Variabilidade genética e sequenciamento de genes associados ao Diabetes mellitus tipo 2 e à obesidade

Orientador : Prof. Dr. Geraldo PichethCoorientadores: Profa. Dra. Fabiane G. de M. Rego. Profa. Dra. Dayane AlbertonTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba, 18/09/2015Inclui referências : f. 114-128Área de concentração: Análises clínicasResumo: O Diabetes mellitus tipo 2 (T2DM) e o Diabetes mellitus gestacional (GDM) estão associados à obesidade (Ob) e são processos patológicos de frequência crescentes no mundo, apresentando elevada morbimortalidade. Múltiplos fatores genéticos estão associados ao T2DM e GDM, embora sejam pouco estudados na população brasileira. Estudar variações genéticas e biomarcadores laboratoriais associados ao risco ou à proteção para o T2DM e GDM, caracteriza o objetivo do presente estudo. O Comitê de Ética e Pesquisa da Universidade Federal do Paraná (UFPR) aprovou esta pesquisa, CAAE: 01038012.2.0000.0102. Os parâmetros antropométricos e laboratoriais (perfis glicêmico e lipídico, bem como de função renal) foram igualmente avaliados. Os polimorfismos foram investigados com sondas fluorescentes (TaqMan®) ou por sequenciamento de DNA (Big Dye). Um estudo com gestantes incluiu 115 saudáveis (CTRL) e 112 portadoras de GDM e o polimorfismo rs2268574:T>C do gene da glucoquinase (GCK). O polimorfismo rs2268574:T>C foi associado à proteção para o GDM (alelo-T, CTRL 48,3% vs GDM 38,4%; P=0,034), com razão de chance de 1,5 (95%IC, 1,03-2,17). Outro estudo envolveu 313 mulheres (idade ? 40 anos) classificadas em grupo controle saudável (CTRL, n=141), T2DM obesas (ObT2DM, n=130; IMC>30kg/m2) e obesas não portadoras de T2DM (Ob, n=42). Os genes e polimorfismos estudados foram: PPARg (rs1801282:C>G), IGF2BP2 (rs4402960:G>T), GCK (rs144723656:C>T, rs2268574:T>C, rs2268575:A>G), GCKR (rs780094:C>T), TCF7L2 (rs7901695:T>C), LEP (rs7799039:A>G), LEPR (rs1137100:A>G, rs1137101:A>G), SLC30A8 (rs13266634:C>T), FTO (rs8050136:A>C, rs1421085:T>C, rs9930506:A>G), e GHRL (rs73125661:A>G, rs57221010:A>C, rs1629816:C>T, rs10490815:A>G, rs27498:C>T, rs27647:G>A, rs26802:A>C, rs55821288:G>A, rs35679:G>A, rs35680:G>A, rs35682:C>T, rs4684677:A>T, rs35683:C>T). Os polimorfismos não foram associados aos grupos em estudo. O gene da preprogrelina (GHRL) foi sequenciado por completo em uma subamostra contendo 20 indivíduos; CTRL=5, ObT2DM=10 e Ob=5). Foram identificados 13 polimorfismos (rs73125661:A>G, rs57221010:A>C, rs1629816:C>T, rs10490815:A>G, rs27498:C>T, rs27647:G>A, rs26802:A>C, rs55821288:G>A, rs35679:G>A, rs35680:G>A, rs35682:C>T, rs4684677:A>T e rs35683:C>T) sem que um marcador molecular (Tagging SNP) para os processos patológicos fosse identificado. Todas as variáveis estudadas avaliando o diabetes tipo 2 e/ou obesidade não mostraram associação (P>0,05) com estes processos patológicos avaliados, assim como parâmetros antropométricos e laboratoriais. Após análises in sílico, as variantes do gene GCK rs144723656:C>T e rs2268574:T>C foram identificadas em uma sequência considerada como facilitadora do processamento do RNA (splicing), enquanto que a variante rs2268575:A>G do mesmo gene, em região com potencial de silenciar este processo. As frequências alélicas dos polimorfismos em estudo foram em geral similares às descritas em outras populações Caucasoides. Vários polimorfismos têm neste estudo