RET 634 germline/gonadal mosaicism generating a second pathogenic amino acid change in multiple endocrine neoplasia type 2A.

Genetic testing for germline RET pathogenic variants, which cause the Multiple Endocrine Neoplasia Type 2 (MEN2) syndrome, has become crucial in managing patients with medullary thyroid carcinoma (MTC). Classically, RET heterozygous missense pathogenic variants are transmitted in a Mendelian autosomal dominant pattern, of which germline/gonadal mosaicism has never been reported. We report the novel occurrence of a MEN2A patient's family in which the siblings inherited three different RET 634 genotypes: wild type (p.Cys634), p.Cys634Gly or p.Cys634Arg heterozygous pathogenic variants. We hypothesized that germline/gonadal mosaicism, derived from an inherited + early somatic mutation in the mother or a double de novo mutation during maternal embryogenesis, led to this rare event in the RET gene. Exome analysis of the proband's deceased mother's paraffin-embedded thyroid tissue confirmed the three nucleotides in the same 634 codon position. For the first time, we describe germline/gonadal mosaicism in RET, generating a second pathogenic amino acid change in the same codon causing MEN2A. Our finding shows that RET parental mosaicism, confirmed by somatic exome sequencing, might explain discrepant genotype cases in siblings with inherited cancers.

Spontaneous fertility and variable spectrum of reproductive phenotype in a family with adult-onset X-linked adrenal insufficiency harboring a novel DAX-1/NR0B1 mutation.

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.

Whole genome and exome sequencing realignment supports the assignment of KCNJ12, KCNJ17, and KCNJ18 paralogous genes in thyrotoxic periodic paralysis locus: functional characterization of two polymorphic Kir2.6 isoforms.

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.

The benefits of a high-intensity aquatic exercise program (HydrOS) for bone metabolism and bone mass of postmenopausal women.

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.

Intelligence Quotient Variability in Klinefelter Syndrome Is Associated With GTPBP6 Expression Under Regulation of X-Chromosome Inactivation Pattern.

Klinefelter syndrome (KS) displays a broad dysmorphological, endocrinological, and neuropsychological clinical spectrum. We hypothesized that the neurocognitive dysfunction present in KS relies on an imbalance in X-chromosome gene expression. Thus, the X-chromosome inactivation (XCI) pattern and neurocognitive X-linked gene expression were tested and correlated with intelligence quotient (IQ) scores. We evaluated 11 KS patients by (a) IQ assessment, (b) analyzing the XCI patterns using both HUMARA and ZDHHC15 gene assays, and (c) blood RT-qPCR to investigate seven X-linked genes related to neurocognitive development (GTPBP6, EIF2S3, ITM2A, HUWE1, KDM5C, GDI1, and VAMP7) and XIST in comparison with 14 (male and female) controls. Considering IQ 80 as the standard minimum reference, we verified that the variability in IQ scores in KS patients seemed to be associated with the XCI pattern. Seven individuals in the KS group presented a random X-inactivation (RXI) and lower average IQ than the four individuals who presented a skewed X-inactivation (SXI) pattern. The evaluation of gene expression showed higher GTPBP6 expression in KS patients with RXI than in controls (p = 0.0059). Interestingly, the expression of GTPBP6 in KS patients with SXI did not differ from that observed in controls. Therefore, our data suggest for the first time that GTPBP6 expression is negatively associated with full-scale IQ under the regulation of the type of XCI pattern. The SXI pattern may regulate GTPBP6 expression, thereby dampening the impairment in cognitive performance and playing a role in intelligence variability in individuals with KS, which warrants further mechanistic investigations.

Increases in summer serum 25-hydroxyvitamin D (25OHD) concentrations in elderly subjects in São Paulo, Brazil vary with age, gender and ethnicity.

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 (chi(2)) 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.

Genotype and phenotype landscape of MEN2 in 554 medullary thyroid cancer patients: the BrasMEN study.

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.

Three years follow-up of pamidronate therapy in two brothers with osteoporosis-pseudoglioma syndrome (OPPG) carrying an LRP5 mutation.

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.

Assessing the clinical and molecular diagnosis of inherited forms of impaired sensitivity to thyroid hormone from a single tertiary center.

AIM: 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. SUBJECTS AND METHODS: 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 (TRß) (non-TR-RTH) were investigated for albumin gene (ALB) mutation. RESULTS: Seventeen patients presented mutations in TRß (RTHß); 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 RTHß 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. CONCLUSION: In the present series, most cases were RTHß 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.

A pioneering RET genetic screening study in the State of Ceará, Brazil, evaluating patients with medullary thyroid cancer and at-risk relatives: experience with 247 individuals.

