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Primary Hyperparathyroidism (PHPT) often leads to bone loss, even in its asymptomatic presentations. Trabecular Bone Score (TBS) is a method to assess the trabecular bone structure of the spine. This study aimed to evaluate TBS measurements combined with Dual X-ray Absorptiometry (DXA) values in the search for more accurate bone fragility risk assessment among PHPT patients. From 2017 to 2019, patients diagnosed with PHPT (n = 64), before surgery, were invited to participate in this study. Bone mineral density (BMD) by DXA at the lumbar spine, total hip, femoral neck, distal third radius, and TBS were determined in patients and controls (n = 63). The vertebral fracture was defined using the Genant method in vertebral images by DXA and vertebral fracture assessment (VFA). Patients and controls did not differ in age, sex, menopausal status, or body mass index (BMI). The PHPT patients presented significantly lower BMD values than the controls in all sites evaluated. The TBS measurements were also statistically lower in PHPT patients than controls (mean TBS PHPT = 1.233 vs TBS controls = 1.280, p = 0.044). Osteoporosis was observed in 50% of PHPT patients and 26.6% of controls (p = 0.02). However, lumbar spine T-Score-2.5 was observed only in 21.8% of PHPT patients. Vertebral fractures were detected in nine individuals (14%) from the PHPT group and four (6.3%) in the controls (p = 0.24). The TBS area under the curve (AUC) was higher than DXA AUC in all sites, for vertebral fracture assessment. The TBS AUC was significant in the PHPT group (0.75, 95% CI 0.62 - 0.88, p = 0.02) and not significant in the DXA analysis. The ROC curve showed that TBS values1.187 are associated with a significantly higher risk of vertebral fracture among PHPT patients (p = 0.02). The TBS used as a complement to DXA measurements is a useful tool which may better assess fragility risk among PHPT patients.

Background Adrenal hypoplasia congenita (AHC) is an X-linked disorder that affects the adrenal cortex and hypothalamus-pituitary-gonadal axis (HPG), leading to primary adrenocortical insufficiency (PAI) and hypogonadotropic hypogonadism. AHC is caused by a mutation in the DAX-1 gene (NR0B1). More commonly, this disease is characterized by early-onset PAI, with symptoms in the first months of life. However, a less severe phenotype termed late-onset AHC has been described, as PAI signs and symptoms may begin in adolescence and adulthood. Here we describe a family report of a novel mutation within NR0B1 gene and variable reproductive phenotypes, including spontaneous fertility, in a very late-onset X-linked AHC kindred. Case presentation Three affected maternal male relatives had confirmed PAI diagnosis between 30 y and at late 64 y. The X-linked pattern has made the endocrinology team to AHC suspicion. Regarding the HPG axis, all males presented a distinct degree of testosterone deficiency and fertility phenotypes, varying from a variable degree of hypogonadism, oligoasthenoteratozoospermia to spontaneous fertility. Interestingly, the other five maternal male relatives unexpectedly died during early adulthood, most likely due to undiagnosed PAI/adrenal crisis as the probable cause of their premature deaths. Sequencing analysis of the NR0B1 gene has shown a novel NR0B1 mutation (p.Tyr378Cys) that segregated in three AHC family members. Conclusions NR0B1 p.Tyr378Cys segregates in an AHC family with a variable degree of adrenal and gonadal phenotypes, and its hemizygous trait explains the disease in affected family members. We recommend that NR0B1 mutation carriers, even those that are allegedly asymptomatic, be carefully monitored while reinforcing education to prevent PAI and consider early sperm banking when spermatogenesis still viable.

SYNE1 gene mutations were identified as a cause of late-onset pure cerebellar syndrome. Non-cerebellar symptoms, including cognitive impairment, were already described in this condition. The aim of this study was to perform a detailed cognitive and psychiatric description of patients with SYNE1 gene mutations. We performed neuropsychological and psychiatric evaluations of six patients with SYNE1 ataxia and compared their performance with 18 normal controls paired for age and education level. SYNE1 ataxia patients present cognitive dysfunction, characterized by impairment in attention and processing speed domains. Otherwise, the psychiatric assessment reported low levels of overall behavioral symptoms with only some minor anxiety-related complaints. Although this is a small sample of patients, these results suggest that SYNE1 ataxia patients may represent a model to investigate effects of cerebellar degeneration in higher hierarchical cognitive functions. For further studies, abstract thinking impairment in schizophrenia may be related to dysfunction in cerebellum pathways.

Background: Vitamin D deficiency is common among PHP patients. While data are limited, some studies suggest that vitamin D deficiency may exacerbates skeletal disease in PHP. TBS is a software-based method for assessment of trabecular bone structure of the spine, based on analysis of pixels obtained in dual energy x-ray absorptiometry (DXA) images. The aim of this study was to evaluate TBS, vitamin D status, clinical and laboratorial measurements in a PHP group of patients in a search for a more accurate bone fragility test for risk assessment in this group of patients. Methods: From June/2017 to January/2019, patients who met the criteria for PHP diagnosis were included in this study. Control group was composed by age and sex-matched healthy individuals. Overall, 64 PHP and 63 controls were enrolled. Bone mineral density (BMD) measured by DXA (Hologic QDR 4500) at the lumbar spine, total hip, femoral neck, and TBS values (InSight™) were determined in both groups. Total and ionized calcium, PTH, 25-hydroxyvitamin D (25(OH)D), creatinine, alkaline phosphatase, P1NP and CTX were measured. None were in use of Vitamin D supplementation. Results: As expected, PHP patients had lower BMD values than controls in all sites (p Conclusion: Serum total calcium presents negative correlation and phosphorus a positive one with TBS in PHP patients. We also found a correlation between TBS and 25(OH)D, both in PHP and in controls. 25(OH)D

Background: PHP is a common disorder, and regardless of being an asymptomatic entity, often leads to bone loss and osteoporosis. TBS is a software-based assessment method of trabecular bone structure of the spine based on the analysis of pixels in dual energy x-ray absorptiometry (DXA) images. The aim of this study was to evaluate TBS measurements in combination with DXA values by searching for more accurate bone fragility risk assessment among PHP patients. Methods: From June/2017 to January/2019, all patients who met the criteria for PHP diagnosis prior to surgery were selected to take part in this study. Control group was composed by non-family related healthy volunteers. Bone mineral density (BMD) by DXA (DXA; Hologic QDR 4500) at the lumbar spine, total hip, femoral neck, distal third of the non-dominant radius and TBS values by InSight were determined in 64 PHP patients and 63 controls. Total and ionized calcium, PTH, 25-hydroxyvitamin D (25(OH)D), creatinine, alkaline phosphatase, P1NP and CTX were measured in all patients and control group. Results: Primary hyperparathyroidism patients presented significant lower BMD values in all evaluated sites compared to controls. TBS measurements were also statistically different among PHP patients and controls (mean TBS PHP=1233 vs TBS controls= 1280, p=0.044). Patients and controls didn’t differ in age, sex, menopausal state or BMI (body mass index). Osteoporosis diagnosis by DXA analysis was observed in 50% of PHP patients and in 26.6% of controls. Self-reported or image-diagnosed fracture occurred in 15 PHP (23.4%) patients and in 11 (17.5%) controls. Among these fractures, vertebral fracture account for 9 (14%) in PHP group and 4 (6.3%) in controls. ROC curve analysis showed that the TBS value < 1187 is associated to significant high risk of vertebral fracture (sensibility 87.5%, specificity 67.3%) among PHP patients. Conclusion: TBS, used as a complement to DXA measurements, is a useful tool to better assess fragility risk among PHP patients.

Resistance to thyroid hormone (RTH), characterized by persistent hyperthyroxinemia with non-suppressed thyrotropin (TSH), is mostly caused by mutations in thyroid hormone receptor beta gene (THRB). Two differential diagnoses should be considered due to similar clinical and laboratory findings: TSH-producing pituitary adenoma (TPA) and Familial Dysalbuminemic Hyperthyroxinemia (FDH). The aim of this study is to describe our single tertiary center experience in the molecular diagnosis of RTH in Brazilian patients, analyzing their clinical and laboratory characteristics and the most common differential diagnosis. We enrolled 30 subjects with clinical and laboratory features of RTH. Patient´s evaluations included clinical examination, thyroid hormone profile and imaging tests. Sequencing analysis for THRB hot spot region was conducted on all patients, and those without mutations in beta isoform of the thyroid hormone receptor (TRβ) (non-TR-RTH) were investigated for albumin gene (ALB) mutation. Seventeen patients presented mutations in TRβ (RTHβ); six were non-TR-RTH, three had a diagnosis of FDH with a mutation in ALB, and four were diagnosed with TPA. Two characteristics were different to what is commonly described in the literature: higher serum TSH levels in RTHβ patients when compared to the non-TR-RTH group, but this difference did not extend to free T4 (FT4) level; also the percentage of non-TR-RTH was higher than what was reported in other series. In the present series, most cases were RTHβ with higher levels of TSH. We described three novel mutations in THRB (p.M313V, p.R320G and p.R438P) and the first patients with FDH molecular diagnosis (p.R242H) documented in Brazil.

