Comprehensive association study of type 2 diabetes and related quantitative traits with 222 candidate genes

OBJECTIVE—Type 2 diabetes is a common complex disorder with environmental and genetic components. We used a candidate gene–based approach to identify single nucleotide polymorphism (SNP) variants in 222 candidate genes that influence susceptibility to type 2 diabetes.RESEARCH DESIGN AND METHODS—In a case-control study of 1,161 type 2 diabetic subjects and 1,174 control Finns who are normal glucose tolerant, we genotyped 3,531 tagSNPs and annotation-based SNPs and imputed an additional 7,498 SNPs, providing 99.9% coverage of common HapMap variants in the 222 candidate genes. Selected SNPs were genotyped in an additional 1,211 type 2 diabetic case subjects and 1,259 control subjects who are normal glucose tolerant, also from Finland.RESULTS—Using SNP- and gene-based analysis methods, we replicated previously reported SNP-type 2 diabetes associations in PPARG, KCNJ11, and SLC2A2; identified significant SNPs in genes with previously reported associations (ENPP1 [rs2021966, P = 0.00026] and NRF1 [rs1882095, P = 0.00096]); and implicated novel genes, including RAPGEF1 (rs4740283, P = 0.00013) and TP53 (rs1042522, Arg72Pro, P = 0.00086), in type 2 diabetes susceptibility.CONCLUSIONS—Our study provides an effective gene-based approach to association study design and analysis. One or more of the newly implicated genes may contribute to type 2 diabetes pathogenesis. Analysis of additional samples will be necessary to determine their effect on susceptibility.Type 2 diabetes is a metabolic disorder characterized by insulin resistance and pancreatic β-cell dysfunction and is a leading cause of morbidity and mortality in the U.S. and worldwide. The incidence of type 2 diabetes is rapidly increasing, with 1.6 million new cases of diabetes diagnosed in individuals aged ≥20 years in the U.S. in 2007 (available at http://www.diabetes.niddk.nih.gov/dm/pubs/statistics/). While environmental factors play a major role in predisposition to type 2 diabetes, substantial evidence supports the influence of genetic factors on disease susceptibility. For example, the twin concordance rate is an estimated 34% for monozygotic twins and 16% for dizygotic twins (1). However, the underlying genetic variants are just beginning to be identified (2).Numerous published reports (3–5) have identified association between type 2 diabetes and common genetic variants in human populations; however, until very recently, variants in only a few genes have been consistently replicated across populations and with large sample sizes. Among these are the Pro12Ala (rs1801282) variant in peroxisome proliferator–activated receptor γ (PPARG) (6), the Glu23Lys (rs5210) variant in the potassium channel gene KCNJ11 (7), and several variants in the Wnt-receptor signaling pathway member TCF7L2 (8).Recent genome-wide studies have implicated many previously unreported genes in type 2 diabetes susceptibility. The first reported genome-wide association (GWA) scan implicated variants at five susceptibility loci that include TCF7L2 and novel loci near the genes SLC30A8, IDE-KIF11-HHEX, LOC387761, and EXT-ALX4 (9). Three companion GWA studies (10–12), including one by our group, replicated evidence for PPARG, KCNJ11, TCF7L2, SLC30A8, and IDE-KIF11-HHEX and provided new evidence for CDKAL1, CDKN2A-CDKN2B, IGF2BP2, FTO, and a region of chromosome 11 with no annotated genes. Additional GWA studies (13–18) provided additional evidence for TCF7L2, CDKAL1, and SLC30A8. The candidate genes WFS1 (19) and TCF2 (20,21) have also been confirmed in large samples, bringing the current list of type 2 diabetes susceptibility loci to at least 10. The recent discovery of these loci still explains only a small fraction (∼2.3%) of the overall risk of type 2 diabetes (12). Therefore, novel susceptibility genes remain to be identified through increasingly comprehensive analyses of both individual genes and the entire genome.The Finland-U.S. Investigation of Type 2 Diabetes Genetics (FUSION) study aims to identify variants influencing susceptibility to type 2 diabetes and related quantitative traits in the Finnish population (22). FUSION has previously identified modest type 2 diabetes association in Finns with variants in HNF4A (23); four genes known to cause maturity-onset diabetes of the young (5,23,24); PPARG, KCNJ11, ENPP1, SLC2A2, PCK1, TNF, IL6 (5), and TCF7L2 (25); and the loci identified in the GWA studies.As a complementary approach to GWA studies, which are conducted without a priori biological hypotheses, we sought to perform an in-depth analysis of >200 genes likely to influence susceptibility to type 2 diabetes and quantitative trait variation that we selected by applying CandidAtE Search And Rank (CAESAR), a text- and data-mining algorithm (26). We aimed to analyze the full spectrum of HapMap-based common variation in each of these candidate genes. The combination of high throughput genotyping, linkage disequilibrium (LD) information from HapMap (27), the ability to impute ungenotyped variants (28), and the improved functional annotation of the genome makes in-depth candidate gene–based association analysis possible.     

