Henrique Ravanhol Frigeri

Polymorphisms in FTO and TCF7L2 genes of Euro-Brazilian women with gestational diabetes

OBJECTIVE To investigate the association between fat mass and obesity-associated (FTO) gene polymorphisms rs8050136C>A and rs9939609T>A, and transcription factor 7-like 2 (TCF7L2) gene polymorphisms rs12255372G>T and rs7903146C>T, in a sample group of pregnant Euro-Brazilian women with or without gestational diabetes mellitus (GDM). METHODS Subjects were classified as either healthy pregnant control (n=200) or GDM (n=200) according to the 2010 criteria of the American Diabetes Association. The polymorphisms were genotyped using fluorescent probes (TaqMan®). RESULTS All groups were in the Hardy-Weinberg equilibrium. The genotype and allele frequencies of the examined polymorphisms did not exhibit significant difference (P>0.05) between the groups. In the healthy and GDM pregnant women groups, the A-allele frequencies (95% CI) of FTO polymorphisms rs8050136 and rs9939609 were 39% (34-44%); 38% (33-43%) and 40% (35-45%); 41% (36-46%), respectively; and the T-allele frequencies of TCF7L2 polymorphisms rs12255372 and rs7903146 were 30% (26-35%), 32% (27-37%) and 29% (25-34%), 36% (31-41%), respectively. CONCLUSION The examined polymorphisms were not associated with GDM in the Euro-Brazilian population studied.

ATP-dependent potassium channels and type 2 diabetes mellitus

Diabetes mellitus is a public health problem, which affects a millions worldwide. Most diabetes cases are classified as type 2 diabetes mellitus, which is highly associated with obesity. Type 2 diabetes is considered a multifactorial disorder, with both environmental and genetic factors contributing to its development. An important issue linked with diabetes development is the failure of the insulin releasing mechanism involving abnormal activity of the ATP-dependent potassium channel, KATP. This channel is a transmembrane protein encoded by the KCNJ11 and ABCC8 genes. Furthermore, polymorphisms in these genes have been linked to type 2 diabetes because of the role of KATP in insulin release. While several genetic variations have been reported to be associated with this disease, the E23K polymorphism is most commonly associated with this pathology, as well as to obesity. Here, we review the molecular genetics of the potassium channel and discusses its most described polymorphisms and their associations with type 2 diabetes mellitus.

The functional polymorphisms -429T > c and -374T > a of the RAGE gene promoter are not associated with gestational diabetes in Euro-Brazilians.

The receptor for advanced glycation end products (RAGE or AGER) is a multiligand member of the immunoglobulin superfamily. RAGE is expressed in several tissues, including human myometrium, chorionic villi and placenta. Advanced glycation end products are the best studied ligands of RAGE; they have pro-inflammatory actions in human gestational tissues, increasing oxidative stress and the release of cytokines and prostaglandins. We investigated the association of RAGE gene promoter polymorphisms -429T>C (rs1800625) and -374T>A (rs1800624) with gestational diabetes. A sample of 750 unrelated European origin pregnant Brazilian women were classified as nondiabetic (control group, N = 600) or having gestational diabetes (N = 150) according to American Diabetes Association 2009 criteria. Genotyping was performed by PCR-RFLP. The frequencies of the rare alleles -429C (6.3 versus 9.1%) and -374A (26 versus 30%) were not significantly different between the gestational diabetes patients and healthy pregnant women. Also, the -429T>C and -374T>A polymorphisms were not associated with body mass index, lipid profile, fasting glycemia, HbA1C, or insulin requirement. We found that functional promoter polymorphisms of the RAGE gene were not associated with gestational diabetes or its complications in these Euro-Brazilian patients.

Low prevalence of glucokinase gene mutations in gestational diabetic patients with good glycemic control.

