RESUMO
PURPOSE OF REVIEW: Multicentric carpotarsal osteolysis (MCTO) is an ultra-rare disorder characterized by osteolysis of the carpal and tarsal bones, subtle craniofacial deformities, and nephropathy. The molecular pathways underlying the pathophysiology are not well understood. RECENT FINDINGS: MCTO is caused by heterozygous mutations in MAFB, which encodes the widely expressed transcription factor MafB. All MAFB mutations in patients with MCTO result in replacement of amino acids that cluster in a phosphorylation region of the MafB transactivation domain and account for a presumed gain-of-function for the variant protein. Since 2012, fewer than 60 patients with MCTO have been described with 20 missense mutations in MAFB. The clinical presentations are variable, and a genotype-phenotype correlation is lacking. Osteolysis, via excessive osteoclast activity, has been regarded as the primary mechanism, although anti-resorptive agents demonstrate little therapeutic benefit. This paper appraises current perspectives of MafB protein action, inflammation, and dysfunctional bone formation on the pathogenesis of the skeletal phenotype in MCTO. More research is needed to understand the pathogenesis of MCTO to develop rational therapies.
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Ossos do Carpo , Osteólise , Humanos , Osteólise/genética , Mutação , Mutação de Sentido Incorreto , Ossos do Carpo/patologia , FenótipoRESUMO
This case describes a child with blindness, recurrent low-impact fractures, low bone mass, and intermittent joint pain who was found to have a novel missense mutation in COL11A1, consistent with Stickler syndrome type II. The case illustrates the phenotypic variability of the syndrome, which may include increased fragility in childhood. INTRODUCTION: Stickler syndrome type II is an autosomal dominant disorder caused by mutations in the gene that encodes the type XI collagen chain α1 (COL11A1). Manifestations include craniofacial dysmorphology and ocular abnormalities that may lead to blindness, hearing loss, and skeletal anomalies that range from joint pain and arthritis to scoliosis and hypermobility. METHODS: Herein, we describe a child who carried the presumed diagnosis of osteoporosis-pseudoglioma syndrome because of the combined findings of recurrent low-impact fractures due to low bone mass and blindness. The child also suffered from joint pain but had no facial dysmorphism or hearing loss. RESULTS: Targeted sequencing and deletion analysis of the LRP5, COL1A1, and COL1A2 genes failed to identify any mutations, and whole exome sequence analysis revealed a novel missense mutation (c.3032C>A:p.P1011Q) in COL11A1, consistent with Stickler type II. CONCLUSION: This case highlights the phenotypic variability of Stickler type II, broadens the list of differential diagnosis of increased bone fragility in childhood, and highlights utility of unbiased genetic testing towards establishing the correct diagnosis in children with frequent fractures.
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Colágeno Tipo XI/deficiência , Doenças do Tecido Conjuntivo/genética , Mutação de Sentido Incorreto , Fraturas por Osteoporose/genética , Descolamento do Vítreo/genética , Criança , Colágeno Tipo XI/genética , Doenças do Tecido Conjuntivo/complicações , Humanos , Masculino , Fraturas por Osteoporose/etiologia , Linhagem , Recidiva , Descolamento do Vítreo/complicaçõesRESUMO
For decades, the internal medicine (IM) subinternship has served as a critical interface between undergraduate and graduate medical education. As such, the vast majority of U.S. medical schools offer this rotation to help students prepare for post-graduate training. Historically an experiential rotation, a formal curriculum with specific learning objectives was eventually developed for this course in 2002. Since then, graduate medical education (GME) has changed significantly with the regulation of duty hours, adoption of competency-based education, and development of training milestones and entrustable professional activities. In response to these and many other changes to residency training and medical practice, in 2010, the Association of Program Directors in Internal Medicine (APDIM) surveyed its members-with input from the Clerkship Directors in Internal Medicine (CDIM) Subinternship Task Force-to determine which core skills program directors expected from new medical school graduates. The results of that survey helped to inform a joint CDIM-APDIM committee's decision to re-evaluate the goals of the IM subinternship in an effort to enhance the transition from medical school to residency. This joint committee defined the minimum expectations of what constitutes an IM subinternship rotation, proposed recommended skills for IM subinterns, and discussed challenges and future directions for this crucial course.
