed overnight urine collection ("gold standard"). One hundred thirty-four children, adolescents, and young adults with insulin-dependent DM Type 1 were studied at 222 outpatient visits. Because of urinary tract infection and/or haematuria, the data of 13 visits were excluded. Finally, 165 timed overnight urine were collected in the remaining 209 visits (79% sample per visit rate). Ten (6.1%) patients presented MA of > or =15 microg/min. In comparison however, 200 spot urine could be screened (96% sample/visit rate) yielding a significant increase in compliance and screening rate (P<.001, McNemar test). Furthermore, at 156 occasions, the gold standard and CM could be directly compared. The sensitivity and the specificity for CM in the spot urine (cut-off > or =30 mg albumin/l) were 0.89 [95% confidence interval (CI) 0.56-0.99] and 0.73 (CI 0.66-0.80), respectively. The positive and negative predictive value were 0.17 (CI 0.08-0.30) and 0.99 (CI 0.95-1.00), respectively. Considering CM albumin-to-creatinine ratio, the results were poorer than with the albumin concentration alone. Using CM instead of quantitative assessment of albuminuria is not cost-effective (35 US dollars versus 60 US dollars/patient/year). In conclusion, to exclude MA, the CM used in the random spot urine is reliable and easy to handle, but positive screening results of > or =30 mg albumin/l must be confirmed by analyses in the timed overnight collected urine. Although the screening compliance is improved, in terms of analysing random morning spot urine for MA, we cannot recommend CM in a paediatric diabetic outpatient setting because the specificity is far too low.
Asunto(s)
Albuminuria/diagnóstico , Albuminuria/orina , Diabetes Mellitus Tipo 1/orina , Nefropatías Diabéticas/diagnóstico , Nefropatías Diabéticas/orina , Manejo de Especímenes/métodos , Adolescente , Edad de Inicio , Niño , Nefropatías Diabéticas/mortalidad , Femenino , Humanos , Fallo Renal Crónico/diagnóstico , Fallo Renal Crónico/mortalidad , Masculino , Tamizaje Masivo/métodosRESUMEN
OBJECTIVE: To study clinical, morphological and molecular characteristics in a Swiss family with autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI). PARTICIPANTS AND METHODS: A 15-month-old girl presenting with symptoms of polydipsia and polyuria was investigated by water deprivation test. Evaluation of the family revealed three further family members with symptomatic vasopressin-deficient diabetes insipidus. T1-weighted magnetic resonance images of the posterior pituitary were taken in two affected adult family members and molecular genetic analysis was performed in all affected individuals. RESULTS: The water deprivation test in the 15-month-old child confirmed the diagnosis of vasopressin-deficient diabetes insipidus and the pedigree was consistent with autosomal dominant inheritance. The characteristic bright spot of the normal vasopressin-containing neurophypophysis was absent in both adults with adFNDI. Direct sequence analysis revealed a new deletion (177-179DeltaCGC) in exon 2 of the AVP-NP II gene in all affected individuals. At the amino acid level, this deletion eliminates cysteine 59 (C59Delta) and substitutes alanine 60 by tryptophan (A60W) in the AVP-NP II precursor; interestingly, the remainder of the reading frame remains unchanged. According to the three-dimensional structure of neurophysin, C59 is involved in a disulphide bond with C65. CONCLUSIONS: Deletion of C59 and substitution of A60W in the AVP-NP II precursor is predicted to disrupt one of the seven disulphide bridges required for correct folding of the neurophysin moiety and thus disturb the function of neurophysin as the vasopressin transport protein. These data are in line with the clinical and morphological findings in the reported family with adFNDI.