Association of common variation in the HNF1alpha gene region with risk of type 2 diabetes

It is currently unclear how often genes that are mutated to cause rare, early-onset monogenic forms of disease also harbor common variants that contribute to the more typical polygenic form of each disease. The gene for MODY3 diabetes, HNF1alpha, lies in a region that has shown linkage to late-onset type 2 diabetes (12q24, NIDDM2), and previous association studies have suggested a weak trend toward association for common missense variants in HNF1alpha with glucose-related traits. Based on genotyping of 79 common SNPs in the 118 kb spanning HNF1alpha, we selected 21 haplotype tag single nucleotide polymorphisms (SNPs) and genotyped them in >4,000 diabetic patients and control subjects from Sweden, Finland, and Canada. Several SNPs from the coding region and 5' of the gene demonstrated nominal association with type 2 diabetes, with the most significant marker (rs1920792) having an odds ratio of 1.17 and a P value of 0.002. We then genotyped three SNPs with the strongest evidence for association to type 2 diabetes (rs1920792, I27L, and A98V) in an additional 4,400 type 2 diabetic and control subjects from North America and Poland and compared our results with those of the original sample and of Weedon et al. None of the results were consistently observed across all samples, with the possible exception of a modest association of the rare (3-5%) A98V variant. These results indicate that common variants in HNF1alpha either play no role in type 2 diabetes, a very small role, or a role that cannot be consistently observed without consideration of as yet unmeasured genetic or environmental modifiers.     

Searching for type 2 diabetes genes on chromosome 20

Genome scans in families with type 2 diabetes identified a putative locus on chromosome 20q. For this study, linkage disequilibrium mapping was used in an effort to narrow a 7.3-Mb region in an Ashkenazi type 2 diabetic population. The region encompassed a 1-logarithm of odds (LOD) interval around the microsatellite marker D20S107, which gave a LOD score of >3 in linkage analysis of a combined Caucasian population. This 7.3-Mb region contained 25 known and 99 predicted genes. Predicted single nucleotide polymorphisms (SNPs) were chosen from public databases and validated. Two SNPs were unique to the Ashkenazi. Here, 91 SNPs with a minor allele frequency of >or=10% were genotyped in pooled DNA from 150 case subjects and 150 control subjects of Ashkenazi Jewish descent. The SNP association study showed that SNP rs2664537 in the TIX1 gene had a significant P value of 0.035, but the finding did not replicate in an additional case pool. In addition, HNF4a and Mybl2 were screened for mutations and new polymorphisms. No mutations were identified, and a new nonsynonymous SNP (R687C in exon 14 of Mybl2) was found. The limits to this type of association study are discussed.     

