RESUMEN
Pituitary adenomas account for 10-15% of primary intracranial tumors. Growth hormone (GH)-secreting adenomas account for 13% of all pituitary adenomas and cause acromegaly. These tumors can be aggressive, invade surrounding structures and are highly recurrent. The objective of this study was to evaluate E-cadherin, Slug and neural cell adhesion molecule (NCAM) expression in GH-secreting pituitary adenomas and its relationship to tumor invasiveness. A cross-sectional study of patients who underwent hypophysectomy due to GH-secreting pituitary adenoma from April 2007 to December 2014 was carried out. The medical records were reviewed to collect clinical data. Immediately after surgery, tumor samples were frozen in liquid nitrogen and stored in a biofreezer at -80°C for assessment of E-cadherin 1 (CDH1), SLUG (SNAI2), and NCAM (NCAM1) by real-time PCR. The samples were fixed in formalin and embedded in paraffin for immunohistochemical analysis of E-cadherin and NCAM. Thirty-five patients with acromegaly were included in the study. Of these, 65.7% had invasive tumors. Immunohistochemically, E-cadherin was expressed in 96.7% of patients, and NCAM in 80% of patients. There was no statistically significant relationship between tumor grade or invasiveness and immunohistochemical expression of these markers. Regarding gene expression, 50% of cases expressed CDH1, none expressed SNAI2, and 53.3% expressed NCAM1. There was no statistically significant relationship between tumor grade or invasiveness and gene expression of CDH1, SNAI2, and NCAM1. The absence of Slug overexpression and of E-cadherin and NCAM suppression suggests that expression of these markers is not associated with tumor invasiveness in GH-secreting pituitary adenomas.
Asunto(s)
Humanos , Masculino , Femenino , Adolescente , Adulto , Persona de Mediana Edad , Anciano , Adulto Joven , Acromegalia/patología , Adenoma/patología , Cadherinas/análisis , Moléculas de Adhesión de Célula Nerviosa/análisis , Factores de Transcripción de la Familia Snail/análisis , Acromegalia/genética , Acromegalia/metabolismo , Inmunohistoquímica , Biomarcadores de Tumor/análisis , Adenoma/genética , Adenoma/química , Expresión Génica , Estudios Transversales , Clasificación del TumorRESUMEN
Pituitary adenomas account for 10-15% of primary intracranial tumors. Growth hormone (GH)-secreting adenomas account for 13% of all pituitary adenomas and cause acromegaly. These tumors can be aggressive, invade surrounding structures and are highly recurrent. The objective of this study was to evaluate E-cadherin, Slug and neural cell adhesion molecule (NCAM) expression in GH-secreting pituitary adenomas and its relationship to tumor invasiveness. A cross-sectional study of patients who underwent hypophysectomy due to GH-secreting pituitary adenoma from April 2007 to December 2014 was carried out. The medical records were reviewed to collect clinical data. Immediately after surgery, tumor samples were frozen in liquid nitrogen and stored in a biofreezer at -80°C for assessment of E-cadherin 1 (CDH1), SLUG (SNAI2), and NCAM (NCAM1) by real-time PCR. The samples were fixed in formalin and embedded in paraffin for immunohistochemical analysis of E-cadherin and NCAM. Thirty-five patients with acromegaly were included in the study. Of these, 65.7% had invasive tumors. Immunohistochemically, E-cadherin was expressed in 96.7% of patients, and NCAM in 80% of patients. There was no statistically significant relationship between tumor grade or invasiveness and immunohistochemical expression of these markers. Regarding gene expression, 50% of cases expressed CDH1, none expressed SNAI2, and 53.3% expressed NCAM1. There was no statistically significant relationship between tumor grade or invasiveness and gene expression of CDH1, SNAI2, and NCAM1. The absence of Slug overexpression and of E-cadherin and NCAM suppression suggests that expression of these markers is not associated with tumor invasiveness in GH-secreting pituitary adenomas.
Asunto(s)
Acromegalia/patología , Adenoma/patología , Cadherinas/análisis , Moléculas de Adhesión de Célula Nerviosa/análisis , Neoplasias Hipofisarias/patología , Factores de Transcripción de la Familia Snail/análisis , Acromegalia/genética , Acromegalia/metabolismo , Adenoma/química , Adenoma/genética , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/análisis , Antígeno CD56/análisis , Estudios Transversales , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Neoplasias Hipofisarias/química , Neoplasias Hipofisarias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Estadísticas no Paramétricas , Adulto JovenRESUMEN
We investigated the presence of mutations/polymorphisms in the FSH receptor (FSHR) gene and their association with phenotype in women with premature ovarian failure (POF) in southern Brazil. Clinical and hormonal variables were determined in 36 46,XX women with primary or secondary amenorrhea before the age of 40 yr, FSH >40 IU/l and ovarian failure. DNA was isolated from peripheral leukocytes. Exons 6, 7, 9, and 10 of the FSHR gene were analyzed by PCR, restriction enzyme analysis, denaturing gradient gel electrophoresis, and direct sequencing. No inactivating mutations were found. Exon 10 had two polymorphisms, Ala307Thr and Ser680Asn (allelic frequency: 52.9 and 35.7%, respectively), which were not related to FSH, LH or estradiol serum levels. Ovarian size and small ovarian follicles on transvaginal sonography were not associated with FSHR genetic variants. In contrast, the last menstruation occurred significantly earlier in patients with the Ala307Thr polymorphism (A: age=33.3+/-7.1 yr vs T: 28.6+/-11.4 yr, p=0.04). In conclusion, we did not identify inactivating mutations in exons 6, 7, 9, and 10 of the FSHR gene. A high frequency of two polymorphisms that are in linkage disequilibrium was found in exon 10 of the FSHR gene. The presence of the Ala307Thr polymorphism may be associated with a more precocious onset of clinical disease.