Asunto(s)
Arginina Vasopresina/deficiencia , Diabetes Insípida/etiología , Diabetes Insípida/genética , Genes Dominantes , Neurofisinas/genética , Precursores de Proteínas/genética , Vasopresinas/genética , Secuencia de Aminoácidos/genética , Secuencia de Bases/genética , Diabetes Insípida/diagnóstico , Femenino , Humanos , Lactante , Imagen por Resonancia Magnética , Biología Molecular/métodos , Datos de Secuencia Molecular , Linaje , SuizaRESUMEN
The human GH gene is 1.7 kilobase pairs (kb) in length and is composed of five exons and four introns. This gene is expressed in the pituitary gland and encodes a 22 kDa protein. In addition to this predominant (75%) form, 5-10% of pituitary GH is present as a 20 kDa protein that has an amino acid (aa) sequence identical to the 22 kDa form except for a 15 aa internal deletion of residues 32-46 as a result of an alternative splicing event. Because it has been reported that non-22-kDa GH isoforms might be partly responsible for short stature and growth retardation in children, the aim of this study was to compare the impact of both 22 kDa and 20 kDa GH on GH receptor gene (GH receptor/GH binding protein (GHR/GHBP)) expression. Various concentrations of 20 kDa and 22 kDa GH (0, 2, 5, 12.5, 25, 50 and 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was measured by quantitative PCR. Addition of either 20 kDa or 22 kDa GH, at low or normal physiological concentrations (0, 2, 5, 12.5, 25 or 50 ng/ml) induced a dose-dependent increase in GHR/GHBP expression. However, a supraphysiological concentration of 20 kDa GH (150 ng/ml) resulted in a significantly lower (P<0.05) downregulation of GHR/GHBP gene transcription compared with the downregulation achieved by this concentration of 22 kDa GH. This difference might be explained by a decreased ability to form a 1 : 1 complex with GHR and/or GHBP, which normally occurs at high concentrations of GH. Nuclear run-on experiments and GHBP determinations confirmed the changes in GHR/GHBP mRNA levels. In conclusion, we report that both 20 kDa and 22 kDa GH, in low and normal physiological concentrations, have the same effect on regulation of GHR/GHBP gene transcription in a human hepatoma cell line. At a supraphysiological concentration of 150 ng/ml, however, 20 kDa GH has a less self-inhibitory effect than the 22 kDa form.
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Carcinoma Hepatocelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/farmacología , Neoplasias Hepáticas/metabolismo , ARN Mensajero/análisis , Receptores de Somatotropina/genética , Proteínas Portadoras/genética , Relación Dosis-Respuesta a Droga , Humanos , Reacción en Cadena de la Polimerasa/métodos , Isoformas de Proteínas/farmacología , Células Tumorales CultivadasRESUMEN
AIMS/HYPOTHESIS: In diabetes mellitus type I, good glycaemic control is crucial in preventing long-term diabetic complications. The aim of this study was to determine the current level of metabolic control in children and adolescents in our diabetes outpatient clinic at the University Children's Hospital, Berne. Furthermore, the impact of different factors such as age, pubertal stage, sex, duration of diabetes and insulin regimen on glycaemic control was studied. METHODS: In a cross-sectional, prospective study 168 children and adolescents with type I diabetes mellitus (f:m = 87:81; prepubertal 48 [mean age 4.4 years, mean duration of diabetes 2.8 years]; pubertal 120 [mean age 9.4 years; mean duration of diabetes 5.2 years]) were studied for three months. Clinical data and HbA1c levels (latex immunoagglutination test) were recorded, statistically analysed and compared with the international literature. RESULTS: In our type I diabetic children and adolescents the overall HbA1c was 8.07 +/- 1.15% (mean +/- SD; test-specific norm for healthy subjects: 4.1-6.1%). Glycaemic control was significantly worse in the pubertal group compared to the prepubertal (HbA1c 8.22 +/- 1.25% vs. 7.81 +/- 0.87%; p < 0.01). In addition, we found better metabolic control in patients with duration of diabetes below 2 years in children and adolescents (HbA1c prepubertal < 2 years: 7.45 +/- 0.67% vs. > 2 years: 8.05 +/- 0.93%, p < 0.05; pubertal < 2 years: 7.62 +/- 0.75% vs. > 2 years: 8.31 +/- 1.29%, p < 0.005). Importantly, sex and insulin regimen did not significantly influence glycaemic control. CONCLUSION/INTERPRETATION: The current level of metabolic control in our children and adolescents with diabetes mellitus type I is comparable to the glycaemic control of the intensively treated adolescent group of the DCCT-study, in whom decreased risk of long-term diabetic complications was found. In contrast, our patients were intensively treated in terms of frequent contacts with the diabetes team, but were not necessarily on an intensified insulin regimen. The impact of biopsychosocial support from multidisciplinary diabetes team on good metabolic control in children and adolescents with type I diabetes mellitus and their families seems to be very important.