Genetic risk scores for body fat distribution attenuate weight loss in women during dietary intervention.

OBJECTIVE: The well-established link between body fat distribution and metabolic health has been suggested to act through an impact on the remodeling capacity of the adipose tissue. Remodeling of the adipose tissue has been shown to affect body fat distribution and might affect the ability to lose weight. We aimed to study the effect of weighted genetic risk scores (GRSs) on weight loss based on single-nucleotide polymorphisms (SNPs) associated with waist-hip-ratio adjusted for body mass index (WHRadjBMI). DESIGN: We included 707 participants (533 women and 174 men) from the NUGENOB multi-center 10-week diet intervention study with weekly weight measurements. We created 3 GRSs, one including all reported WHRadjBMI SNPs (GRStotal), one including only SNPs with genome-wide significance in women or with significantly greater effect in women (GRSwomen), and one excluding SNPs in the GRSwomen (GRSmen). The data were analyzed in a mixed linear model framework. RESULTS: The GRStotal and GRSwomen attenuated weight loss in women. The effect was strongest for the GRSwomen with an effect of 2.21 g per risk allele per day (95% confidence intereval (CI) (0.90;3.52), P=0.0009). Adjustment for WHR, basal metabolic rate or diet compliance did not affect the result. The GRSs had no effect on weight loss in men. The VEGFA rs1358980-T strongly attenuated weight loss in both men and women (ß=15.95 g per risk allele per day, (3.16;26.74), P=0.013) and (ß=15.95 g per risk allele per day, (2.58;13.53), P=0.004), respectively). CONCLUSION: Our findings suggest that genetic variants influencing body fat distribution attenuate weight loss in women independently on the effect on WHR. The stronger effect of the GRSwomen implies heterogenic effects of the WHRadjBMI variants on weight loss. A strong effect of rs1358980-T in the VEGFA locus suggests that angiogenesis plays a role, but this needs confirmation from functional studies.

Estimating the causal effect of body mass index on hay fever, asthma and lung function using Mendelian randomization.

BACKGROUND: Observational studies have shown that body mass index (BMI) is positively associated with asthma. However, observational data are prone to confounding and reverse causation. In Mendelian randomization, genetic variants are used as unconfounded markers of exposures to examine causal effects. We examined the causal effect of BMI on asthma, hay fever, allergic sensitization, serum total immunoglobulin E (IgE), forced expiratory volume in one-second (FEV1) and forced vital capacity (FVC). METHODS: We included 490 497 participants in the observational and 162 124 participants in the genetic analyses. A genetic risk score (GRS) was created using 26 BMI-associated single nucleotide polymorphisms (SNPs). Results were pooled in meta-analyses and expressed as odds ratios (ORs) or ß-estimates with 95% confidence interval (CI). RESULTS: The GRS was significantly associated with asthma (OR=1.009; 95% CI: 1.004, 1.013), but not with hay fever (OR= 0.998; 95% CI: 0.994, 1.002) or allergic sensitization (OR=0.999; 95% CI: 0.986, 1.012) per BMI-increasing allele. The GRS was significantly associated with decrease in FEV1: ß=-0.0012 (95% CI: -0.0019, -0.0006) and FVC: ß=-0.0022 (95% CI: -0.0031, -0.0014) per BMI-increasing allele. Effect sizes estimated by instrumental variable analyses were OR=1.07 (95% CI: 1.03, 1.10) for asthma, a 9 ml decrease in FEV1 (95% CI: 2.0-15 mL decrease) and a 16 ml decrease in FVC (95% CI: 7.0-24 mL decrease) per 1 kg/m2 higher BMI. CONCLUSIONS: The results support the conclusion that increasing BMI is causally related to higher prevalence of asthma and decreased lung function, but not with hay fever or biomarkers of allergy.

Association studies of genetic scores of serum vitamin B12 and folate levels with symptoms of depression and anxiety in two danish population studies.

