| PartI Genetic variants in SLC6A20 gene and the risk of type 2 diabetesObjective:SLC6A20 (solute carrier family 6 (proline IMINO transporter), member 20) is an essential component in the metabolism of proline. Proline plays an important role in glucose and energy homeostasis by regulating the metabolism of glutamine and arginine. We investigated if polymorphisms in SLC6A20 gene were associated with the susceptibility of type 2 diabetes.Methods:We first investigated the association of polymorphisms in SLC6A20 gene with type 2 diabetes in the Rotterdam Study, a prospective, population-based cohort (n=5974). Secondly, positive findings in the Rotterdam Study were replicated in an independent Dutch population (n=3133) with case-control study design. Finally, positive findings in the Dutch case-control study were replicated in a Chinese Han population (n=2279) with case-control study design.Data of anthropometric parameters, blood pressure, fasting plasma glucose, oral glucose tolerance test (OGTT) and lipid profiles were collected.We selected tagging single nucleotide polymorphisms (SNPs) with R2=l and minor allele frequency (MAF)>0.05 across the region of SLC6A20 gene (include 20kb upstream and 1 0kb downstream of the gene) from HapMap Phase II, using pairwise tagging model. We used genotyping data available in Illumina 550K genotyping BeadChip version3 in 5974 subjects from the Rotterdam Study. In the Dutch case-control study, we genotyped the associated SNPs in individuals from the DiaGene Study, using TaqMan allelic discrimination assays. Genotypes were imputed for participants from the extension of the cohort of the Rotterdam Study. In the Chinese case-control study, we genotyped the associated SNPs by matrix-assisted laser desorption ionization-time of flight mass spectroscopy. Deviation from Hardy-Weinberg equilibrium was assessed by means of x2 testing. SNPs that were not in Hardy-Weinberg equilibrium were excluded from further analyses. Pairwise linkage disequilibrium (LD) including D'and r2 were estimated by Haploview. Haplotypes estimating from population genotype data were performed in Haplo. Stats. In the Rotterdam Study, we tested the association of the polymorphisms or haplotypes with type 2 diabetes risk in Cox proportional hazards models. In replication study, we tested the association of the polymorphisms with type 2 diabetes using logistic regression. We tested the association of the polymorphisms or haplotypes with clinical variables using multiple linear regression and general linear model. All models were adjusted for year of birth and sex. Additional models were adjusted for BMI. We used Bonferroni correction for multiple testing. Meta-analysis was performed in Comprehensive Meta Analysis.Results:In the Rotterdam Study,22 tagging SNPs were studied.19 SNPs were in Hardy-Weinberg equilibrium in the total population and in individuals without type 2 diabetes. Three SNPs-rs6770261, rs2531747 and rs12488144 deviated from Hardy-Weinberg equilibrium (x2 testing p＜0.001, p=0.01 and p=0.02 respectively) and were excluded from further analyses.7 SNPs were associated with the risk of type 2 diabetes. The minor alleles of rs13062383, rs10461016 and rs2286489 increased the risk of type 2 diabetes adjusted for year of birth and sex (HR=1.34,95% CI 1.13-1.60, p=0.001; HR=1.30,95%CI 1.09-1.54, p=0.003; HR=1.20,95%CI 1.07-1.35, p=0.003). The minor alleles of rsl7279465, rs2191028, rs2191027 and rs4299518 decreased the risk of type 2 diabetes adjusted for year of birth and sex (HR=0.86,95%CI 0.76-0.97, p=0.01; HR=0.84,95%CI 0.74-0.95, p=0.01; HR=0.85, 95%CIO.74-0.97, p=0.02; HR=0.86,95%CI 0.76-0.97, p=0.02). The associations remained similar after additional adjustment for BMI. The association of rs1 3062383, rs10461016 and rs2286489 with type 2 diabetes were significant after Bonferroni correction. However, the association of rs2191028, rs2191027 and rs4299518 with type 2 diabetes were not significant anymore after Bonferroni correction.Several SNPs were associated with clinical variables, including total cholesterol, high density lipoprotein cholesterol, BMI, waist circumference, waist to hip ratio, systolic blood pressure and diastolic blood pressure. But these associations were not significant anymore after Bonferroni correction. The haplotype blocks were constructed for 19 SNPs in SLC6A20. The 19 SNPs