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Competência Clínica/normas , Currículo , Educação de Graduação em Medicina/normas , Medicina Interna/educação , Internato e Residência , Educação Baseada em Competências , Educação de Pós-Graduação em Medicina , Humanos , Avaliação das Necessidades , Inquéritos e Questionários , Estados UnidosRESUMO
BACKGROUND: Natural health products (NHP) are increasingly being used by patients concomitantly receiving prescription drugs, which can result in potentially serious drug-herb interactions. This is particularly true for St. John's wort. OBJECTIVE: This study was conducted to evaluate pharmacists and natural health product retailers' knowledge on this topic. METHODS: An interviewer approached 24 pharmacists and 6 natural health product retailers to obtain information regarding St. John's wort. If asked, the interviewer indicated that they were currently using cyclosporine to treat a problem of proteinuria. The response of the pharmacist or NHP retailer to a series of questions was recorded after the encounter. RESULTS: 90% of respondents indicated that St. John's wort was useful for treating depression. Two-thirds of the respondents required prompting by the interviewer before providing any comments pertaining to safety. 60% of the respondents inquired about concurrent medications and 40% made statements regarding potential drug-herb interactions. CONCLUSION: Despite the potential for a serious drug-herb interaction involving St. John's wort and cyclosporine, less than half of the pharmacists and natural health product retailers that were encountered in the study addressed this topic with the prospective patient/client. Individuals selling natural health products need to communicate more information to their patients/clients regarding potential drug-herb interactions.
Assuntos
Conhecimentos, Atitudes e Prática em Saúde , Interações Ervas-Drogas , Hypericum/química , Farmacêuticos/normas , Comércio , Ciclosporina/efeitos adversos , Coleta de Dados , Humanos , Hypericum/efeitos adversos , Educação de Pacientes como Assunto/normas , Fitoterapia/efeitos adversosRESUMO
BACKGROUND: This trial investigated the efficacy and safety of weekly cetuximab combined with two different schedules of paclitaxel/carboplatin for stage IIIB/IV non-small-cell lung cancer (NSCLC). METHODS: A total of 168 patients with previously untreated stage IIIB/IV NSCLC were randomized to arm A, cetuximab (400 mg/m(2) day 1 followed by weekly 250 mg/m(2)) + paclitaxel (Taxol) (225 mg/m(2))/carboplatin (AUC6) day 1 every 3 weeks or arm B, same cetuximab regimen plus paclitaxel (100 mg/m(2)) days 1, 8, and 15 every 3 weeks and carboplatin (AUC6) day 1 every 4 weeks. Treatment continued for a four-cycle maximum. Patients with a complete response, partial response, or stable disease after four cycles could receive cetuximab 250 mg/m(2)/week until disease progression or unacceptable toxicity. The primary end point was to evaluate progression-free survival (PFS). RESULTS: Median PFS was 4.7 and 4.3 months for arms A and B, respectively (6-month PFS, 27.3% versus 30.9%). Median overall survival was 11.4 versus 9.8 months for arms A and B, respectively; estimated 1-year survival, 47.7% versus 39.3%; and objective response rate, 29.6% versus 25%. The regimen was well tolerated with rash and hematologic toxicity being most common. CONCLUSIONS: This study did not meet the prespecified benchmark of 35% 6-month PFS rate; both combination schedules of cetuximab plus paclitaxel/carboplatin were feasible and equivalent for treating advanced NSCLC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Carboplatina/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/patologia , Cetuximab , Relação Dose-Resposta a Droga , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Paclitaxel/administração & dosagemRESUMO
AIMS: Monitoring treated patients with thyroid cancer for recurrent or metastatic disease is currently based upon the serial measurement of circulating plasma thyroglobulin (Tg) concentrations. However, the clinical usefulness of Tg immunoassays is limited by poor sensitivity and interference from anti-Tg antibodies. This study investigated whether the detection of Tg mRNA in peripheral blood, using reverse transcriptase polymerase chain reaction (RT-PCR), is of value in the biochemical surveillance of patients with thyroid cancer. METHODS: RNA was extracted from peripheral blood of five normal controls, six patients with abnormal thyroid function tests, and 28 patients who had undergone thyroidectomy for well differentiated thyroid cancer. From each, an 87 bp product from base pair 262 to 348 in the cDNA sequence of the thyroglobulin gene was amplified by RT-PCR. RESULTS: Tg mRNA was detected in normal individuals and patients with thyroid cancer. In the group of patients studied, identification of metastatic thyroid tissue by radioiodine scanning correlated better with Tg mRNA assay results than with serum Tg concentrations (accuracy 84% v 75%). No interference from circulating Tg antibodies was apparent. In patients studied prospectively over a 12 month period, there was a significant correlation between detectable Tg mRNA in peripheral blood and the presence or absence of metastatic disease, as demonstrated by radioiodine scanning. CONCLUSIONS: These results suggest that detection of Tg mRNA in blood is a more sensitive marker for metastatic thyroid disease than Tg immunoassay, and appears to be unaffected by the presence of circulating anti-Tg antibodies.