BACKGROUND/OBJECTIVES: Observational studies have suggested low serum levels of vitamin B12 or folate to be risk factors of depression and anxiety. However, these results may be biased by confounding and reverse causation. Mendelian randomization studies are not subject to these limitations. The aim was to examine the association of genetic scores of vitamin B12 and folate-associated alleles with depression and anxiety. SUBJECTS/METHODS: The study included 4126 participants from two Danish population-based studies. Serum vitamin B12 and folate were measured. Weighed allele scores were calculated as the sum of weights (genetic effect sizes) for 12 and two variants increasing circulating levels of vitamin B12 and folate, respectively. Symptoms of depression and anxiety were assessed by the Symptom Check List (SCL)-90-R, and self-reported doctor-diagnosed depression and anxiety. RESULTS: An increased weighed allele score for serum vitamin B12 was associated with decreased odds of a SCL-90-R score above the 90th percentile (OR 0.540 (95%CI 0.302-0.967)) in Health2006 but not in Inter99, in the pooled analysis (OR 0.817 (95%CI 0.331-2.018)) or with other outcomes. The weighed allele score for serum folate was not associated with any of the measured outcome variables: SCL-90-R scores of depression (pooled OR 0.603 (95%CI 0.101-3.602)), anxiety (pooled OR 0.619 (95%CI 0.110-3.495)), combined score or history of doctor-diagnosed depression or anxiety. CONCLUSION: Our results do not provide evidence for a causal effect of circulating folate or vitamin B12 on the risk of depression or anxiety. However, we cannot rule out small to moderate effects, and thus large scale studies are needed.

Mendelian randomisation study of the associations of vitamin B12 and folate genetic risk scores with blood pressure and fasting serum lipid levels in three Danish population-based studies.

BACKGROUND/OBJECTIVES: The aim was to examine the association of genetic risk scores (GRSs) of vitamin B12 and folate-associated variants with blood pressure and lipids. SUBJECTS/METHODS: The study included 12 532 adults from three population-based studies (Inter99, Health2006 and Dan-MONICA10) conducted in Denmark. GRSs were calculated by summarising the number of vitamin B12 and folate increasing alleles. Weighted GRSs were calculated as the sum of weights for each allele corresponding to genetic effects sizes. RESULTS: GRSs for serum vitamin B12 and folate were associated with serum vitamin B12 and folate, respectively. The ß coefficients (95% confidence interval (CI), P-value) for regression of log-transformed serum B12/folate on the weighted GRSs were 0.57 (0.54, 0.61), P<0.001 and 0.85 (0.70, 1.01), P<0.01. No associations were observed between the vitamin B12 GRSs and any of the blood pressure and lipid-related outcomes in the combined analyses. Increasing number of folate increasing alleles was associated with increased high-density lipoprotein (HDL) cholesterol concentrations (ß coefficient (95% CI, P-value) for regression of log-transformed HDL on the weighted GRSs, 0.081 (0.015, 0.148), P=0.017), but not with blood pressure, triglyceride, and low-density lipoprotein and total cholesterol levels. CONCLUSIONS: GRSs were not associated with blood pressure and lipid levels, except for an association between the GRS for folate and HDL cholesterol. Further studies are needed to determine whether a causal association between folate and HDL cholesterol exists.

A genetic risk score of 45 coronary artery disease risk variants associates with increased risk of myocardial infarction in 6041 Danish individuals.

BACKGROUND: In Europeans, 45 genetic risk variants for coronary artery disease (CAD) have been identified in genome-wide association studies. We constructed a genetic risk score (GRS) of these variants to estimate the effect on incidence and clinical predictability of myocardial infarction (MI) and CAD. METHODS: Genotype was available from 6041 Danes. An unweighted GRS was constructed by making a summated score of the 45 known genetic CAD risk variants. Registries provided information (mean follow-up = 11.6 years) on CAD (n = 374) and MI (n = 124) events. Cox proportional hazard estimates with age as time scale was adjusted for sex, BMI, type 2 diabetes mellitus and smoking status. Analyses were also stratified either by sex or median age (below or above 45 years of age). We estimated GRS contribution to MI prediction by assessing net reclassification index (NRI) and integrated discrimination improvement (IDI) added to the European SCORE for 10-year MI risk prediction. RESULTS: The GRS associated significantly with risk of incident MI (allele-dependent hazard ratio (95%CI): 1.06 (1.02-1.11), p = 0.01) but not with CAD (p = 0.39). Stratification revealed association of GRS with MI in men (1.06 (1.01-1.12), p = 0.02) and in individuals above the median of 45.11 years of age (1.06 (1.00-1.12), p = 0.03). There was no interaction between GRS and gender (p = 0.90) or age (p = 0.83). The GRS improved neither NRI nor IDI. CONCLUSION: The GRS of 45 GWAS identified risk variants increase the risk of MI in a Danish cohort. The GRS did not improve NRI or IDI beyond the performance of conventional European SCORE risk factors.

Sex-specific effects of naturally occurring variants in the dopamine receptor D2 locus on insulin secretion and type 2 diabetes susceptibility.

