Variabilidade do gene de Glucoquinase no diabetes gestacional

Abstract

Resumo: 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.