Assuntos
Biomarcadores Tumorais/sangue , RNA Mensageiro/sangue , RNA Neoplásico/sangue , Tireoglobulina/genética , Neoplasias da Glândula Tireoide/sangue , Adenocarcinoma Folicular/diagnóstico , Adenocarcinoma Folicular/secundário , Adenocarcinoma Papilar/diagnóstico , Adenocarcinoma Papilar/secundário , Autoanticorpos/sangue , Progressão da Doença , Seguimentos , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tireoglobulina/imunologiaRESUMO
The aim of this study was to provide confirmation that once-weekly dosing with 70 mg of alendronate (seven times the daily oral dose) and twice-weekly dosing with 35 mg is equivalent to the 10-mg once-daily regimen and to gain more extensive safety experience with this new dosing regimen. Twelve hundred fifty-eight postmenopausal women (aged 42-95 years) with osteoporosis (bone mineral density [BMD] of either lumbar spine or femoral neck at least 2.5 SDs below peak young adult mean or prior vertebral or hip fracture) were assigned to receive oral once-weekly alendronate, 70 mg (n = 519); twice-weekly alendronate, 35 mg (n = 369); or daily alendronate 10 mg (n = 370) for a total of 2 years of double-blind experience. Mean BMD increases from baseline (95% CI) at 24 months in the once-weekly, twice-weekly, and daily treatment groups, respectively, were 6.8% (6.4, 7.3), 7.0% (6.6,7.5), and 7.4% (6.9,7.8) at the lumbar spine and 4.1% (3.8,4.5), 4.3% (3.9,4.7), and 4.3% (3.9,4.7) at the total hip. These increases in BMD as well as the BMD increases at the femoral neck, trochanter, and total body and the reductions of biochemical markers of bone resorption (urinary cross-linked N-telopeptides of type I collagen [NTx]) and bone formation (serum bone-specific alkaline phosphatase [BSAP]) were similar for the three dosing regimens. All treatment regimens were well tolerated with a similar incidence of upper gastrointestinal (GI) adverse experiences. The incidence rates of clinical fractures, captured as adverse experiences, were similar among the groups. The 2-year results confirm the conclusion reached after 1 year that once-weekly alendronate is therapeutically equivalent to daily dosing, providing patients with a more convenient dosing option that may potentially enhance adherence to therapy.