sua primeira descrição quanto à suas frequências em uma amostra da população brasileira. Palavras-chave: Diabetes mellitus Gestacional, Diabetes mellitus tipo 2, obesidade, genes-alvo, variabilidade genética.Abstract: The Type 2 Diabetes mellitus (T2DM) and Gestational Diabetes mellitus (GDM) are associated with obesity (Ob) and are pathological processes of increasing frequency in the world, with high morbidity and mortality. Multiple genetic factors are associated with T2DM and GDM, although little of them are studied in the Brazilian population. The study of genetic variations and laboratory biomarkers associated with increased risk or protection for T2DM and GDM characterizes the aim of this study. The Ethics Committee of the Federal University of Paraná (UFPR) approved this research, CAAE search: 01038012.2.0000.0102. Anthropometric and laboratory parameters (glycemic and lipid profiles and kidney function) were assessed. The polymorphisms were investigated with fluorescent probes (TaqMan®) or by DNA sequencing (BigDye). The study included 115 healthy pregnant women (CTRL) and 112 women with GDM and the polymorphism rs2268574:T>C of the glucokinase gene (GCK). The polymorphism rs2268574:T>C was associated with protection for GDM (T-allele, CTRL 48.3% vs GDM 38.4%; P = 0.034), with an odds ratio of 1.5 (95% CI, 1.03 -2.17). The other study investigated 313 women (age ? 40 years) classified in healthy control group (CTRL, n = 141), obese T2DM (ObT2DM, n = 130; BMI> 30kg/m2) and obese non T2DM carriers (Ob, n = 42). The studied genes and polymorphisms were: PPAR (rs1801282C>G), IGF2BP2 (rs4402960:G>T), GCK (rs144723656:C>T, rs2268574:T>C, rs2268575:A>G), GCKR (rs780094:C>T), TCF7L2 (rs7901695:T>C), LEP (rs7799039:A>G), LEPR (rs1137100:A>G, rs1137101:A>G), SLC30A8 (rs13266634:C>T), FTO (rs8050136:A>C, rs1421085:T>C, rs9930506:A>G), and GHRL (rs73125661:A>G, rs57221010:A>C, rs1629816:C>T, rs10490815:A>G, rs27498:C>T, rs27647:G>A, rs26802:A>C, rs55821288:G>A, rs35679:G>A, rs35680:G>A, rs35682:C>T, rs4684677:A>T, rs35683:C>T) polymorphisms were not associated to the study groups. The preproghrelin gene (GHRL) was completely sequenced on a subsample composed by 20 subjects, CTRL = 5, ObT2DM = 10 and Ob = 5). In this gene were identified 13 polymorphisms (rs73125661:A>G, rs57221010:A>C, rs1629816:C>T, rs10490815:A>G, rs27498:C>T, rs27647:G>A, rs26802:A>C, rs55821288:G>A, rs35679:G>A, rs35680:G>A, rs35682:C>T, rs4684677:A>T and rs35683:C>T) without identification of a molecular marker (Tagging SNP) for the studied pathological processes. All variables evaluating type 2 diabetes and/or obesity were not associated (P>0.05) with the studied pathological processes, as well as anthropometric and laboratory parameters. After in silico analyzes, variants of GCK gene (rs144723656:C>T and rs2268574:T>C were identified in a sequence considered as a enhancer of RNA processing (splicing), while the variant rs2268575:A>G at the same gene, in a potential region to silence this process. The allelic frequencies of the polymorphisms in the study were generally similar to those described in other Caucasian populations. Several polymorphisms have first described as to their frequencies in a sample of the Brazilian population. Keywords: Gestational Diabetes mellitus, Type 2 Diabetes mellitus, obesity, target-genes, genetic variability

