Contribution of variants in the small heterodimer partner gene to birthweight,adiposity, and insulin levels: mutational analysis and association studies in multiple populations

Loss of function mutations in the small heterodimer partner (SHP) gene have been reported to cause obesity and increased birth weight. We examined the relation between genetic variation in SHP and birth weight, adiposity, and insulin levels in three independent populations. The coding regions and 562 bases of the SHP promoter were screened for mutations in 329 subjects with severe early-onset obesity. Two novel missense mutations, R34G and R36C, were identified; these were not found in control subjects and did not cosegregate with obesity in family studies. Two common polymorphisms, G171A and -195CTGAdel, were found in 12 and 16% of subjects, respectively. Within the obese cohort, G171A and -195CTGAdel carriers had higher and lower birth weights, respectively, than wild-type subjects, the rare homozygotes for G171A being particularly large at birth. In a U.K. population-based cohort of 1,079 children, the 171A allele was associated with higher BMI (P < 0.05) and waist circumference (P = 0.001). Children carrying the G171A variant had higher 30-min insulin responses to a glucose load (P = 0.03). In conclusion, although mutations in SHP are not a common cause of severe human obesity, genetic variation in the SHP locus may influence birth weight and have effects on BMI, possibly through effects on insulin secretion.     

Positional candidate gene analysis of Lim domain homeobox gene (Isl-1) on chromosome 5q11-q13 in a French morbidly obese population suggests indication for association with type 2 diabetes

The Lim domain homeobox gene (Isl-1) is a positional candidate gene for obesity that maps on chromosome 5q11-q13, a locus linked to BMI and leptin levels in French Caucasians. Isl-1 might be involved in body weight regulation and glucose homeostasis via the activation of proglucagon gene expression, which encodes for glucagon and glucagon-like peptides. By mutation screening of 72 obese subjects, we identified three single-nucleotide polymorphisms (SNPs) in the Isl1 gene. The allele frequencies in the morbidly obese group did not differ from that of the control group. In the obese group, the -47G allele was associated with a decreased risk of type 2 diabetes (odds ratio 0.41, P = 0.019). The AG bearers displayed a higher maximal BMI than the AA bearers in the whole obese group (P = 0.026) as well as in the type 2 diabetic obese subgroup (P = 0.014). In obese families, this allele was not preferentially transmitted from heterozygous parents to their obese siblings, indicating that Isl-1 does not contribute to the linkage with obesity on 5cen-q. However, in French Caucasian morbidly obese subjects, the Isl1-47A-->G SNP may modulate the risk for type 2 diabetes and may increase body weight in diabetic morbidly obese subjects.     

No evidence of association of ENPP1 variants with type 2 diabetes or obesity in a study of 8,089 U.K. Caucasians

Ectoenzyme nucleotide pyrophosphate phosphodiesterase 1 (ENPP1) is an inhibitor of insulin-induced activation of the insulin receptor. There is strong evidence from several previous studies that a common coding variant of ENPP1 (K121Q) and a three-marker haplotype (Q121, IVS20delT-11, and G+1044TGA) are associated with type 2 diabetes and obesity. We examined the impact of ENPP1 variation on type 2 diabetes and obesity in a large U.K. genetic association study. We genotyped the three previously associated polymorphisms in 2,363 type 2 diabetic case and 4,045 control subjects, as well as 1,681 subjects from 529 type 2 diabetic families. We used the same subjects for morbid and moderate obesity association studies. For type 2 diabetes, moderate and morbid obesity, and for both the Q121 and three-marker haplotype, our results exclude with >95% confidence the effect sizes from previous studies (Q121 allele: odds ratio 1.02 [95% CI 0.93-1.12], P = 0.61; 1.00 [0.85-1.18], P = 0.99; and 0.92 [0.70-1.20], P = 0.41; three-marker haplotype: 1.10 [0.96-1.26], P = 0.17; 0.97 [0.77-1.23], P = 0.81; and 0.86 [0.57-1.30], P = 0.46 for type 2 diabetes, moderate, and morbid obesity, respectively). A K121Q type 2 diabetes meta-analysis of all previously published studies remained significant after the inclusion of this study (1.25 [1.10-1.43], P = 0.0007), although there was some evidence of publication bias. In conclusion, we find no evidence that previously associated variants of ENPP1 are associated with type 2 diabetes or obesity in the U.K. population.     