Glucokinase (GCK) plays a key role in glucose homeostasis. Gestational diabetes mellitus increases the risk of gestational complications in pregnant women and fetuses. We screened for mutations in coding and flanking regions of the GCK gene in pregnant women with or without gestational diabetes in a Brazilian population. A sample of 200 pregnant women classified as healthy (control, N = 100) or with gestational diabetes (N = 100) was analyzed for mutations in the GCK gene. All gestational diabetes mellitus patients had good glycemic control maintained by diet alone and no complications during pregnancy. Mutations were detected by single-strand conformation polymorphism and DNA sequencing. Thirteen of the 200 subjects had GCK gene mutations. The mutations detected were in intron 3 (c.43331A>G, new), intron 6 (c.47702T>C, rs2268574), intron 9 (c.48935C>T, rs2908274), and exon 10 (c.49620G>A, rs13306388). None of these GCK mutations were found to be significantly associated with gestational diabetes mellitus. In summary, we report a low frequency of GCK mutations in a pregnant Brazilian population and describe a new intronic variation (c.43331A>G, intron 3). We conclude that mutations in GCK introns and in non-translatable regions of the GCK gene do not affect glycemic control and are not correlated with gestational diabetes mellitus.

The polymorphism rs2268574 in Glucokinase gene is associated with gestational Diabetes mellitus.

Dear Editor:Glucokinase (GCK, EC 2.7.1.2) catalyzes the conversion of glucose toglucose-6-phosphate, the first step in glucose metabolism. GCK is con-sidered a glucose sensor in the pancreatic β-cells [1]. The Glucokinasegene (GCK gene; HGNC:4195) is located in chromosome 7 (7p15.3-p15.1). More than 600 mutations in the GCK gene have been reportedand about 2–5% of all Caucasian gestational Diabetes mellitus (GDM)cases are due to Single Nucleotide Polymorphisms (SNPs) in this gene[2]. In previous study, we showed that GDM patients with good glyce-mic control are associated with a low prevalence of GCK mutations de-tected with PCR-SSCP [3]. We focus now on patients that requiresinsulinuse(poorglycemiccontrol)afterGDMdiagnosticsandanalyzedby DNA sequencing, the exons and introns 5 and 6 of the GCK gene forpolymorphisms, in a case–control study. This selected region encodesaminoacids(Exon5:T168,K169;Exon6:N204,D205)forglucosebind-ing in the Glucokinase active site. Healthy Euro-Brazilian pregnantwomen (Control, n = 115) and patients with gestational Diabetesmellitus (GDM, n = 112) were classified according to the American Di-abetesAssociationcriteria[4].Patientswithovertdiabeteswereexclud-ed. The Ethics Committee on Human Research of our institutionapproved this study. PCR (PF: 5′-TCTGAGCCTGTTTCCTCAGC-3′ and PR:5 ′-GGCCCTTGAAGCCTGTTGTA-3 ; 505 bp amplicon) for exons 5 and 6,and flanking regions were performed as described elsewhere [5]. Allsamplesweresequenced(BigDye,3500XL,AppliedBiosystems).Allse-quences showed base-calling quality of more than Q30. The SNPs wereidentified and aligned using CodonCode Alligner v.4.1.1 (CodonCodeCorporation), BlastSNP and Reference SNP database (http://www.ncbi.nlm.nih.gov/).TheSNPgenotypewasnotassociatedwithanthropomet-ric and laboratory parameters (regression analysis, data not shown)in either group. No difference was observed in genotype distribution(P = 0.054). However, a significant difference (P = 0.034) and OddsRatio (CI 95%) 1.5 (1.03–2.17) regarding minor allele (T)wasobservedin the studied population (Table 1). The T-allele frequency observed inhealthy pregnant women (48%) was similar to that for Caucasians(~46%) and higher than for Orientals (~35%) and Sub-Saharan African(~27%) according to HapMap (http://www.hapmap.org/). Although nolinkage disequilibrium for the rs2268574 polymorphism, compared toothers, SNPs in the GCK gene was identified (HaploView v4.2, Day Lab,Cambridge, USA), we observed the rs2268574T N C is located in a puta-tive splicing region (Human Splicing Finder v.2.4.1; http://www.umd.be/HSF/). We hypothesize that the SNP could affect gene transcriptionor more plausibly, it could be in linkage disequilibrium with some