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 1/sangre , Hemoglobina Glucada/análisis , Adolescente , Factores de Edad , Edad de Inicio , Índice de Masa Corporal , Niño , Estudios Transversales , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Femenino , Humanos , Insulina/uso terapéutico , Masculino , PubertadRESUMEN
We identified a new point mutation in the CYP19 gene responsible for aromatase (P450arom) deficiency in a 46,XY male infant with unremarkable clinical findings at birth. This boy is homozygote for a 1-bp (C) deletion in exon 5 of the aromatase gene causing a frame-shift mutation. The frame-shift results in a prematurely terminated protein that is inactive due to the absence of the functional regions of the enzyme. Aromatase deficiency was suspected prenatally because of the severe virilization of the mother during the early pregnancy, and the diagnosis was confirmed shortly after birth. Four weeks after birth, the baby boy showed extremely low levels of serum estrogens, but had a normal level of serum free testosterone; in comparison with the high serum concentration of androstenedione at birth, a striking decrease occurred by 4 weeks postnatally. We previously reported elevated basal and stimulated FSH levels in a female infant with aromatase deficiency in the first year of life. In contrast, in the male infant, basal FSH and peak FSH levels after standard GnRH stimulation tests were normal. This finding suggests that the contribution of estrogen to the hypothalamic-pituitary gonadotropin-gonadal feedback mechanism is different in boys and girls during infancy and early childhood. In normal girls, serum estradiol concentrations strongly correlate with circulating inhibin levels, and thus, low inhibin levels may contribute to the striking elevation of FSH in young girls with aromatase deficiency. In contrast, estradiol levels are physiologically about a 7-fold lower in boys than in girls, and serum inhibin levels remain elevated even though levels of FSH, LH, and testosterone are decreased.
Asunto(s)
Aromatasa/deficiencia , Aromatasa/genética , Estrógenos/deficiencia , Hormona Folículo Estimulante/sangre , Mutación del Sistema de Lectura , Hormona Luteinizante/sangre , Androstenodiona/sangre , Secuencia de Bases , Estrógenos/sangre , Femenino , Hormona Liberadora de Gonadotropina , Heterocigoto , Homocigoto , Humanos , Recién Nacido , Masculino , Linaje , Embarazo , Complicaciones del Embarazo , Testosterona/sangre , Virilismo/genéticaRESUMEN
AIMS/HYPOTHESIS: The aims of this study were to analyse the changes of serum leptin in newly diagnosed children and adolescents with Type I (insulin-dependent) diabetes mellitus after insulin treatment and to examine the possible impact of ketoacidosis on these changes. METHODS: Baseline serum leptin concentrations were measured in 28 newly diagnosed Type I diabetic patients [age 8.75 +/- 4.05 years (means +/- SD); BMI 15.79 +/- 2.47 kg/m(2); HbA(1 c) 11.3 +/- 1.9 %] with (n = 18) and without (n = 10) ketoacidosis before commencement of insulin treatment, at the time of diagnosis. Thereafter, during a 4-day course of continuous intravenous insulin injection to gain and maintain euglycaemia, serum leptin concentrations were assessed. RESULTS: Baseline serum leptin concentrations, adjusted to age, BMI, sex and pubertal stage, differed among these patients. There was, however, an increase of leptin in all subjects from 1.37 +/- 0.56 ng/ml (mean +/- SD) up to 2.97 +/- 1.52 ng/ml by 117 % (p < 0.0001) after insulin therapy. On average, peak serum leptin concentration was obtained after 42 h of insulin treatment. Further, there was no difference in the mean increase of serum leptin concentrations in the two groups, namely with and without ketoadicosis, of insulin-dependent diabetic children and adolescents. In addition, there was no correlation between serum leptin concentrations and correction of ketoacidosis during insulin treatment. CONCLUSIONS/INTERPRETATION: Insulin increases serum leptin, within 1 day, in children and adolescents with newly diagnosed Type I diabetes. Ketoacidosis does not influence this interaction between insulin and leptin.