PTPN22 R620W functional variant in type 1 diabetes and autoimmunity related traits

The PTPN22 gene, encoding the lymphoid-specific protein tyrosine phosphatase, a negative regulator in the T-cell activation and development, has been associated with the susceptibility to several autoimmune diseases, including type 1 diabetes. Based on combined case-control and family-based association studies, we replicated the finding of an association of the PTPN22 C1858T (R620W) functional variant with type 1 diabetes, which was independent from the susceptibility status at the insulin gene and at HLA-DR (DR3/4 compared with others). The risk contributed by the 1858T allele was increased in patients with a family history of other autoimmune diseases, further supporting a general role for this variant on autoimmunity. In addition, we found evidence for an association of 1858T allele with the presence of GAD autoantibodies (GADA), which was restricted to patients with long disease duration (>10 years, P < 0.001). This may help define a subgroup of patients with long-term persistence of GADA. The risk conferred by 1858T allele on GAD positivity was additive, and our meta-analysis also supported an additive rather than dominant effect of this variant on type 1 diabetes, similar to previous reports on rheumatoid arthritis and systemic lupus erythematosus.     

Genetic polymorphisms of cytokine genes in type 2 diabetes mellitus

Diabetes mellitus is a combined metabolic disorder which includes hyperglycemia, dyslipidemia, stroke and several other complications. Various groups all over the world are relentlessly working out the possible role of a vast number of genes associated with type 2 diabetes (T2DM). Inflammation is an important outcome of any kind of imbalance in the body and is therefore an indicator of several diseases, including T2DM. Various ethnic populations around the world show different levels of variations in single nucleotide polymorphisms (SNPs). The present review was undertaken to explore the association of cytokine gene polymorphisms with T2DM in populations of different ethnicities. This will lead to the understanding of the role of cytokine genes in T2DM risk and development. Association studies of genotypes of SNPs present in cytokine genes will help to identify risk haplotype(s) for disease susceptibility by developing prognostic markers and alter treatment strategies for T2DM and related complications. This will enable individuals at risk to take prior precautionary measures and avoid or delay the onset of the disease. Future challenges will be to understand the genotypic interactions between SNPs in one cytokine gene or several genes at different loci and study their association with T2DM.     

Genetic variation of the GLUT10 glucose transporter (SLC2A10) and relationships to type 2 diabetes and intermediary traits

The SLC2A10 gene encodes the GLUT10 facilitative glucose transporter, which is expressed in high amounts in liver and pancreas. The gene is mapped to chromosome 20q12-q13.1, a region that has been shown to be linked to type 2 diabetes. The gene was examined in 61 Danish type 2 diabetic patients, and a total of six variants (-27C-->T, Ala206Thr, Ala272Ala, IVS2 + 10G-->A, IVS4 + 18T-->G, and IVS4 + 26G-->A) were identified and investigated in an association study, which included 503 type 2 diabetic patients and 510 glucose-tolerant control subjects. None of the variants were associated with type 2 diabetes. Interestingly, carriers of the codon 206 Thr allele had 18% lower fasting serum insulin levels (P = 0.002) and 20% lower insulinogenic index (P = 0.03) than homozygous carriers of the Ala allele. These results suggest that variation in the coding region of SLC2A10 does not contribute substantially to the pathogenesis of type 2 diabetes in the examined study population. However, the codon 206 polymorphism may be related to the interindividual variation in fasting and oral glucose-induced serum insulin levels.     

Evaluation of common variants in the six known maturity-onset diabetes of the young (MODY) genes for association with type 2 diabetes

