RESUMEN

We have sought the molecular diagnosis of OI in 38 Brazilian cases through targeted sequencing of 15 candidate genes. While 71% had type 1 collagen-related OI, defects in FKBP10, PLOD2 and SERPINF1, and a potential digenic P3H1/WNT1 interaction were prominent causes of OI in this underrepresented population. INTRODUCTION: Defects in type 1 collagen reportedly account for 85-90% of osteogenesis imperfecta (OI) cases, but most available molecular data has derived from Sanger sequencing-based approaches in developed countries. Massively parallel sequencing (MPS) allows for systematic and comprehensive analysis of OI genes simultaneously. Our objective was to obtain the molecular diagnosis of OI in a single Brazilian tertiary center cohort. METHODS: Forty-nine individuals (84% adults) with a clinical diagnosis of OI, corresponding to 30 sporadic and 8 familial cases, were studied. Sixty-three percent had moderate to severe OI, and consanguinity was common (26%). Coding regions and 25-bp boundaries of 15 OI genes (COL1A1, COL1A2, IFITM5 [plus 5'UTR], SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, PLOD2, BMP1, SP7, TMEM38B, WNT1, CREB3L1) were analyzed by targeted MPS and variants of interest were confirmed by Sanger sequencing or SNP array. RESULTS: A molecular diagnosis was obtained in 97% of cases. COL1A1/COL1A2 variants were identified in 71%, whereas 26% had variants in other genes, predominantly FKBP10, PLOD2, and SERPINF1. A potential digenic interaction involving P3H1 and WNT1 was identified in one case. Phenotypic variability with collagen defects could not be explained by evident modifying variants. Four consanguineous cases were associated to heterozygous COL1A1/COL1A2 variants, and two nonconsanguineous cases had compound PLOD2 heterozygosity. CONCLUSIONS: Novel disease-causing variants were identified in 29%, and a higher proportion of non-collagen defects was seen. Obtaining a precise diagnosis of OI in underrepresented populations allows expanding our understanding of its molecular landscape, potentially leading to improved personalized care in the future.

Asunto(s)

Osteogénesis Imperfecta , Adulto , Brasil , Colágeno Tipo I/genética , Heterocigoto , Humanos , Mutación , Osteogénesis Imperfecta/genética , Proteínas de Unión a Tacrolimus/genética

RESUMEN

The congenital adrenal hyperplasia population seems to have an intrinsic tendency to a high frequency of low bone mass. However in this single-center and long-term evaluated cohort, the simplified corticoid regimen, with exclusive dexamethasone single dose reposition during adulthood, did not represent a risk factor for decrease in bone health. INTRODUCTION: The impact of long-term and supposedly physiological doses of gluco and mineralocorticoid (GC/MC) on bone mineral density (BMD) in congenital adrenal hyperplasia (CAH) remains discordant among studies, which contain different clinical forms and corticoid regimens. Our aim was to evaluate the BMD in CAH adults receiving similar GC regimen since childhood and to correlate it with GC/MC cumulative doses. METHODS: Only patients with good compliance, who used cortisone acetate (CA) during childhood and dexamethasone after the final height achievement. Cumulative GC/MC doses were calculated from diagnosis until last evaluation. BMD was analyzed by the first and last energy X-ray absorptiometry (DXA) scans performed. RESULTS: Twenty simple virilizing (SV) and 14 salt wasting (WS) whose mean age was 26 ± 6 years, mean CA, dexamethasone, and fludrocortisone cumulative doses were 63,813 ± 32,767, 812 ± 558, and 319 ± 325 mg/m2, respectively. Based on the last DXA, low BMD was observed in 11% of patients, total hip Z-score was lower in the SW than SV form (p = 0.04). Cumulative CA dose had an inverse correlation with femoral neck Z-score (p < 0.01). Total cumulative GC and MC doses had an inverse correlation with total hip Z-score (p < 0.01). In the analysis of sequential BMD during dexamethasone therapy, no association was observed among cumulative GC/MC doses, clinical forms, sex, and lumbar Z-score delta. CONCLUSIONS: Even though a low CA regimen during growth periods in addition to MC replacement appears to have an influence on BMD at femoral sites, interestingly a low dexamethasone one does not seem to be deleterious for bone health in adulthood.