AIMS: Modulation of dopamine receptor D2 (DRD2) activity affects insulin secretion in both rodents and isolated pancreatic ß-cells. We hypothesized that single nucleotide polymorphisms in the DRD2/ANKK1 locus may affect susceptibility to type 2 diabetes in humans. METHODS: Four potentially functional variants in the coding region of the DRD2/ANKK1 locus (rs1079597, rs6275, rs6277, rs1800497) were genotyped and analysed for type 2 diabetes susceptibility in up to 25 000 people (8148 with type 2 diabetes and 17687 control subjects) from two large independent Dutch cohorts and one Danish cohort. In addition, 340 Dutch subjects underwent a 2-h hyperglycaemic clamp to investigate insulin secretion. Since sexual dimorphic associations related to DRD2 polymorphisms have been previously reported, we also performed a gender-stratified analysis. RESULTS: rs1800497 at the DRD2/ANKK1 locus was associated with a significantly increased risk for type 2 diabetes in women (odds ratio 1.14 (1.06-1.23); P = 4.1*104) but not in men (odds ratio 1.00 (95% CI 0.93-1.07); P = 0.92) or the combined group. Although rs1800497 was not associated with insulin secretion, we did find another single nucleotide polymorphism in this locus, rs6275, to be associated with increased first-phase glucose-stimulated insulin secretion in women (P = 5.5*104) but again not in men (P = 0.34). CONCLUSION: The present data identify DRD2/ANKK1 as a potential sex-specific type 2 diabetes susceptibility gene.

Replication and meta-analysis of common variants identifies a genome-wide significant locus in migraine.

BACKGROUND AND PURPOSE: Genetic factors contribute to the aetiology of the prevalent form of migraine without aura (MO) and migraine with typical aura (MTA). Due to the complex inheritance of MO and MTA, the genetic background is still not fully established. In a population-based genome-wide association study by Chasman et al. (Nat Genet 2011: 43: 695-698), three common variants were found to confer risk of migraine at a genome-wide significant level (P < 5 × 10(-8) ). We aimed to evaluate the top association single nucleotide polymorphisms (SNPs) from the discovery set by Chasman et al. in a primarily clinic-based Danish and Icelandic cohort. METHODS: The top association SNPs were assessed in 2523 cases and 38,170 controls, and a meta-analysis was performed, combining the discovery set with all the follow-up studies. Finally the confirmed SNPs were assessed in a genotype-phenotype analysis. RESULTS: Two out of three SNPs that showed genome-wide significant associations in the previous study: rs10166942 (near TRPM8) and rs11172113 (in LRP1) were significantly associated with migraine in the present study. The meta-analysis confirmed the previous three genome-wide significant associated SNPs (rs2651899, rs10166942 and rs11172113) to confer risk of migraine. In addition, the C-allele of rs2078371 (near TSPAN-2) also reached genome-wide significance for association with migraine [OR = 1.14; CI = (1.09-1.20); P = 2.55 × 10(-8) ]. CONCLUSION: TSPAN-2 encodes an integral membrane protein involved in oligodendrogenesis. This new finding supports the plausible implication of neuroglia in the pathophysiology of MO and MTA.

Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes.

AIMS/HYPOTHESIS: Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. METHODS: The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. RESULTS: Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). CONCLUSIONS/INTERPRETATION: We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.

Association studies of novel obesity-related gene variants with quantitative metabolic phenotypes in a population-based sample of 6,039 Danish individuals.

AIMS/HYPOTHESIS: Genome-wide association studies have identified novel WHR and BMI susceptibility loci. The aim of this study was to elucidate if any of these loci had an effect on quantitative measures of glucose homeostasis, including estimates of insulin release and insulin sensitivity in an epidemiological setting. METHODS: By applying an additive genetic model, 14 WHR-associated gene variants and 18 BMI-associated variants were investigated for their relationships with glucose-related metabolic traits in treatment-naive individuals from the population-based Inter99 study sample (n = 6,039). RESULTS: Of the variants associated with BMI, the QPCTL rs2287019 C allele was associated with an increased insulinogenic index of 7.4% per risk allele (p = 4.0 × 10⁻7) and increased disposition index of 5.6% (p = 6.4 × 10⁻5). The LRP1B rs2890652 C allele was associated with insulin resistance, showing a 3.3% increase (p = 0.0011) using the HOMA-insulin resistance (HOMA-IR) index and a 2.2% reduction (p = 0.0014) with the Matsuda index. Of the variants associated with WHR, LYPLAL1/SLC30A10 rs4846567 G allele carriers showed a 5.2% lower HOMA-IR (p = 0.00086) in women, indicating improved insulin sensitivity. Female carriers of the VEGFA rs6905288 A allele were insulin resistant, with a 3.7% increase in HOMA-IR (p = 0.00036) and 4.0% decrease in Matsuda index (p = 2 × 10⁻4). CONCLUSIONS: Our correlative findings from analysing single-locus data suggest that some variation in validated BMI and WHR loci are associated with either increased or decreased insulin sensitivity and thereby potentially with metabolically healthy or metabolically unhealthy subsets of obesity. The results call for testing in larger study samples and for further physiological exploration of the possible metabolic implications of these loci.

