No correspondence between resistance mutations in the HCV-NS3 protease at baseline and early telaprevir-based triple therapy.

Direct-acting antiviral (DAA)-based therapy is the new standard treatment for chronic hepatitis C virus (HCV) infection. However, protease inhibitor (PI)-resistant viral variants have been often described. This study aimed to examine HCV-NS3 protease variants at baseline and at 4 weeks under triple therapy. To this end, we analyzed the presence of variants in HCV-NS3 protease region from peripheral blood samples of 16 patients infected with HCV-1 at baseline and at 4 weeks of combined therapy with telaprevir, pegylated interferon, and ribavirin, using next-generation sequencing. Several variants with synonymous and non-synonymous amino acid substitutions were detected at both time points. Variants detected at low frequency corresponded to 74% (HCV-1a) and 35% (HCV-1b) of non-synonymous substitutions. We found nine PI-resistance-associated variants (V36A, T54S, V55I, Q80K, Q80R, V107I, I132V, D168E, M175L) in HCV-NS3 of 10 patients. There was no correspondence of resistance-associated variant profile between baseline and at 4 weeks. Moreover, these resistance variants at baseline and short-term treatment are not good predictors of outcome under triple therapy. Our study also shows a large number of others minor and major non-synonymous variants in HCV-NS3 early in telaprevir-based therapy that can be important for further drug resistance association studies with newly developed PI agents.

Polymorphisms in IL-10 and INF-γ genes are associated with early atherosclerosis in coronary but not in carotid arteries: A study of 122 autopsy cases of young adults.

Atherosclerosis is a complex disease, involving both genetic and environmental factors. However, the influence of genetic variations on its early development remains unclear. This study examined the association of 12 different polymorphisms with atherosclerosis severity in anterior descending coronary (DA, n = 103) and carotid arteries (CA, n = 66) of autopsied young adults (< 30 years old). Histological sections (H-E) were classified according to the American Heart Association. Polymorphisms in ACE, TNF-α (- 308G/A and - 238 G/A), IFN-γ (+ 874 A/T), MMP-9 (- 1562 C/T), IL-10 (- 1082 A/G and - 819 C/T), NOS3 (894 G/T), ApoA1 (rs964184), ApoE (E2E3E4 isoforms), and TGF-ß (codons 25 and 10) genes were genotyped by gel electrophoresis or automatic DNA sequencing. Firearm projectile or car accident was the main cause of death, and no information about classical risk factors was available. Histological analysis showed high prevalence of type III atherosclerotic lesions in both DA (69%) and CA (39%) arteries, while severe type IV and V lesions were observed in 14% (DA) and 33% (CA). Allele frequencies and genotype distributions were determined. Among the polymorphisms studied, IFN-γ and IL-10 (- 1082 A/G) were related to atherosclerosis severity in DA artery. No association between genotypes and lesion severity was found in CA. In conclusion, we observed that the high prevalence of early atherosclerosis in young adults is associated with IFN-γ (p < 0.001) and IL-10 (p = 0.013) genotypes. This association is blood vessel dependent. Our findings suggest that the vascular system presents site specialization, and specific genetic variations may provide future biomarkers for early disease identification.

Evidences for the involvement of cell surface glycans in stem cell pluripotency and differentiation.

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development, several issues regarding their pluripotency, differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study, we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that, upon spontaneous differentiation, iPS-MBMC present high amounts of terminal ß-galactopyranoside residues, pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation, prompting an ectoderm commitment. Exposed ß-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface, which could modulate intercellular or intracellular interactions. Together, our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and, therefore, the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation.

AT1 and aldosterone receptors blockade prevents the chronic effect of nandrolone on the exercise-induced cardioprotection in perfused rat heart subjected to ischemia and reperfusion.