Background: Low levels of vitamin D 25OHD are frequently described in PHP patients. The aim of this study was to evaluate bone parameters and vitamin D status in PHP patients and controls. Methods: Prior to surgery, 64 PHP patients and 63 healthy matched control subjects regarding age, gender and body mass index were enrolled in this study along 18 months. 25OHD and PTH were measured using Roche® Immunoassays. Bone mineral density (BMD) by dual X-ray absorptiometry (DXA) (Hologic QDR 4500) and TBS (InSight™) were determined in all patients and controls. Distribution of total, bioavailable and free (calculated) 25OH and its correlation with TBS and DXA in both groups was evaluated. DBP (vitamin D binding protein) SNPs genetic analysis was performed by ABI 7500 real time PCR System. None of the patients and controls were taking vitamin D supplements before the study. Results: PHP patients had lower BMD values than controls in all sites (p

ObjectivesAbout one-quarter of patients with medullary thyroid cancer (MTC) have inherited disease due to mutations in theRETgene. A rare mutation in exon 8 (G533C) ofRET, previously described in a large Brazilian family with MEN2A, also appeared to be clustering in Greece, whereas it was rarely reported in other ethnic groups. The aim of this study was to identify a possible common ancestry between these carriers.Patients and methodsTwelveRETG533C mutation carriers, four randomly selected from the Brazilian cohort and eight from apparently unrelated Greek families, were studied for a possible common ancestral origin.RETflanking microsatellite markers at chromosome 10q (D10S197, D10S196, D10S1652 and D10S537) were used.ResultsGenomic DNA analysis using these markers showed that many of these apparently unrelated individuals shared a common haplotype indicating a common ancestral origin.ConclusionOur data suggest that Brazilian and Greek patients with MTC carrying the G533C mutation in exon 8 ofRETgene originate from a common ancestor. Due to historical reasons, we speculate that the more plausible explanation for the origin of this mutation is in Greece.

Inward rectifying potassium – Kir – channels drive the resting potential to potassium reversal potential and, when disrupted, might be related to muscular diseases. Recently, Thyrotoxic Periodic Paralysis (TPP) has emerged as a channelopathy related to mutations in KCNJ18 gene, which encodes Kir2.6 channel. TPP is a neuromuscular disorder characterized by a triad of muscle weakness, hypokalemia, and thyrotoxicosis, the latter being essential for the crisis. Direct sequencing revealed two heterozygous mutations – D252N and R386C – in two TPP patients. KCNJ18 cDNAs were cloned into mammalian expression plasmids and transiently expressed in HEK 293T cells to investigate the functional effects of Kir2.6 mutations. Patch-clamp and confocal laser scanning microscopy experiments were carried out, comparing the WT channel to its mutants. D252N mutation down-regulates the Kir2.6 activity, decreasing the K+ current density (∼34%) when compared to the WT channel; whereas the mutation R386C shows no significant changes from WT. The mutant D252N Kir2.6 channel also showed a substantial reduction of ∼51% in membrane abundance relative to WT channel. Our study describes the functional consequences of a single amino acid change in Kir2.6 channel. Further analysis regarding hormonal conditions and Kir channel expression are required to provide new clues about the TPP pathophysiology.

Thyrotoxic periodic paralysis (TPP) is a life-threatening neuromuscular complication of thyrotoxicosis characterized by muscle weakness and hypokalemia and with an unclear etiopathogenesis. However, the 17q24.3 locus had been genetically linked to TPP, in which the genetic variant rs312691 (TC genotype) in long intergenic noncoding RNA (lincRNA) CTD-2378E21.1 is located downstream of inward-rectifier potassium (Kir) channel genes [KCNJ2 and its antisense KCNJ2 (AS-KCNJ2)]. A TPP patient with a suppressed thyroid-stimulating hormone level, a high free thyroxine level of (5.8 ng/dL), and low serum potassium level of (2 mEq/L) was evaluated for Kir channel expression during and after recovery from thyrotoxicosis. We observed that circulating lincRNA and Kir expression varied in accordance with thyroid status and TC genotype. To endorse this association of a lincRNA-rs312691 variant with a genetic risk of TPP, an additional series of 37 patients with TPP and 32 patients with thyrotoxic without paralysis (TWP) were assessed. We verified that the risk of minor allele C was greater in TPP than in TWP (odds ratio, 5.289; P = 0.0062), and protective major allele T was more frequent than observed in the 1000 genome controls (odds ratio, 11.90; P < 0.0001). AS-KCNJ2 was downregulated during thyrotoxicosis in the TWP controls carrying allele T and were upregulated in those with TPP with risk allele C. Moreover, KCNJ2 (Kir2.1) expression was reduced during thyrotoxicosis and restored in euthyroid status. We further excluded any other coding variant by performing targeted exome sequencing mutational screening in 17q24.3. Our data suggest that high lincRNA AS-KCNJ2 and CDT-2378E21.1 expression, possibly driven by the triiodothyronine regulatory mechanism, reduces the Kir2.1 expression observed during thyrotoxicosis. This finding could contribute to the understanding of the reduced inward-rectifying current observed during muscle weakness in genetically susceptible TPP patients., Our study provides in vivo evidence for diminished inward rectifying potassium channel gene expression during thyrotoxic muscle attack that might be regulated by a novel long intergenic noncoding RNA.

Germline mutations in codon 918 of exon 16 of theRETgene (M918T) are classically associated with multiple endocrine neoplasia type 2B (MEN 2B) with highly aggressive medullary thyroid cancer (MTC), pheochromocytoma and a unique phenotype. The objectives of this study are to describe the rare M918VRETmutation discovered in 8 MTC kindreds from Brazil lacking the MEN 2B phenotype classically observed in M918T patients and to investigate the presence of a founder effect for this germline mutation. Eight apparently sporadic MTC cases were diagnosed with the germline M918VRETmutation. Subsequently, their relatives underwent clinical and genetic assessment (n = 113), and M918V was found in 42 of them. Until today, 20/50 M918V carriers underwent thyroidectomy and all presented MTC/C-cell hyperplasia; the remainder carriers are on clinical follow-up. None of the M918V carriers presented clinical features of MEN 2B. Their clinical presentation was heterogeneous, and the age at tumor diagnosis ranged from 24 to 59 years. Lymph node metastases were present in 12/20 patients, and presumable distant metastases in 2/20; in contrast, we observed a carrier of up to 87 years of age without evidence of MTC. Ethnographic fieldwork and haplotype analyses suggested that the founder mutation first settled in that area fifteen generations ago and originated from Portugal. Our study is the first to demonstrate theRETM918V mutation co-segregating in 8 familial MTC kindreds with validated evidence of a founder effect. We suggest that M918V MTC should be clinically considered an American Thyroid Association (ATA) moderate-risk category.

Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant genetic disease caused by RET gene germline mutations that is characterized by medullary thyroid carcinoma (MTC) associated with other endocrine tumors. Several reports have demonstrated that the RET mutation profile may vary according to the geographical area. In this study, we collected clinical and molecular data from 554 patients with surgically confirmed MTC from 176 families with MEN2 in 18 different Brazilian centers to compare the type and prevalence of RET mutations with those from other countries. The most frequent mutations, classified by the number of families affected, occur in codon 634, exon 11 (76 families), followed by codon 918, exon 16 (34 families: 26 with M918T and 8 with M918V) and codon 804, exon 14 (22 families: 15 with V804M and 7 with V804L). When compared with other major published series from Europe, there are several similarities and some differences. While the mutations in codons C618, C620, C630, E768 and S891 present a similar prevalence, some mutations have a lower prevalence in Brazil, and others are found mainly in Brazil (G533C and M918V). These results reflect the singular proportion of European, Amerindian and African ancestries in the Brazilian mosaic genome.