Common variation in oxidative phosphorylation genes is not a major cause of insulin resistance or type 2 diabetes.

AIMS/HYPOTHESIS: There is substantial evidence that mitochondrial dysfunction is linked to insulin resistance and is present in several tissues relevant to the pathogenesis of type 2 diabetes. Here, we examined whether common variation in genes involved in oxidative phosphorylation (OxPhos) contributes to type 2 diabetes susceptibility or influences diabetes-related metabolic traits. METHODS: OxPhos gene variants (n = 10) that had been nominally associated (p < 0.01) with type 2 diabetes in a recent genome-wide meta-analysis (n = 10,108) were selected for follow-up in 3,599 type 2 diabetic and 4,956 glucose-tolerant Danish individuals. A meta-analysis of these variants was performed in 11,729 type 2 diabetic patients and 43,943 non-diabetic individuals. The impact on OGTT-derived metabolic traits was evaluated in 5,869 treatment-naive individuals from the Danish Inter99 study. RESULTS: The minor alleles of COX10 rs9915302 (p = 0.02) and COX5B rs1466100 (p = 0.005) showed nominal association with type 2 diabetes in our Danish cohort. However, in the meta-analysis, none of the investigated variants showed a robust association with type 2 diabetes after correction for multiple testing. Among the alleles potentially associated with type 2 diabetes, none negatively influenced surrogate markers of insulin sensitivity in non-diabetic participants, while the minor alleles of UQCRC1 rs2228561 and COX10 rs10521253 showed a weak (p < 0.01 to p < 0.05) negative influence on indices of glucose-stimulated insulin secretion. CONCLUSIONS/INTERPRETATION: We cannot rule out the possibility that common variants in or near OxPhos genes may influence beta cell function in non-diabetic individuals. However, our quantitative trait studies and a sufficiently large meta-analysis indicate that common variation in proximity to the examined OxPhos genes is not a major cause of insulin resistance or type 2 diabetes.

Homozygous carriers of the G allele of rs4664447 of the glucagon gene (GCG) are characterised by decreased fasting and stimulated levels of insulin, glucagon and glucagon-like peptide (GLP)-1.

AIMS/HYPOTHESIS: The glucagon gene (GCG) encodes several hormones important for energy metabolism: glucagon, oxyntomodulin and glucagon-like peptide (GLP)-1 and -2. Variants in GCG may associate with type 2 diabetes, obesity and/or related metabolic traits. METHODS: GCG was re-sequenced as a candidate gene in 865 European individuals. Twenty-nine variants were identified. Four variants that were considered to have a likelihood for altered functionality: rs4664447, rs7581952, Ile158Val and Trp169Ter, were genotyped in 17,584 Danes. RESULTS: When examined in 5,760 treatment-naive individuals, homozygous carriers of the low frequency (minor allele frequency 2.3%) G allele of rs4664447, predicted to disrupt an essential splice enhancer binding site, had lower levels of fasting plasma glucose (mean ± SD, 4.8 ± 1.2 vs 5.5 ± 0.8 mmol/l, p = 0.004); fasting serum insulin (22 ± 14 vs 42 ± 27 pmol/l, p = 0.04); glucose-stimulated serum insulin (159 ± 83 vs 290 ± 183 pmol/l, p = 0.01) and adult height (165 ± 10 vs 172 ± 9 cm, p = 0.0009) compared with A allele carriers. During oral glucose tolerance and hyperglycaemic arginine stimulation tests, the plasma AUC for GLP-1 (730 ± 69 vs 1,334 ± 288 pmol/l × min, p = 0.0002) and basal and stimulated levels of serum insulin and plasma glucagon were ∼50% decreased (p < 0.001) among three homozygous carriers compared with nine matched wild-type carriers. rs7581952, Ile158Val and Trp169Ter (where 'Ter' indicates 'termination') variants of GCG did not significantly associate or co-segregate with the metabolic traits examined. CONCLUSIONS/INTERPRETATION: Re-sequencing of GCG revealed a low frequency intronic variant, rs4664447, and follow-up physiological studies suggest that this variant in homozygous form may cause decreased fasting and stimulated levels of insulin, glucagon and GLP-1. Overall, our findings suggest that variation in GCG has no major impact on carbohydrate metabolism in the study populations examined.