Asunto(s)

Hiperplasia Suprarrenal Congénita , Densidad Ósea , Absorciometría de Fotón , Hiperplasia Suprarrenal Congénita/tratamiento farmacológico , Adulto , Niño , Glucocorticoides/efectos adversos , Humanos , Estudios Retrospectivos , Adulto Joven

RESUMEN

Testotoxicosis is a rare cause of peripheral precocious puberty in boys caused by constitutively activating mutations of the LHCG receptor. Affected males usually have normal gonadotropin profiles and fertility in their adult life. Here, we described the long-term follow-up of a 24-year-old young man with severe testotoxicosis due to a de novo activating mutation in the third transmembrane helix of the LHCGR (p.Leu457Arg). This patient was treated with different medications, including medroxyprogesterone acetate, ketoconazole, cyproterone acetate and aromatase inhibitor from age 2.5 to 9.5 years. His basal and GnRH-stimulated gonadotropin levels were continually suppressed during and after medical treatment. At adulthood, extremely high serum testosterone levels (>35 nmol/L), undetectable gonadotropin levels (LH < 0.15 IU/L and FSH < 0.6 IU/L) and oligozoospermia were evidenced. Despite his suppressed FSH levels and an unfavorable spermogram, the patient fathered a healthy girl and biological paternity was confirmed through analysis of microsatellites. Spontaneous fertility in a young man with severe testotoxicosis and chronic suppression of FSH levels reinforces the key role of high intratesticular testosterone levels in human spermatogenesis.

Asunto(s)

Fertilidad/genética , Pubertad Precoz/genética , Receptores de HL/genética , Testosterona/sangre , Adulto , Humanos , Masculino , Mutación , Pubertad Precoz/sangre

RESUMEN

Targeted massively parallel sequencing (TMPS) has been used in genetic diagnosis for Mendelian disorders. In the past few years, the TMPS has identified new and already described genes associated with primary ovarian insufficiency (POI) phenotype. Here, we performed a targeted gene sequencing to find a genetic diagnosis in idiopathic cases of Brazilian POI cohort. A custom SureSelectXT DNA target enrichment panel was designed and the sequencing was performed on Illumina NextSeq sequencer. We identified 1 homozygous 1-bp deletion variant (c.783delC) in the GDF9 gene in 1 patient with POI. The variant was confirmed and segregated using Sanger sequencing. The c.783delC GDF9 variant changed an amino acid creating a premature termination codon (p.Ser262Hisfs*2). This variant was not present in all public databases (ExAC/gnomAD, NHLBI/EVS and 1000Genomes). Moreover, it was absent in 400 alleles from fertile Brazilian women screened by Sanger sequencing. The patient's mother and her unaffected sister carried the c.783delC variant in a heterozygous state, as expected for an autosomal recessive inheritance. Here, the TMPS identified the first homozygous 1-bp deletion variant in GDF9. This finding reveals a novel inheritance pattern of pathogenic variant in GDF9 associated with POI, thus improving the genetic diagnosis of this disorder.

Asunto(s)

Factor 9 de Diferenciación de Crecimiento/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Insuficiencia Ovárica Primaria/genética , Adulto , Alelos , Brasil , Codón sin Sentido/genética , Femenino , Homocigoto , Humanos , Mutación , Linaje , Insuficiencia Ovárica Primaria/fisiopatología , Eliminación de Secuencia/genética , Adulto Joven

RESUMEN

PURPOSE: Pituitary macroadenomas (MACs) represent 10-30 % of Cushing's disease (CD) cases. The aim of this study was to report the clinical, laboratorial and imaging features and postsurgical outcomes of microadenoma (MIC) and MAC patients. METHODS: Retrospective study with 317 CD patients (median 32 years old, range 9-71 years) admitted between 1990 and 2014, 74 (23.3 %) of whom had MAC. RESULTS: Hirsutism, plethora facial, muscular weakness and muscular atrophy were more frequent in the MIC patients. Nephrolithiasis, osteopenia, hyperprolactinaemia and galactorrhoea were more prevalent in MAC patients. The morning serum cortisol (Fs), nocturnal salivary cortisol (NSC), nocturnal Fs (Fs 2400 h), low- and high-dose dexamethasone suppression test results and CRH and desmopressin test results were similar between the subgroups. MIC patients showed higher urinary cortisol at 24 h (UC), and MAC patients presented higher ACTH levels but lower Fs/ACTH, Fs 2400 h/ACTH, NSC/ACTH and UC/ACTH ratios. There were negative correlations of tumour size with Fs/ACTH, Fs 2400 h/ACTH, NSC/ACTH and UC/ACTH ratios. Overall, the postsurgical remission and recurrence rates were similar between MIC and MAC. However, patients in remission (MIC + MAC) showed smaller tumour diameters and a lower prevalence of invasion and extension on MRI. CONCLUSIONS: Despite exhibiting higher plasma ACTH levels, CD patients with MAC presented lower cortisol/ACTH ratios than did patients with MIC, with a negative correlation between tumour size and cortisol/ACTH ratios. The overall postsurgical remission and recurrence rates were similar between MIC and MAC patients, with those with larger and/or invasive tumours showing a lower remission rate.