Leptin (rs7799039) and solute carrier family 30 zinc transporter (rs13266634) polymorphisms in Euro-Brazilian pregnant women with gestational diabetes

Leptin (LEP), a protein that plays a fundamental role in the metabolism of energy reserves, and the solute carrier family 30 A8 zinc transporter (SLC30A8) have been consistently associated with diabetes. Women with gestational diabetes are at moderate risk of developing diabetes type 1 and 2 after pregnancy, in addition to complications to the fetus. We investigated the association of the polymorphisms rs7799039 (LEP) and rs13266634 (SLC30A8) in a case-control study in Euro-Brazilians with gestational diabetes (GDM, N = 134) and healthy pregnant women (control, N = 180). Real-time PCR with fluorescent probes (TaqMan system) was applied to genotyping. All polymorphisms were in Hardy-Weinberg equilibrium. The minor allele frequencies, for healthy and GDM, respectively, for the A-allele (LEP gene rs7799039) were 40.3% (95%CI = 35-45%) vs 36.6% (95%CI = 31-42%), P = 0.345; and for the T-allele (SLC30A8 gene rs13266634) were 27.8% (95%CI = 23-32%) vs 23.5% (95%CI = 18-29%), P = 0.227. Genotype comparisons for both polymorphisms showed no significant difference (P > 0.05). The polymorphisms rs7799039 and rs13266634 were not associated with GDM in the population studied (P > 0.05). The minor allele frequencies for both polymorphisms were similar to those of other Caucasian populations.

Von Willebrand Disease Lab Diagnosis.

AbstractThe hemorrhagic diseases are characterized by bleeding which can vary considerably according to their severity. The von Willebrand disease (VWD) is the most frequent hereditary hemorrhagic disease and the prevalence of clinically significant disease is probably closer to 1:1000, being an extremely heterogeneous and complex disorder that is related to the deficiency in concentration, structure or function of von Willebrand factor (VWF). The VWD is divided into type 1, with partial deficiency of the VWF, type 2, with qualitative defects in the molecule with four subdivisions, and type 3, with very low or undetectable levels of plasma and platelet VWF and ristocetin cofactor activity. The laboratory diagnosis of VWD is complex. Specific tests that assess the functionality and concentrations of the VWF and FVIII are needed. The routine tests are the bleeding time, the activated partial thromboplastin time and the platelet count, however, singly, they may not suggest the diagnosis of VWD, requiring further specific tests, such as VWF function evaluation through its ristocetin cofactor assay (VWF:RCo), VWF protein concentration immunoassay (VWF:Ag), the factor VIII coagulation assay (FVIII:C), VWF binding to immobilized collagen (VWF:CB), ristocetin-induced platelet aggregation (RIPA), VWF multimers patterns, factor VIII binding of immobilized VWF (VWF:FVIIIB), among others. From the moment the diagnosis is confirmed, the appropriate treatment for each patient is sought, with the purpose of increasing plasma concentrations of the deficient protein, both in bleeding episodes, as for invasive procedures. Although diagnosis facilitates treatment other approach in the present scenario is prenatal diagnosis which, is the need of the hour.

Specific Obesity-related Adipokines

Obesity is defined as abnormal or excessive fat accumulation that presents a risk to health [1]. The most important associated elements of this tissue are the fatty acids, however the adipocytes might secrete immunological components, such as prostaglandins. In the last 12 years, the endocrine role of the adipose tissue by its ability to secrete molecules with biological effects has also been discovered. Furthermore, this tissue was considered, by many researchers, as a dynamic organ involved in a variety of metabolic and physiological processes [2]. Apart from that, adipocytes might secrete a variety of bioactive peptides called adipokines. An alteration on adipokines secretion can be caused by excessive growth of adipose tissue, which can leads to adipocyte hypertrophy [3].