Next-generation sequencing (NGS) has enriched the understanding of the human genome. However, homologous or repetitive sequences shared among genes frequently produce dubious alignments and can puzzle NGS mutation analysis, especially for paralogous potassium channels. Potassium inward rectifier (Kir) channels are important to establish the resting membrane potential and regulating the muscle excitability. Mutations in Kir channels cause disorders affecting the heart and skeletal muscle, such as arrhythmia and periodic paralysis. Recently, a susceptibility muscle channelopathy-thyrotoxic periodic paralysis (TPP)-has been related to Kir2.6 channel (KCNJ18 gene). Due to their high nucleotide sequence homology, variants found in the potassium channels Kir2.6 and Kir2.5 have been mistakenly attributable to Kir2.2 polymorphisms or mutations. We aimed at elucidating nucleotide misalignments by performing realignment of whole exome sequencing (WES) and whole genome sequencing (WGS) reads to specific Kir2.2, Kir2.5, and Kir2.6 cDNA sequences using BWA-MEM/GATK pipeline. WES/WGS reads correctly aligned 26.9/43.2, 37.6/31.0, and 35.4/25.8 % to Kir2.2, Kir2.5, and Kir2.6, respectively. Realignment was able to reduce over 94 % of misalignments. No putative mutations of Kir2.6 were identified for the three TPP patients included in the cohort of 36 healthy controls using either WES or WGS. We also distinguished sequences for a single Kir2.2, a single Kir2.5 sequence, and two Kir2.6 isoforms, which haplotypes were named RRAI and QHEV, based on changes at 39, 40, 56, and 249 residues. Electrophysiology records on both Kir2.6_RRAI and _QHEV showed typical rectifying currents. In our study, the reduction of misalignments allowed the elucidation of paralogous gene sequences and two distinct Kir2.6 haplotypes, and pointed the need for checking the frequency of these polymorphisms in other populations with different genetic background.

Sao Paulo State Research Foundation Medullary thyroid carcinoma (MTC), a neuroendocrine tumor originating from thyroid parafollicular cells, has been demonstrated to be associated with mutations in RET, HRAS, KRAS and NRAS. However, the role of other genes involved in the oncogenesis of neural crest tumors remains to be fully investigated in MTC. The current study aimed to investigate the presence of somatic mutations in BRAF, CDKN2A and PI3KCA in MTC, and to investigate the correlation with disease progression. DNA was isolated from paraffin-embedded tumors and blood samples from patients with MTC, and the hotspot somatic mutations were sequenced. A total of 2 novel HRAS mutations, p.Asp33Asn and p.His94Tyr, and polymorphisms within the 3' untranslated region (UTR) of CDKN2A (rs11515 and rs3088440) were identified, however, no mutations were observed in other genes. It was suggested that somatic point mutations in BRAF, CDKN2A and PI3KCA do not participate in the oncogenesis of MTC. Further studies are required in order to clarify the contribution of the polymorphisms identified in the 3' UTR of CDKN2A in MTC. Univ Fed Sao Paulo, Dept Med, Escola Paulista Med, Lab Mol & Translat Endocrinol, 669 Rua Pedro Toledo, BR-04039032 Sao Paulo, Brazil Univ Fed Sao Paulo, Escola Paulista Med, Dept Biochem, BR-04039032 Sao Paulo, Brazil Univ Fed Sao Paulo, Escola Paulista Med, Dept Pathol, BR-04039032 Sao Paulo, Brazil Univ Fed Sao Paulo, Dept Med, Escola Paulista Med, Lab Mol & Translat Endocrinol, 669 Rua Pedro Toledo, BR-04039032 Sao Paulo, Brazil Univ Fed Sao Paulo, Escola Paulista Med, Dept Biochem, BR-04039032 Sao Paulo, Brazil Univ Fed Sao Paulo, Escola Paulista Med, Dept Pathol, BR-04039032 Sao Paulo, Brazil FAPESP: 2012/11036-3 FAPESP: 2012/02465-8 FAPESP: 2012/01628-0 FAPESP: 2009/50575-4 FAPESP: 2012/00079-3 FAPESP: 2011/20747-8 Web of Science

INTRODUCTION: Several methods have been proposed to improve operative success in renal hyperparathyroidism. OBJECTIVE: To evaluate stereomicroscopy in parathyroid tissue selection for total parathyroidectomy with autotransplantation in secondary (SHPT)/tertiary (THPT) hyperparathyroidism. METHODS: 118 renal patients underwent surgery from April of 2000 to October 2009. They were divided into two groups: G1, 66 patients operated from April of 2000 to May of 2005, with tissue selection based on macroscopic observation; G2, 52 patients operated from March of 2008 to October 2009 with stereomicroscopy for tissue selection searching for the presence of adipose cells. All surgeries were performed by the same surgeon. Patients presented SHPT (dialysis treatment) or THPT (renal-grafted). Follow-up was 12-36 months. Intra-operative parathyroid hormone (PTH) was measured in 100/118 (84.7%) patients. RESULTS: Data are presented as means. G1 included 66 patients (38 SHPT, 24 females/14 males; 40.0 years of age; 28 THPT, 14 females/14 males; 44 years of age). G2 included 52 patients (29 SHPT, 11 females/18 males; 50.7 years of age; 23 THPT, 13 females/10 males, 44.4 years of age). SHPT patients from G2 presented preoperative serum calcium higher than those of SHPT patients in G1 (p < 0.05), suggesting a more severe disease. Definitive hypoparathyroidism was found in seven of 118 patients (5.9%). Graft-dependent recurrence occurred in four patients, two in each group. All occurred in dialysis patients. CONCLUSION: Stereomicroscopy in SHPT/THPT surgical treatment may be a useful tool to standardize parathyroid tissue selection. INTRODUÇÃO: Diversos métodos têm sido propostos com intuito de melhorar índices de sucesso cirúrgico no tratamento do hiperparatiroidismo associado à doença renal crônica (DRC). OBJETIVOS: Avaliar uso do estereomicroscópio na seleção de tecido paratiroideano na paratiroidectomia total com autoimplante em pacientes com DRC. MÉTODOS: 118 pacientes DRC operados entre 04/2000-10/2009 foram divididos em: G1-66 pacientes operados entre 04/2000-05/2005 cuja seleção de tecido foi realizada por método convencional (macroscopia); G2-52 pacientes operados entre 03/2008-10/2009, cuja seleção de tecido foi realizada com uso da estereomicroscopia: Leica-Stereomicroscope (amplificação: 10×-80×). Pacientes foram ainda categorizados em hiperparatiroidismo secundário (HPS) ou terciário (HPT) (HPS-diálise/HPT-transplantados renais). Seguimento pós-operatório: 12-36 meses. PTH intraoperatório medido 100/118 pacientes (84.7%). Todos pacientes foram operados pelo mesmo cirurgião. RESULTADOS: Dados em média. G1, 66 pacientes (38 HPS, 24f/14m; 40 anos; 28 HPT, 14f/14m; 44 anos). G2, 52 pacientes (29 HPS, 11f/18m; 50,7 anos; 23 HPT, 13f/10m; 44,4 anos). Pacientes dialíticos do G2 apresentaram cálcio pré-operatório maior que G1 (p < 0,05), sugerindo doença mais severa. Hipoparatiroidismo definitivo: 7/118 (5,9%) pacientes: G1, 4/66 (6%); G2, 3/52 (5,7%). Recorrência do hiperparatiroidismo no autoimplante: 4 pacientes, 2 em cada grupo. Todas foram em pacientes em diálise. CONCLUSÃO: Estereomicroscopia no tratamento do hiperparatiroidismo associado à DRC é útil na padronização da técnica de seleção de tecido para o autoimplante.

Six Brazilian families with mild familial hyperglycaemia have been screened for glucokinase (GCK) mutations. All had mutations that co-segregated with the phenotype. One of the mutations, the deletion 96_98delAAG (p.Lys32del), had not been previously described, reinforcing the worldwide prevalence of GCK MODY and widespread existence of undetected new mutations.

OBJECTIVE: Adequate isolation of nucleic acids from peripheral blood, fine-needle aspiration cells in stained slides, and fresh and formalin-fixed/paraffin-embedded tissues is crucial to ensure the success of molecular endocrinology techniques, especially when samples are stored for long periods, or when no other samples can be collected from patients who are lost to follow-up. Here, we evaluate several procedures to improve current methodologies for DNA (salting-out) and RNA isolation. MATERIALS AND METHODS: We used proteinase K treatment, heat shock, and other adaptations to increase the amount and quality of the material retrieved from the samples. RESULTS: We successfully isolated DNA and RNA from the samples described above, and this material was suitable for PCR, methylation profiling, real-time PCR and DNA sequencing. CONCLUSION: The techniques herein applied to isolate nucleic acids allowed further reliable molecular analyses. Arq Bras Endocrinol Metab. 2012;56(9):618-26