Type 2 diabetes risk allele near CENTD2 is associated with decreased glucose-stimulated insulin release.

AIMS/HYPOTHESIS: By combining multiple genome-wide association (GWA) studies and comprehensive replication efforts, 12 novel type 2 diabetes associated loci have recently been discovered. Here we evaluate the effect of lead variants of these loci on estimates of insulin release and insulin resistance derived from an oral glucose tolerance test. METHODS: We examined 12 lead variants in or near HMGA2, CENTD2 (also known as ARAP1), KLF14, PRC1, TP53INP1, ZBED3, ZFAND6, CHCHD9, DUSP9, KCNQ1, BCL11A and HNF1A in 5,722 middle-aged people from the population-based Inter99 sample. RESULTS: Carriers of the major diabetogenic allele of rs1552224 in CENTD2 had increased 30-min plasma glucose values (2.0%, p = 2 × 10(-5)) as well as 4.2% reduced insulin release 30 min after an oral glucose load (p = 0.001). Risk allele carriers also had decreased BIGTT-acute insulin release (AIR), which is a surrogate measure of insulin release where sex, BMI, plasma glucose and serum insulin are integrated (5.3%, p = 8 × 10(-7)). In addition, a decreased corrected insulin response (CIR; 9.9%, p = 3 × 10(-8)) was observed. For rs5945326 near DUSP9 on the X-chromosome we stratified according to sex. Male carriers of the risk allele showed nominally decreased BIGTT-AIR (2.6%, p = 0.01). No associations with intermediate metabolic traits were found in women. For the remaining ten lead variants no consistent associations were demonstrated. CONCLUSIONS/INTERPRETATION: Of the lead variants from 12 novel type 2 diabetes associated loci, CENTD2 significantly associated with increased plasma glucose values and decreased glucose-stimulated insulin release, suggesting that the diabetogenic effect of this locus is mediated through an impaired pancreatic beta cell function.

The diabetogenic VPS13C/C2CD4A/C2CD4B rs7172432 variant impairs glucose-stimulated insulin response in 5,722 non-diabetic Danish individuals.

AIMS/HYPOTHESIS: A genome-wide association study in the Japanese population reported two genome-wide significant loci associated with type 2 diabetes of which the VPS13C/C2CD4A/C2CD4B locus was replicated in Europeans. We looked for potential associations between the diabetogenic VPS13C/C2CD4A/C2CD4B rs7172432 variant and diabetes-related intermediary traits. METHODS: We genotyped the rs7172432 variant in the population-based Inter99 cohort (n = 6,784) and analysed quantitative diabetes-related traits in 5,722 non-diabetic participants who all were examined by an OGTT. RESULTS: The diabetes-associated A allele was associated with 0.60 cm higher waist circumference (p = 0.004), 0.037 mmol/l higher fasting plasma glucose (p = 4 × 10(-5)) and 0.11 mmol/l higher plasma glucose at 30 min during an OGTT (p = 4 × 10(-4)). In analyses adjusted for concomitant insulin sensitivity levels the diabetogenic allele was associated with a lower acute glucose-stimulated insulin response (GSIR) as estimated by 30 min serum insulin (ß = -0.039, p = 2 × 10(-7)), insulinogenic index (ß = -0.057, p = 1 × 10(-8)) and BIGTT-acute insulin release (ß = -0.041, p = 9 × 10(-9)). As rs7172432 is situated in a region previously associated with glycaemic traits, we tested linkage disequilibrium (LD) with the reported regional lead single-nucleotide polymorphisms for fasting (rs11071657) and 2 h plasma glucose (rs17271305), and performed conditional analyses of rs7172432. Rs7172432 showed moderate LD with rs11071657 and rs17271305 (R (2) < 0.34) and we found strong association by almost unchanged effect sizes of rs7172432 with plasma glucose and estimates of GSIR in analyses conditional on rs11071657 and rs17271305. CONCLUSIONS/INTERPRETATION: The diabetogenic VPS13C/C2CD4A/C2CD4B rs7172432 A allele associates with GSIR in non-diabetic individuals from the general population, suggesting an impaired beta cell function as an intermediary diabetes-related trait.

Glucose tolerance, insulin sensitivity and insulin release in European non-diabetic carriers of a polymorphism upstream of CDKN2A and CDKN2B.