Asunto(s)

Adenoma/sangre , Hormona Adrenocorticotrópica/sangre , Hidrocortisona/sangre , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/complicaciones , Neoplasias Hipofisarias/sangre , Adenoma/etiología , Adenoma/patología , Adolescente , Adulto , Anciano , Niño , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/fisiopatología , Neoplasias Hipofisarias/etiología , Neoplasias Hipofisarias/patología , Estudios Retrospectivos , Adulto Joven

RESUMEN

Type 1 insulin-like growth factor receptor (IGF-1R) is overexpressed in a variety of human cancers, including adrenocortical tumors. The aim of the work was to investigate the effects of IGF-1R downregulation in a human adrenocortical cell line by small interfering RNA (siRNA). The human adrenocortical tumor cell line NCI H295R was transfected with 2 specific IGF1R siRNAs (# 1 and # 2) and compared with untreated cells and a negative control siRNA. IGF1R expression was determined by quantitative reverse-transcription PCR (qRTPCR) and Western blot. The effects of IGF-1R downregulation on cell proliferation and apoptosis were assessed. IGF-1R levels were significantly decreased in cells treated with IGF-1R siRNA # 1 or # 2. Relative expression of IGF1R mRNA decreased approximately 50% and Western blot analysis revealed a 30% of reduction in IGF-1R protein. Downregulation of this gene resulted in 40% reduction in cell growth in vitro and 45% increase in apoptosis using siRNA # 2. These findings demonstrate that decreasing IGF-1R mRNA and protein expression in NCI H295R cells can partially inhibit adrenal tumor cell growth in vitro. Targeting IGF1R is a promising therapy for pediatric malignant adrenocortical tumor and can still be an option for adult adrenocortical cancer based on personalized genomic tumor profiling.

Asunto(s)

Corteza Suprarrenal/citología , Silenciador del Gen , Receptor IGF Tipo 1/metabolismo , Apoptosis/genética , Línea Celular , Proliferación Celular , Regulación hacia Abajo/genética , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Receptor IGF Tipo 1/genética

RESUMEN

DAX1 transcription factor is a key determinant of adrenogonadal development, acting as a repressor of SF1 targets in steroidogenesis. It was recently demonstrated that DAX1 regulates pluripotency and differentiation in murine embryonic stem cells. In this study, we investigated DAX1 expression in adrenocortical tumors (ACTs) and correlated it with SF1 expression and clinical parameters. DAX1 and SF1 protein expression were assessed in 104 ACTs from 34 children (25 clinically benign and 9 malignant) and 70 adults (40 adenomas and 30 carcinomas). DAX1 gene expression was studied in 49 ACTs by quantitative real-time PCR. A strong DAX1 protein expression was demonstrated in 74% (25 out of 34) and 24% (17 out of 70) of pediatric and adult ACTs, respectively (χ(2)=10.1, p=0.002). In the pediatric group, ACTs with a strong DAX1 expression were diagnosed at earlier ages than ACTs with weak expression [median 1.2 (range, 0.5-4.5) vs. 2.2 (0.9-9.4), p=0.038]. DAX1 expression was not associated with functional status in ACTs. Interestingly, a positive correlation was observed between DAX1 and SF1 protein expression in both pediatric and adult ACTs (r=0.55 for each group separately; p<0.0001). In addition, DAX1 gene expression was significantly correlated with SF1 gene expression (p<0.0001, r=0.54). In conclusion, DAX1 strong protein expression was more frequent in pediatric than in adult ACTs. Additionally, DAX1 and SF1 expression positively correlated in ACTs, suggesting that these transcription factors might cooperate in adrenocortical tumorigenesis.

Asunto(s)

Neoplasias de la Corteza Suprarrenal/metabolismo , Carcinogénesis/metabolismo , Receptor Nuclear Huérfano DAX-1/metabolismo , Factor Esteroidogénico 1/metabolismo , Neoplasias de la Corteza Suprarrenal/genética , Adenoma Corticosuprarrenal/genética , Adenoma Corticosuprarrenal/metabolismo , Carcinoma Corticosuprarrenal/genética , Carcinoma Corticosuprarrenal/metabolismo , Adulto , Carcinogénesis/genética , Niño , Preescolar , Receptor Nuclear Huérfano DAX-1/genética , Femenino , Expresión Génica , Humanos , Lactante , Masculino , Persona de Mediana Edad , Factor Esteroidogénico 1/genética

RESUMEN

Male-to-female transsexual persons use oestrogens + antiandrogens to adapt their physical bodies to the female sex. Doses are usually somewhat higher than those used by hypogonadal women receiving oestrogen replacement. Particularly in cases of self-administration of cross-sex hormones, doses may be very high. Oestrogens are powerful stimulators of synthesis and release of prolactin and serum prolactin levels are usually somewhat increased following oestrogen treatment. Prolactinomas have been reported in male-to-female transsexual persons, both after use of high and conventional doses of oestrogens but remain rare events. We report two new cases of prolactinomas in male-to-female transsexual persons, one in a 41-year-old subject who had used nonsupervised high-dose oestrogen treatment since the age of 23 years and another one in a 42 year old who had initiated oestrogen treatment at the age of 17 years. Their serum prolactin levels were strongly increased, and the diagnosis of a pituitary tumour was confirmed by imaging techniques. Both cases responded well to treatment with cabergoline treatment whereupon serum prolactin normalised. Our two cases are added to the three cases of prolactinomas in the literature in persons who had used supraphysiological doses of oestrogens.