Sao Paulo State Research Foundation-FAPESP FAPESP Federal Agency of Support and Evaluation of Postgraduate Education (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior) National Council for Scientific and Technological Development Context: Calcitonin (CT) is a sensitive marker of medullary thyroid carcinoma (MTC) and is used for primary diagnosis and follow-up after thyroidectomy. However, persistently elevated CT is observed even after complete surgical removal without evidence of a recurrent or persistent tumor. Objective: To investigate the presence of assay interference in the serum CT of MTC patients who are apparently without a structural disease. Patients and Methods: We studied three index MTC cases for CT assay interference and 14 patients with metastatic MTC. The CT level was measured using an immunofluorometric assay. Screening for assay interference was performed by determination of CT levels before and after serum treatment with polyethylene glycol. Additionally, samples were analyzed by chromatography on ultra-performance liquid chromatography and protein A-Sepharose. Results: Patients with biochemical and structural disease showed CT mean recovery of 84.1% after polyethylene glycol treatment, whereas patients suspected of interference showed recovery from 2-7%. The elution profile on UPLC showed that the immunometric CT from these three patients behaved like a high molecular mass aggregate (>300 kDa). Additionally, when these samples were applied to the protein A-Sepharose, CT immunoreactivity was retained on the column and was only released after lowering the pH. Conclusions: For the first time, our results show the presence of a novel pitfall in the CT immunoassay: "macrocalcitonin." Its etiology, frequency, and meaning remain to be defined, but its recognition is of interest and can help clinicians avoid unnecessary diagnostic investigations and treatment during the follow-up of MTC. Univ Fed Sao Paulo, Escola Paulista Med, Div Endocrinol, Dept Med,Thyroid Dis Ctr, BR-04039032 Sao Paulo, SP, Brazil Univ Fed Sao Paulo, Escola Paulista Med, Div Endocrinol, Dept Med,Lab Mol & Translat Endocrinol, BR-04039032 Sao Paulo, SP, Brazil Univ Fed Sao Paulo, Escola Paulista Med, Dept Biochem, Div Mol Biol, BR-04044020 Sao Paulo, SP, Brazil Univ Fed Sao Paulo, Escola Paulista Med, Div Endocrinol, Dept Med,Thyroid Dis Ctr, BR-04039032 Sao Paulo, SP, Brazil Univ Fed Sao Paulo, Escola Paulista Med, Div Endocrinol, Dept Med,Lab Mol & Translat Endocrinol, BR-04039032 Sao Paulo, SP, Brazil Univ Fed Sao Paulo, Escola Paulista Med, Dept Biochem, Div Mol Biol, BR-04044020 Sao Paulo, SP, Brazil FAPESP: 2006/60402-1 FAPESP: 2010/51547-1 FAPESP: 2010/19478 Web of Science

Objective Consuming a low-iodine diet (LID) is a widely accepted practice before administering radioiodine (131I) to evaluate and to treat thyroid disease. Although this procedure is well established for the management of patients with differentiated thyroid cancer, its use in patients with benign disease is unclear. So, we aimed to evaluate the influence of a LID on the outcome in patients with Graves’ disease (GD) treated with131I. Subjects and methods We evaluated 67 patients with GD who were divided into 2 groups: one group (n = 31) consumed a LID for 1-2 weeks, and the second group (n = 36) was instructed to maintain a regular diet (RD). Results The LID group experienced a 23% decrease in urinary iodine after 1 week on the diet and a significant 42% decrease after 2 weeks on the diet. The majority (53%) of the patients in the LID group had urinary iodine levels that were consistent with deficient iodine intake. However, there was no difference in the rate of hyperthyroidism’s cure between the LID and the RD groups 6 months after 131I therapy. Furthermore, the therapeutic efficacy did not differ in patients with varying degrees of sufficient iodine intake (corresponding urinary iodine levels: < 10 μg/dL is deficient; 10-29.9 μg/dL is sufficient; and > 30 μg/dL is excessive). Conclusion In the present study, we demonstrated that although a LID decreased urinary iodine levels, those levels corresponding with sufficient or a mild excess in iodine intake did not compromise the therapeutic efficacy of131I for the treatment of GD.

Osteoporosis Pseudoglioma (OPPG) is characterized by severe juvenile-onset osteoporosis and ocular abnormalities. It is caused by one of several inactivating mutations in LRP5, a gene importantly involved in bone formation. The objective of this study was to evaluate the efficacy of teriparatide in a young man with OPPG. The subject of this case report is a 19-year-old man with congenital blindness and low trauma fractures because of OPPG. A 2-year course of teriparatide, 20 µg/day, was initiated after a 6-year course of intravenous pamidronate infusions, the latter 3 years of which had minimal effects on bone mineral density (BMD). Measurements in serum were made of C-terminal telopeptide of type I collagen (CTX), N-terminal propeptide of type I collagen (P1NP), total and ionized calcium, phosphate, uric acid, complete blood count, and renal and liver function tests. Urinary calcium/creatinine ratio was determined. BMD was measured by DXA yearly. BMD increased by 9.7% in lumbar spine and 10.2% in right femur hip. CTX rose early, peaking in month 3, followed by an increase in P1NP, peaking in month 9. Both indices returned to baseline by month 24. The increase in CTX followed by P1NP is an unusual time course when teriparatide is used to treat osteoporosis but may be typical of low bone turnover states. There were no adverse events. In a patient with OPPG, teriparatide markedly increased BMD in the lumbar spine and femur hip.

Ion channels serve diverse cellular functions, mainly in cell signal transduction. In endocrine cells, these channels play a major role in hormonal secretion, Ca2+-mediated cell signaling, transepithelial transport, cell motility and growth, volume regulation and cellular ionic content and acidification of lysosomal compartments. Ion channel dysfunction can cause endocrine disorders or endocrine-related manifestations, such as pseudohypoaldosteronism type 1, Liddle syndrome, Bartter syndrome, persistent hyperinsulinemic hypoglycemia of infancy, neonatal diabetes mellitus, cystic fibrosis, Dent's disease, hypomagnesemia with secondary hipocalcemia, nephrogenic diabetes insipidus and, the most recently genetically identified channelopathy, thyrotoxic hypokalemic periodic paralysis. This review briefly recapitulates the membrane action potential in endocrine cells and offers a short overview of known endocrine channelopathies with focus on recent progress regarding the pathophysiological mechanisms and functional genetic defects. Canais iônicos auxiliam diferentes funções celulares, principalmente na transdução de sinal. Nas células endócrinas, esses canais têm funções importantes na secreção hormonal, sinalização do Ca2+, transporte transepitelial, regulação da motilidade, volume e conteúdo iônico celular e da acidificação do compartimento lisossomal (pH). Como esperado, as alterações nos canais iônicos podem causar distúrbios endocrinológicos, como pseudo-hipoaldosteronismo tipo 1, síndrome de Liddle, síndrome de Bartter, hipoglicemia hiperinsulinêmica da infância, diabetes melito neonatal, fibrose cística, doença de Dent, hipomagnesemia com hipocalcemia secundária, diabetes insípido nefrogênico e paralisia periódica tirotóxica hipocalêmica. Este artigo propõe uma breve revisão das canalopatias endócrinas conhecidas, com foco particular nos recentes progressos no conhecimento dos mecanismos fisiopatológicos adquirido a partir das alterações funcionais encontradas.

Desenvolvemos método semi-automatizado em placa para a determinação de iodo urinário; utilizamos, primeiramente, a digestão das amostras de urina com persulfato de amônio e, a seguir, estimamos a quantidade de iodo pela redução do sulfato cérico amoniacal. O método foi validado no inquérito nacional de monitoração da deficiência de iodo, realizado em 1994, que empregou um sistema de amostragem mista da população brasileira e analisou a iodúria em 16.803 amostras de urina de escolares obtidas por coleta casual. Em 401 municípios estudados encontramos 4 com deficiência de iodo de grau moderado (Almas, Arraias e Paraná, em Tocantins, e Cocos, na Bahia) e 116 de grau leve. Desta forma, este estudo mostrou a presença de regiões com deficiência de iodo em 1994, a despeito do programa de iodação do sal. Dados recentes de outros autores, com número menor de municípios, indicam excesso de ingestão de iodo. Assim, num país de dimensões continentais e heterogêneo como o Brasil, é necessária a realização de avaliações periódicas de amplitude nacional para a verificação da ingestão de iodo da população. O método apresentado neste estudo apresenta as características de simplicidade e eficiência necessários para este tipo de estudo populacional. In this study we developed a semi-automated method for the measurement of urinary iodine using firstly ammonium persulfate for digestion of urine followed by estimation of iodine content in the Sandell-Kolthoff reaction, in which iodine acts as a catalyst for the reduction of cerium. This method was validated in the 3rd Brazilian National Survey of iodine deficiency in 1994. We studied 16,803 casual urine samples from schoolchildren of 401 cities and found 4 moderately-deficient towns (Almas, Arraias, and Parana, in the State of Tocantins, and Cocos, in the State of Bahia), and 116 mildly-deficient. This work suggests that despite the salt iodization program, there was some iodine-deficient areas in Brazil in 1994. Recent surveys, involving less cities, are indicating an excess of iodine ingestion. Therefore, in a country of continental dimensions and very heterogeneous in terms of public health, periodical evaluations are necessary to monitor the real situation of iodine nutrition in Brazil. The method developed in this paper is suitable for these surveys.