OBJECTIVE: Initial diagnosis of medullary thyroid carcinoma (MTC) is frequently associated with advanced stages and a poor prognosis. Thus, the need for earlier diagnoses and detection in relatives at risk for the disease has led to increased use of RET genetic screening. SUBJECTS AND METHODS: We performed RET screening in 247 subjects who were referred to the Brazilian Research Consortium for Multiple Endocrine Neoplasia (BRASMEN) Center in the State of Ceará. Direct genetic sequencing was used to analyze exons 8, 10, 11, and 13-16 in MTC index cases and specific exons in at risk relatives. Afterward, clinical follow-up was offered to all the patients with MTC and their affected relatives. RESULTS: RET screening was performed in 60 MTC index patients and 187 at-risk family members. At the initial clinical assessment of the index patients, 54 (90%) were diagnosed with apparently sporadic disease and 6 (10%) diagnosed with hereditary disease. After RET screening, we found that 31 (52%) index patients had sporadic disease, and 29 (48%) had hereditary disease. Regarding at-risk relatives, 73/187 were mutation carriers. Mutations in RET codon 804 and the rare p.M918V mutation were the most prevalent. CONCLUSIONS: Performing RET screening in Ceará allowed us to identify a different mutation profile in this region compared with other areas. RET screening also enabled the diagnosis of a significant number of hereditary MTC patients who were initially classified as sporadic disease patients and benefited their relatives, who were unaware of the risks and the consequences of bearing a RET mutation.

[Development of a semi-automated method for measuring urinary iodine and its application in epidemiological studies in Brazilian schoolchildren]. / Desenvolvimento de um método para a determinação da iodúria e sua aplicação na excreção urinária de iodo em escolares brasileiros.

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.

Down-regulation of Kir2.6 channel by c-termini mutation D252N and its association with the susceptibility to Thyrotoxic Periodic Paralysis.

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.

Novel lincRNA Susceptibility Gene and Its Role in Etiopathogenesis of Thyrotoxic Periodic Paralysis.

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.

Evidence for the founder effect of RET533 as the common Greek and Brazilian ancestor spreading multiple endocrine neoplasia 2A.

OBJECTIVES: About one-quarter of patients with medullary thyroid cancer (MTC) have inherited disease due to mutations in the RET gene. A rare mutation in exon 8 (G533C) of RET, 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 METHODS: Twelve RET G533C mutation carriers, four randomly selected from the Brazilian cohort and eight from apparently unrelated Greek families, were studied for a possible common ancestral origin. RET flanking microsatellite markers at chromosome 10q (D10S197, D10S196, D10S1652 and D10S537) were used. RESULTS: Genomic DNA analysis using these markers showed that many of these apparently unrelated individuals shared a common haplotype indicating a common ancestral origin. CONCLUSION: Our data suggest that Brazilian and Greek patients with MTC carrying the G533C mutation in exon 8 of RET gene 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.

Analysis of somatic mutations in BRAF, CDKN2A/p16 and PI3KCA in patients with medullary thyroid carcinoma.

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 paraffin­embedded 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.

M918V RET mutation causes familial medullary thyroid carcinoma: study of 8 affected kindreds.

Germline mutations in codon 918 of exon 16 of the RET gene (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 M918V RET mutation 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 M918V RET mutation. 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 the RET M918V 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.

Macrocalcitonin Is a Novel Pitfall in the Routine of Serum Calcitonin Immunoassay.

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.

Low iodine diet does not improve the efficacy of radioiodine for the treatment of Graves' disease.

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 with 131I. 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 of 131I for the treatment of GD.

[Parathyroid hormone values obtained with immunometric assays depend on the amino-terminal antibody specificity]. / Valores de paratormônio obtidos com ensaios imunométricos dependem da especificidade do anticorpo amino terminal empregado.

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 (P<0.0001) and medians of 35.0 and 51.0 ng/L (P<0.0001). In a second study, 252 samples were analyzed using our assay and an immunochemiluminometric assay, resulting in a correlation of 0.883 (P<0.0001) and medians of 36.0 and 45.5 ng/L (P<0.0001). 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.

The insulin-sensitivity sulphonylurea receptor variant is associated with thyrotoxic paralysis.

Thyrotoxicosis is the most common cause of the acquired flaccid muscle paralysis in adults called thyrotoxic periodic paralysis (TPP) and is characterised by transient hypokalaemia and hypophosphataemia under high thyroid hormone levels that is frequently precipitated by carbohydrate load. The sulphonylurea receptor 1 (SUR1 (ABCC8)) is an essential regulatory subunit of the ß-cell ATP-sensitive K(+) channel that controls insulin secretion after feeding. Additionally, the SUR1 Ala1369Ser variant appears to be associated with insulin sensitivity. We examined the ABCC8 gene at the single nucleotide level using PCR-restriction fragment length polymorphism (RFLP) analysis to determine its allelic variant frequency and calculated the frequency of the Ala1369Ser C-allele variant in a cohort of 36 Brazilian TPP patients in comparison with 32 controls presenting with thyrotoxicosis without paralysis (TWP). We verified that the frequency of the alanine 1369 C-allele was significantly higher in TPP patients than in TWP patients (61.1 vs 34.4%, odds ratio (OR)=3.42, P=0.039) and was significantly more common than the minor allele frequency observed in the general population from the 1000 Genomes database (61.1 vs 29.0%, OR=4.87, P<0.005). Additionally, the C-allele frequency was similar between TWP patients and the general population (34.4 vs 29%, OR=1.42, P=0.325). We have demonstrated that SUR1 alanine 1369 variant is associated with allelic susceptibility to TPP. We suggest that the hyperinsulinaemia that is observed in TPP may be linked to the ATP-sensitive K(+)/SUR1 alanine variant and, therefore, contribute to the major feedforward precipitating factors in the pathophysiology of TPP.