Assuntos
Alendronato/administração & dosagem , Osteoporose Pós-Menopausa/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Alendronato/efeitos adversos , Fosfatase Alcalina/sangue , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea , Colágeno/urina , Colágeno Tipo I , Método Duplo-Cego , Esquema de Medicação , Feminino , Fraturas Ósseas/etiologia , Gastroenteropatias/induzido quimicamente , Humanos , Vértebras Lombares/efeitos dos fármacos , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/complicações , Peptídeos/urina , Resultado do TratamentoRESUMO
BACKGROUND: In clinical practice, warfarin is underused for the prevention of stroke in individuals with atrial fibrillation despite unequivocal evidence of benefit and acceptable safety. OBJECTIVE: To ascertain, from primary care physicians, their beliefs and preferences regarding the use of warfarin in patients with atrial fibrillation. MATERIALS AND METHODS: A questionnaire was mailed to a random sample of 1000 primary care physicians in Ontario. Physician prescribing preferences from among treatment options available (warfarin, acetylsalicylic acid, ticlopidine, no therapy and other) were recorded for four separate scenarios of atrial fibrillation with varying degrees of risk for stroke. Physician perception of the risks associated with warfarin use and their awareness of the evidence of benefit were assessed. RESULTS: Three hundred twenty-four physicians returned completed questionnaires. Among the four scenarios, physicians choosing not to use warfarin were three to six times more likely than physicians choosing to use warfarin to believe that there was inadequate evidence of benefit of warfarin for stroke prophylaxis, and they were four to six times more likely to be concerned about the risks of hemorrhage. These beliefs did not change significantly with scenarios describing patients with a high risk of stroke. CONCLUSIONS: Physician reluctance to use warfarin is associated with a false understanding of the benefit to risk ratio, which arises from a low appreciation of therapeutic benefits and a high concern regarding hemorrhage.
Assuntos
Anticoagulantes/uso terapêutico , Fibrilação Atrial/tratamento farmacológico , Transtornos Cerebrovasculares/prevenção & controle , Padrões de Prática Médica , Varfarina/uso terapêutico , Fibrilação Atrial/complicações , Transtornos Cerebrovasculares/etiologia , Coleta de Dados , Tomada de Decisões , Uso de Medicamentos/estatística & dados numéricos , Hemorragia/induzido quimicamente , Humanos , Ontário , Médicos de Família , Fatores de Risco , Inquéritos e QuestionáriosRESUMO
Hypoparathyroidism is characterized by hypocalcemia, hyperphosphatemia, and absent or markedly reduced circulating concentrations of parathyroid hormone. The transcription factor GCMB is predominantly, if not exclusively, expressed in parathyroid cells and is critical for development of the parathyroid glands in mice. Thus, in the present study we examined the GCMB gene, mapped to 6p23-24, as a candidate for isolated hypoparathyroidism. We defined the boundaries of the five exons of the human GCMB gene and then identified a large intragenic mutation in the GCMB genes of the proband of an extensive kindred with isolated hypoparathyroidism. Her parents and several other unaffected relatives were heterozygous for the mutation. Despite an absence of any history of consanguinity, microsatellite analysis showed shared genotypes that flanked the GCMB gene over a span of 5 cM, suggesting that both of the proband's GCMB alleles had been derived from a single common ancestor. Analysis of additional, unrelated cases did not disclose the same mutation. We conclude that homozygous loss of function of the GCMB gene impairs normal parathyroid gland embryology and is responsible for isolated hypoparathyroidism in a subset of patients with this disease.
Assuntos
Hipoparatireoidismo/genética , Mutação , Neuropeptídeos/genética , Fatores de Transcrição/genética , Alelos , Animais , Sequência de Bases , Pré-Escolar , Primers do DNA/genética , Éxons , Feminino , Humanos , Íntrons , Masculino , Camundongos , Proteínas Nucleares , Linhagem , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
GH-releasing peptides (GHRPs) are synthetic peptides that bind to specific receptors and thereby stimulate the secretion of pituitary GH. In vivo it is uncertain whether these peptides act directly on somatotroph cells or indirectly via release of GHRH from the hypothalamus. In this study we compared the pituitary hormone response to GHRP-2 in 11 individuals with isolated GH deficiency (GHD) due to a homozygous mutation of the GHRH receptor (GHRH-R) gene and in 8 normal unrelated controls. Basal serum GH levels were lower in the GHD group compared with controls [0.11 +/- 0.11 (range, <0.04 to 0.38) vs. 0.59 +/- 0.76 microg/L (range, 0.04-2.12 microg/L); P = 0.052]. After GHRP-2 administration there was a 4.5-fold increase in serum GH relative to baseline values in the GHD group (0.49 +/- 0.41 vs. 0.11 +/- 0.11 microg/L; P = 0.002), which was significantly less than the 79-fold increase in the control group (46.8 +/- 17.6 vs. 0.59 +/- 0.76 microg/L; P = 0.008). Basal and post-GHRP-2 serum levels of ACTH, cortisol, and PRL were similar in both groups. Basal levels of serum TSH were significantly higher in the GHD group than in the control group (3.23 +/- 2.21 vs. 1.37 +/- 0.34 microIU/mL; P = 0.003). TSH levels in both groups did not change after GHRP-2 administration. These results suggest that an intact GHRH signaling system is not an absolute requirement for GHRP-2 action on GH secretion and that GHRP-2 has a GHRH-independent effect on pituitary somatotroph cells.