Introduction: Hyperparathyroidism is a frequent complication of chronic kidney disease (CKD). Total parathyroidectomy (PTX) with parathyroid tissue autotransplantation (AT) is a treatment option in those individuals that do not respond to clinical management. Objective: To evaluate grafted parathyroid tissue response during induced hypocalcemia among CKD patients who underwent total PTX with AT. Methods: Eighteen patients with renal hyperparathyroidism were submitted to total PTX with parathyroid AT selected by stereomicroscopy between April and October 2008. Eleven (eight with successful kidney transplantation, 2 in peritoneal dialysis and 1 in hemodialysis) were clinically stable and eligible for testing. Hypocalcemia was induced using sodium bicarbonate infusion in 5 healthy controls and in patients 6-12 months after surgery. Results: Among controls, hypocalcemia elicited a major rise in intact PTH (iPTH) levels 4 minutes after bicarbonate infusion. In patients, a significant decrease in ionized calcium concentration was observed [from 1.17 ± 0.12 to 1.09 ± 0.11 mean (± SE) mmol/L] in the 4th minute (p < 0.001) illustrating the nadir point. In the 10thminute, ionized calcium did not show a statistical increase compared to the 4th minute (p = 0.451). The iPTH levels ranged from 34.8 ± 18.6 to 34.1 ± 18.8 pg/mL (similar values between base line and 4thminute p = 0.087) and did not change in the 10th minute (33.3 ± 19,6 pg/ mL p = 0.693). Conclusion: Among CKD patients tested 6-12 months after surgery, grafted parathyroid tissue revealed a blunted secretory capacity during bicarbonate induced hypocalcemia with no changes in iPTH levels

A forma mais eficaz e segura de se diagnosticar o hipotireoidismo congênito (HC) é através dos programas de rastreamento neonatal com dosagens sanguíneas do hormônio tireotrófico (TSH). Está estabelecido que a incidência de HC varia entre 1:2.300 - 1:5.000 nascimentos, sendo mais elevada em áreas de carência iódica. Complementando o programa de rastreamento para HC desenvolvido entre a Faculdade de Medicina de Ribeirão Preto (FMRP-USP) e a Universidade Federal de São Paulo (UNIFESP), dosou-se iodo em amostras urinárias de 141 recém-nascidos (RN) de Ribeirão Preto, sendo 87 meninos e 54 meninas. Os resultados de TSH não indicaram casos de HC, embora um elevado número de crianças (0,96%) tenha sido convocado para novos testes. As iodúrias variaram entre 2,1 e 194µg/l, (média de 58,3±36,2µg/l). Não foram observadas diferenças entre os valores de iodo urinário dos RN segundo o sexo ou idade gestacional. Foi encontrada correlação negativa entre iodúria e TSH de sangue de cordão umbilical (r= -0,20, p= 0,02).

Familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) are consequent to inactivating mutations of the calcium-sensing receptor (CaR) gene. FHH is usually associated with heterozygous inactivating mutations of the CaR gene, whereas NSHPT is usually due to homozygous inactivation of the CaR gene. FHH is generally asymptomatic and is characterized by mild to moderate lifelong hypercalcemia, relative hypocalciuria, and normal intact PTH, whereas individuals with NSHPT frequently show life-threatening hypercalcemia. In this study, we report a novel inactivating mutation of the CaR gene, identified in a 9-yr-old Brazilian girl who was found to be severely hypercalcemic during investigation of a 6-month history of headaches and vomits. Direct sequencing of the CaR gene from this patient showed a novel homozygous mutation (L13P) in exon 2. Functional characterization by intracellular calcium measurement by fluorometry showed that the mutant receptor had a dose-response curve shifted to the right relative to that of wild type. The proband's consanguineous parents, who had mild asymptomatic hypercalcemia, showed the same mutation in the heterozygous form. The mutation described in this study is the inactivating missense mutation present at the most N-terminal end among the known CaR missense mutations. This study reinforces the fact that patients with homozygous inactivation of the CaR gene may present with severe hypercalcemia in different phases of life.

Thirty-two Brazilian families with MODY phenotype were screened for GCK and HNF1A mutations. GCK mutations were found in 8 families, all patients with mild asymptomatic hyperglycaemia; 3 of them are novel: p.Asp365Asn, p.Gly81Asp and p.Val253Leu. Previously described mutations in HNF1A were found in 2 families.

In patients with uremia, intact parathyroid hormone (PTH) measurement appears to overestimate the biologically active hormone in circulation. The recent description of the accumulation in these patients of a non-intact PTH form measured by the standard immunometric assays, re-opened the question. In this study we submitted serum samples from 7 patients with primary hyperparathyroidism (PHP) and from 10 patients with hyperparathyroidism secondary to chronic renal failure (SHP) to preparative HPLC in order to discriminate the molecular forms measured by our currently used immunofluorometric assay for intact PTH. The elution profile obtained with the HPLC system showed two clearly defined peaks, the first one corresponding to a lower molecular weight form, and the second to the intact PTH (1-84) form. In patients with SHP the area under the curve for the first peak (mean 29.5%, range 20.6 to 40.4%) was significantly greater than that observed for patients with PHP (mean 15.6%, range 5.6 to 21.9%). This confirms previous studies showing accumulation of molecular forms of slightly lower molecular weight, presumably PTH (7-84), in patients with SHP and, to a lesser extent, in patients with PHP. The real necessity of assays that discriminate between these two molecular forms is debatable.

Univ Fed Rio Grande do Sul, Hosp Clin Porto Alegre, Endocrinol Unit, Postgrad Fellowship Program Endocrinol, Porto Alegre, RS, Brazil

This study aimed to evaluate the 24-week effects of a high-intensity aquatic exercise program on bone remodeling markers and bone mass of postmenopausal women. In this randomized, controlled trial we studied 108 women (58.8 ± 6.4 years), randomized into Aquatic Exercise Group (AEG), n = 64, performing 24 weeks of aquatic exercises, and Control Group (CG), n = 44, sedentary. They had their fasting morning blood sample collected for the measures of intact parathyroid hormone (iPTH), procollagen type 1 amino-terminal propeptide (P1NP) and carboxy-terminal cross-linking telopeptide of type I collagen (CTx). Bone mass was measured by dual-energy X-ray absorptiometry before and after the intervention. Participants of both groups received a daily supplementation of 500 mg of elementary calcium and 1,000 IU of vitamin D (cholecalciferol). Results showed an augment in bone formation marker (P1NP) only in the AEG (15.8 %; p = 0.001), and although both groups experienced significant enhancements in bone resorption marker (CTx), this increase was less considerable in the AEG (15 % in the AEG and 29 % in the CG). IPTH was increased by 19 % in the CG (p = 0.003) at the end. The femoral trochanter BMD presented a 1.2 % reduction in the CG (p = 0.009), whereas in the AEG no change was observed (p = 0.069). The proposed aquatic exercise program was efficient in attenuating bone resorption raise and enhancing bone formation, which prevented the participants in the AEG from reducing the femoral trochanter BMD, as happened in the CG.

We hereby report two patients with parathyroid carcinoma presenting extremely high calcium and PTH levels, severe bone disease, and palpable neck mass at diagnosis. They both underwent parathyroidectomy, and one of them evolved to lung metastasis. Important hypocalcemia was observed after surgery in both: after parathyroidectomy in one patient, and only after surgical removal of the metastasis in the other. Both required intravenous calcium replacement, thus revealing hungry bone syndrome (HBS). HBS usually reflects rapid mineralization after correction of hyperparathyroidism. The more severe the bone disease before surgery, the more prone the patient is to HBS after surgery. Despite being an unfavorable outcome, HBS state suggests that surgical removal of hypersecretory parathyroid tissue was accomplished. In this study, HBS was observed in both patients, who presented severe bone disease prior to surgery. HBS would be expected post-operatively in successful parathyroid carcinoma removal. O presente artigo descreve o relato de dois pacientes com carcinoma de paratiroide que apresentavam valores intensamente elevados de cálcio sérico e de PTH, associado a doença óssea e presença de nódulo cervical palpável ao diagnóstico. Ambos foram submetidos à paratiroidectomia, sendo que um evoluiu com metástases pulmonares. Hipocalcemia importante foi observada após a paratiroidectomia em um paciente e somente após remoção cirúrgica das metástases pulmonares em outro. Ambos necessitaram de reposição endovenosa de cálcio, revelando, assim, o estado de fome óssea (FO). A presença da FO usualmente reflete rápida mineralização óssea após correção do hiperparatiroidismo; assim, quanto mais severa a doença óssea previa à cirurgia, maior será a FO observada no pós-operatório desses pacientes. Embora inicialmente considerada um evento indesejável, a FO representa a bem-sucedida remoção cirúrgica do tecido paratiroideano hipersecretor. Fome óssea deve ser esperada no pós-operatório do tratamento cirúrgico bem-sucedido do carcinoma de paratiroide.