PURPOSE: Myocardial tolerance to ischaemia/reperfusion (I/R) injury is improved by exercise training, but this cardioprotection is impaired by the chronic use of anabolic androgenic steroids (AAS). The present study evaluated whether blockade of angiotensin II receptor (AT1-R) with losartan and aldosterone receptor (mineralocorticoid receptor, MR) with spironolactone could prevent the deleterious effect of AAS on the exercise-induced cardioprotection. METHODS AND RESULTS: Male Wistar rats were exercised and treated with either vehicle, nandrolone decanoate (10 mg/kg/week i.m.) or the same dose of nandrolone plus losartan or spironolactone (20 mg/kg/day orally) for 8 weeks. Langendorff-perfused hearts were subjected to I/R and evaluated for the postischaemic recovery of left ventricle (LV) function and infarct size. mRNA and protein expression of angiotensin II type 1 receptor (AT1-R), mineralocorticoid receptor (MR), and KATP channels were determined by reverse-transcriptase polymerase chain reaction and Western blotting. Postischaemic recovery of LV function was better and infarct size was smaller in the exercised rat hearts than in the sedentary rat hearts. Nandrolone impaired the exercise-induced cardioprotection, but this effect was prevented by losartan (AT1-R antagonist) and spironolactone (MR antagonist) treatments. Myocardial AT1-R and MR expression levels were increased, and the expression of the KATP channel subunits SUR2a and Kir6.1 was decreased and Kir6.2 increased in the nandrolone-treated rat hearts. The nandrolone-induced changes of AT1-R, MR, and KATP subunits expression was normalized by the losartan and spironolactone treatments. CONCLUSION: The chronic nandrolone treatment impairs the exercise-induced cardioprotection against ischaemia/reperfusion injury by activating the cardiac renin-angiotensin-aldosterone system and downregulating KATP channel expression.

The heat shock proteins of Trypanosoma cruzi.

Trypanosoma cruzi is the causal agent of Chagas' disease, a debilitating disorder affecting millions of people in several countries. A flagellated protozoan parasite, T. cruzi has a complex life cycle that involves infecting an insect and a mammalian host. During its life cycle, the parasite undergoes several kinds of stress, prominent among which is heat stress. To deal with this environmental challenge, molecular chaperones and proteases, also known as heat shock proteins (HSPs), are induced as part of the stress response. Several families of HSPs are synthesized by T. cruzi, including members of the major HSP classes such as HSP70, HSP90, HSP100, HSP40, chaperonins and small HSPs, and these proteins show conserved and unique features. In this review we describe these proteins and the corresponding gene expression patterns and discuss their relevance to the biology of the parasite.

Interleukin 28B-related polymorphisms: a pathway for understanding hepatitis C virus infection?

AIM: To analyze the role of rs12979860 and rs8099917 polymorphisms in hepatitis C virus (HCV) genotype 1 infection of Brazilians. METHODS: A total of 145 adult patients diagnosed with genotype 1 chronic hepatitis C (CHC) who had completed a 48-wk regimen of pegylated-interferon α-2a or -2b plus ribavirin combination therapy were recruited from six large urban healthcare centers and 199 healthy blood donors (controls) from a single site between January 2010 and January 2012. Data on the patients' response to treatment was collected. Polymerase chain reaction-restriction fragment length polymorphism genotyping of the interleukin (IL)28B gene fragment encompassing the single nucleotide polymorphisms (SNPs) rs12979860 (C/T) and rs8099917 (T/G) was carried out for 79 of the CHC patients and 199 of the controls. Bi-directional amplicon sequencing of the two SNPs was carried out for the remaining 66 CHC patients. RESULTS: SNP rs12979860 genotyping was successful in 99.5% of the controls and 97.2% of the CHC patients, whereas the SNP rs8099917 genotyping was successful in 95.5% of the controls and 100% of the CHC patients. The genotype and allele distributions for both rs12979860 and rs8099917 were significantly different between the control and CHC patient groups, with significantly higher genotype frequencies of CC and TT in the controls (P = 0.037 and 0.046, respectively) and of TT and GG in the CHC patients (P = 0.0009 and 0.0001, respectively). Analysis of the CHC patients who achieved sustained virological response (SVR) to treatment (n = 55) indicated that the rs12979860 C allele and CC genotype were predictors of SVR (P = 0.02). No significant correlation was found between rs8099917 genotypes and treatment response, but carriers of the T allele showed significantly higher rates of SVR (P = 0.02). Linkage disequilibrium analysis of the group that achieved SVR showed a significant association between rs12979860 and rs8099917 (P = 0.07). CONCLUSION: The higher allele frequency of rs12979860 C and rs8099917 T observed in non-HCV-infected individuals may indicate a potential protective role for these IL28B-related polymorphisms.