Human resistin gene, obesity, and type 2 diabetes: mutation analysis and population study

The hormone resistin has been suggested to link obesity to type 2 diabetes by modulating steps in the insulin-signaling pathway and inducing insulin resistance. Thus, the resistin gene represents a potential candidate for the etiology of insulin resistance and type 2 diabetes. In this study, we analyzed the coding sequence of the three exons of the resistin gene, together with its 5' regulatory region and 3' untranslated region (UTR), by single-strand conformation polymorphism (SSCP) in 58 type 2 diabetic subjects, 59 obese subjects, and 60 normal subjects. Only one sequence variant was detected in the resistin gene. Sequencing of this variant revealed the presence of a single nucleotide substitution (SNP) in the 3'-UTR of exon 3 (G1326A) [corrected]. Because 3'-UTR SNPs have been shown to affect gene expression, we examined the frequency of this SNP in 591 subjects (198 obese subjects, 207 diabetic subjects, and 186 control subjects) by PCR amplification and BseRI digestion. No significant association was found between the G1326A [corrected] variant and diabetes and obesity. Comparison of clinical and metabolic parameters between G1326A [corrected] carriers and noncarriers again showed no significant difference. In conclusion, our data suggest that genetic defects of the resistin gene are unlikely to play a role in the etiology of these common disorders in our population.     

Association between tumour necrosis factor-alpha G-308A polymorphism and risk of nephropathy in obese Chinese type 2 diabetic patients.

BACKGROUND: The G-308A polymorphism in the promoter region of the tumor necrosis factor alpha (TNF-alpha) gene has been reported to be associated with insulin resistance and obesity, both of which may increase the risk of diabetic nephropathy. We hypothesized that this polymorphism might interact with obesity to affect development of diabetic nephropathy. METHODS: A consecutive cohort of 1281 Chinese type 2 diabetic patients was enrolled for analysis. Genotyping of TNF-alpha G-308A polymorphism was performed using a PCR-based RFLP method with NcoI digestion. The mean value of the albumin creatinine ratio (ACR) of a random spot urine sample and a timed urinary collection was used to determine albuminuric status. Diabetic nephropathy was defined as serum creatinine > or =150 micromol/L and/or mean ACR > or =25 mg/mmol. Obesity was defined as body mass index > or =25 kg/m2 using Asian criteria. RESULTS: The G-308A polymorphism was not associated with either obesity or nephropathy. Clinical characteristics were similar between GG and GA/AA genotype carriers. Amongst the obese patients, GG genotype carriers had a higher median (interquartile range) urinary ACR [3.16 (0.70, 59.10) vs 1.28 (0.48, 12.28) mg/mmol; p = 0.01] and albumin excretion rate [38.7 (12.1, 620.3) vs 21.4 (8.9, 224.0) microg/min, p = 0.03] than GA/AA carriers. On multiple logistic regression analysis, compared with non-obese GA/AA carriers, obese subjects with the GG genotype had a 2.5-fold increased risk (95% CI: 1.04-6.03; P = 0.04) of nephropathy after adjustment for confounding factors. Other independent factors for diabetic nephropathy included male sex, systolic blood pressure, triglycerides (logarithmically transformed value), and the presence of cardiovascular and microvascular complications. CONCLUSION: Our findings suggest that the GG genotype of TNF-alpha G-308A polymorphism or a genetic variant in close linkage disequilibrium may interact with obesity to increase the risk of nephropathy in Chinese Type 2 diabetic patients. Apart from the need for replication of these results, functional studies are required to clarify its significance.     

A putative functional polymorphism in the IGF-I gene: association studies with type 2 diabetes,adult height,glucose tolerance,and fetal growth in U.K. populations

IGF-I has a critical role in growth and metabolism. A microsatellite polymorphism 1 kb upstream to the IGF-I gene has recently been associated with several adult phenotypes. In a large Dutch cohort, the absence of the commonest allele (Z) was associated with reduced serum IGF-I levels, reduced height, and an increased risk of type 2 diabetes and myocardial infarction. This result has not been replicated, and the role of this polymorphism in these traits in U.K. subjects is not known. We sought further evidence for the involvement of this variant in type 2 diabetes using a case-control study and IGF-I and diabetes-related traits in a population cohort of 640 U.K. individuals aged 25 years. Absence of the common allele was not associated with type 2 diabetes (odds ratio 0.70, 95% CI 0.47-1.04 for X/X versus Z/Z genotype, chi(2) test for trend across genotypes, P = 0.018). In the population cohort, the common allele (Z) was associated with decreased IGF-I levels (P = 0.01), contrary to the Dutch study, but not with adult height (P = 0.23), glucose tolerance (P = 0.84), oral glucose tolerance test-derived values of beta-cell function (P = 0.90), or insulin resistance (P = 0.66). There was no association with measures of fetal growth, including birth weight (P = 0.17). Our results do not support the previous associations and suggest that the promoter microsatellite is unlikely to be functionally important.     