Luminol na ciência forense

In a crime scene, the collection of evidence and a subsequent laboratory analysis compose the fundamental steps to allow the expert to reveal the truth for the final verdict in a jury and to bring back the comfort to the victim’s family. Bloodstains are usually found and sent to laboratories as a vestige to unravel the origin of the material. However, some scenes are modified in order to conceal the real culprit for the criminal act. For these cases, the luminol reagent can be useful. This test is very often used to visualize occult blood. Luminol is considered the most sensitive test once it can identify the blood presence in scale of nanograms. When this reagent comes into contact with blood,the light emission occurs through a phenomenon known as  chemiluminescence. This luminescence can be produced by other interfering compounds, leading to a misinterpretation for the presence of blood. Despite this shortcoming, the present review article highlights the indispensability of the reagent luminol on a crime scene.Na cena do crime, a coleta de evidências e uma análise laboratorial subsequente compõem as etapas fundamentais para permitir que o especialista revele a verdade do veredicto final em um júri e traga de volta o conforto à família da vítima. As manchas de sangue são geralmente encontradas e enviadas aos laboratórios como um vestígio para desvendar a origem do material. No entanto, algumas cenas são modificadas para ocultar o verdadeiro culpado pelo ato criminoso. Para esses casos, o reagente luminol pode ser útil. Este teste é frequentemente usado para visualizar sangue oculto. O luminol é considerado o teste mais sensível, pois pode identificar a presença de sangue na escala de nanogramas. Quando este reagente entra em contato com o sangue, a emissão de luz ocorre através de um fenômeno conhecido como quimioluminescência. Essa luminescência pode ser produzida por outros compostos interferentes, levando a uma má interpretação da presença de sangue. Apesar dessa deficiência, o presente artigo de revisão destaca a indispensabilidade do luminol do reagente na cena do crime

Variabilidade do gene de Glucoquinase no diabetes gestacional

Orientador : Prof. Dr. Geraldo PichethCo-orientadores : Profa. Drª Fabiane G. de M. Rego, Prof. Dr. Emanuel M. de SouzaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba,18/02/2011Bibliografia: fls. 88-95Área de concentração: Insumos, medicamentos e correlatosResumo: O Diabetes mellitus Gestacional (DMG) e definido como qualquer grau de intolerancia a glucose com primeira deteccao durante a gravidez. A patologia tem prevalencia em cerca de 7% das gestacoes e pode promover complicacoes para gestante e feto. A Glucoquinase (GCK) e uma enzima que fosforila a glucose na posicao 6 e atua como sensor para liberacao de insulina do pancreas. Polimorfismos no gene da GCK estao associados ao diabetes monogenico do tipo MODY-2 (Maturity-Onset Diabetes of the Young Type 2). O objetivo deste trabalho foi procurar polimorfismos no gene da GCK em exons e suas regioes flanqueadoras. O estudo tem a aprovacao do Comite de Etica e Pesquisa da Universidade Federal do Parana (UFPR). Foram analisadas por PCR-SSCP (polimorfismo de conformacao de fita simples) e sequenciamento de DNA em 200 amostras de gestantes classificadas em grupo controle (Saudaveis, n=100, CTRL) e diabeticas gestacionais (DMG, n=100). Os grupos apresentaram idade media de 24,9„b6,2, 31,7„b6,3, respectivamente para CTRL e DMG. Enquanto o grupo CTRL foi composto apenas por Euro-Brasileiras, o grupo DMG contempla 10% de Afrodescendentes. As gestantes diabeticas apresentaram IMC superior ao