Dynamics of resistance mutations to NS3 protease inhibitors in a cohort of Brazilian patients chronically infected with hepatitis C virus (genotype 1) treated with pegylated interferon and ribavirin: a prospective longitudinal study.

UNLABELLED: About sixty thousand new cases of Hepatitis C virus (HCV) infection are recorded in Brazil each year. These cases are currently treated with pegylated interferon (PEG-IFN) and ribavirin (RBV) with an overall success rate of 50%. New compounds for anti-HCV therapy targeted to the HCV NS3 protease are being developed and some already form the components of licensed therapies. Mapping NS3 protease resistance mutations to protease inhibitors or anti-viral drug candidates is important to direct anti-HCV drug treatment. METHODS: Sequence analysis of the HCV NS3 protease was conducted in a group of 68 chronically infected patients harboring the HCV genotype 1. The patients were sampled before, during and after a course of PEG-IFN-RBV treatment. RESULTS: Resistance mutations to the protease inhibitors, Boceprevir and Telaprevir were identified in HCV isolated from three patients (4.4%); the viral sequences contained at least one of the following mutations: V36L, T54S and V55A. In one sustained virological responder, the T54S mutation appeared during the course of PEG-IFN and RBV therapy. In contrast, V36L and V55A mutations were identified in virus isolated from one relapsing patient before, during, and after treatment, whereas the T54S mutation was identified in virus isolated from one non-responding patient, before and during the treatment course. CONCLUSIONS: The incidence and persistence of protease resistance mutations occurring in HCV from chronically infected patients in Brazil should be considered when using protease inhibitors to treat HCV disease. In addition, patients treated with the current therapy (PEG-IFN and RBV) that are relapsing or are non-responders should be considered candidates for protease inhibitor therapy.

Sexual dimorphism of thyroid reactive oxygen species production due to higher NADPH oxidase 4 expression in female thyroid glands.

BACKGROUND: Dual oxidases (DUOX1 and DUOX2) are NADPH oxidases (NOX) involved in hydrogen peroxide production necessary for thyroid hormonogenesis, but recently, the NOX4 has also been described in the thyroid gland. The prevalence of thyroid disease is higher in women, and the basis for this difference might involve a higher oxidative stress level in the female thyroid gland. Hence, we aimed at evaluating whether the function and the expression of enzymes involved in the thyroid redox balance differ between females and males. METHODS: DUOX1, DUOX2, NOX4, glutathione peroxidase (GPx), and catalase activities and expression levels were evaluated in the thyroids of prepubertal and adult male and female rats. The mRNA levels of DUOXA1 and DUOXA2, the DUOX maturation factors, and of p22phox and Poldip2 (subunits of NOX4) were also determined. RESULTS: A higher calcium-independent H(2)O(2) production was detected in the adult female rat thyroid, being higher in the estrous phase of the cycle. Moreover, the expression of NOX4 and Poldip2 mRNA was higher in the thyroids of adult female rats, as well as in PCCL3 cells treated with 17ß-estradiol. The GPx1 mRNA expression was higher in adult female thyroids, while GPx2 and GPx3 mRNA and total GPx activity were not significantly different. Catalase mRNA expression and activity, together with thyroid thiol levels were significantly lower in the adult female rat thyroid. CONCLUSIONS: Taken together, our results show that the thyroid gland of female rats is exposed to higher oxidative stress levels due both to increased reactive oxygen species (ROS) production through NOX4, and decreased ROS degradation.

A single nucleotide polymorphism, rs129679860, in the IL28B locus is associated with the viral kinetics and a sustained virological response in a chronic, monoinfected hepatitis C virus genotype-1 Brazilian population treated with pegylated interferon-ribavirin.

Single nucleotide polymorphisms (SNPs) in the interleukin (IL)28B locus have been associated with a sustained virological response (SVR) in interferon-ribavirin (IFN-RBV)-treated chronic hepatitis C virus (HCV)-infected patients in European and African populations. In this study, the genotype frequency of two IL28B SNPs (rs129679860 and rs8099917) in a cohort of chronic HCV-monoinfected patients in Brazil was evaluated and the SNP sufficient to predict the treatment response outcome was determined. A total of 66 naïve genotype-1 chronic HCV-infected patients were genotyped and the associated viral kinetics and SVR were assessed. The overall SVR was 38%. Both the viral kinetics and SVR were associated with rs129679860 genotypes (CC = 62% vs. CT = 33% vs. TT = 18%, p = 0.016). However, rs8099917 genotypes were only associated with SVR (TT = 53% vs. TG = 33% vs. GG = 18%; p = 0.032). In this population, the analysis of a single SNP, rs12979860, successfully predicts SVR in the IFN-RBV treatment of HCV.