An important question in human genetics is the extent to which genes causing monogenic forms of disease harbor common variants that may contribute to the more typical form of that disease. We aimed to comprehensively evaluate the extent to which common variation in the six known maturity-onset diabetes of the young (MODY) genes, which cause a monogenic form of type 2 diabetes, is associated with type 2 diabetes. Specifically, we determined patterns of common sequence variation in the genes encoding Gck, Ipf1, Tcf2, and NeuroD1 (MODY2 and MODY4-MODY6, respectively), selected a comprehensive set of 107 tag single nucleotide polymorphisms (SNPs) that captured common variation, and genotyped each in 4,206 patients and control subjects from Sweden, Finland, and Canada (including family-based studies and unrelated case-control subjects). All SNPs with a nominal P value <0.1 for association to type 2 diabetes in this initial screen were then genotyped in an additional 4,470 subjects from North America and Poland. Of 30 nominally significant SNPs from the initial sample, 8 achieved consistent results in the replication sample. We found the strongest effect at rs757210 in intron 2 of TCF2, with corrected P values <0.01 for an odds ratio (OR) of 1.13. This association was observed again in an independent sample of 5,891 unrelated case and control subjects and 500 families from the U.K., for an overall OR of 1.12 and a P value <10(-6) in >15,000 samples. We combined these results with our previous studies on HNF4alpha and TCF1 and explicitly tested for gene-gene interactions among these variants and with several known type 2 diabetes susceptibility loci, and we found no genetic interactions between these six genes. We conclude that although rare variants in these six genes explain most cases of MODY, common variants in these same genes contribute very modestly, if at all, to the common form of type 2 diabetes.     

八段锦运动干预对2型糖尿病患者相关指标的影响

目的 探讨适宜中老年2型糖尿病患者的运动方式,以全面改善患者的身心状况.方法 将80例中老年2型糖尿病患者随机分为对照组和观察组各40例.两组均予糖尿病常规运动和降血糖治疗.观察组在此基础上由糖尿病专科护士指导八段锦练习,1次/d,1 h/次;每周集中练习1次.连续4个月后评价效果.结果 干预后观察组中医症状积分、焦虑评分、抑郁评分、心踝指数、踝肱指数、餐后2h血糖、糖化血红蛋白、三酰甘油值均显著优于对照组(P＜0.05,P＜0.01).结论 八段锦运动对中老年2型糖尿病患者有较好的辅助治疗作用,能有效改善患者的身心状况.     

Mapping genes influencing type 2 diabetes risk and BMI in Japanese subjects

We have carried out an autosomal genome scan for genes contributing to the development of type 2 diabetes and affecting BMI in the Japanese population (164 families, 256 affected sib-pairs). We found 12 regions that showed nominally significant multipoint evidence of linkage with type 2 diabetes (i.e. logarithm of odds [LOD] score >0.59, P < 0.05): chromosome 1 29.9 cM; chromosome 2 169.6 and 236.8 cM; chromosome 4 104.9 cM; chromosome 5 114.8 cM; chromosome 6 42.3 cM; chromosome 8 15.3 and 93.3 cM; chromosome 9 140.0 cM; chromosome 11 131.6 cM; chromosome 17 36.1 cM; and chromosome 21 48.0 cM. Twelve regions showed nominal multipoint evidence for linkage with log-transformed BMI (lnBMI): chromosome 2 167.9 and 210.5 cM; chromosome 3 185.7 cM; chromosome 4 118.9 and 145.6 cM; chromosome 5 131.9 cM; chromosome 7 7.4 cM; chromosome 10 70.0 cM; chromosome 15 12.8 cM; chromosome 16 30.0 cM; and chromosome 17 47.8 and 100.2 cM. Although none of the regions achieved genome-wide levels of significance, simulation studies showed that we observed more linkage signals than expected if there were no loci contributing to type 2 diabetes or BMI. Eight of the regions showing nominal evidence for linkage with type 2 diabetes have been reported in other genome scans, and seven of the regions showing linkage with lnBMI have shown linkage with BMI and BMI-related traits in other studies. Thus, our results may replicate findings in other studies. They may also indicate new regions of the genome that are involved in the regulation of blood glucose levels or body weight.     