New polymorphism of ENPP1 (PC-1) is associated with increased risk of type 2 diabetes among obese individuals

The K121Q polymorphism in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is associated with type 2 diabetes and obesity. The possibility of other ENPP1 polymorphisms influencing these phenotypes has received little attention. Our aim was to examine the associations of tagging single nucleotide polymorphisms (SNPs) and haplotypes of the linkage disequilibrium (LD) block containing K121Q polymorphism with type 2 diabetes in a Polish population, controlling for any effect of obesity. We genotyped 426 type 2 diabetic case and 370 control subjects for seven SNPs in ENPP1. In the total group, neither type 2 diabetes nor obesity was significantly associated with any SNP. However, in obese subjects, two SNPs were significantly associated with type 2 diabetes: the Q allele of K121Q (odds ratio 1.6 [95% CI 1.003-2.6]) and T allele of rs997509 (4.7 [1.6-13.9]). In the LD block, four SNPs plus the K121Q polymorphism distinguished six haplotypes, three of which carried the Q allele. Interestingly, the T allele of rs997509 sufficed to distinguish a 121Q-carrying haplotype that was significantly more associated with type 2 diabetes than the other two (4.2 [1.3-13.5]). These other two 121Q-carrying haplotypes were not associated with type 2 diabetes. In conclusion, we found a new SNP, rs997509, in intron 1 that is strongly associated with risk of type 2 diabetes in obese individuals. The molecular mechanisms underlying this association are unknown.     

Influence of obesity and type 2 diabetes on gluconeogenesis and glucose output in humans: a quantitative study

The contribution of gluconeogenesis (GNG) to endogenous glucose output (EGO) in type 2 diabetes is controversial. Little information is available on the separate influence of obesity on GNG. We measured percent GNG (by the 2H2O technique) and EGO (by 6,6-[2H]glucose) in 37 type 2 diabetic subjects (9 lean and 28 obese, mean fasting plasma glucose [FPG] 8.3 +/- 0.3 mmol/l) and 18 control subjects (6 lean and 12 obese) after a 15-h fast. Percent GNG averaged 47 +/- 5% in lean control subjects and was significantly increased in association with both obesity (P < 0.01) and diabetes (P = 0.004). By multivariate analysis, percent GNG was independently associated with BMI (partial r = 0.27, P < 0.05, with a predicted increase of 0.9% per BMI unit) and FPG (partial r = 0.44, P = 0.0009, with a predicted increase of 2.7% per mmol/l of FPG). In contrast, EGO was increased in both lean and obese diabetic subjects (15.6 +/- 0.5 micromol x min(-1) x kg(-1) of fat-free mass, n = 37, P = 0.002) but not in obese nondiabetic control subjects (13.1 0.7, NS) as compared with lean control subjects (12.4 +/- 1.4). Consequently, gluconeogenic flux (percent GNG x EGO) was increased in obesity (P = 0.01) and markedly elevated in diabetic subjects (P = 0.0004), whereas glycogenolytic flux was reduced only in association with obesity (P = 0.05). Fasting plasma glucagon levels were significantly increased in diabetic subjects (P < 0.05) and positively related to EGO, whereas plasma insulin was higher in obese control subjects than lean control subjects (P = 0.05) and unrelated to measured glucose fluxes. We conclude that the percent contribution of GNG to glucose release after a 15-h fast is independently and quantitatively related to the degree of overweight and the severity of fasting hyperglycemia. In obese individuals, reduced glycogenolysis ensures a normal rate of glucose output. In diabetic individuals, hyperglucagonemia contributes to inappropriately elevated rates of glucose output from both GNG and glycogenolysis.     