Assuntos
Hormônio do Crescimento Humano/deficiência , Hormônio do Crescimento Humano/metabolismo , Mutação , Oligopeptídeos/farmacologia , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Hormônio Adrenocorticotrópico/sangue , Hormônios/farmacologia , Hidrocortisona/sangue , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Prolactina/sangue , Tireotropina/sangueRESUMO
OBJECTIVE: Mutations in the GH releasing hormone receptor (GHRH-R) have recently been shown to cause autosomal recessive isolated GH deficiency (IGHD). Patients who are homozygous for GHRH-R mutations have a subnormal GH response to pharmacological agents that stimulate GH secretion and an appropriate response to exogenous GH therapy. We searched for mutations in the GHRH-R gene in a family in which two of three siblings were affected by IGHD. DESIGN: We sequenced the 13 coding exons, the intron-exon boundaries and 327 bases of the promoter of the GHRH-R gene from peripheral blood cell genomic DNA of an index patient. RESULTS: Both affected individuals were compound heterozygotes for two previously undescribed GHRH-R mutations: a change in codon 137 that replaces histidine with leucine (H137L), and a 5 bp deletion in exon 11 (Del 1140-1144). The patients' father was heterozygous for the H137L mutation, and the mother was heterozygous for the exon 11 deletion. We used site-directed mutagenesis to create the mutants in wild-type GHRH-R cDNA. Transient transfection of GHRH-R cDNAs in Chinese hamster ovary cells showed that cells transfected with both mutant receptors failed to increase cyclic AMP after treatment with GHRH. CONCLUSIONS: We describe a family in which two siblings with IGHD were compound heterozygotes for two new mutations in the GHRH-R gene. These results suggest that mutant alleles for GHRH-R gene may be more common than previously suspected.
Assuntos
Hormônio do Crescimento/deficiência , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Animais , Células CHO , Criança , Pré-Escolar , Cricetinae , AMP Cíclico/metabolismo , Análise Mutacional de DNA , Feminino , Deleção de Genes , Hormônio Liberador de Hormônio do Crescimento , Heterozigoto , Humanos , Masculino , Mutação , TransfecçãoRESUMO
Oncogenic osteomalacia (OOM), X-linked hypophosphatemia (XLH), and autosomal dominant hypophosphatemic rickets (ADHR) are phenotypically similar disorders characterized by hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum calcitriol concentrations, normal serum concentrations of calcium and parathyroid hormone, and defective skeletal mineralization. XLH results from mutations in the PHEX gene, encoding a membrane-bound endopeptidase, whereas ADHR is associated with mutations of the gene encoding FGF-23. Recent evidence that FGF-23 is expressed in mesenchymal tumors associated with OOM suggests that FGF-23 is responsible for the phosphaturic activity previously termed "phosphatonin." Here we show that both wild-type FGF-23 and the ADHR mutant, FGF-23(R179Q), inhibit phosphate uptake in renal epithelial cells. We further show that the endopeptidase, PHEX, degrades native FGF-23 but not the mutant form. Our results suggest that FGF-23 is involved in the pathogenesis of these three hypophosphatemic disorders and directly link PHEX and FGF-23 within the same biochemical pathway.