Mutations in the glucokinase gene (GCK) account for many cases of monogenic diabetes featuring maturity-onset diabetes of the young type 2 (MODY2). The clinical pattern of this form of hyperglycemia is rather stable, with a slight elevation in blood glucose, which is usually not progressive. Patients rarely require pharmacological interventions and microvascular complications related to diabetes are unusual. We describe the clinical follow-up of two cases of MODY2 with two different mutations in GCK gene, one in exon 7, p.Glu265Lys (c.793 G> A), which has been previously described, and a novel one, in exon 2, p.Ser69Stop (c. 206C> G). The clinical course of both cases shows similarity in metabolic control of this form of diabetes over the years. Arq Bras Endocrinol Metab. 2012;56(8):490-5 Mutações no gene da glicoquinase (GCK) são determinantes de uma forma de diabetes monogênico denominada de MODY2 (maturity-onset diabetes of the young, tipo 2). O padrão clínico dessa forma de distúrbio glicêmico é bastante estável, com hiperglicemia leve, geralmente não progressiva. Intervenções farmacológicas raramente são necessárias e complicações crônicas secundárias ao diabetes são infrequentes. Descrevemos o acompanhamento clínico de dois casos de MODY2 com duas mutações diferentes, uma no éxon 7, p.Glu265Lys (c.793 G>A) já descrita anteriormente, e outra inédita no éxon 2 p.Ser69Stop (c. 206C>G). A evolução clínica de ambos os casos demonstra uma semelhança no padrão metabólico dessa forma de diabetes ao longo dos anos. Arq Bras Endocrinol Metab. 2012;56(8):490-5

RET sequencing has become an important tool in medullary thyroid cancer (MTC) evaluation and should be performed even in the absence of family history of MTC. The most commonly studied exons in index cases are 8, 10, 11, and 13–16. To address the ATA guidelines regarding the sequencing of the entire coding region of RET, we selected 50 patients with sporadic MTC (sMTC) without mutations in the hot spot regions of RET for extended investigation of exons 1–7, 9, 12, 17, 18, and 19. Twenty-seven of 50 patients presented with one or more features suggesting familial disease. We found only a new RET variant (p.Gly550Glu) in one patient with MTC. Several polymorphisms were observed, and their frequency was histogram scaled by exons and introns. Eight patients were also included for somatic mutation search. We estimated the sequencing cost by stratifying into four investigation approaches: (1) hot spot exons in a new patient, (2) the remaining exons if the hot spots are negative in a patient with suspected familial disease, (3) a relative of a carrier for a known RET mutation, and (4) tumor sequencing. In spite of the increasing number of variants being described in MTC, it appears that there is no direct clinical benefit in extending RET germ line analysis beyond the hot spot regions in sMTC. The cost evaluation in apparent sMTC using a tiered approach may help clinicians make more suitable decisions regarding the benefits of investigating only the hot spots against the entire coding region of RET.

Background Maturity-Onset Diabetes of the Young (MODY) is a group of monogenic forms of diabetes mellitus (DM) caused by mutations in at least 13 genes. Mutations in Glucokinase (GCK) and Hepatocyte nuclear factor-1 homeobox A (HNF1A) are the most common. Frequency of subtypes varies according to studied population. A broader view of MODY in Brazil is needed to improve diagnosis, epidemiological registry, and clinical care, also paving the way for future research.

OBJETIVO: O objetivo deste estudo é avaliar se a medida perioperatória do hormônio da paratireoide (PTH) pode identificar os pacientes com maior risco de desenvolver sintomas de hipocalcemia. SUJEITOS E MÉTODOS: Foram estudados quarenta pacientes submetidos à tireodiectomia total. A medida do cálcio sérico e do PTH foi feita após a indução anestésica, uma hora (PTH1) e um dia após a cirurgia. Os pacientes foram avaliados quanto à presença de sintomas de hipocalcemia e tratados com suplementação de cálcio e vitamina D quando necessário. RESULTADOS: Dezesseis pacientes apresentaram sintomas de hipocalcemia. Os pacientes sintomáticos apresentaram PTH1 significantemente menor e queda no PTH significativamente maior. Usando o valor de 12,1 ng/L como corte, conseguimos distinguir pacientes com e sem sintomas de hipocalcemia com sensibilidade de 93,7% e especificidade de 91,6%. Utilizando como corte a queda de 73,5% no valor do PTH, temos sensibilidade de 91,6% e especificidade de 87,5%. CONCLUSÃO: O PTH1 e a queda no PTH são bons preditores de hipocalcemia no pós-operatório de tireoidectomia total OBJECTIVE: The purpose of this study was to evaluate if the measurement of peri-operative parathyroid hormone (PTH) is able to identify patients with increased risk of developing symptoms of hypocalcemia. SUBJECTS AND METHODS: Forty patients who underwent total thyroidectomy were studied prospectively. Ionized serum calcium and PTH were measured after induction of anesthesia, one hour (PTH1) and one day after surgery (PTH24). Patients were evaluated for symptoms of hypocalcemia and treated with calcium and vitamin D supplementation as necessary. RESULTS: Symptomatic hypocalcemia developed in 16 patients. Symptomatic patients had significant lower PTH1 and greater drops in PTH levels. The selection of 12.1 ng/L as PTH1 level cutoff level divided patients with and without symptoms with 93.7% sensitivity and 91.6% specificity. The selection of 73.5% as the cutoff value for PTH decrease resulted in 91.6% sensitivity and 87.5% specificity. CONCLUSION: PTH1 levels and the drop in PTH levels are reliable predictors of developing symptomatic hypocalcemia after total thyroidectomy. Universidade Federal de São Paulo (UNIFESP) UNIFESP Universidade Federal de São Paulo, EPM, São Paulo, Brazil SciELO

OBJECTIVE: To assess bone turnover markers (BTM) and bone mineral density (BMD) after discontinuation of alendronate treatment used for five or more years. SUBJECTS AND METHODS: 40 patients (pt) with post-menopausal osteoporosis treated with alendronate (10 mg/d) for at least five years (Group 1, G1) had their medication discontinued. Group 2 (G2): 25 pt treated with alendronate for at least one year. Group 3 (G3): 23 treatment-naïve osteoporotic pt. BMD was evaluated in G1 and G2 at baseline and after 12 months. Collagen type I cross-linked C-telopeptide (CTX) and procollagen type 1 N-terminal propeptide (P1NP) levels were measured in all pt at baseline, and in G1 and G2 every three months for 12 months. Data were analyzed using ANOVA on ranks and Mann-Whitney tests. RESULTS: Mean BMD values in G1 and G2 did not differ during follow-up. However, 16 pt (45.7%) in G1 and one (5.2%) in G2 lost BMD (P < 0.001). BTM at baseline was not different between G1 and G2, and both were lower than G3. A significant increase in BTM levels was detected in G1 pt after three months, but not in G2. CONCLUSION: Observed BMD loss and BTM rise after alendronate withdrawal imply that bone turnover was not over suppressed, and alendronate discontinuation may not be safe. OBJETIVO: Avaliar a evolução dos marcadores de metabolismo ósseo (MMO) e da densidade mineral óssea (DMO) após cinco anos de uso de alendronato em mulheres osteoporóticas na pós-menopausa. SUJEITOS E MÉTODOS: 40 pacientes (pct) osteoporóticas, na pós-menopausa, em uso de alendronato (10 mg/dia) por pelo menos 5 anos (Grupo 1 − G1) tiveram o uso do bisfosfonato suspenso. O grupo 2 (G2): 25 mulheres na pós-menopausa, em uso de alendronato (10 mg/dia) há pelo menos 1 ano. Grupo 3 (G3): 23 pct osteoporóticas, controles ainda sem tratamento. G1 e G2 submeteram-se à avaliação da DMO por DXA (basal e após 12 meses de seguimento). Todas as pct colheram amostras basais de CTX e P1NP, e G1 e G2 submeteram-se a coletas trimestrais de CTX e P1NP durante 1 ano. Resultados foram analisados por ANOVA on ranks e Mann-Whitney. RESULTADOS: Níveis médios de DMO não variaram em G1 ou G2 durante o estudo; no entanto, 16 pct (45,7%) no G1 e 1 pct (5,2%) no G2 apresentaram redução clinicamente significativa de DMO (P < 0,001). Níveis basais de CTX e P1NP não diferiram entre G1 e G2, com ambos inferiores aos níveis de G3. Em G1, observou-se elevação significativa de CTX e P1NP após 3 meses. Os níveis de CTX e P1NP em G2 permaneceram estáveis durante todo o seguimento. CONCLUSÃO: Não parece haver supressão excessiva do metabolismo ósseo na prática clínica. A suspensão temporária do alendronato após seu uso prolongado pode não ser segura.