grupo controle, respectivamente, 33,5„b6,3 e 25,2„b4,2. As concentracoes de glucose em jejum, perfil lipidico foram significativamente maiores no grupo com diabetes exceto para o LDL-colesterol que nao diferiu. As determinacoes de ureia e creatinina apontam que ambos os grupos nao demonstram evidencia de lesao renal. A analise molecular obtida de 14 amplicons que cobriram as regioes genicas em estudo mostrou variacoes, todas do tipo transicao, em quatro regioes intronica em 13 das amostras estudadas (6,5%). As variacoes identificadas foram: c.43441A>G (Intro3, nao descrita), c.44702T>C (Intron6, rs2268574), c.48935C>T (Intron9, rs2908274) e c.49620G>A (Exon10, regiao nao codificadora, rs13306388). As frequencias estimadas, bem como 95% de Intervalo de Confianca (95% IC) para os alelos raros das variantes descritas foram: alelo-G, 0,3% (0-1%); alelo-C, 3,3% (2-5%), alelo-T, 0,8% (0-2%) e alelo-A, 1% (0-2%), respectivamente para as variantes detectadas nos Introns4, 6, 9 e Exon10. Para nenhuma das variantes foi observado associacao com processos patologicos. Em sintese, o gene da Glucoquinase nao apresentou variante do tipo ¡§hotspot¡¨ associada com o diabetes gestacional na populacao em estudo e uma nova variante, no Intron3 (c.43441A>G), foi identificada no presente estudo.Abstract: Gestational Diabetes mellitus (GDM) is defined as any degree of glucose intolerance with first detection during pregnancy. The disease is prevalent in about 7% of pregnancies and may lead to complications for mother and fetus. Glucokinase (GCK) is an enzyme that phosphorylates glucose in position 6 and acts as a sensor for the release of insulin from the pancreas. GCK gene polymorphisms are associated with monogenic diabetes type MODY-2 (Maturity Onset Diabetes of the Young Type 2). The aim of this study was investigate for GCK gene polymorphisms in exons and their flanking regions. The study was approved by the Ethics Committee by Federal University of Parana (UFPR). Were analyzed by PCR-SSCP (single strand conformation polymorphism) and DNA sequencing 200 samples from pregnant women classified in control group (healthy, n=100, CTRL) and gestational diabetes (GDM, n=100). The groups had a mean age of 24.9„b6.2, 31.7„b6.3, respectively for CTRL and DMG. While the CTRL group was composed only of Euro-Brazilian, DMG group has 10% for Afro-descendants. Diabetic women had a BMI (kg/m2) greater than the control group, respectively, 33.5„b6.3 and 25.2„b4.2. The concentrations of fasting glucose and lipid levels were significantly higher in the group with diabetes except for LDL-cholesterol that did not differ. Measurements of urea and creatinine showed that in both groups there was no evidence of renal failure. Molecular analysis obtained from 14 amplicons that comprehends the genetic regions studied showed variations, all in transition form, in four noncoding regions in 13 of the studied samples (6.5%). The variations were identified: c.43441A> G (intron3, not described), c.44702T>C (Intron6, rs2268574), c.48935C>T (Intron9, rs2908274) and c.49620G>A (Exon10 noncoding region, rs13306388). Estimated frequencies and 95% confidence interval (95% CI) for the rare alleles of the variants described were: G-allele, 0.3% (0-1%), C-allele, 3.3% ( 2-5%), T-allele, 0.8% (0-2%) and allele-A, 1% (0-2%) respectively for the variants found in Introns 3, 6, 9 and Exon10. For none of the variants were observed association with disease processes. In summary, the Glucokinase gene variant did not show associated-"hotspot" variant with gestational diabetes in the study population and a new variant in Intron3 (c.43441A>G) was identified in this study