Asunto(s)
Fenotipo , Insuficiencia Ovárica Primaria/diagnóstico , Insuficiencia Ovárica Primaria/genética , Receptores de HFE/genética , Adolescente , Adulto , Brasil/epidemiología , Femenino , Frecuencia de los Genes/genética , Humanos , Polimorfismo Genético/genética , Insuficiencia Ovárica Primaria/epidemiologíaRESUMEN
Several genes that influence the development and function of the hypothalamic-pituitary-gonadal-axis (HPG) have been identified. These genes encode an array of transcription factors, matrix proteins, hormones, receptors, and enzymes that are expressed at multiple levels of the HPG. We report the experience of a single Endocrinology Unit in the identification and characterization of naturally occurring mutations in families affected by HPG disorders, including forms of precocious puberty, hypogonadism and abnormal sexual development due to impaired gonadotropin function. Eight distinct genes implicated in HPG function were studied: KAL, SF1, DAX1, GnRH, GnRHR, FSHá, FSHR, and LHR. Most mutations identified in our cohort are described for the first time in literature. New mutations in SF1, DAX1 and GnRHR genes were identified in three Brazilian patients with hypogonadism. Eight boys with luteinizing hormone- (LH) independent precocious puberty due to testotoxicosis were studied, and all have their LH receptor (LHR) defects elucidated. Among the identified LHR molecular defects, three were new activating mutations. In addition, these mutations were frequently associated with new clinical and hormonal aspects, contributing significantly to the knowledge of the molecular basis of reproductive disorders. In conclusion, the naturally occurring genetic mutations described in the Brazilian families studied provide important insights into the regulation of the HPG.
Asunto(s)
Humanos , Masculino , Trastornos Gonadales , Sistema Hipotálamo-Hipofisario , Mutación , Marcadores Genéticos , Trastornos Gonadales , GonadotropinasRESUMEN
Several genes that influence the development and function of the hypothalamic-pituitary-gonadal-axis (HPG) have been identified. These genes encode an array of transcription factors, matrix proteins, hormones, receptors, and enzymes that are expressed at multiple levels of the HPG. We report the experience of a single Endocrinology Unit in the identification and characterization of naturally occurring mutations in families affected by HPG disorders, including forms of precocious puberty, hypogonadism and abnormal sexual development due to impaired gonadotropin function. Eight distinct genes implicated in HPG function were studied: KAL, SF1, DAX1, GnRH, GnRHR, FSHbeta, FSHR, and LHR. Most mutations identified in our cohort are described for the first time in literature. New mutations in SF1, DAX1 and GnRHR genes were identified in three Brazilian patients with hypogonadism. Eight boys with luteinizing hormone- (LH) independent precocious puberty due to testotoxicosis were studied, and all have their LH receptor (LHR) defects elucidated. Among the identified LHR molecular defects, three were new activating mutations. In addition, these mutations were frequently associated with new clinical and hormonal aspects, contributing significantly to the knowledge of the molecular basis of reproductive disorders. In conclusion, the naturally occurring genetic mutations described in the Brazilian families studied provide important insights into the regulation of the HPG.
Asunto(s)
Trastornos Gonadales/genética , Sistema Hipotálamo-Hipofisario , Mutación/genética , Marcadores Genéticos/genética , Trastornos Gonadales/fisiopatología , Gonadotropinas/genética , Humanos , MasculinoRESUMEN
OBJECTIVE: To search for germline activating mutations of the FSH receptor in girls with gonadotropin-independent precocious puberty. DESIGN: Molecular studies in human tissue. SETTING: Four girls with polycystic ovaries and gonadotropin-independent isosexual precocious puberty without clinical and molecular features of McCune-Albright syndrome. INTERVENTION(S): Peripheral blood was used for DNA extraction. The alpha-subunit of the Gs gene and the entire exon 10 of FSH receptor gene were amplified by polymerase chain reaction (PCR). Gs-alpha mutations characteristic of McCune-Albright syndrome were excluded by denaturating gradient gel electrophoresis (DGGE) and allele-specific PCR. Exon 10 of the FSH receptor gene was analyzed by DGGE and direct sequencing. MAIN OUTCOME MEASURE(S): Results of DGGE and direct sequencing. RESULT(S): No germline activating mutations were detected in exon 10 of our patients. Instead, two previously described polymorphisms were found, leading to the substitution of alanine for threonine at position 307 and of serine for asparagine at position 680 of the FSH receptor molecule. CONCLUSION(S): Germline activating mutations were not found in exon 10 of the FSHR gene in any of our patients. Further studies, preferably in ovarian tissue, will be required to exclude the presence of somatic activating mutations of the FSH receptor in these patients.