A single nucleotide polymorphism, rs129679860, in the IL28B locus is associated with the viral kinetics and a sustained virological response in a chronic, monoinfected hepatitis C virus genotype-1 Brazilian population treated with pegylated interferon-ribavirin

Single nucleotide polymorphisms (SNPs) in the interleukin (IL)28B locus have been associated with a sustained virological response (SVR) in interferon-ribavirin (IFN-RBV)-treated chronic hepatitis C virus (HCV)-infected patients in European and African populations. In this study, the genotype frequency of two IL28B SNPs (rs129679860 and rs8099917) in a cohort of chronic HCV-monoinfected patients in Brazil was evaluated and the SNP sufficient to predict the treatment response outcome was determined. A total of 66 naïve genotype-1 chronic HCV-infected patients were genotyped and the associated viral kinetics and SVR were assessed. The overall SVR was 38%. Both the viral kinetics and SVR were associated with rs129679860 genotypes (CC = 62% vs. CT = 33% vs. TT = 18%, p = 0.016). However, rs8099917 genotypes were only associated with SVR (TT = 53% vs. TG = 33% vs. GG = 18%; p = 0.032). In this population, the analysis of a single SNP, rs12979860, successfully predicts SVR in the IFN-RBV treatment of HCV.

Association of IL-10, IL-4, and IL-28B gene polymorphisms with spontaneous clearance of hepatitis C virus in a population from Rio de Janeiro.

BACKGROUND: Cytokines play an important role in the regulation of the immune response. In hepatitis C virus (HCV) infection, cytokine levels may influence the outcome of acute HCV infection. Polymorphisms in cytokine genes have been associated to different expression levels in response to infection. This study was carried out to investigate the association of several cytokine gene polymorphisms with disease outcome in HCV-infected patients. FINDINGS: Patients with chronic or spontaneously resolved HCV infection were included in a cross-sectional study. A comparative analysis was performed between the groups regarding frequency distribution of the following cytokines' gene polymorphisms: IL-10 (-1082 A/G; -819 T/C; -592 A/C), IL-4 (+33C/T), IFN-γ (+874 T/A), TNF-α (-238 G/A and -308 G/A) and IL-28B (rs12979860 C/T and rs8099917 T/G). RESULTS: Eighteen patients with spontaneous viral clearance and 161 with chronic HCV infection were included. In the comparative analysis, the GG genotype of the IL-10 polymorphism -1082A/G was more frequent in patients with spontaneous viral clearance when compared to patients with chronic HCV (41.2% vs 6.2%; p = 0.001). This association was also found for the CC genotype of the IL-4 polymorphism +33C/T (72.2% vs 36.7%; p = 0.017) and the CC and TT genotypes of the IL-28B polymorphisms rs 12979860 and rs 8099917 (88.9% vs 30.3%; p < 0.001 and 88.9% vs 49.6%; p = 0.002). The IL10 (A-1082 G) and IL-28B (Crs12979860T) gene polymorphisms showed odds ratios of 12.848 and 11.077, respectively, and thus may have a greater influence on HCV spontaneous viral clearance. The IFN-γ (+874 T/A), TNF-α (-238 G/A and -308 G/A) polymorphisms did not show significant association with spontaneous viral clearance or chronicity. CONCLUSION: The G allele for IL-10 (-1082 A/G), the C allele for IL-4 (+3 C/T) and the C and T alleles for IL-28B (rs12979860 and rs8099917, respectively) are associated with spontaneous viral clearance in hepatitis C infection.

Polymorphisms upstream of the melanocortin-1 receptor coding region are associated with human pigmentation variation in a Brazilian population.