Asunto(s)

Estrógenos/efectos adversos , Neoplasias Hipofisarias/diagnóstico , Prolactinoma/diagnóstico , Personas Transgénero , Adulto , Antineoplásicos/uso terapéutico , Cabergolina , Ergolinas/uso terapéutico , Estrógenos/uso terapéutico , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Neoplasias Hipofisarias/inducido químicamente , Neoplasias Hipofisarias/tratamiento farmacológico , Prolactina/sangre , Prolactinoma/inducido químicamente , Prolactinoma/tratamiento farmacológico

RESUMEN

There is a strong correlation between the severity of genotypes and 17OH-progesterone levels in patients with the nonclassical form of 21-hydroxylase deficiency (NC-CAH); however, there are few studies regarding the correlation with clinical signs. The aim of the study was to evaluate whether genotypes correlate with the severity of the hyperandrogenic phenotype. A cohort of 114 NC-CAH patients were diagnosed by stimulated-17OHP ≥10 ng/ml. CYP21A2 genotypes were divided into 2 groups according to the severity of enzymatic impairment; mild and severe. Clinical data and hormonal profiles were compared between the 2 groups. Age at onset of manifestations did not differ between children or adults carrying both mild and severe genotypes. Frequencies of precocious pubarche and hirsutism, with or without menstrual abnormalities, were similar between the 2 groups. There were no differences in basal testosterone levels of adult symptomatic females carrying both genotypes, but there were differences between adult females with (92.9±49.5 ng/dl) and without hirsutism (43.8±38 ng/dl) (p=0.0002). Similar frequencies of both genotypes were observed in asymptomatic females and in those with clitoromegaly. Nonclassical genotypes do not predict the severity of phenotype. Asymptomatic and virilized females carrying the same genotype suggest that there is a modulatory effect of genes involved in the androgen pathway on the phenotype.

Asunto(s)

Hiperplasia Suprarrenal Congénita/sangre , Hiperplasia Suprarrenal Congénita/genética , Genotipo , Hiperandrogenismo/sangre , Hiperandrogenismo/genética , Esteroide 21-Hidroxilasa/sangre , Esteroide 21-Hidroxilasa/genética , Adolescente , Hiperplasia Suprarrenal Congénita/complicaciones , Adulto , Edad de Inicio , Andrógenos/sangre , Niño , Preescolar , Estudios de Cohortes , Femenino , Hirsutismo/sangre , Hirsutismo/complicaciones , Hirsutismo/genética , Humanos , Hiperandrogenismo/complicaciones , Testosterona/sangre

RESUMEN

The 21-hydroxylase deficiency (21OHD) is caused by CYP21A2 mutations resulting in severe or moderate enzymatic impairments. 21OHD females carrying similar genotypes present different degrees of external genitalia virilization, suggesting the influence of other genetic factors. Single nucleotide variants (SNVs) in the CYP3A7 gene and in its transcription factors, related to fetal 19-carbon steroid metabolism, could modulate the genital phenotype. To evaluate the influence of the 21OHD genotypes and the CYP3A7, PXR and CAR SNVs on the genital phenotype in 21OHD females. Prader scores were evaluated in 183 patients. The CYP3A7, PXR and CAR SNVs were screened and the 21OHD genotypes were classified according to their severity: severe and moderate groups. Patients with severe genotype showed higher degree of genital virilization (Prader median III, IQR III-IV) than those with moderate genotype (III, IQR II-III) (p < 0.001). However, a great overlap was observed between genotype groups. Among all the SNVs tested, only the CAR rs2307424 variant correlated with Prader scores (r(2) = 0.253; p = 0.023). The CYP21A2 genotypes influence the severity of genital virilization in 21OHD females. We also suggest that the CAR variant, which results in a poor metabolizer phenotype, could account for a higher degree of external genitalia virilization.

Asunto(s)

Hiperplasia Suprarrenal Congénita/genética , Genitales/metabolismo , Polimorfismo de Nucleótido Simple , Receptores Citoplasmáticos y Nucleares/genética , Esteroide 21-Hidroxilasa/genética , Virilismo/genética , Hiperplasia Suprarrenal Congénita/complicaciones , Hiperplasia Suprarrenal Congénita/patología , Alelos , Hidrocarburo de Aril Hidroxilasas/genética , Niño , Preescolar , Receptor de Androstano Constitutivo , Citocromo P-450 CYP3A , Femenino , Frecuencia de los Genes , Genitales/patología , Genotipo , Humanos , Recién Nacido , Receptor X de Pregnano , Receptores de Esteroides/genética , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Virilismo/complicaciones , Virilismo/patología

RESUMEN

There is a high incidence of pituitary-dependent hyperadrenocorticism (PDH) in Poodle dogs, with family members being affected by the disease, suggesting a genetic involvement. Tpit is an obligate transcription factor for the expression of pro-opiomelanocortingene and for corticotroph terminal differentiation. The aim of the present study was to screen the Tpit gene for germline mutations in Poodles with PDH. Fifty Poodle dogs (33 female, 8.71±2.8 years) with PDH and 50 healthy Poodle dogs (32 females, 9.4241 2.8 years) were studied. Genomic DNA was isolated from peripheral blood, amplified by PCR and submitted to automatic sequence. No mutation in the coding region of Tpit was found, whereas the new single nucleotide polymorphism p.S343G, in heterozygous state, was found in the same frequency in both PDH and control groups. We concluded that Tpit gain-of-function mutations are not involved in the etiology of PDH in Poodle dogs.

