Investigation Of Type 2 Diabetes Mellitus Related Gene

Objective: Obesity(OB) and diabetes mellitus(DM) are metabolic diseases induced by abnormal genes expression and the cffect of environment. Genetic factor participates pathogenesis in defferent types of OB and DM. A lot of research results indicate that gene polymorphism is related closely to pathogenesis of OB and DM.Mitochondrie contents genes and system of protein synthesis. Mitochondrie is unique cell organ taking along genetic maiteral besides cell nucleus. Mitochondrial gene(mtDNA) and/or nuclear gene (nDNA) are interdependency and interactive each other.Great researches in the relation of mtDNA and/or nDNA mutation and pathogenisis of DM were achieved in the last several years. But now, investigator have been focused on the relation of the morbidity of OB and life style and food structure. The research about the OB predisposing genes screening mainly focus on the nDNA without mtDNA.Foxa2 is an important activating transcription factor(ATF) in the liver. The gene can be inhibited by insulin. Foxo1 is a key switch regulation maiteral to fatty acid decomposition in the liver in the condition of empty stomach, and Foxa2 is mainly promote the glyconeogenesis. Low insulin level can activate Foxo1 and Foxa2. Foxo1 activation may lead to increase of gluconeogenesis. Foxa2 activation might lead to oxidation of fatty acidβand production of ketobodies and very low density lipoprotein(VLDL). In the state of insulin resistance, Foxa2 was inhibited by lower insulin signal but Foxo1 was not, which induce to an increase of gluconeogenesis and decrases of oxidation of fatty acidβoxidation production of ketobodies and very low density lipoprotein. All of these metabolic disturbances are related closely to pathogenesis of T2DM.Great advance is achieved in the role of mtDNA mutation leading to DM in the last several years.More than ten of mtDNA mutations related with DM were found. But no correlation research on mtDNA mutation leading to OB is reported in the world. Mitochondrial oxidative phosphorylation is related closely to human energy metabolism. Decrease and dysfunction of polypeptide in the mitochondrial respiratory chain can affect oxidative phosphorylation and interfere TCA cycle. So mitochondrial dysfunction may be have some effects on lipid metabolism and morbidity of OB.Therefore, we investigated the relationship between the foxa2 gene variant in nDNA and type 2 diabetes mellitus .In the mtDNA, A3537G,A4824G,A5351G,C6960T were investigated which were found in Japanese T2DM patiens sequencing (www.mitomap.org), but never reported that these gens are related with DM or OB by now. Part 1: Association of the foxa2 gene variant with type 2 diabetes mellitusMethods: 145 patients with T2DM includs 63 men and 82 women were examined. Mean age was 56.56±13.62yers. 334 non-diabetic subjects with fasting glucose less than 5.6 mmol/L or OGTT glucose less than 7.8 mmol/L includs 188 men and 146 women. Mean age was 52.31±10.59 yers. In Dalian Han nationality were selected. DNA was isolated from peripheral blood cell according to standard procedures. According to the sequence of Foxa2 Sense and anti-sense primers of the promoter and exon were designed according to the sequence of Foxa2 gene 1253bp,195bp,1677bp fragments and fragments were amplified by polymerase chain reaction. Those areas were sequencinged to look for SNP.Result: The SNP in the areas of Foxa2's promoter and exon was not found in patients with T2DM of Dalian Han nationality.Conclusions: Foxa2 is a conservatism gene. It is not related with the morbidity of type 2 diabetes mellitus in Dalian Han nationality .The other transcription regulatory factor of the compounds maybe have effect in inducing type 2 diabetes mellitus.Part 2:Study of correlations among mitochondrial DNA A3537G, A4824G, A5351G, C6960T polymorphisms and obesity and type 2 diabetes mellitusSubjects: 496 Guangxi officials who were received health examination and diagnosed as overweight or obesity were enrolled in the study. 96 with overweight including 60 men and 36 women were examined. Mean age was 46.33±6.25 years. 400 with obesity including 280 men and 120 women were examined. Mean age was 47.52±3.85 years. 137 healthy people who were received health examination in Guangxi province were enrolled as controls.Methods: Genomic DNA was extracted from peripheral blood karyocytes, primer was designed according to mitochondrial DNA sequence. Fragments including A3537G, A4824G, A5351G, C6960T gene mutation sites were amplified by polymerase chain reaction (PCR), the length of the fragments were 557bP, 658bP, 505bP and 541bP respectively, then were digested with AluI, BstEII, HhaI, BgⅡincision enzymes respectively in 37℃thermotank for 12 hours.The products were separated by electrophoresis on a 8% polyacrylamide gel (PAGE), genotypes were identified(AA, GG, CC, TT), and were compared among verweight group, obesity group and control group, the 4 sites constructed haplotypes were analyzed at the same time.Results: (1).The classification of overweight and obesity for Guangxi population should be the Asia adult BMI classification proposed by 2000 international obesity special team. (2). In the study of correlations among mtDNA A3537G, A4824G, A5351G, C6960T polymorphisms and obesity, significant difference was only found in A5351G point mutation between pure OB group and healthy group (X2=7.41 , P=0.006 ,OR=3.53,95%CI=1.35-6.29).but the A5351G mutation was silent mutation, it suggest that some linkage disequilibrium risk genetic mutation may exist point mutation beside A5357G associated with obesity in Guangxi population. (3). In the study of correlations among mtDNA A3537G, A4824G, A5351G, C6960T polymorphisms and OB accompanied with DM, no significant difference was found in the variant rate of the 4 point mutation between OB+DM group and healthy group(X2=7.41,P=0.006,OR=3.53,95%CI=1.35-6.29).It suggest the 4 point mutation has no correlation with DM in the obesity patients. (4). In the study of correlations among mtDNA A3537G, A4824G, A5351G, C6960T polymorphisms and obesity accompanied with IGT, significant difference was only found in A5351G point mutation between OB+IGT group and control group(X2=8.296,P=0.004,OR=4.40,95%CI=1.49-9.63).but the A5351G mutation was silent mutation, it suggest that some linkage disequilibrium risk genetic mutation may exist point mutation beside A5357G associated with IGT in Guangxi population. (5). In the study of correlations among mtDNA A3537G, A4824G, A5351G, C6960T polymorphisms and overweight accompanied with DM/IGT, no significant difference was found in the variant rate of the 4 point mutation among OW+DM/IGT group and healthy group(X2=7.41 ,P=0.006,OR=3.53,95%CI=1.35-6.29).It suggest the 4 point mutation has no correlation with OW+DM/IGT in the obesity patients.(6). Haplotypes were detected from the mtDNA A3537G.A4824G,A5351G,C6960T mutations, only AAGC haplotype (G was 5351 A mutated to G) has significant distribution in case-control groups.Conclusions: mtDNA A5351G mutation site can be used as genetic mark in Guangxi population with OB or OB+IGT. Some linkage disequilibrium risk genetic mutation maybe exist beside A5357G associated with OB and OB+IGT in Guangxi population.AAGC haplotype is a high risk genotype. So, the risk of T2DM could increase with the living standard improving in population with AAGC haplotype.