OBJECTIVE: We describe an association of two SNPs, rs3212345:C>T and rs3212346:G>A, located approximately 2.5 kb upstream of the melanocortin-1 receptor (MC1R) translation initiation codon, with pigmentation phenotype variation in a Southeast Brazilian miscegenated population. METHODS: One hundred thirty-eight genetically unrelated subjects, with multicolor phenotype, were selected from the southeast region of Brazil. Skin, hair and eye color, and tanning ability were rated. Genotypes for each SNP (rs3212345:C>T and rs3212346:G>A) were determined. A logistic regression analysis was performed with the additive model to determine which of the polymorphisms contributed to a specific phenotype. RESULTS: We found that the rs3212345:C>T is associated with light skin, red hair, and poor tanning ability, while the rs3212346:G>A is associated with dark skin, black hair, and strong tanning ability. The presence of rs3212345-C and rs3212346-A alleles in human, chimpanzee, gorilla, orangutan, and marmoset genomes suggests that they are the ancestral alleles. CONCLUSION: These data suggest that the rs3212345-T and rs3212346-G alleles may have contributed to lighter pigmentation phenotypes in modern humans. Genotyping for these SNPs may prove useful to the fields of molecular anthropology and forensic genetics.

Human menstrual blood-derived mesenchymal cells as a cell source of rapid and efficient nuclear reprogramming.

Induced pluripotent stem cells (iPSCs) were originally generated by forced ectopic expression of four transcription factors genes-OCT4, KLF4, SOX2, and c-MYC-in fibroblasts. However, the efficiency of iPSCs obtention is extremely low, and reprogramming takes about 20 days. We reasoned that adult cells showing basal expression of core embryonic stem (ES) cell regulator genes could be a better cell source for reprogramming. Menstrual blood-derived mesenchymal cells (MBMCs) are multipotent cells that show detectable levels of some of the core ES cells regulators. The aim of this study was to determine whether reprogramming efficiency could be increased by using MBMCs as a cell source to generate iPSCs. MBMCs were transduced with recombinant retroviruses expressing the coding regions of OCT4, SOX2, and KLF4 genes. Cells with high nucleus/cytoplasm ratio can be detected about 5 days of posttransduction, and colonies of typical ES-like cells begun to appear after 7 days. At day 15, colonies were picked up and expanded for characterization. Most of the clones were morphologically identical to ES cells and positive at the mRNA and protein levels for all pluripotency markers tested. The clones are capable of forming embryoid bodies and to differentiate in vitro into cells of the three germ cell layers. Our results show that the reprogramming was faster and with efficiency around 2-5%, even in the absence of ectopic expression of c-MYC. To date, this is the first study showing MBMCs as a cell source for nuclear reprogramming.

Polimorfismo do gene da enzima conversora de angiotensina e avaliação cardiovascular em adultos com deficiência do hormônio do crescimento / Angiotensin converting enzyme gene polymorphisms and cardiovascular evaluation in growth hormone deficient adults

Fundamentos: A enzima conversora da angiotensina (ECA) é importante reguladora da pressão arterial (PA). Polimorfismos no gene da ECA estão associados a alterações na PA. Não existem ainda estudos sobre areposição de hormônio do crescimento (GH) em adultos com deficiência do hormônio do crescimento (DGH) deacordo com os genótipos da ECA.Objetivo: Avaliar a resposta cardiovascular ao GH em adultos com DGH de acordo com o seu genótipo da ECA. Métodos: Avaliados 18 pacientes com hipopituitarismo de acordo com o genótipo da ECA no basal e 24 mesesapós reposição com GH de acordo com parâmetros clínicos e cardiovasculares.Resultados: Dez mulheres e 8 homens foram avaliados (média de idade 44,9±10,9 anos). Distribuição genotípicaencontrada: genótipo DD: 7 (38,9%) pacientes; genótipo ID: 11 (61,1%) pacientes. Frequência cardíaca, PA sistólica e diastólica, carga pressórica sistólica e diastólica, e funções sistólica e diastólica foram normais em todos ospacientes. Na avaliação basal, pacientes com genótipo DD demonstraram níveis de PA sistólica, diastólica diurna e nas 24 horas mais altos (p<0,05); carga pressórica diastólica maior (p<0,05). Comparando-se os dois genótipos ao final do estudo, os pacientes com genótipo DD evidenciaram: redução estatisticamente significativa da pressão diastólica diurna e nas 24 horas, da carga pressórica sistólica e diastólica diurna e nas 24 horas; e aumento da frequência cardíaca noturna (p<0,005).Conclusão: Os resultados sugerem que pacientes com DGH e genótipo DD apresentam maiores benefícios coma reposição com GH em relação ao controle da PA.