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O hiperadrenocorticismo ACTH-dependente (HACAD) apresenta elevada incidência em cães da raça Poodle, sendo que membros da mesma família têm sido acometidos pela doença, sugerindo envolvimento genético. Tpit é um fator de transcrição obrigatório para a expressão do gene da pro-opiomelanocortina e para a diferenciação terminal dos corticotrofos. O objetivo deste trabalho foi pesquisar mutações germinativas no gene Tpit em Poodles com HACAD. Cinquenta cães da raça Poodle, 33 fêmeas, média de idade de 8,71±2,8 anos, com HACAD, e 50 cães Poodles saudáveis, 32 fêmeas, média de idade de 9,4±2,8 anos, foram estudados. Mutações na região codificadora do gene Tpit não foram identificadas. Foi observado um novo polimorfismo em heterozigose, p.S343G, com a mesma frequência no grupo de cães com HACAD e no grupo-controle. Conclui-se que a mutação ativadora no gene Tpit não está envolvida na patogênese do hiperadrenocorticismo ACTH-dependente em cães da raça Poodle.

Asunto(s)

Animales , Perros , Mutación de Línea Germinal , Hiperfunción de las Glándulas Suprarrenales/veterinaria , Polimorfismo Genético , Corticotrofos , Proopiomelanocortina

RESUMEN

Insulin-like growth factor type 1 (IGF1) is a mediator of growth hormone (GH) action, and therefore, IGF1 is a candidate gene for recombinant human GH (rhGH) pharmacogenetics. Lower serum IGF1 levels were found in adults homozygous for 19 cytosine-adenosine (CA) repeats in the IGF1 promoter. The aim of this study was to evaluate the influence of (CA)n IGF1 polymorphism, alone or in combination with GH receptor (GHR)-exon 3 and -202 A/C insulin-like growth factor binding protein-3 (IGFBP3) polymorphisms, on the growth response to rhGH therapy in GH-deficient (GHD) patients. Eighty-four severe GHD patients were genotyped for (CA)n IGF1, -202 A/C IGFBP3 and GHR-exon 3 polymorphisms. Multiple linear regressions were performed to estimate the effect of each genotype, after adjustment for other influential factors. We assessed the influence of genotypes on the first year growth velocity (1st y GV) (n=84) and adult height standard deviation score (SDS) adjusted for target-height SDS (AH-TH SDS) after rhGH therapy (n=37). Homozygosity for the IGF1 19CA repeat allele was negatively correlated with 1st y GV (P=0.03) and AH-TH SDS (P=0.002) in multiple linear regression analysis. In conjunction with clinical factors, IGF1 and IGFBP3 genotypes explain 29% of the 1st y GV variability, whereas IGF1 and GHR polymorphisms explain 59% of final height-target-height SDS variability. We conclude that homozygosity for IGF1 (CA)19 allele is associated with less favorable short- and long-term growth outcomes after rhGH treatment in patients with severe GHD. Furthermore, this polymorphism exhibits a non-additive interaction with -202 A/C IGFBP3 genotype on the 1st y GV and with GHR-exon 3 genotype on adult height.

Asunto(s)

Hormona del Crecimiento/deficiencia , Hormona del Crecimiento/genética , Factor I del Crecimiento Similar a la Insulina , Proteínas Recombinantes/administración & dosificación , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Niño , Preescolar , Femenino , Genotipo , Hormona del Crecimiento/administración & dosificación , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Repeticiones de Microsatélite/genética , Farmacogenética , Polimorfismo Genético , Regiones Promotoras Genéticas/genética , Resultado del Tratamiento

RESUMEN

There is a high incidence of pituitary-dependent hyperadrenocorticism (PDH) in Poodle dogs, with family members being affected by the disease, suggesting a genetic involvement. Tpit is an obligate transcription factor for the expression of pro-opiomelanocortingene and for corticotroph terminal differentiation. The aim of the present study was to screen the Tpit gene for germline mutations in Poodles with PDH. Fifty Poodle dogs (33 female, 8.71±2.8 years) with PDH and 50 healthy Poodle dogs (32 females, 9.4241 2.8 years) were studied. Genomic DNA was isolated from peripheral blood, amplified by PCR and submitted to automatic sequence. No mutation in the coding region of Tpit was found, whereas the new single nucleotide polymorphism p.S343G, in heterozygous state, was found in the same frequency in both PDH and control groups. We concluded that Tpit gain-of-function mutations are not involved in the etiology of PDH in Poodle dogs.(AU)