AIMS/HYPOTHESIS: The aim of this study was to investigate the association of the rs10811661 polymorphism near the CDKN2B/CDKN2A genes with glucose tolerance, insulin sensitivity and insulin release in three samples of white people with European ancestry. METHODS: Sample 1 comprised 845 non-diabetic offspring of type 2 diabetes patients recruited in five European centres participating in the EUGENE2 study. Samples 2 and 3 comprised, respectively, 864 and 524 Italian non-diabetic participants. All individuals underwent an OGTT. Screening for the rs10811661 polymorphism was performed using a TaqMan allelic discrimination assay. RESULTS: The rs10811661 polymorphism did not show a significant association with age, BMI and insulin sensitivity. Participants carrying the TT genotype showed a significant reduction in insulin release, measured by an OGTT-derived index, compared with carriers of the C allele, in the three samples. When these results were pooled with those of three published studies, and meta-analysed with a random-effects model, the T allele was significantly associated with reduced insulin secretion (-35.09 [95% CI 14.68-55.52], p = 0.0008 for CC+CT vs TT; and -29.45 [95% CI 9.51-49.38], p = 0.0038, for the additive model). In addition, in our three samples, participants carrying the TT genotype exhibited an increased risk for impaired glucose tolerance (IGT) compared with carriers of the C allele (OR 1.55 [95% CI 1.20-1.95] for the meta-analysis of the three samples). CONCLUSIONS/INTERPRETATION: Our data, together with the meta-analysis of previously published studies, show that the rs10811661 polymorphism is associated with impaired insulin release and IGT, suggesting that this variant may contribute to type 2 diabetes by affecting beta cell function.

Carriers of the TCF7L2 rs7903146 TT genotype have elevated levels of plasma glucose, serum proinsulin and plasma gastric inhibitory polypeptide (GIP) during a meal test.

AIMS/HYPOTHESIS: The transcription factor 7-like 2 (TCF7L2) rs7903146 T allele associates with type 2 diabetes in several populations, possibly mediated via decreased incretin secretion and/or action and altered beta and alpha cell function. We aimed to study circulating levels of glucose, proinsulin, insulin, C-peptide, glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and gastric inhibitory polypeptide (GIP) among individuals carrying the high-risk rs7903146 TT genotype and low-risk CC genotype following a meal test. METHODS: A meal challenge was performed in 31 glucose-tolerant men (age 54 ± 7 years and BMI 26 ± 3 kg/m²) with rs7903146 TT genotype and 31 glucose-tolerant age- and BMI-matched men with CC genotype (age 53 ± 6 years and BMI 26 ± 3 kg/m²). Serum proinsulin, insulin, C-peptide and plasma glucose, glucagon, GLP-1, GLP-2 and GIP were obtained 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 180, 210, and 240 min after ingestion of a standardised breakfast meal. RESULTS: An elevated incremental AUC for plasma glucose was observed among TT genotype carriers (CC carriers 21.8 ± 101.9 mmol/l × min vs TT carriers 97.9 ± 89.2 mmol/l × min, p = 0.001). TT carriers also had increased AUCs for proinsulin (CC carriers 6,030 ± 3,001 pmol/l × min vs TT carriers 6,917 ± 4,820 pmol/l × min, p = 0.03), C-peptide (CC carriers 397.6 ± 131.9 nmol/l × min vs TT carriers 417.1 ± 109.3 nmol/l × min, p = 0.04) and GIP (CC carriers 12,310 ± 3,840 pmol/l × min vs TT carriers 14,590 ± 5,910 pmol/l × min, p = 0.004). CONCLUSIONS/INTERPRETATION: Middle-aged normoglycaemic individuals carrying the rs7903146 TCF7L2 risk TT genotype show early signs of dysregulated glucose metabolism, decreased processing of proinsulin and elevated GIP secretion following a meal challenge.

Type 2 diabetes risk alleles near ADCY5, CDKAL1 and HHEX-IDE are associated with reduced birthweight.