Cysteine proteases in differentiation of embryonic stem cells into neural cells.

Glycosylated mouse cystatin C (mCysC), an endogenous inhibitor of cysteine cathepsin proteases (CP), has been suggested as a cofactor of ß-FGF to induce the differentiation of mouse embryonic stem cells into neural progenitor cells (NPCs). To investigate the possible role of CP in neural differentiation, we treated embryoid bodies (EBs) with (i) E64, an inhibitor of papain-like CP and of calpains, (ii) an inhibitor of cathepsin L (iCatL), (iii) an inhibitor of calpains (iCalp), or (iv) cystatins, and their ability to differentiate into neural cells was assessed. We show that the inhibition of CP induces a significant increase in Pax6 expression in EBs, leading to an increase in the number of nestin-positive cells after 3 days. Fourteen days after E64 treatment, we observed increased numbers of ß-III-tubulin-positive cells, showing greater percentage of immature neurons, and this feature persisted up to 24 days. At this point, we encountered higher numbers of neurons with inward Na(+) current compared with untreated EBs. Further, we show that mCysC and iCatL, but not unglycosylated egg white cystatin or iCalp, increased the numbers of NPCs. In contrast to E64 and iCatL, mCysC did not inhibit CP in EBs and its neural-inducing activity required ß-FGF. We propose that the inhibition of CP induces the differentiation of mouse embryonic stem cells into NPCs and neurons through a mechanism that is distinct from CysC-induced neural differentiation.

Investigação de variantes gênicas de canais iônicos em pacientes com síndrome do QT longo / Investigation of ion channel gene variants in patients with long QT syndrome / Investigación de variantes génicas de canales iónicos en pacientes con síndrome del QT largo

FUNDAMENTO: A síndrome do QT longo (SQTL) é uma síndrome arrítmica herdada com aumento do intervalo QT e risco de morte súbita. Mutações nos genes KCNQ1, KCNH2 e SCN5A respondem por 90 por cento dos casos com genótipo determinado, e a genotipagem é informativa para aconselhamento genético e melhor manejo da doença. OBJETIVO: Investigação molecular e análise computacional de variantes gênicas de KCNQ1, KCNH2 e SCN5A associadas à SQTL em famílias portadoras da doença. MÉTODOS: As regiões codificantes dos genes KCNQ1, KCNH2 e SCN5A de pacientes com SQTL e familiares foram sequenciadas e analisadas utilizando o software Geneious ProTM. RESULTADOS: Foram investigadas duas famílias com critérios clínicos para SQTL. A probanda da Família A apresentava QTC = 562 ms, Escore de Schwartz = 5,5. A genotipagem identificou a mutação G1714A no gene KCNH2. Foi observado QTC = 521 ± 42 ms nos familiares portadores da mutação contra QTC = 391 ± 21 ms de não portadores. A probanda da Família B apresentava QTc = 551 ms, Escore de Schwartz = 5. A genotipagem identificou a mutação G1600T, no mesmo gene. A análise dos familiares revelou QTC = 497 ± 42 ms nos portadores da mutação, contra QTC = 404 ± 29 ms nos não portadores. CONCLUSÃO: Foram encontradas duas variantes gênicas previamente associadas à SQTL em duas famílias com diagnóstico clínico de SQTL. Em todos os familiares portadores das mutações foi observado o prolongamento do intervalo QT. Foi desenvolvida uma estratégia para identificação de variantes dos genes KCNQ1, KCNH2 e SCN5A, possibilitando o treinamento de pessoal técnico para futura aplicação na rotina diagnóstica.