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O hiperadrenocorticismo ACTH-dependente (HACAD) apresenta elevada incidência em cães da raça Poodle, sendo que membros da mesma família têm sido acometidos pela doença, sugerindo envolvimento genético. Tpit é um fator de transcrição obrigatório para a expressão do gene da pro-opiomelanocortina e para a diferenciação terminal dos corticotrofos. O objetivo deste trabalho foi pesquisar mutações germinativas no gene Tpit em Poodles com HACAD. Cinquenta cães da raça Poodle, 33 fêmeas, média de idade de 8,71±2,8 anos, com HACAD, e 50 cães Poodles saudáveis, 32 fêmeas, média de idade de 9,4±2,8 anos, foram estudados. Mutações na região codificadora do gene Tpit não foram identificadas. Foi observado um novo polimorfismo em heterozigose, p.S343G, com a mesma frequência no grupo de cães com HACAD e no grupo-controle. Conclui-se que a mutação ativadora no gene Tpit não está envolvida na patogênese do hiperadrenocorticismo ACTH-dependente em cães da raça Poodle.(AU)

Asunto(s)

Animales , Perros , Mutación de Línea Germinal , Hiperfunción de las Glándulas Suprarrenales/veterinaria , Polimorfismo Genético , Corticotrofos , Proopiomelanocortina

RESUMEN

CONTEXT: Loss-of-function mutations of the kisspeptin-1 receptor gene, KISS1R, have been identified in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH). OBJECTIVE: To investigate KISS1R defects in patients with absent or delayed puberty. PATIENTS: We investigated KISS1R gene defects in a cohort of 99 Brazilian patients with nIHH or constitutional delay of puberty (CDP). METHODS: The entire coding region of KISS1R was amplified by PCR followed by automatic sequencing. In addition, screening for KISS1R exonic deletions was performed by multiplex ligation-dependent probe amplification. RESULTS: One novel homozygous KISS1R mutation was identified in two siblings with nIHH. This variant was an insertion/deletion (indel) mutation characterized by the deletion of three nucleotides (GCA) at position -2 to -4, and by the insertion of seven nucleotides (ACCGGCT) at the same position, within the 3' splice acceptor site of intron 2 of KISS1R. The brothers who carried this KISS1R mutation had no clinical evidence of pubertal development at the ages of 14 and 20 years. Computational analysis of this indel mutation predicted the generation of an abnormal protein. In addition, a new heterozygous KISS1R variant (p.E252Q) was identified in a male patient with sporadic nIHH. However, in vitro studies of this variant did not demonstrate functional impairment. Only known polymorphisms were identified in patients with CDP. CONCLUSION: Loss-of-function mutations of KISS1R represents a rare cause of nIHH, and was absent in patients with CDP. We have described a novel KISS1R homozygous splice acceptor site mutation in the familial form of nIHH.

Asunto(s)

Homocigoto , Hipogonadismo/genética , Sitios de Empalme de ARN/genética , Receptores Acoplados a Proteínas G/genética , Animales , Brasil , Células COS , Chlorocebus aethiops , Femenino , Humanos , Masculino , Mutación/genética , Pubertad Tardía/genética , Receptores de Kisspeptina-1 , Hermanos

RESUMEN

CONTEXT: Kisspeptin, encoded by the KISS1 gene, is a key stimulatory factor of GnRH secretion and puberty onset. Inactivating mutations of its receptor (KISS1R) cause isolated hypogonadotropic hypogonadism (IHH). A unique KISS1R-activating mutation was described in central precocious puberty (CPP). OBJECTIVE: Our objective was to investigate KISS1 mutations in patients with idiopathic CPP and normosmic IHH. PATIENTS: Eighty-three children with CPP (77 girls) and 61 patients with IHH (40 men) were studied. The control group consisted of 200 individuals with normal pubertal development. METHODS: The promoter region and the three exons of KISS1 were amplified and sequenced. Cells expressing KISS1R were stimulated with synthetic human wild-type or mutant kisspeptin-54 (kp54), and inositol phosphate accumulation was measured. In a second set of experiments, kp54 was preincubated in human serum before stimulation of the cells. RESULTS: Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in three unrelated children with idiopathic CPP. Both mutations were absent in 400 control alleles. The p.P74S mutation was identified in the heterozygous state in a boy who developed CPP at 1 yr of age. The p.H90D mutation was identified in the homozygous state in two unrelated girls with CPP. In vitro studies revealed that the capacity of the P74S and H90D mutants to stimulate IP production was similar to the wild type. After preincubation of wild-type and mutant kp54 in human serum, the capacity to stimulate signal transduction was significantly greater for P74S compared with the wild type, suggesting that the p.P74S variant is more stable. Only polymorphisms were found in the IHH group. CONCLUSION: Two KISS1 mutations were identified in unrelated patients with idiopathic CPP. The p.P74S variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype.