in SLC6A20 fall into four blocks with high LD and different sizes. Haplotype analysis revealed significant associations with diabetes risk in block 1 and block 2. The haplotype H3 (GGAC) in block 1 was associated with a HR of 1.24 (95%CI 1.04-1.49, p=0.02) for type 2 diabetes compared with the reference group (haplotype H1 (GAAC)) adjusted for year of birth and sex. The haplotype H3 (AA) in block 2 was associated with a HR of 1.28 (95%CI 1.07-1.54, p=0.01) for type 2 diabetes compared with the reference group (haplotype H1 (AG)) adjusted for year of birth and sex. The association remained similar after additional adjustment for BMI. There was no significant association with haplotypes in block 3 and block 4. The association of block 1 and block 2 with type 2 diabetes became non-significant after Bonferroni correction. The association of the haplotypes with clinical variables were not significant after Bonferroni correction.In the Dutch replication study, rs13062383 and rs10461016 were in Hardy-Weinberg equilibrium in the total population and in individuals without type 2 diabetes. The A allele of rs13062383 increased the risk of type 2 diabetes adjusted for year of birth and sex (OR=1.45,95%CI 1.19-1.76, p<0.001). The association remained similar after additional adjustment for BMI. The association of rs10461016 with type 2 diabetes was not significant (OR=0.95,95%CI 0.80-1.13, p=0.55).In the Chinese replication study, rs13062383 was in Hardy-Weinberg equilibrium in the total population and in individuals without type 2 diabetes. The A allele of rs13062383 increased the risk of type 2 diabetes adjusted for year of birth and sex (OR=1.21,95%CI 1.03-1.42, p=0.02). The association was more significant after additional adjustment for BMI (OR=1.26,95%CI 1.07-1.49, p=0.007).Meta-analysis showed a significant association of rs1 3062383 with type 2 diabetes, with an OR of 1.35 (95%CI 1.21-1.50, p=0.000).Conclusions:Polymorphisms of the SLC6A20 gene were associated with the risk of type 2 diabetes. SLC6A20 gene was the susceptible gene of type 2 diabetes. Part II Genetic variants in GCKR gene and the risk of type 2 diabetesObjective:Glucokinase (GCK) is the key glucose phosphorylation enzyme responsible for the first rate-limiting step in the glycolysis pathway and regulates glucose-stimulated insulin secretion from pancreatic beta cells and glucose metabolism in the liver. Its activity is modulated by glucokinase regulatory protein (GCKR). The GCKR rs780094 polymorphism has been reported to be associated with type 2 diabetes. The aim of this study was to replicate this association in Han Chinese individuals and to identify the potential mechanisms underlying these associations.Methods:We investigated the association of polymorphisms in GCKR gene with type 2 diabetes in a Chinese Han population (n=2279) with case-control study design.Data of anthropometric parameters, blood pressure, fasting plasma glucose, oral glucose tolerance test (OGTT) and lipid profiles were collected. Arginine stimulation test was performed in diabetic patients, and acute insulin response (AIR) and acute C-peptide response (ACPR) were calculated.We selected tagging single nucleotide polymorphisms (SNPs) with R2=l and minor allele frequency (MAF)>0.05 across the region of GCKR gene (include 20kb upstream and 9kb downstream of the gene) from HapMap Phase II, using pairwise tagging model. We genotyped the SNPs by matrix-assisted laser desorption ionization-time of flight mass spectroscopy.Deviation from Hardy-Weinberg equilibrium was assessed by means of x2 testing. SNPs that were not in Hardy-Weinberg equilibrium were excluded from further analyses. Pairwise linkage disequilibrium (LD) including D'and r2 were estimated by Haploview. Haplotypes estimating from population genotype data were performed in Haplo.Stats. We tested the association of the polymorphisms and haplotypes with type 2 diabetes using logistic regression. We tested the association of the polymorphisms and haplotypes with clinical variables using multiple linear regression and general linear model. All models were adjusted for year of birth and sex. Additional models were adjusted for BMI. We used Bonferroni correction for multiple testing.Results:4 tagging SNPs were studied. All four SNPs were in Hardy-Weinberg equilibrium in the total population and in individuals without type 