Background Hypovitaminosis D is a common condition among elderly individuals in temperate-climate countries, with a clear seasonal variation on 25 hydroxyvitamin D levels, increasing after summer and decreasing after winter, but there are few data from sunny countries such as Brazil. Many factors can interfere on vitamin D cutaneous synthesis. We aimed at studying the 25OHD variations during winter and summer in an outdoor physically active elderly population living in São Paulo city, and analysed their determining factors. Methods Ninety-nine individuals (52 women and 47 men, from 55 to 83 years old) from different ethnic groups were selected from an outdoor physical activity group. Data are reported as Mean ± SD, and we used Pearson Linear Correlation, Student's t-test for non-related samples, Chi-square (χ²) test and One-way ANOVA for analysis. Results Mean 25OHD value for the whole group was 78.9 ± 30.9 nmol/L in the winter and 91.6 ± 31.7 nmol/L in the summer (p = 0.005). Mean winter serum 25OHD concentrations were not different between men and women (81.2 ± 30.1 nmol/L vs. 76.7 ± 31.8 nmol/L, respectively), and 19.2% of the individuals showed values < 50 nmol/L. In the summer, we noticed an increase only for men (107.6 ± 31.4 nmol/L) compared to women (76.7 ± 24.0 nmol/L), and 6.5% showed values < 50 nmol/L. A decrease in the mean PTH in the summer compared to the winter was noticed, with PTH levels showing a relationship with 25OHD concentrations only in the winter (r = -0.208, p = 0.041). White individuals showed an increase in mean serum 25OHD in the summer (p = 0.016) which was not noticed for other ethnic groups (Asians, native Brazilians and blacks). An increase in 25OHD values in the summer was observed in the age groups ranging from 51-60 and 61-70 years old (p < 0.05), but not in the age group from 71 years old on. Conclusions 25OHD values increased during the summer in elderly residents of São Paulo, but to different extents depending on ethnicity, gender and age. This season-dependent increase was noticed only among men, white and who were in the youngest group of individuals.

OBJECTIVE: To study carboxyl-terminal (COOH) parathyroid hormone (PTH) circulating forms in patients with hyperparathyroidism due to end stage renal disease (ESRD). METHODS: An immunometric assay that recognizes both intact and COOH PTH forms was developed. The assay, in conjunction with an intact assay, was used to measure PTH in serum samples obtained from 25 patients with hyperparathyroidism due to ESRD. Samples were also submitted to gel filtration chromatography in a Superdex® 30 1.6 x 60 cm column, and the PTH content in the elution tubes, measured using both assays. RESULTS: Values from 39.000 to 232.300 ng/mL (mean ± sd = 101.680 ± 45.330 ng/mL) were found using the COOH assay (PTH 39-84 was used as standard). Values obtained by the intact PTH assay ranged from 318 to 3.307 ng/mL (1.769 ± 693 ng/mL) with a correlation between assays of 0.462 (p = 0.02). The elution profile obtained using the COOH assay showed a preponderance of forms with MW ranging from 8.500 to 4.500 daltons. The profiles obtained from the 25 patients were very similar. CONCLUSIONS: In patients with hyperparathyroidism due to ESRD circulating PTH levels contain a broad range of molecular forms including COOH with MW ranging from 8.500 to 4.500 daltons. These forms are not recognized by the standard intact PTH assays. The correlation of these findings to the clinical aspects of bone disease in ESRD patients remains to be studied. OBJETIVO: Estudar as formas carboxi-terminal (COOH) circulantes de paratormônio (PTH) em pacientes com hiperparatiroidismo devido à insuficiência renal crônica (IRC) terminal. MÉTODOS: Foi desenvolvido um ensaio imunométrico que reconhece formas intactas e COOH longas de PTH. Esse ensaio foi utilizado, em conjunto com um ensaio para molécula intacta de PTH, em amostras de 25 pacientes com hiperparatiroidismo devido à IRC. As amostras também foram submetidas à cromatografia de gel filtração em coluna de Superdex® 30 de 1,6 x 60 cm, e o conteúdo de PTH nos tubos de eluato foi medido, empregando-se os dois ensaios. RESULTADOS: Valores entre 39.000 e 232.300 ng/mL (média ± dp = 101,680 ± 45,330 ng/mL) foram obtidos usando-se o ensaio COOH (PTH 39-84 foi utilizado como padrão). Com o ensaio para PTH intacto, os valores distribuíram-se entre 318 e 3,307 ng/mL (1,769 ± 693 ng/mL) com correlação entre ambos de 0,462 (p = 0,02). O perfil cromatográfico obtido com o ensaio COOH mostrou predomínio de formas com PM entre 8.500 e 4.500 daltons. Os perfis cromatográficos dos 25 pacientes foram bastante semelhantes. CONCLUSÕES: Em pacientes com hiperparatiroidismo devido à IRC, os níveis circulantes de PTH contêm um espectro de formas moleculares que incluem formas carboxi-terminais, com PM entre 8.500 e 4.500 daltons. Essas formas não são reconhecidas pelos ensaios de rotina utilizados para a medida de PTH intacto. A correlação entre esses achados e os aspectos clínicos da doença óssea em pacientes com IRC necessita de maiores estudos.

UNLABELLED Osteoporosis-pseudoglioma (OPPG) is a rare syndrome characterized by severe osteoporosis and ocular defects caused by homozygotic inactivation mutations in the LRP5 gene. Bisphosphonate has been demonstrated to improve bone mineral density (BMD) in children with OPPG. We present here a 3 years follow-up of two brothers with OPPG carrying a novel mutation in the LRP5 gene, who were treated with intravenous pamidronate. PATIENT REPORT We looked for a mutation in the LRP5 gene in two brothers (12 and 4 years old) with clinical features of OPPG (blindness, low BMD and fragility fractures) and in their consanguineous parents to confirm the diagnosis of OPPG. The patients were treated with bisphosphonate for 3 years. They received 1 mg/kg/day of pamidronate for 2 consecutive days, every 3 months during the first year, and every 4 months in subsequent years. Calcium, phosphorus, total alkaline phosphatase, parathyroid hormone, hepatic transaminases, creatinine and hemogram tests were performed before each infusion. Bone densitometry was performed at baseline and at the end of the follow-up. RESULTS AND CONCLUSION The affected brothers carry a missense mutation in the third codon of exon 8 (AAT-->ATT) that led to the exchange of an asparagine for an isoleucine (N531I). Both parents were found to be heterozygous for this mutation. The intravenous pamidronate therapy was safe for up to 3 years of use. Moreover, increased BMD and decreased fracture rate were observed in our patients with OPPG.

BACKGROUND: The hereditary form of medullary thyroid carcinoma may occur isolated as a familial medullary thyroid carcinoma (FMTC) or as part of Multiple Endocrine Neoplasia 2A (MEN2A) and 2B (MEN2B). MEN2B is a rare syndrome, its phenotype may usually, but not always, be noted by the physician. In the infant none of the MEN2B characteristics are present, except by early gastrointestinal dysfunction caused by intestinal neuromas. When available, genetic analysis confirms the diagnosis and guides pre-operative evaluation and extent of surgery. Here we report four cases of MEN2B in which the late diagnosis had a significant impact in clinical evolution and, potentially, in overall survival. CASE REPORT: We report four cases, 2 men and 2 women, with differences in their phenotypes and with a late diagnosis. The first case has a history of severe gastrointestinal obstruction requiring a surgery intervention two days after his birth. The second told had nodules in the oral mucosa and constipation since childhood. The third case referred a history of constipation from birth until 5 months of life. The fourth has had a history of chronic constipation since childhood. DISCUSSION: New concepts have emerged since the RET oncogene was identified in 1993 as the responsible gene for hereditary medullary thyroid carcinoma. The majority of MEN2B individuals have M918T mutation in the exon 16 of RET, with a few cases having a mutation A883F or the association of V804M with E805K, Y806C or S904C mutations. The consensus classifies the RET mutation in codon 918 as of highest risk and recommends total thyroidectomy and central lymph node dissection until 6 months after birth. A fast and precise diagnosis is essential to reach these goals. The identification of early manifestations such as intestinal ganglioneuromatosis and oral mucosal neuromas should prompt the physician to initiate an investigation for multiple endocrine neoplasia type 2B. CONCLUSION: The diagnosis of MEN2B is very important to allow appropriate investigation of associated diseases and to allow counseling and appropriate screening of relatives for a RET mutation. Even patients with MEN2B, which often have typical physical features, may not be properly recognized and be followed as a sporadic case. Based on this, all suspicious cases of multiple endocrine neoplasia should undergo a molecular genetic test. A forma hereditária do carcinoma medular da tiróide pode ocorrer de modo isolado, o carcinoma medular da tiróide familiar (FMTC), ou como parte das neoplasias endócrinas múltiplas tipo 2A (MEN2A) e 2B (MEN2B). MEN2B é uma síndrome rara e seu fenótipo é usualmente, mas nem sempre, notado pelo médico. Na infância, nenhuma das características de MEN2B estão presentes, exceto pela disfunção gastrintestinal precoce, causada pelos neuromas intestinais. Quando disponível, a análise genética confirma o diagnóstico e orienta a avaliação pré-operatória e extensão da cirurgia. Neste artigo, apresentamos quatro casos de MEN2B, nos quais o diagnóstico tardio teve impacto significativo na evolução clínica e, potencialmente, na mortalidade em geral. APRESENTAÇÃO DOS CASOS: Apresentamos quatro casos, dois homens e duas mulheres, com diferenças em seus fenotipos e com diagnóstico tardio. O primeiro caso tem história de obstrução gastrintestinal importante em que foi necessária cirurgia dois dias após o nascimento. O segundo paciente apresentava nódulos na mucosa oral e constipação desde a infância. O terceiro referia história de constipação desde o nascimento até 5 meses de idade. O quarto tinha história de constipação intestinal desde a infância. DISCUSSÃO: Novos conceitos emergiram desde que o oncogene RET foi identificado, em 1993, como o gene responsável pelo carcinoma medular da tiróide hereditário. A maioria dos indivíduos apresenta a mutação M918T no éxon 16 do RET, enquanto poucos casos apresentam a mutação A883F ou a associação de V804M com E805K, Y806C ou S904C. O consenso recomenda a tiroidectomia total com dissecção dos linfonodos no compartimento central até os 6 meses após o nascimento. O diagnóstico rápido e preciso é essencial para o atingir os objetivos. CONCLUSÃO: O diagnóstico precoce de MEN2B é muito importante para propiciar a investigação apropriada de doenças associadas e para permitir aconselhamento e rastreamento dos parentes para uma mutação do RET. Pacientes com MEN2B, que apresentam frequentemente achados típicos ao exame físico, podem não ser reconhecidos e seguidos como casos esporádicos. Por causa disso, todos os casos de neoplasia endócrina múltipla devem ser avaliados pelo teste genético para mutações do RET.