Asunto(s)

Hipogonadismo/genética , Pubertad Precoz/genética , Pubertad/genética , Receptores Acoplados a Proteínas G/genética , Proteínas Supresoras de Tumor/genética , Exones/genética , Femenino , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Lactante , Kisspeptinas , Masculino , Mutación , Pene/anomalías , Receptores de Kisspeptina-1

RESUMEN

Neonatal screening for congenital adrenal hyperplasia (CAH) is useful in diagnosing salt wasting form (SW). However, there are difficulties in interpreting positive results in asymptomatic newborns. The main objective is to analyze genotyping as a confirmatory test in children with neonatal positive results. Patients comprised 23 CAH children and 19 asymptomatic infants with persistently elevated 17-hydroxyprogesterone (17OHP) levels. CYP21A2 gene was sequenced and genotypes were grouped according to the enzymatic activity of the less severe allele: A1 null, A2 < 2%, B 3-7%, C > 20%. Twenty-one children with neonatal symptoms and/or 17OHP levels > 80 ng/ml carried A genotypes, except two virilized girls (17OHP < 50 ng/ml) without CAH genotypes. Patients carrying SW genotypes (A1, A2) and low serum sodium levels presented with neonatal 17OHP > 200 ng/ml. Three asymptomatic boys carried simple virilizing genotypes (A2 and B): in two, the symptoms began at 18 months; another two asymptomatic boys had nonclassical genotypes (C). The remaining 14 patients did not present CAH genotypes, and their 17OHP levels were normalized by 14 months of age. Molecular analysis is useful as a confirmatory test of CAH, mainly in boys. It can predict clinical course, identify false-positives and help distinguish between clinical forms of CAH.

Asunto(s)

Hiperplasia Suprarrenal Congénita/diagnóstico , Hiperplasia Suprarrenal Congénita/enzimología , Tamizaje Neonatal , Esteroide 21-Hidroxilasa/genética , 17-alfa-Hidroxiprogesterona/sangre , Hiperplasia Suprarrenal Congénita/sangre , Niño , Preescolar , Femenino , Estudios de Seguimiento , Heterocigoto , Humanos , Lactante , Recién Nacido , Masculino

RESUMEN

Ectopic ACTH secretion represents 8-18% of the cases of endogenous hypercortisolism. Pheochromocytomas correspond to 2-25% of the cases and surgery is the indicated treatment. We describe a case of ACTH-secreting pheochromocytoma treated with percutaneous ethanol injection (PEI) guided by computed tomography (CT). A 71-yr-old man presented with diabetes, severe hypokalemia, weight loss, muscle weakness, and hypertension. Hormonal evaluation revealed elevated levels of urinary cortisol, ACTH, catecholamines, and urinary metanephrines. There was no cortisol or ACTH response to desmopressin stimulation test. Magnetic resonance revealed bilateral adrenal nodules, larger on the left side. The suspected diagnosis was ectopic ACTH syndrome caused by pheochromocytoma. Ketoconazole treatment resulted in reduction of urinary cortisol levels but was followed by severe cholestasis and hepatic dysfunction, preventing surgery; it was substituted by octreotide with reduction of ACTH and cortisol levels, but without improvement of cholestasis. The patient presented cachexia and developed multiple pulmonary abscesses that also prevented surgical treatment, thus he was treated with percutaneous ethanol injection guided by CT of the left adrenal tumor. During the procedure, the patient had an increase in blood pressure controlled by the infusion of sodium nitroprusside followed by hypotension that required infusion of dopamine and volume expansion. Afterwards, he presented hormonal normalization, normal catecholamines levels, and clinical improvement. Histological tissue analysis confirmed pheochromocytoma. We concluded that CT-guided PEI represents an efficient alternative therapy to ectopic ACTH-secreting pheochromocytomas in patients without clinical conditions for surgery.

Asunto(s)

Síndrome de ACTH Ectópico/tratamiento farmacológico , Síndrome de ACTH Ectópico/etiología , Neoplasias de las Glándulas Suprarrenales/complicaciones , Etanol/uso terapéutico , Feocromocitoma/complicaciones , Solventes/uso terapéutico , Síndrome de ACTH Ectópico/diagnóstico , Hormona Adrenocorticotrópica/orina , Anciano , Catecolaminas/orina , Humanos , Hidrocortisona/orina , Inyecciones Subcutáneas , Masculino , Metanefrina/orina , Tomografía Computarizada por Rayos X