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Endopeptidases/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Túbulos Renais/metabolismo , Mesenquimoma/genética , Mutação , Osteomalacia/genética , Fosfatos/metabolismo , Proteínas/metabolismo , Transcrição Gênica , Substituição de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Rim , Túbulos Renais/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Gambás , Endopeptidase Neutra Reguladora de Fosfato PHEX , Proteínas Recombinantes/farmacologia , Especificidade por Substrato , Transfecção , Células Tumorais CultivadasRESUMO
Paget's disease of bone (PDB) is a common disorder characterized by focal areas of increased and disorganized osteoclastic bone resorption, leading to bone pain, deformity, pathological fracture, and an increased risk of osteosarcoma. Genetic factors play an important role in the pathogenesis of Paget's disease. In some families, the disease has been found to be linked to a susceptibility locus on chromosome 18q21-22, which also contains the gene responsible for familial expansile osteolysis (FEO)--a rare bone dysplasia with many similarities to Paget's disease. Insertion mutations of the TNFRSF11A gene encoding Receptor Activator of NF kappa B (RANK) have recently been found to be responsible for FEO and rare cases of early onset familial Paget's disease. Loss of heterozygosity (LOH) affecting the PDB/FEO critical region has also been described in osteosarcomas suggesting that TNFRSF11A might also be involved in the development of osteosarcoma. In order to investigate the possible role of TNFRSF11A in the pathogenesis of Paget's disease and osteosarcoma, we conducted mutation screening of the TNFRSF11A gene in patients with familial and sporadic Paget's disease as well as DNA extracted from Pagetic bone lesions, an osteosarcoma arising in Pagetic bone and six osteosarcoma cell lines. No specific abnormalities of the TNFRSF11A gene were identified in a Pagetic osteosarcoma, the osteosarcoma cell lines, DNA extracted from Pagetic bone lesions, or DNA extracted from peripheral blood in patients with familial or sporadic Paget's disease including several individuals with early onset Paget's disease. These data indicate that TNFRSF11A mutations contribute neither to the vast majority of cases of sporadic or familial PDB, nor to the development of osteosarcoma.
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Neoplasias Ósseas/genética , Predisposição Genética para Doença , Glicoproteínas/genética , Osteíte Deformante/genética , Osteossarcoma/genética , Receptores Citoplasmáticos e Nucleares/genética , Adulto , DNA/análise , Análise Mutacional de DNA , Primers do DNA/química , Testes Genéticos , Humanos , Osteoprotegerina , Mutação Puntual , Reação em Cadeia da Polimerase , Receptores do Fator de Necrose TumoralRESUMO
OBJECTIVE: GH-releasing hormone (GHRH) is a potent stimulator of somatotroph cell proliferation and GH secretion. GHRH acts via binding to a G-protein coupled receptor (GPCR) (GHRH-R), that activates adenylyl cyclase (AC) and increases growth and function of somatotroph cells. Indeed, a subset (30--40%) of somatotrophic adenomas contain somatic mutations of the GNAS1 gene that encodes the alpha subunit of the G-protein (G(s)alpha) that stimulates AC. As activating mutations of other GPCRs cause development of endocrine tumours, we hypothesized that somatic activating mutations of the GHRH-R might provide the molecular basis for somatotroph cell proliferation in a subset of human GH-secreting pituitary adenomas. DESIGN: We analysed genomic DNA isolated from 26 somatotrophinomas, 17 of which lacked activating mutations in the GNAS1 gene. We individually amplified via polymerase chain reaction all 13 coding exons and the exon-intron boundaries of the GHRH-R gene. We used denaturing gradient gel electrophoresis to search for abnormalities in exons 1 through 11. Abnormally migrating bands were subjected to direct sequencing. Exons 12 and 13, encoding for the intracellular C-terminal domain, were subjected to direct sequencing. RESULTS: Mutations were not detected in any of the tumours, but a rare polymorphism in codon 225 corresponding to the third transmembrane domain (V225I) was discovered. CONCLUSIONS: GHRH-R mutations are absent or rare in somatotrophinomas, and other mechanisms must explain the somatotroph cell proliferation in the adenomas that lack activating mutations in the GNAS1 gene.