AIMS/HYPOTHESIS: The fetal insulin hypothesis suggests that variation in the fetal genotype influencing insulin secretion or action may predispose to low birthweight and type 2 diabetes. We examined associations between 25 confirmed type 2 diabetes risk variants and birthweight in individuals from the Danish Inter99 population and in meta-analyses including Inter99 data and reported studies. METHODS: Midwife records from the Danish State Archives provided information on mother's age and parity, as well as birthweight, length at birth and prematurity of the newborn in 4,744 individuals of the population-based Inter99 study. We genotyped 25 risk alleles showing genome-wide associations with type 2 diabetes. RESULTS: Birthweight was inversely associated with the type 2 diabetes risk alleles of ADCY5 rs11708067 (beta = -33 g [95% CI -55, -10], p = 0.004) and CDKAL1 rs7756992 (beta = -22 g [95% CI -43, -1], p = 0.04). The association for the latter locus was confirmed in a meta-analysis (n = 24,885) (beta = -20 g [95% CI -29, -11], p = 5 x 10(-6)). The HHEX-IDE rs1111875 variant showed no significant association among Danes (p = 0.09); however, in a meta-analysis (n = 25,164) this type 2 diabetes risk allele was associated with lower birthweight (beta = -16 g [95% CI -24, -8], p = 8 x 10(-5)). On average, individuals with high genetic risk (>or=25 type 2 diabetes risk alleles) weighed marginally less at birth than those with low genetic risk (<25 type 2 diabetes risk alleles) (beta = -35 g [95% CI -69, -2], p = 0.037). CONCLUSIONS/INTERPRETATION: We report a novel association between the fetal ADCY5 type 2 diabetes risk allele and decreased birthweight, and confirm in meta-analyses associations between decreased birthweight and the type 2 diabetes risk alleles of HHEX-IDE and CDKAL1. No strong general effect on birthweight can be ascribed to the 25 common type 2 diabetes risk alleles.

Variants at DGKB/TMEM195, ADRA2A, GLIS3 and C2CD4B loci are associated with reduced glucose-stimulated beta cell function in middle-aged Danish people.

AIMS/HYPOTHESIS: A meta-analysis of 21 genome-wide association studies identified 11 novel genetic loci implicated in fasting glucose homeostasis. We aimed to evaluate the impact of these variants on insulin release and insulin sensitivity estimated from OGTTs. METHODS: Eleven variants in or near DGKB/TMEM195, ADCY5, MADD, ADRA2A, FADS1, CRY2, SLC2A2, GLIS3, PROX1, C2CD4B and IGF1 were genotyped in 6,784 middle-aged participants of the population-based Inter99 cohort. Association studies of quantitative estimates of insulin release and insulin sensitivity were performed in 5,722 non-diabetic Danish participants on whom an OGTT was performed. RESULTS: Assuming an additive genetic model, carriers of the alleles increasing fasting glucose in DGKB/TMEM195, ADRA2A, GLIS3 and C2CD4B showed decreased glucose-stimulated insulin release as assessed by the BIGTT-acute insulin response index (2.7-3.5%; p < 0.005 for all) and by corrected insulin response (2.8-5.9%; p < 0.03 for all). In addition, the PROX1 glucose-raising allele showed a 2.9% decreased corrected insulin response (p = 0.03), while the hyperglycaemic allele of variants in or near ADRA2A, FADS1, CRY2 and C2CD4B were associated with a 2.6% to 9.3% decrease in one or both of two different OGTT-based disposition indices (p < 0.02 for all). After correction for multiple testing, variants in the DGKB/TMEM195, ADRA2A, GLIS3 and C2CD4B loci were associated with estimates of beta cell function. CONCLUSIONS/INTERPRETATION: We found that the lead variants at the DGKB/TMEM195, ADRA2A, GLIS3 and C2CD4B loci were associated with decreased glucose-stimulated insulin response. This association underlines the importance of pancreatic beta cell dysfunction in the genetic predisposition to hyperglycaemia and type 2 diabetes.

A variant in the G6PC2/ABCB11 locus is associated with increased fasting plasma glucose, increased basal hepatic glucose production and increased insulin release after oral and intravenous glucose loads.