RESUMEN

INTRODUCTION AND HYPOTHESIS: Androgen insensitivity syndrome (AIS) constitutes a natural model to study effects of androgens and estrogens on growth and bone density. We evaluated height and bone density in patients with AIS with mutations in the androgen receptor (AR) gene. METHODS: A retrospective analysis was conducted of eight subjects with complete AIS (CAIS) and four with partial AIS (PAIS) submitted to gonadectomy followed by estrogen replacement, and three with PAIS who did not undergo gonadectomy. Standing height and bone mineral apparent density (BMAD) by DXA were measured and compared with male (z (m)) and female (z (f)) reference populations. The z-scores were compared with a value of zero using the one-sample t-test. RESULTS: Final heights of patients with CAIS and PAIS were intermediate between those predicted for females and males. BMAD of the lumbar spine in CAIS and PAIS after gonadectomy and estrogen replacement (z (f) = - 1.56 +/- 1.04, P = 0.006, and z (m) = - 0.75 +/- 0.89, P = 0.04) indicated vertebral bone deficit, whereas BMAD at the femoral neck was normal. No patient reported fractures. CONCLUSION: Subjects with AIS had mean final height intermediate between mean normal male and female, and decreased bone mineral density in the lumbar spine. These data suggest an important role for androgens in normal male growth and bone density not replaced by estrogens.

Asunto(s)

Síndrome de Resistencia Androgénica/fisiopatología , Estatura/genética , Densidad Ósea/genética , Mutación , Receptores Androgénicos/genética , Absorciometría de Fotón , Adolescente , Adulto , Síndrome de Resistencia Androgénica/genética , Síndrome de Resistencia Androgénica/cirugía , Castración , Terapia de Reemplazo de Estrógeno , Femenino , Cuello Femoral/fisiopatología , Humanos , Vértebras Lumbares/fisiopatología , Masculino , Receptores Androgénicos/fisiología , Estudios Retrospectivos

RESUMEN

OBJECTIVE: PROP1 mutations are the most common cause of genetic combined pituitary hormone deficiency (CPHD). The aim of this study was to investigate the PROP1 gene in two siblings with CPHD. DESIGN: Pituitary function and imaging assessment and molecular analysis of PROP1. PATIENTS: Two siblings, born to consanguineous parents, presented with GH deficiency associated with other pituitary hormone deficiencies (TSH, PRL and gonadotrophins). The male sibling also had an evolving cortisol deficiency. METHODS: Pituitary size was evaluated by magnetic resonance imaging (MRI). PROP1 gene analysis was performed by polymerase chain reaction (PCR), automatic sequencing and Southern blotting. Amplification of sequence tag sites (STS) and the Q8N6H0 gene flanking PROP1 were performed to define the extension of PROP1 deletion. RESULTS: MRI revealed a hypoplastic anterior pituitary in the girl at 14 years and pituitary enlargement in the boy at 18 years. The PROP1 gene failed to amplify in both siblings, whereas other genes were amplified. Southern blotting analysis revealed the PROP1 band in the controls and confirmed complete PROP1 deletion in both siblings. The extension of the deletion was 18.4 kb. The region flanking PROP1 contains several Alu core sequences that might have facilitated stem-loop-mediated excision of PROP1. CONCLUSIONS: We report here a complete deletion of PROP1 in two siblings with CPHD phenotype.

Asunto(s)

Enanismo Hipofisario/genética , Proteínas de Homeodominio/genética , Hipopituitarismo/genética , Adolescente , Southern Blotting , Consanguinidad , Enanismo Hipofisario/patología , Femenino , Eliminación de Gen , Homocigoto , Humanos , Hipopituitarismo/patología , Masculino , Adenohipófisis/patología , Hermanos

RESUMEN

The R337H TP53 mutation is a low-penetrance molecular defect that predisposes to adrenocortical tumour (ACT) formation in Brazilian and possibly other populations. Additional genetic defects may be responsible for the variable expression of ACTs in these cases. The inhibin alpha-subunit gene (INHA) on 2q33-qter has been implicated in mouse adrenocortical tumourigenesis. We studied 46 pediatric patients with ACTs from Brazil for INHA genetic alterations; 39 of these patients were heterozygous carriers of the R337H TP53 mutation. We first mapped the INHA gene by radiation hybrid analysis and determined 10 linked microsatellite markers in an area flanked by D2S1371 and D2S206 on 2q33-qter. These markers were then used for loss of heterozygozity (LOH) studies in nine paired germline and tumour DNA samples. Mapping placed the INHA gene in close proximity to D2S2848 (SHGC11864) with a log of odds (LOD) score of 5.84. LOH for at least one marker in the region was identified in 8/9 tumours (89%). Six patients were heterozygous for three INHA mutations: one in exon 1, 127C>G, and two in exon 2, 3998G>A and 4088G>A, all leading to amino acid substitutions (P43A, G227R, and A257T, respectively). A257T is located in a conserved INHA region, highly homologous to transforming growth factor-beta; both G227R and A257T change polarity, and, in addition, G227R changes the pH. We conclude that these sequence alterations and the detected 2q allelic changes suggest that INHA may be one of the contributing factors needed for ACT formation in pediatric patient carriers of the R337H TP53 mutation.

Asunto(s)

Neoplasias de la Corteza Suprarrenal/genética , Genes p53 , Inhibinas/genética , Mutación , Sustitución de Aminoácidos , Niño , Mapeo Cromosómico , Análisis Mutacional de ADN , Heterocigoto , Humanos , Pérdida de Heterocigocidad