Common variants in the ENPP1 gene are not reproducibly associated with diabetes or obesity

The common missense single nucleotide polymorphism (SNP) K121Q in the ectoenzyme nucleotide pyrophosphate phosphodiesterase (ENPP1) gene has recently been associated with type 2 diabetes in Italian, U.S., and South-Asian populations. A three-SNP haplotype, including K121Q, has also been associated with obesity and type 2 diabetes in French and Austrian populations. We set out to confirm these findings in several large samples. We genotyped the haplotype K121Q (rs1044498), rs1799774, and rs7754561 in 8,676 individuals of European ancestry with and without type 2 diabetes, in 1,900 obese and 930 lean individuals of European ancestry from the U.S. and Poland, and in 1,101 African-American individuals. Neither the K121Q missense polymorphism nor the putative risk haplotype were significantly associated with type 2 diabetes or BMI. Two SNPs showed suggestive evidence of association in a meta-analysis of our European ancestry samples. These SNPs were rs7754561 with type 2 diabetes (odds ratio for the G-allele, 0.85 [95% CI 0.78-0.92], P = 0.00003) and rs1799774 with BMI (homozygotes of the delT-allele, 0.6 [0.42-0.88], P = 0.007). However, these findings are not supported by other studies. We did not observe a reproducible association between these three ENPP1 variants and BMI or type 2 diabetes.     

Differential association of basal and postprandial plasma ghrelin with leptin, insulin, and type 2 diabetes

To gain further insight into the regulatory role of insulin and leptin on plasma ghrelin, 56 normal weight, 128 normoinsulinemic obese, 121 hyperinsulinemic obese, and 30 type 2 diabetic normoinsulinemic and 75 type 2 diabetic hyperinsulinemic obese patients were examined. In the obese subjects, basal hyperinsulinemia was associated with significantly lower ghrelin independent of BMI, age, and leptin. In normoinsulinemic (normal weight and normoinsulinemic obese) subjects, ghrelin was inversely related to stepwise increasing leptin. Multiple regression analysis and matching for insulin revealed a significant negative interaction of ghrelin with leptin but not insulin. In type 2 diabetic normoinsulinemic subjects, ghrelin was significantly lower compared with that in normoinsulinemic obese subjects. In type 2 diabetic hyperinsulinemic subjects, ghrelin was significantly lower than in normoinsulinemic subjects, whereas no further reduction was observed compared with hyperinsulinemic obese subjects. The postprandial decrease was significantly attenuated in normoinsulinemic obese and hyperinsulinemic obese subjects (-214.8 +/- 247 pg/ml [normal weight], -137.6 +/- 107 pg/ml [normoinsulinemic obese], -85.5 +/- 69 pg/ml [hyperinsulinemic obese], P < 0.001; mean +/- SD), whereas type 2 diabetes had no independent postprandial effect. In conclusion, the present data support the concept that leptin could be of importance for suppression of basal ghrelin during moderate weight gain in normoinsulinemic subjects, whereas hyperinsulinemia but not leptin is responsible in more severe obesity. Postprandial suppression of ghrelin is attenuated by as yet unknown mechanisms that are related to body weight but not to insulin or type 2 diabetes.     

The insulin gene variable number tandem repeat class I/III polymorphism is in linkage disequilibrium with birth weight but not Type 2 diabetes in the Pima population

The insulin gene variable number tandem repeat (INS-VNTR) is proposed to exert pleiotropic genetic effects on birth weight and diabetes susceptibility. In our study, we examined the influence of a polymorphism in tight linkage disequilibrium with INS-VNTR (-23Hph1) on birth weight and type 2 diabetes in the Pima population. A parent-offspring "trio" design was used to assess parent-of-origin effects and population stratification. The presence of the -23Hph1 T-allele was associated with lower birth weight (n = 192; -140 g per copy of the T-allele; P = 0.04), even after adjustment for effects of population stratification (P = 0.03). The effects of paternally transmitted T-alleles were greater than those of maternally transmitted alleles (paternally transmitted: -250 g, P = 0.05; maternally transmitted: -111 g, P = 0.43), but this difference was not statistically significant (P = 0.50). The -23Hph1 T-allele was associated with an increased prevalence of type 2 diabetes (P = 0.009), which family-based association analysis suggested was attributable to population structure (P = 0.04) without significant evidence of linkage disequilibrium between diabetes prevalence and genotype (P = 0.86). Thus allelic variation of the INS gene is associated with lower birth weight and increased prevalence of type 2 diabetes. Significant linkage disequilibrium was found between -23Hph1 and birth weight but not type 2 diabetes, an observation that supports a potential functional role of INS polymorphisms in the regulation of birth weight.     