Avaliação de Valores de Normalidade da Contagem de Reticulócitos Utilizando o Contador Hematológico Cell-Dyn 3500

A Pimenta Preta (Piper nigrun L.), conhecida popularmente como Pimenta-do-Reino, pertence a familia Piperaceae, e trepadeira, arbustifera perene, originaria das regioes tropicais da India, e que foi introduzida no Brasil pelos escravos na epoca da colonizacao. Suas sementes sao utilizadas ha seculos como condimento no preparo de alimentos, e na medicina popular, em preparacoes do tipo cataplasmas, pomadas e cremes para o tratamento de diversos agravos na saude, dada suas acoes terapeuticas sob o organismo. O presente estudo teve por objetivo investigar, por meio de uma revisao de literatura, as acoes biologicas da Piper nigrun L. A literatura pesquisada evidenciou que os principios ativos encontrados nesta especie, especialmente a piperina, possui acao anti-inflamatoria, antioxidante, analgesica dentre outras capazes de otimizar processos cicatriciais, circulacao sanguinea, bem como combater os sinais do envelhecimento ocasionados por radicais livres quando consumida em quantidades apropriadas. Observou-se tambem, que esta planta pode ser rica em vitaminas, minerais e outros compostos; pode ser alimento benefico para o bom funcionamento e desenvolvimento do organismo humano. A literatura mostrou ser bastante escassa no que diz respeito a publicacao de estudo sobre as acoes biologicas desta planta que nao a antiinflamatoria e antioxidante, assim como sobre os efeitos terapeuticos e nutricionais, tanto no tratamento como na prevencao de agravos na saude. Ao termino do estudo concluiu-se que e importante que se amplie pesquisas e estudos experimentais a respeito da acao biologica desta planta.

Correlation and comparison of immunohistochemistry for HER2/neu, using the antibody SP3 and chromogenic in situ hybridization in breast carcinomas samples

Introducao: Avancos no campo da biologia molecular tem proporcionado a diferenciacao dos subtipos moleculares das neoplasias mamarias, fornecendo melhor prognostico e ferramentas importantes para a terapeutica de pacientes com câncer de mama. Entre esses subtipos, as alteracoes ocorridas no gene receptor tipo 2 do fator de crescimento epidermico humano (HER2/neu) amplificam o seu numero de copias e geram o aumento da proteina HER2. Estudos mostram que pacientes portadoras de câncer de mama HER2/neu amplificado tendem a ter recaida mais cedo e tempo de sobrevida menor, sendo o anticorpo monoclonal Trastuzumab a terapia indicada. A elegibilidade das pacientes para a terapia e feita inicialmente pela tecnica de imuno-histoquimica (IHQ), que avalia o nivel de expressao da proteina HER2. Apos essa avaliacao, os casos que apresentam diagnosticos equivocos (escore 2+) sao encaminhados para uma tecnica mais precisa, a hibridizacao cromogena in situ (CISH). Objetivo: Analisar a sensibilidade e a especificidade do anticorpo SP3, alem de determinar o seu nivel de concordância com a tecnica de CISH. Material e metodos: Estudo retrospectivo no banco de dados de um laboratorio anatomopatologico, em laudos de exames de CISH para HER2/neu. Conclusao: Os resultados revelaram que o clone SP3 apresentou 100% de especificidade e 92% de sensibilidade. A IHQ revela variabilidade em seus resultados, porem e sabido que a tecnica e uma importante ferramenta na rotina diaria dos laboratorios, contribuindo na triagem inicial das pacientes portadoras de câncer de mama, que, posteriormente, mostram resultados satisfatorios quando comparados com a tecnica de CISH.