Assuntos
Adenoma/genética , DNA de Neoplasias/análise , Hormônio do Crescimento/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Hipofisárias/genética , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Adenoma/metabolismo , Animais , Células CHO , Cricetinae , Mutação em Linhagem Germinativa , Humanos , Mutagênese Sítio-Dirigida , Neoplasias Hipofisárias/metabolismoRESUMO
Isolated GH deficiency (IGHD) is familial in 5-30% of cases. The majority of patients have the type IB form, characterized by autosomal recessive transmission, low but measurable serum concentrations of GH, and responsiveness to exogenous GH therapy. Unique mutations in the gene encoding the GHRH receptor (GHRHR) have previously been described in 2 kindreds with IGHD IB. However, the prevalence of GHRHR mutations in patients with IGHD IB is unknown. We analyzed 30 families with IGHD IB in which more than 1 member was affected. Linkage analysis was performed in 28 of the families, and in 3 families sibling pair analysis indicated linkage to the GHRHR gene locus. These 3 families as well as 2 families in which linkage analysis was not performed were screened for mutations in the 13 coding exons, the intron-exon boundaries, and 327 bases of the promoter of the GHRHR gene. We identified novel GHRHR missense mutations in 2 of the 3 kindreds with informative linkage and in 1 family in which linkage had not been performed. In 1 family affected members were homozygous for a mutation in codon 144 that replaces leucine with histidine (L144H). Affected subjects in a second family were compound heterozygotes, carrying both the L144H mutation and a second mutation in codon 242 that replaces phenylalanine with cysteine. Affected subjects in a third family were homozygous for a mutation that replaces alanine at codon 222 with glutamic acid. All 3 mutations segregated with the IGHD phenotype. All 3 mutant receptors were expressed in CHO cells, and each failed to show a cAMP response after treatment of the cells with GHRH. These results demonstrate that missense mutations in the GHRHR gene are a cause of IGHD IB, and that defects in the GHRHR gene may be a more common cause of GH deficiency than previously suspected.
Assuntos
Hormônio do Crescimento Humano/deficiência , Mutação/genética , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Adolescente , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Células CHO , Pré-Escolar , Cricetinae , Humanos , Dados de Sequência Molecular , Linhagem , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismoRESUMO
Adipocyte beta-adrenergic sensitivity is compromised in animal models of obesity and type 2 diabetes. Although changes in the membrane concentrations of G-protein alpha subunits (Galpha) have been implicated, it remains to be determined how these changes are affected by insulin resistance in the different animal models. Because previous studies used young animals, we measured the concentrations of Galpha and Gbeta subunits in epididymal fat from aged (48 weeks old) db/db mice and from their lean littermates to more closely reproduce the model of type 2 diabetes mellitus. Levels of immunoreactive Galphas, Galphai(1/2), Galphao and Galphaq/11 were all significantly greater in adipocyte membranes from the db/db mice than in membranes from their lean non-diabetic littermate controls. Levels of Galphai(1) and Galphai(2) were also individually determined and although they appeared to be slightly higher in db/db membranes, these differences were not significant. Although the levels of both Galphas isoforms were elevated, levels of the 42 and 46 kDa proteins rose by approximately 42% and 20% respectively, indicating differential protein processing of Galphas. By contrast, levels of Galphai3 were similar in the two groups. The levels of common Gbeta and Gbeta2 were also elevated in db/db mice, whereas Gbeta1 and Gbeta4 levels were not different. To determine whether these changes were due to insulin resistance per se or to elevated glucocorticoid production, G-protein subunit levels were quantified in whole cell lysates from 3T3-L1 adipocytes that were stimulated with different concentrations of either insulin or corticosterone. Although none of the subunit levels was affected by insulin, the levels of both Galphas isoforms were increased equally by corticosterone in a concentration-dependent manner. Since glucocorticoids are known regulators of Galphas gene expression in many cell types and in adipocytes from diabetic rodents, the results presented herein appear to more accurately reflect diabetic pathophysiology than do those of previous studies which report a decrease in Galphas levels. Taken together, these results indicate that most of the selective changes in G-protein subunit production in adipocytes from this animal model of type 2 diabetes may not be due to diminished insulin sensitivity, but may be due to other endocrine or metabolic abnormalities associated with the diabetic phenotype.