Asunto(s)
Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Cetoacidosis Diabética/sangre , Cetoacidosis Diabética/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Proteínas/metabolismo , Adolescente , Glucemia/metabolismo , Niño , Preescolar , Femenino , Humanos , Lactante , Cinética , Leptina , Masculino , Proteínas/efectos de los fármacosRESUMEN
As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). Although pit-1 was 1 of the first factors identified as a cause of CPHD in mice, many other homeodomain and transcription factors have been characterized as being involved in different developmental stages of pituitary gland development, such as prophet of pit-1 (prop-1), P-Lim, ETS-1, and Brn 4. The aims of the present study were first to screen families and patients suffering from different forms of CPHD for PROP1 gene alterations, and second to define possible hot spots and the frequency of the different gene alterations found. Of 73 subjects (36 families) analyzed, we found 35 patients, belonging to 18 unrelated families, with CPHD caused by a PROP1 gene defect. The PROP1 gene alterations included 3 missense mutations, 2 frameshift mutations, and 1 splice site mutation. The 2 reported frameshift mutations could be caused by any 2-bp GA or AG deletion at either the 148-GGA-GGG-153 or 295-CGA-GAG-AGT-303 position. As any combination of a GA or AG deletion yields the same sequencing data, the frameshift mutations were called 149delGA and 296delGA, respectively. All but 1 mutation were located in the PROP1 gene encoding the homeodomain. Importantly, 3 tandem repeats of the dinucleotides GA at location 296-302 in the PROP1 gene represent a hot spot for CPHD. In conclusion, the PROP1 gene seems to be a major candidate gene for CPHD; however, further studies are needed to evaluate other genetic defects involved in pituitary development.
Asunto(s)
Proteínas de Homeodominio/genética , Hormonas Hipofisarias/deficiencia , Hormonas Hipofisarias/genética , Factores de Transcripción/genética , Hormona Adrenocorticotrópica/deficiencia , Adulto , Empalme Alternativo , Animales , Femenino , Hormona Folículo Estimulante/deficiencia , Mutación del Sistema de Lectura , Eliminación de Gen , Genoma , Gonadotropinas/deficiencia , Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/genética , Humanos , Hormona Luteinizante/deficiencia , Masculino , Ratones , Mutación Missense , Linaje , Fenotipo , Polimorfismo Genético , Prolactina/deficiencia , Tirotropina/deficienciaRESUMEN
As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). A mutation in a novel, tissue-specific, paired-like homeodomain transcription factor, termed Prophet of Pit-1 (PROP1), has been identified as causing the Ames dwarf (df) mouse phenotype, and thereafter, different PROP1 gene alterations have been found in humans with CPHD. We report on the follow-up of two consanguineous families (n = 12), with five subjects affected with CPHD (three males and two females) caused by the same nucleotide C to T transition, resulting in the substitution of Arg-->Cys in PROP1 at codon 120. Importantly, there is a variability of phenotype, even among patients with the same mutation. The age at diagnosis was dependent on the severity of symptoms, ranging from 9 months to 8 yr. Although in one patient TSH deficiency was the first symptom of the disorder, all patients became symptomatic by exhibiting severe growth retardation and failure to thrive, which was mainly caused by GH deficiency (n = 4). The secretion of the pituitary-derived hormones (GH, PRL, TSH, LH, and FSH) declined gradually with age, following a different pattern in each individual; therefore, the deficiencies developed over a variable period of time. All of the subjects entered puberty spontaneously, and the two females also experienced menarche and periods before a replacement therapy was necessary.