2 diabetes. rs3817588 and rs780094 were associated with the risk of type 2 diabetes. The G alleles of rs3817588 and rs780094 increased the risk of type 2 diabetes adjusted for year of birth and sex (OR=1.21,95%CI 1.06-1.39, p=0.004;OR=1.19,95%CI 1.05-1.34, p=0.005). The associations remained similar after additional adjustment for BMI.In non-diabetic controls, rs3817588 and rs780094 were associated with plasma triglyceride level adjusted for year of birth, sex and BMI (p＜0.001, p=0.02). The G alleles of rs3817588 and rs780094 contributed to lower plasma triglyceride level. The association of rs3817588 with plasma triglyceride level remained significant after Bonferroni correction. However, the association of rs780094 with plasma triglyceride level was not significant anymore after Bonferroni correction. The associations of rs8179206 and rs2293572 with plasma triglyceride level were not significant. rs3817588 was associated with waist circumference (p=0.01) adjusted for year of birth and sex. The association was not significant anymore after Bonferroni correction. The associations of other polymorphisms with waist circumference were not significant. The associations of all four polymorphisms with BMI, fasting plasma glucose, total cholesterol, HDL-C, LDL-C, systolic blood pressure and diastolic blood pressure were not significant.The G allele of rs3817588 decreased the risk of hypertriglyceridemia adjusted or year of birth, sex and BMI (OR=0.80,95%CI 0.65-1.00, p=0.05). However, the association became non-significant after Bonferroni correction. The association of rs780094 with hypertriglyceridemia was not significant.The diabetic group was classified into 4 subgroups according to quartile of diabetic course. The G allele of rs8179206 contributed to lower level of AIR and ACPR in the second quartile subgroup with 1-7 years of diabetes adjusted for year of birth, sex and BMI (p=0.03, p=0.01). The G allele of rs780094 contributed to lower level of AIR in the third quartile subgroup with 8-11 years of diabetes adjusted for year of birth, sex and BMI (p=0.03). The G allele of rs3817588 contributed to lower level of AIR and ACPR in the fourth quartile subgroup with more than 11 years of diabetes adjusted for year of birth, sex and BMI (p=0.03, p=0.03). However, these associations were not significant anymore after Bonferroni correction.The haplotype block was constructed for 4 SNPs in GCKR.2 SNPs-rs3817588 and rs780094 fell into one block. The block was associated with type 2 diabetes adjusted for year of birth and sex (p for trend=0.016). The haplotype GG was associated with an OR of 1.24 for type 2 diabetes compared to the haplotype AA adjusted for year of birth and sex. The association remained similar after additional adjustment for BMI.In non-diabetic controls, the haplotype block was associatied with plasma triglyceride level adjusted for year of birth, sex and BMI (p for trend=0.001). The haplotype GG was associated with a lower plasma triglyceride level compared to the haplotype AA (p<0.001). The association remained significant after Bonferroni correction. The haplotype block was associatied with waist circumference adjusted for year of birth and sex (p for trend=0.03). The haplotype GG was associated with a higher waist circumference compared to the haplotype AA (p=0.01). The association became non-significant after Bonferroni correction. BMI, fasting plasma glucose, total cholesterol, HDL-C, LDL-C, systolic blood pressure and diastolic blood pressure were not significantly different between haplotypes.The association of haplotype block with hypertriglyceridemia was not significant adjusted for year of birth, sex and BMI.In diabetic cases, the association of haplotype block with AIR and ACPR was not significant adjusted for year of birth, sex and BMI.Conclusions:rs780094 A/G and rs3817588 A/G polymorphisms of the GCKR gene were associated with the risk of type 2 diabetes in Chinese Han population. rs3817588 A/G polymorphism of the GCKR gene was associated with plasma triglyceride level in Chinese Han population. GCKR gene was the susceptible gene of type 2 diabetes of Chinese Han population. The associations of rs780094 A/G and rs3817588 A/G polymorphisms of the GCKR gene with type 2 diabetes were probably mediated by impaired isletβcell function. |
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