BACKGROUND: The long QT syndrome (LQTS) is an inherited arrhythmia syndrome with increased QT interval and risk of sudden death. Mutations in genes KCNQ1, KCNH2 and SCN5A account for 90 percent of cases with genotype determined, and genotyping is informative for genetic counseling and better disease management. OBJECTIVE: Molecular investigation and computational analysis of gene variants of KCNQ1, KCNH2 and SCN5A associated with LQTS, in families with the disease. METHODS: The coding regions of genes KCNQ1, KCNH2 and SCN5A in patients with LQTS and their family members were sequenced and analyzed using Geneious ProTM software. RESULTS: Two families with clinical criteria for LQTS were investigated. The proband of Family A had QTC = 562 ms, Schwartz Score = 5.5. The genotyping identified the G1714A mutation in the KCNH2 gene. QTC = 521 ± 42 ms was observed in family members carrying the mutation against QTC = 391 ± 21 ms for non-carriers. The proband of Family B had QTc = 551 ms, Schwartz Score = 5.5. The genotyping identified the G1600T mutation, in the same gene. The analysis of family members revealed QTC = 497 ± 42 ms in mutation carriers, compared with QTC = 404 ± 29 ms in non-carriers. CONCLUSION: Two gene variants previously associated with LQTS were found in two families clinically diagnosed with LQTS. The prolongation of the QT interval was observed in all family members carrying the mutations. A strategy was developed to identify variants of genes KCNQ1, KCNH2 and SCN5A, making it possible to train technical staff for future application to diagnosis routine.

FUNDAMENTO: El síndrome del QT largo (SQTL) es un síndrome arrítmico heredado con aumento del intervalo QT y riesgo de muerte súbita. Mutaciones en los genes KCNQ1, KCNH2 y SCN5A responden por 90 por ciento de los casos con genotipo determinado, y el genotipaje es informativo para aconsejamiento genético y mejor manejo de la enfermedad. OBJETIVO: Investigación molecular y análisis computacional de variantes génicas de KCNQ1, KCNH2 y SCN5A asociadas a la SQTL en familias portadoras de la enfermedad. MÉTODOS: Las regiones codificantes de los genes KCNQ1, KCNH2 y SCN5A de pacientes con SQTL y familiares fueron secuenciadas y analizadas utilizando el software Geneious Pro®. RESULTADOS: Fueron investigadas dos familias con criterios clínicos para SQTL. La probanda de la Familia A presentaba QT C = 562 ms, Escore de Schwartz = 5,5. El genotipaje identificó la mutación G1714A en el gen KCNH2. Fue observado QT C = 521 ± 42 ms en los familiares portadores de la mutación contra QT C = 391 ± 21 ms de no portadores. La probanda de la Familia B presentaba QT C = 551 ms, Escore de Schwartz = 5. El genotipaje identificó la mutación G1600T, en el mismo gen. El análisis de los familiares reveló QT C = 497 ± 42 ms en los portadores de la mutación, contra QT C = 404 ± 29 ms en los no portadores. CONCLUSIÓN: Fueron encontradas dos variantes génicas previamente asociadas a la SQTL en dos familias con diagnóstico clínico de SQTL. En todos los familiares portadores de las mutaciones fue observada la prolongación del intervalo QT. Fue desarrollada una estrategia para identificación de variantes de los genes KCNQ1, KCNH2 y SCN5A, posibilitando el entrenamiento de personal técnico para futura aplicación en la rutina diagnóstica.

Investigation of ion channel gene variants in patients with long QT syndrome.

BACKGROUND: The long QT syndrome (LQTS) is an inherited arrhythmia syndrome with increased QT interval and risk of sudden death. Mutations in genes KCNQ1, KCNH2 and SCN5A account for 90% of cases with genotype determined, and genotyping is informative for genetic counseling and better disease management. OBJECTIVE: Molecular investigation and computational analysis of gene variants of KCNQ1, KCNH2 and SCN5A associated with LQTS, in families with the disease. METHODS: The coding regions of genes KCNQ1, KCNH2 and SCN5A in patients with LQTS and their family members were sequenced and analyzed using Geneious ProTM software. RESULTS: Two families with clinical criteria for LQTS were investigated. The proband of Family A had QTC = 562 ms, Schwartz Score = 5.5. The genotyping identified the G1714A mutation in the KCNH2 gene. QTC = 521 ± 42 ms was observed in family members carrying the mutation against QTC = 391 ± 21 ms for non-carriers. The proband of Family B had QTc = 551 ms, Schwartz Score = 5.5. The genotyping identified the G1600T mutation, in the same gene. The analysis of family members revealed QTC = 497 ± 42 ms in mutation carriers, compared with QTC = 404 ± 29 ms in non-carriers. CONCLUSION: Two gene variants previously associated with LQTS were found in two families clinically diagnosed with LQTS. The prolongation of the QT interval was observed in all family members carrying the mutations. A strategy was developed to identify variants of genes KCNQ1, KCNH2 and SCN5A, making it possible to train technical staff for future application to diagnosis routine.