Analysis of genetic variation in Akt2/PKB-beta in severe insulin resistance, lipodystrophy, type 2 diabetes, and related metabolic phenotypes

We previously reported a family in which a heterozygous missense mutation in Akt2 led to a dominantly inherited syndrome of insulin-resistant diabetes and partial lipodystrophy. To determine whether genetic variation in AKT2 plays a broader role in human metabolic disease, we sequenced the entire coding region and splice junctions of AKT2 in 94 unrelated patients with severe insulin resistance, 35 of whom had partial lipodystrophy. Two rare missense mutations (R208K and R467W) were identified in single individuals. However, insulin-stimulated kinase activities of these variants were indistinguishable from wild type. In two large case-control studies (total number of participants 2,200), 0 of 11 common single nucleotide polymorphism (SNPs) in AKT2 showed significant association with type 2 diabetes. In a quantitative trait study of 1,721 extensively phenotyped individuals from the U.K., no association was found with any relevant intermediate metabolic trait. In summary, although heterozygous loss-of- function mutations in AKT2 can cause a syndrome of severe insulin resistance and lipodystrophy in humans, such mutations are uncommon causes of these syndromes. Furthermore, genetic variation in and around the AKT2 locus is unlikely to contribute significantly to the risk of type 2 diabetes or related intermediate metabolic traits in U.K. populations.     

A genome-wide linkage scan for genes controlling variation in urinary albumin excretion in type II diabetes

The main hallmark of diabetic nephropathy is elevation in urinary albumin excretion. We performed a genome-wide linkage scan in 63 extended families with multiple members with type II diabetes. Urinary albumin excretion, measured as the albumin-to-creatinine ratio (ACR), was determined in 426 diabetic and 431 nondiabetic relatives who were genotyped for 383 markers. The data were analyzed using variance components linkage analysis. Heritability (h2) of ACR was significant in diabetic (h2=0.23, P=0.0007), and nondiabetic (h2=0.39, P=0.0001) relatives. There was no significant difference in genetic variance of ACR between diabetic and nondiabetic relatives (P=0.16), and the genetic correlation (rG=0.64) for ACR between these two groups was not different from 1 (P=0.12). These results suggested that similar genes contribute to variation in ACR in diabetic and nondiabetic relatives. This hypothesis was supported further by the linkage results. Support for linkage to ACR was suggestive in diabetic relatives and became significant in all relatives for chromosome 22q (logarithm of odds, LOD=3.7) and chromosome 7q (LOD=3.1). When analyses were restricted to 59 Caucasian families, support for linkage in all relatives increased and became significant for 5q (LOD=3.4). In conclusion, genes on chromosomes 22q, 5q and 7q may contribute to variation in urinary albumin excretion in diabetic and nondiabetic individuals.     

Genetic variation at the adiponectin locus and risk of type 2 diabetes in women

Previous data suggesting that polymorphisms in the adiponectin gene were associated with insulin resistance or type 2 diabetes have been inconsistent. We assessed the relationship between five common haplotype-tagging single nucleotide polymorphisms (SNPs) in the adiponectin gene (-11365C>G, -4034A>C, -3964A>G, +45T>G, and +276G>T), haplotypes defined by these SNPs, and the risk of type 2 diabetes by conducting a nested case-control study of 642 incident cases of type 2 diabetes and 995 matching control subjects in the Nurses' Health Study. Overall, we did not observe significant differences in genotype or allele frequencies for the five SNPs between the case and control subjects. After adjustment for diabetes risk factors, the -4034 C/C genotype was associated with a reduced risk of diabetes (odds ratio [OR] compared with the A/A genotype = 0.70, 95% CI 0.50-0.99, P = 0.04). In subgroup analyses, the +276 genotype was significantly associated with diabetes risk only among subjects with peroxisome proliferator-activated receptor-gamma (PPAR gamma) variant 12Ala allele (OR comparing +276 T alleles with the G/G genotype = 1.69, 1.04-2.75, P = 0.035) or among obese subjects (1.46, 1.03-2.08, P = 0.03). These data suggest a potential interaction between the adiponectin genotype and PPAR gamma genotype or obesity, but these analyses should be considered exploratory and require further investigation in larger studies.     