In this study we developed a semi-automated method for the measurement of urinary iodine using firstly ammonium persulfate for digestion of urine followed by estimation of iodine content in the Sandell-Kolthoff reaction, in which iodine acts as a catalyst for the reduction of cerium. This method was validated in the 3rd Brazilian National Survey of iodine deficiency in 1994. We studied 16,803 casual urine samples from schoolchildren of 401 cities and found 4 moderately-deficient towns (Almas, Arraias, and Parana, in the State of Tocantins, and Cocos, in the State of Bahia), and 116 mildly-deficient. This work suggests that despite the salt iodization program, there was some iodine-deficient areas in Brazil in 1994. Recent surveys, involving less cities, are indicating an excess of iodine ingestion. Therefore, in a country of continental dimensions and very heterogeneous in terms of public health, periodical evaluations are necessary to monitor the real situation of iodine nutrition in Brazil. The method developed in this paper is suitable for these surveys.

O metabolismo do PTH e complexo e as formas circulantes incluem o PTH 1-84, assim como fragmentos C-terminal. A primeira geração de ensaios para o PTH incluía vários ensaios competitivos com especificidades para as regiões carboxi, meio da molécula e amino-terminal. A limitação destes ensaios e a evolução metodológica, levaram ao desenvolvimento dos ensaios não competitivos de 2ª. geração no final dos anos 80, baseados no reconhecimento por dois anticorpos diferentes, contra a porção amino e carboxi-terminal respectivamente. A observação que em algumas circunstâncias segmentos carboxiterminais longos também eram detectados, levou ao desenvolvimento dos ensaios de 3ª. geração, baseados em anticorpos específicos para a porção aminoterminal com maior especificidade para os primeiros aminoácidos, e assim mensurando apenas a forma molecular que ativa o PTH1R. As vantagens práticas e o custo-benefício deste ensaio ainda e motivo de debate. A observação recente de que fragmentos carboxiterminais têm atividade biológica via receptor distinto, aponta para a necessidade futura de mais de um ensaio para avaliar a função do paratormônio. PTH metabolism is complex and the circulating forms include the intact 1-84 molecule as well as several carboxyl-terminal fragments. The first generation of PTH assays included several types of competitive assays, with specificities that spanned carboxyl, mid-region and amino-terminal portions of the molecule. The limitations of these assays and the methodological evolution led to the description of 2nd generation non-competitive immunometric assays for PTH in the late 80's, based on the recognition of the PTH molecule by two different antibodies, one directed against de amino-terminal and other against the carboxyl-terminal segments. The observation that in some circumstances long carboxyl-terminal segments were also measured by 2nd generation assays led to the development of 3rd generation assays based on amino-terminal specific antibodies that are specific for the first amino acids, measuring only the molecular forms that activate PTH1R. The practical and cost-benefit advantages of these assays are still debatable. The recent observation that carboxyl-terminal fragments of PTH have biological activity via a distinct receptor than PTH1R, points to the future need of more than one assay in order to evaluate parathyroid hormone function. Laboratorio Fleury Universidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM) Division of Endocrinology UNIFESP, EPM, Division of Endocrinology SciELO

The lack of vitamin D is a major changeable factor involved in the pathophysiology of osteoporosis. Since the major source for this hormone is its cutaneous synthesis via ultraviolet radiation (UVR), we studied the serum level of 25-hydroxyvitamin D (25OHD) in 250 free-living elderly people (79.1 years old) from a subtropical region according to the UVR incidence and its correlations with parathormone (PTH) and ionized calcium. UVR and 25OHD differed according to the season of the year (P

Introduction of 2nd generation immunometric assays for the measurement of serum parathyroid hormone (PTH), turned them more available, simple and rapid. These methods, based on double identification of the PTH molecule, supposedly measure the intact, bioactive molecule, with the sequence 1-84. Recent works showed that they also measure forms with amino-terminal deletions, like the 7-84 form, which are not able to activate the traditional PTH receptor (PTH1R). Thus, an important practical aspect is the definition of the PTH forms measured by the immunometric assays, a fact that depends on the specificity of the antibodies employed. In this report we compare the results obtained with an in-house immunofluorometric assay that presents a cross-reactivity of 50% with the 7-84 PTH sequence, and two commercial 2nd generation assays, that react 100%. In a first study, 135 samples were measured using our assay and an electrochemiluminescent assay, resulting in a correlation coefficient of 0.961 (P0.0001) and medians of 35.0 and 51.0 ng/L (P0.0001). In a second study, 252 samples were analyzed using our assay and an immunochemiluminometric assay, resulting in a correlation of 0.883 (P0.0001) and medians of 36.0 and 45.5 ng/L (P0.0001). In both studies results obtained with the in-house assay were significantly lower, as expected by the specificity of the anti-amino-terminal antibody employed. Our data support the need of a precise description of the specificity of the amino-terminal antibodies employed in 2nd generation PTH assays in order to better compare results and define normal ranges.

A introdução de ensaios imunométricos (EIM) de 2ª geração, tornaram a medida de paratormônio (PTH) sérico mais disponível, simples e rápida, aumentando sua utilização. Esses métodos, baseados em dupla identificação da molécula de PTH, mediriam supostamente a molécula intacta, bioativa, de seqüência 1-84. Recentes trabalhos mostraram que eles também medem formas com deleções amino-terminais, como a forma 7-84, que não ativam o receptor tradicional de PTH (PTH1R). Em função disto, um aspecto prático importante é a definição das formas de PTH medidas pelos EIM, sendo que estas dependem da especificidade dos anticorpos empregados. Neste trabalho, comparamos um ensaio imunofluorométrico por nós desenvolvido, que apresenta reatividade cruzada de 50% com a seqüência 7-84 do PTH, com dois ensaios comerciais de 2ª geração, que reagem 100%. Numa 1ª. comparação, 135 amostras de soro foram dosadas com o nosso ensaio e com um ensaio eletroquimioluminescente, obtendo-se uma correlação de 0,961 (P

Amiodarone, a drug extensively used as an antiarrhythmic agent, contains 37% iodine and causes several thyroid abnormalities. The transplacental passage of amiodarone occurs with chronic therapy; we describe in this report the outcome of 9 pregnant women who used amiodarone (200 mg/day) for treatment of resistant tachycardia and the follow-up of their newborns. All women were clinically euthyroid at the 3rd trimester and showed expected values of thyroid hormones (mean +/- SD: total T4, 228 +/- 45 nmol/L; total T3, 4.0 +/- 0.65 nmol/L; TSH, 4.0 +/- 1.8 mU/L; negative thyroid antibodies). At birth all newborns were normal on routine examination with no goiter or corneal changes. T4 and TSH, measured on dried umbilical blood spots were normal or borderline-normal in 8 of 9 babies. Only 1 neonate presented clearly abnormal values of T4 and TSH (96 mU/L); on clinical grounds the baby was normal, without signs of hypothyroidism. At 1 month of life, T4 and TSH were normal. Follow-ups at 3, 6, and 12 months were normal. We conclude that is not necessary to discontinue treatment with amiodarone in pregnant women with resistant tachycardia, but it is imperative to evaluate the thyroid function of the newborn, since transient hypothyroidism may occur.