GAS6 intron 8 c.834 + 7G > A gene polymorphism in diabetic nephropathy

Abstract

Background - Aim: In animal experiments, growth arrest-specific 6 (Gas6) protein plays a key role in the development of mesangial cell and glomerular hypertrophy in the early phase of diabetic nephropathy, and diabetic nephropathy is prevented by warfarin-induced inhibition of GAS6 protein. It was shown that GAS6 intron 8 c.834?+?7G?>?A polymorphism is protective against type 2 diabetes mellitus, and AA genotype is associated with higher blood levels of GAS6 protein. Our aim is to investigate whether this polymorphism is a risk factor for diabetic nephropathy in type 2 diabetes mellitus. Method: Eighty-seven patients with diabetic nephropathy were compared with 66 non-diabetic controls in terms of GAS6 intron 8 c.834?+?7G?>?A polymorphism. Patients with history of stroke, ischemic heart disease were excluded. Each patient was examined by the ophthalmologist to determine diabetic retinopathy. Results: Frequency of GG, GA and AA genotypes are similar in diabetic nephropathy and control groups according to GAS6 intron 8 c.834?+?7G?>?A polymorphism (p?=?0.837). Rate of diabetic retinopathy was 54.02%. In the subgroup analysis, GA genotype was significantly more frequent than GG genotype in patients with diabetic retinopathy when compared to without diabetic retinopathy (p?=?0.010). Conclusion: In our study, GAS6 intron 8 c.834?+?7G?>?A polymorphism was not associated with diabetic nephropathy in type 2 diabetes mellitus. However, heterozygous state of this polymorphism may be a risk factor for diabetic retinopathy in patients with diabetic nephropathy.     

Islet cell autoimmunity in a triethnic adult population of the Third National Health and Nutrition Examination Survey

Markers of humoral islet cell autoimmunity, such as autoantibodies (AAs) against the 65-kDa isoform of GAD (GAD65), serve as determinants of risk for autoimmune diabetes. Despite the high prevalence of diabetes in U.S. racial and ethnic minority adult populations, little is known concerning the prevalence of GAD65 AA in these groups. We estimated the prevalence of GAD65 AA in 1,064 diabetic and 1,036 nondiabetic participants who were 40-90 years of age from the Third National Health and Nutrition Examination Survey (NHANES III), which provides a representative ethnic sample of the U.S. diabetic population. The prevalence of GAD65 AA was higher in diabetic participants compared with nondiabetic participants in non-Hispanic whites (n = 920; 6.3% vs. 2.0%; P = 0.001) and non-Hispanic blacks (n = 534; 3.7% vs. 1.3%; P = 0.08) but not in Mexican Americans (n = 646; 1.2% vs. 2.6%; P = 0.18). Among diabetic non-Hispanic whites and non-Hispanic blacks, being GAD65 AA positive was associated with lower BMI and C-peptide (P < 0.05). These results may reflect the outcome of an autoimmune process leading to beta-cell destruction/dysfunction in non-Hispanic white and non-Hispanic black adult diabetic patients as it occurs in a similar manner in type 1 diabetes. Among diabetic Mexican Americans, the lower prevalence of GAD65 AA suggests a lower frequency of autoimmune-related diabetes.     

Common Polymorphisms in the Adiponectin Gene ACDC Are Not Associated With Diabetes in Pima Indians

Adiponectin is an abundant adipose tissue-derived protein with important metabolic effects. Plasma adiponectin levels are decreased in obese individuals, and low adiponectin levels predict insulin resistance and type 2 diabetes. Two variants in the adiponectin gene ACDC have been previously associated with plasma adiponectin levels, obesity, insulin resistance, and type 2 diabetes. To determine the role of genetic variation in ACDC in susceptibility to obesity and type 2 diabetes in Pima Indians, we screened the promoter, exons, and exon-intron boundaries of the gene to identify allelic variants. We identified 17 informative polymorphisms that comprised four common (minor allele frequency >15%) linkage disequilibrium clusters consisting of 1-4 variants each. We genotyped one representative polymorphism from each cluster in 1,338 individuals and assessed genotypic association with type 2 diabetes, BMI, serum lipid levels, serum adiponectin levels, and measures of insulin sensitivity and secretion. None of the ACDC variants were associated with type 2 diabetes, BMI, or measures of insulin sensitivity or secretion. One variant, single nucleotide polymorphism (SNP)-12823, was associated with serum adiponectin levels (P = 0.002), but this association explained only 2% of the variance of serum adiponectin levels. Our findings suggest that these common ACDC polymorphisms do not play a major role in susceptibility to obesity or type 2 diabetes in this population.