Asunto(s)
Mutación de Línea Germinal , Quistes Ováricos/genética , Pubertad Precoz/genética , Receptores de HFE/genética , Sustitución de Aminoácidos , Niño , Preescolar , Electroforesis/métodos , Femenino , Heterocigoto , HumanosRESUMEN
To establish normative data and determine the value of fluorometric AutoDELFIA assays (Wallac Oy) in the investigation of precocious puberty, we determined serum levels of LH, FSH, testosterone, and estradiol under basal and GnRH-stimulated conditions in 277 normal subjects at various pubertal stages and in 77 patients with precocious puberty. A substantial overlap was observed in basal and GnRH-stimulated gonadotropin levels in normal individuals of both sexes with pubertal Tanner stages 1 and 2. The 95th percentile of the normal prepubertal population was the cut-off limit between prepubertal and pubertal levels. These limits were 0.6 IU/L in both sexes for basal LH, 9.6 IU/L in boys and 6.9 IU/L in girls for peak LH after GnRH stimulation, 19 ng/dL in boys for basal testosterone, and 13.6 pg/mL in girls for basal estradiol. Basal and peak LH exceeding these limits were considered positive tests for the diagnosis of gonadotropin-dependent precocious puberty. According to these criteria, the sensitivities of basal and peak LH for the latter diagnosis were 71.4% and 100% in boys, and 62.7% and 92.2% in girls. The specificity and positive predicted value were 100% in both sexes for basal and peak LH levels. The negative predicted values for basal and peak LH were 62.5% and 100% in boys, and 40.6% and 76.5% in girls. Basal and GnRH-stimulated FSH levels overlapped among the various pubertal stages in normal subjects and were, in general, not helpful in the differential diagnosis of precocious puberty. In conclusion, basal LH levels were sufficient to establish the diagnosis of gonadotropin-dependent precocious puberty in 71.4% of boys and 62.7% of girls. In the remaining patients, a GnRH stimulation test was still necessary to confirm this diagnosis. Finally, suppressed LH and FSH levels after GnRH stimulation indicate gonadotropin-independent sexual steroid production.
Asunto(s)
Fluorometría/normas , Pubertad Precoz/diagnóstico , Adolescente , Adulto , Niño , Preescolar , Femenino , Hormona Liberadora de Gonadotropina , Gonadotropinas/sangre , Humanos , Masculino , Pubertad Precoz/sangre , Valores de Referencia , Esteroides/sangreRESUMEN
OBJECTIVE: To investigate the presence of FSH receptor gene mutations in women with premature ovarian failure (POF). DESIGN: Clinical and molecular studies. SETTING: Research laboratory in a university setting. PATIENT(S): Fifteen 46,XX women with POF and 42 normal fertile controls. INTERVENTION(S): Exon 7 was amplified and digested with BsmI to screen for the previously described inactivating mutation C566T. Exon 10 was screened for mutations by denaturing gradient gel electrophoresis and direct sequencing. MAIN OUTCOME MEASURE(S): Polymerase chain reaction followed by restriction enzyme analysis, denaturing gradient gel electrophoresis, and direct sequencing. RESULT(S): No inactivating mutations were identified in exons 7 and 10 of the FSH receptor gene in women with familial or sporadic POF. Exon 10 had two polymorphisms, G919A and G2039A, whose allelic frequencies were 46.7% and 56.6%, respectively, in women with POF. The allelic frequency of both polymorphisms was 59.5% in normal fertile controls. CONCLUSION(S): No inactivating mutations in exons 7 and 10 of the FSH receptor gene were identified in Brazilian women with POF. A high frequency of two polymorphisms that are in linkage disequilibrium was found in exon 10 of this gene.
Asunto(s)
Periodicidad , Insuficiencia Ovárica Primaria/genética , Receptores de HFE/genética , Adolescente , Adulto , Brasil , Estudios de Casos y Controles , Femenino , Humanos , Mutación , Reacción en Cadena de la PolimerasaRESUMEN
The radioimmunoassay of, urinary cortisol extracted by organic solvent (non-conjugated cortisol) has been used in the diagnosis of hypercortisolism for a long time. With the development of more specific antisera it became possible to measure urinary cortisol without extraction (total urinary cortisol). We compared both methods in the hypercortisolism diagnosis. Basal 24-hour urinary cortisol levels from 43 normal subjects, 53 obese patients and 53 Cushing's syndrome patients were measured. Urinary cortisol levels were not statistically different in normal and obese group in either methods (p > 0.05). There was a significant difference for the Cushing's syndrome group in relation to the normal and obese groups in both methods (p < 0.05). Sensitivity was similar for both methods and a positive correlation was noticed in the three groups. We conclude that both methods are efficient for the diagnosis of hypercortisolism but we suggest that total urinary cortisol should be the first choice to diagnose Cushing's syndrome considering its advantages such as low cost and feasibility.