Variabilidade do gene de Glucoquinase no diabetes gestacional

Orientador : Prof. Dr. Geraldo PichethCo-orientadores : Profa. Drª Fabiane G. de M. Rego, Prof. Dr. Emanuel M. de SouzaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba,18/02/2011Bibliografia: fls. 88-95Área de concentração: Insumos, medicamentos e correlatosResumo: O Diabetes mellitus Gestacional (DMG) e definido como qualquer grau de intolerancia a glucose com primeira deteccao durante a gravidez. A patologia tem prevalencia em cerca de 7% das gestacoes e pode promover complicacoes para gestante e feto. A Glucoquinase (GCK) e uma enzima que fosforila a glucose na posicao 6 e atua como sensor para liberacao de insulina do pancreas. Polimorfismos no gene da GCK estao associados ao diabetes monogenico do tipo MODY-2 (Maturity-Onset Diabetes of the Young Type 2). O objetivo deste trabalho foi procurar polimorfismos no gene da GCK em exons e suas regioes flanqueadoras. O estudo tem a aprovacao do Comite de Etica e Pesquisa da Universidade Federal do Parana (UFPR). Foram analisadas por PCR-SSCP (polimorfismo de conformacao de fita simples) e sequenciamento de DNA em 200 amostras de gestantes classificadas em grupo controle (Saudaveis, n=100, CTRL) e diabeticas gestacionais (DMG, n=100). Os grupos apresentaram idade media de 24,9„b6,2, 31,7„b6,3, respectivamente para CTRL e DMG. Enquanto o grupo CTRL foi composto apenas por Euro-Brasileiras, o grupo DMG contempla 10% de Afrodescendentes. As gestantes diabeticas apresentaram IMC superior ao grupo controle, respectivamente, 33,5„b6,3 e 25,2„b4,2. As concentracoes de glucose em jejum, perfil lipidico foram significativamente maiores no grupo com diabetes exceto para o LDL-colesterol que nao diferiu. As determinacoes de ureia e creatinina apontam que ambos os grupos nao demonstram evidencia de lesao renal. A analise molecular obtida de 14 amplicons que cobriram as regioes genicas em estudo mostrou variacoes, todas do tipo transicao, em quatro regioes intronica em 13 das amostras estudadas (6,5%). As variacoes identificadas foram: c.43441A>G (Intro3, nao descrita), c.44702T>C (Intron6, rs2268574), c.48935C>T (Intron9, rs2908274) e c.49620G>A (Exon10, regiao nao codificadora, rs13306388). As frequencias estimadas, bem como 95% de Intervalo de Confianca (95% IC) para os alelos raros das variantes descritas foram: alelo-G, 0,3% (0-1%); alelo-C, 3,3% (2-5%), alelo-T, 0,8% (0-2%) e alelo-A, 1% (0-2%), respectivamente para as variantes detectadas nos Introns4, 6, 9 e Exon10. Para nenhuma das variantes foi observado associacao com processos patologicos. Em sintese, o gene da Glucoquinase nao apresentou variante do tipo ¡§hotspot¡¨ associada com o diabetes gestacional na populacao em estudo e uma nova variante, no Intron3 (c.43441A>G), foi identificada no presente estudo.Abstract: Gestational Diabetes mellitus (GDM) is defined as any degree of glucose intolerance with first detection during pregnancy. The disease is prevalent in about 7% of pregnancies and may lead to complications for mother and fetus. Glucokinase (GCK) is an enzyme that phosphorylates glucose in position 6 and acts as a sensor for the release of insulin from the pancreas. GCK gene polymorphisms are associated with monogenic diabetes type MODY-2 (Maturity Onset Diabetes of the Young Type 2). The aim of this study was investigate for GCK gene polymorphisms in exons and their flanking regions. The study was approved by the Ethics Committee by Federal University of Parana (UFPR). Were analyzed by PCR-SSCP (single strand conformation polymorphism) and DNA sequencing 200 samples from pregnant women classified in control group (healthy, n=100, CTRL) and gestational diabetes (GDM, n=100). The groups had a mean age of 24.9„b6.2, 31.7„b6.3, respectively for CTRL and DMG. While the CTRL group was composed only of Euro-Brazilian, DMG group has 10% for Afro-descendants. Diabetic women had a BMI (kg/m2) greater than the control group, respectively, 33.5„b6.3 and 25.2„b4.2. The concentrations of fasting glucose and lipid levels were significantly higher in the group with diabetes except for LDL-cholesterol that did not differ. Measurements of urea and creatinine showed that in both groups there was no evidence of renal failure. Molecular analysis obtained from 14 amplicons that comprehends the genetic regions studied showed variations, all in transition form, in four noncoding regions in 13 of the studied samples (6.5%). The variations were identified: c.43441A> G (intron3, not described), c.44702T>C (Intron6, rs2268574), c.48935C>T (Intron9, rs2908274) and c.49620G>A (Exon10 noncoding region, rs13306388). Estimated frequencies and 95% confidence interval (95% CI) for the rare alleles of the variants described were: G-allele, 0.3% (0-1%), C-allele, 3.3% ( 2-5%), T-allele, 0.8% (0-2%) and allele-A, 1% (0-2%) respectively for the variants found in Introns 3, 6, 9 and Exon10. For none of the variants were observed association with disease processes. In summary, the Glucokinase gene variant did not show associated-"hotspot" variant with gestational diabetes in the study population and a new variant in Intron3 (c.43441A>G) was identified in this study

The rs10885122 polymorphism of the adrenoceptor alpha 2A (ADRA2A) gene in Euro-Brazilians with type 2 diabetes mellitus

Objective To investigate the association of the rs10885122G>T polymorphism in the ADRA2A gene in a Euro-Brazilian sample of healthy (controls) and type 2 diabetic (T2D) subjects. Subjects and methods We used fluorescent probes (TaqMan) to genotype 241 subjects, that is, 121 healthy and 120 T2D subjects, who were classified based on the Brazilian Diabetes Association (2013) and American Diabetes Association (2014) criteria. Results The genotype and allele frequencies showed no significant (P > 0.05) difference between the two studied groups. The minor allele (T) frequencies (95%CI) for rs10885122 were 19% (14-24%) and 20% (15-26%) for healthy and T2D groups, respectively. Carriers of the T allele (genotypes GT+TT) were significantly associated (P = 0.016) with approximately a 7-kg body weight reduction compared with the genotype GG, which was only found in the T2D group. Conclusion The rs10885122G>T polymorphism of the ADRA2A gene was not associated with T2D in Euro-Brazilians, and carriers of the T allele had lower body weight in the presence of T2D. Arch Endocrinol Metab. 2015;59(1):29-3