AIMS/HYPOTHESIS: An association between elevated fasting plasma glucose and the common rs560887 G allele in the G6PC2/ABCB11 locus has been reported. In Danes we aimed to examine rs560887 in relation to plasma glucose and serum insulin responses following oral and i.v. glucose loads and in relation to hepatic glucose production during a hyperinsulinaemic-euglycaemic clamp. Furthermore, we examined rs560887 for association with impaired fasting glycaemia (IFG), impaired glucose tolerance (IGT), type 2 diabetes and components of the metabolic syndrome. METHODS: rs560887 was genotyped in the Inter99 cohort (n = 5,899), in 366 young, healthy Danes, in non-diabetic relatives of type 2 diabetic patients (n = 196), and in young and elderly twins (n = 159). Participants underwent an OGTT, an IVGTT or a 2 h hyperinsulinaemic-euglycaemic clamp. RESULTS: The rs560887 G allele associated with elevated fasting plasma glucose (p = 2 x 10(-14)) but not with plasma glucose levels at 30 min (p = 0.9) or 120 min (p = 0.9) during an OGTT. G allele carriers had elevated levels of serum insulin at 30 min during an OGTT (p = 1 x 10(-4)) and relatives of type 2 diabetes patients carrying the G allele had an increased acute insulin response (p = 4 x 10(-4)) during an IVGTT. Among elderly twins, G allele carriers had higher basal hepatic glucose production (p = 0.04). Finally, the G allele associated with the risk of having IFG (OR 1.26, 95% CI 1.08-1.47, p = 0.002), but not with IGT (OR 0.94, 95% CI 0.82-1.08, p = 0.4) or type 2 diabetes (OR 0.93, 95% CI 0.84-1.04, p = 0.2). CONCLUSIONS/INTERPRETATION: The common rs560887 G allele in the G6PC2/ABCB11 locus is associated with increased fasting glycaemia and increased risk of IFG, associations that may be partly related to an increased basal hepatic glucose production rate.

Combined analysis of 19 common validated type 2 diabetes susceptibility gene variants shows moderate discriminative value and no evidence of gene-gene interaction.

AIMS/HYPOTHESIS: The list of validated type 2 diabetes susceptibility variants has recently been expanded from three to 19. The variants identified are common and have low penetrance in the general population. The aim of the study is to investigate the combined effect of the 19 variants by applying receiver operating characteristics (ROC) to demonstrate the discriminatory value between glucose-tolerant individuals and type 2 diabetes patients in a cross-sectional population of Danes. METHODS: The 19 variants were genotyped in three study populations: the population-based Inter99 study; the ADDITION study; and additional type 2 diabetic patients and glucose-tolerant individuals. The case-control studies involved 4,093 type 2 diabetic patients and 5,302 glucose-tolerant individuals. RESULTS: Single-variant analyses demonstrated allelic odds ratios ranging from 1.04 (95% CI 0.98-1.11) to 1.33 (95% CI 1.22-1.45). When combining the 19 variants, subgroups with extreme risk profiles showed a threefold difference in the risk of type 2 diabetes (lower 10% carriers with < or =15 risk alleles vs upper 10% carriers with > or =22 risk alleles, OR 2.93 (95% CI 2.38-3.62, p = 1.6 x 10(-25)). We calculated the area under a ROC curve to estimate the discrimination rate between glucose-tolerant individuals and type 2 diabetes patients based on the 19 variants. We found an area under the ROC curve of 0.60. Two-way gene-gene interaction showed few nominal interaction effects. CONCLUSIONS/INTERPRETATION: Combined analysis of the 19 validated variants enables detection of subgroups at substantially increased risk of type 2 diabetes; however, the discrimination between glucose-tolerant and type 2 diabetes individuals is still too inaccurate to achieve clinical value.

Variation in the peroxisome proliferator-activated receptor delta gene in relation to common metabolic traits in 7,495 middle-aged white people.

AIMS/HYPOTHESIS: Studies in animals reveal that peroxisome proliferator-activated receptor delta (PPARdelta) regulates glucose metabolism and insulin sensitivity in both the liver and skeletal muscles. Moreover, PPARdelta augments physical endurance and increases oxidative metabolism, thereby averting obesity. Thus, we hypothesised that common variation in the PPARD gene is associated with insulin resistance and metabolic traits. MATERIALS AND METHODS: We studied variation in the exonic region of PPARD. Based upon the results of variant detection and information derived from the HapMap data resource, we selected common variants and tag single-nucleotide polymorphisms for genotyping in 7,495 white subjects, including 1,416 patients with type 2 diabetes. RESULTS: Fourteen nucleotide variants were identified and a total of 12 variants capturing the common variation of PPARD were genotyped. In the population-based Inter99 (ClinicalTrials.gov ID no: NCT00289237) sample we observed no robust association with homeostasis model assessment of insulin resistance (HOMA-IR), adiposity measures or fasting serum lipids. Similarly, no association with type 2 diabetes or the metabolic syndrome was found. CONCLUSIONS/INTERPRETATION: Based on thorough investigation, we conclude that common variation in PPARD does not significantly affect the risk of metabolic disease in the population studied. Given the confidence intervals that were found for effect size estimates, we can effectively rule out an increase in HOMA-IR of any tag SNP above 7% per allele, assuming an additive model. Likewise, we can exclude an odds ratio of type 2 diabetes above 1.27 per allele.