Chronic treatment with anabolic steroids induces ventricular repolarization disturbances: cellular, ionic and molecular mechanism.

The illicit use of supraphysiological doses of androgenic steroids (AAS) has been suggested as a cause of arrhythmia in athletes. The objectives of the present study were to investigate the time-course and the cellular, ionic and molecular processes underlying ventricular repolarization in rats chronically treated with AAS. Male Wistar rats were treated weekly for 8 weeks with 10mg/kg of nandrolone decanoate (DECA n=21) or vehicle (control n=20). ECG was recorded weekly. Action potential (AP) and transient outward potassium current (I(to)) were recorded in rat hearts. Expression of KChIP2, Kv1.4, Kv4.2, and Kv4.3 was assessed by real-time PCR. Hematoxylin/eosin and Picrosirius red staining were used for histological analysis. QTc was greater in the DECA group. After DECA treatment the left, but not right, ventricle showed a longer AP duration than did the control. I(to) current densities were 47.5% lower in the left but not in the right ventricle after DECA. In the right ventricle the I(to) inactivation time-course was slower than in the control group. After DECA the left ventricle showed lower KChIP2 ( approximately 26%), Kv1.4 ( approximately 23%) and 4.3 ( approximately 70%) expression while the Kv 4.2 increased in 4 ( approximately 250%) and diminished in 3 ( approximately 30%) animals of this group. In the right ventricle the expression of I(to) subunits was similar between the treatment and control groups. DECA-treated hearts had 25% fewer nuclei and greater nuclei diameters in both ventricles. Our results strongly suggest that supraphysiological doses of AAS induce morphological remodeling in both ventricles. However, the electrical remodeling was mainly observed in the left ventricle.

Trypanosoma cruzi: modulation of HSP70 mRNA stability by untranslated regions during heat shock.

Gene regulation in trypanosomatids occurs mainly by post-transcriptional mechanisms modulating mRNA stability and translation. We have investigated heat shock protein (HSP) 70 gene regulation in Trypanosoma cruzi, the causal agent of Chagas' disease. The HSP70 mRNA's half-life increases after heat shock, and the stabilization is dependent on protein synthesis. In a cell-free RNA decay assay, a U-rich region in the 3' untranslated region (UTR) is a target for degradation, which is reduced when in the presence of protein extracts from heat shocked cells. In a transfected reporter gene assay, both the 5'- and 3'-UTRs confer temperature-dependent regulation. Both UTRs must be present to increase mRNA stability at 37 degrees C, indicating that the 5'- and 3'-UTRs act cooperatively to stabilize HSP70 mRNA during heat shock. We conclude that HSP70 5'- and 3'-UTRs regulate mRNA stability during heat shock in T. cruzi.

Voltage-dependent calcium and chloride currents in S17 bone marrow stromal cell line.

The bone marrow stromal cell line S17 has been used to study hematopoiesis in vitro. In this study, we demonstrate the presence of calcium and chloride currents in cultured S17 cells. Calcium currents were of low amplitude or barely detectable (50-100 pA). Hence to amplify the currents, we have used barium as a charge carrier. Barium currents were identified based on their distinct voltage-dependence, and sensitivity to dihydropyridines. S17 cells also exhibited a slowly activating outward current without inactivation, most commonly seen when the sodium of the extracellular solution was replaced either by TEA (TEA/Cs saline) or NMDG (NMDG saline), or by addition of amiloride to the extracellular solution. This current was abolished either by 500 microM SITS (4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid) or 500 microM DPC (diphenylamine-2-carboxylic acid) a cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel blocker, identifying it as a Cl(-) current. RT-PCR identified the presence of ENaC and CFTR transcripts. CFTR blockade reduced cell proliferation, suggesting that this channel plays a physiological role in regulation of S17 cell proliferation.