Impact of type 2 diabetes on nitric oxide and adrenergic modulation of myocardial perfusion

Type 2 diabetic patients are characterized by a reduced adenosine-induced hyperemic myocardial perfusion, which may contribute to their increased cardiovascular morbidity. We hypothesized that the reduced hyperemia can be explained by functional changes in endothelial or autonomic nervous regulation. In 12 type 2 diabetic patients without signs of ischemic heart disease and 14 age-matched control subjects, myocardial perfusion was measured at rest, during adenosine, and during adenosine and alpha-receptor blockade (phentolamine) using positron emission tomography on two separate days: 1) with, and 2) without nitric oxide (NO) inhibition with N(G)-nitro-L-arginine methyl ester. Myocardial perfusion during adenosine was lower in type 2 diabetic patients compared with control subjects (P = 0.05). No significant effect of NO inhibition on myocardial perfusion during adenosine was found in any of the groups. In control subjects, alpha-receptor blockade increased hyperemic myocardial vascular resistance during NO inhibition, whereas no effect was observed in type 2 diabetic patients. At rest, a significant correlation was observed between rate-pressure product and myocardial perfusion in control subjects. NO inhibition and type 2 diabetes abolished this correlation. Endothelial and cardiac autonomic nerve function seems to play only a minimal role in the reduced hyperemic myocardial perfusion in type 2 diabetic patients. However, the linear correlation between resting perfusion and cardiac work appears to be abolished in type 2 diabetes and during NO synthase inhibition.     

105例Ⅱ型糖尿病患者骨密度变化的分析

目的 研究Ⅱ型糖尿病患骨密度的变化规律和临床特点，更深入了解糖尿病对骨代谢的影响。方法 使用双能X线骨密度测定仪测定105例Ⅱ型糖尿病患腰椎L1～L4、Ward’s三角区、股骨颈、股骨大转子区的骨密度(BMD)，并以同年龄、同性别的健康的骨密度比较，研究其骨密度的变化规律和临床特点。结果 ①女性Ⅱ型糖尿病患BMD测定值与同年龄对照组比较差异无显性。②男性Ⅱ型糖尿病患BMD测定值除了51～60岁年龄组差异有显性外，61岁以上年龄组差异无显性。③男性Ⅱ型糖尿病患BMD测定值明显高于同年龄的女性。各年龄组Ward’s三角区BMD测定值明显低于腰椎和股骨颈及股骨大转子区。结论 ①雌激素水平下降是绝经后妇女骨质疏松的主要原因。②Ⅱ型糖尿病能使部分老年患易患有骨质疏松症。③骨质疏松患最早出现骨密度改变在Ward’s三角区。     

Evidence for different susceptibility genes for proteinuria and ESRD in type 2 diabetes

Proteinuria and impaired kidney function are 2 major traits of diabetic nephropathy that aggregate (are heritable) in families of diabetic individuals. Although both traits are heritable, they are not genetically correlated. These findings not only support the hypothesis that the development of diabetic nephropathy consists of 2 distinct disease processes (ie, increasing proteinuria and declining kidney function) but also strongly justify searches for the putative genes that separately determine variation in these processes. These searches have used both genome-wide scans and candidate-gene approaches. By use of genome-scan approaches, several research groups have identified genetic regions on chromosomes 7q, 18q, and 22q that harbor genes that determine either variation in urinary albumin excretion or susceptibility to proteinuria in families who have type 2 diabetes. The evidence for linkage in these 3 genetic regions was suggestive or strong, but, except for 7q, the regions did not overlap across studies. Two genome scans performed in families who have type 2 diabetes identified genetic regions on chromosome 3q, 6q, 7p, and 18q that harbor susceptibility genes that determine variation in glomerular filtration rate or susceptibility to the development of end-stage renal disease (ESRD). The region on 7p overlapped in both studies. Optimism is growing that a positional cloning approach applied to these putative genetic regions will lead to the isolation of the susceptibility genes for proteinuria and ESRD. Meanwhile, significant efforts that make use of the candidate-gene approach have been directed to the search for susceptibility genes for diabetic nephropathy. Unfortunately, positive findings have not been consistent.