General Framework for Meta-Analysis of Haplotype Association Tests.

For complex traits, most associated single nucleotide variants (SNV) discovered to date have a small effect, and detection of association is only possible with large sample sizes. Because of patient confidentiality concerns, it is often not possible to pool genetic data from multiple cohorts, and meta-analysis has emerged as the method of choice to combine results from multiple studies. Many meta-analysis methods are available for single SNV analyses. As new approaches allow the capture of low frequency and rare genetic variation, it is of interest to jointly consider multiple variants to improve power. However, for the analysis of haplotypes formed by multiple SNVs, meta-analysis remains a challenge, because different haplotypes may be observed across studies. We propose a two-stage meta-analysis approach to combine haplotype analysis results. In the first stage, each cohort estimate haplotype effect sizes in a regression framework, accounting for relatedness among observations if appropriate. For the second stage, we use a multivariate generalized least square meta-analysis approach to combine haplotype effect estimates from multiple cohorts. Haplotype-specific association tests and a global test of independence between haplotypes and traits are obtained within our framework. We demonstrate through simulation studies that we control the type-I error rate, and our approach is more powerful than inverse variance weighted meta-analysis of single SNV analysis when haplotype effects are present. We replicate a published haplotype association between fasting glucose-associated locus (G6PC2) and fasting glucose in seven studies from the Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium and we provide more precise haplotype effect estimates.

Joint effect of unlinked genotypes: application to type 2 diabetes in the EPIC-Potsdam case-cohort study.

Analyzing multiple single nucleotide polymorphisms (SNPs) is a promising approach to finding genetic effects beyond single-locus associations. We proposed the use of multilocus stepwise regression (MSR) to screen for allele combinations as a method to model joint effects, and compared the results with the often used genetic risk score (GRS), conventional stepwise selection, and the shrinkage method LASSO. In contrast to MSR, the GRS, conventional stepwise selection, and LASSO model each genotype by the risk allele doses. We reanalyzed 20 unlinked SNPs related to type 2 diabetes (T2D) in the EPIC-Potsdam case-cohort study (760 cases, 2193 noncases). No SNP-SNP interactions and no nonlinear effects were found. Two SNP combinations selected by MSR (Nagelkerke's R² = 0.050 and 0.048) included eight SNPs with mean allele combination frequency of 2%. GRS and stepwise selection selected nearly the same SNP combinations consisting of 12 and 13 SNPs (Nagelkerke's R² ranged from 0.020 to 0.029). LASSO showed similar results. The MSR method showed the best model fit measured by Nagelkerke's R² suggesting that further improvement may render this method a useful tool in genetic research. However, our comparison suggests that the GRS is a simple way to model genetic effects since it does not consider linkage, SNP-SNP interactions, and no non-linear effects.

Hemochromatosis (HFE) gene mutations and risk of gastric cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

Hereditary hemochromatosis (HH) is a strong risk factor for hepatocellular cancer, and mutations in the HFE gene associated with HH and iron overload may be related to other tumors, but no studies have been reported for gastric cancer (GC). A nested case-control study was conducted within the European Prospective Investigation into Cancer and Nutrition (EPIC), including 365 incident gastric adenocarcinoma and 1284 controls matched by center, sex, age and date of blood collection. Genotype analysis was performed for two functional polymorphisms (C282Y/rs1800562 and H63D/rs1799945) and seven tagSNPs of the HFE genomic region. Association with all gastric adenocarcinoma, and according to anatomical localization and histological subtype, was assessed by means of the odds ratio (OR) and 95% confidence interval (CI) estimated by unconditional logistic regression adjusted for the matching variables. We observed a significant association for H63D with OR (per rare allele) of 1.32 (CI = 1.03-1.69). In subgroup analyses, the association was stronger for non-cardia anatomical subsite (OR = 1.60, CI = 1.16-2.21) and intestinal histological subtype (OR = 1.82, CI = 1.27-2.62). Among intestinal cases, two tagSNPs (rs1572982 and rs6918586) also showed a significant association that disappeared after adjustment for H63D. No association with tumors located in the cardia or with diffuse subtype was found for any of the nine SNPs analyzed. Our results suggest that H63D variant in HFE gene seems to be associated with GC risk of the non-cardia region and intestinal type, possibly due to its association with iron overload although a role for other mechanisms cannot be entirely ruled out.

Prostate stem-cell antigen gene is associated with diffuse and intestinal gastric cancer in Caucasians: results from the EPIC-EURGAST study.

A genome-wide study performed in a Japanese population identified a strong association between SNP rs2294008 (Met1Thr) in the Prostate Stem Cell Antigen gene (PSCA) and diffuse-type gastric cancer (GC). This association was validated in different Asian populations, and, very recently, a study has been published in Caucasians. In this study, we analyzed the association between PSCA variation and GC risk in Caucasians from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Six tagSNPs covering the PSCA gene region were genotyped in 411 incident gastric adenocarcinoma cases and 1530 matched controls from a nested case-control study in the EPIC cohort. Associations were analyzed by unconditional logistic regression, adjusting for age, sex and country. The T allele of rs2294008 in PSCA was found to be a highly significant risk factor for GC (per allele OR = 1.42, 95% CI: 1.23-1.66, p-value = 6.5 × 10(-6) ), particularly of the noncardia-type (per allele OR = 1.47, 95% CI: 1.19-1.81, p-value = 3 × 10(-4) ). At contrast with previous studies, no significant differences were observed between the diffuse (per allele OR = 1.54, 95% CI: 1.20-1.96, p-value = 5 × 10(-4) ) and the intestinal (per allele OR = 1.52, 95% CI: 1.20-1.93, p-value = 5 × 10(-4) ) GC histological subtypes. Although rs12155758 and rs9297976 were also found associated with GC, this association appeared to be due to linkage disequilibrium with rs2294008. Haplotype analysis did not provide additional information. These results confirm the association between variation in the promoter region of PSCA and GC risk in Caucasians and also indicate that the rs2294008 variant is a similar risk factor for both the diffuse and intestinal-types of GC.

Adiponectin level as a consequence of genetic variation, but not leptin level or leptin: adiponectin ratio, is a risk factor for carotid intima-media thickness.

BACKGROUND AND PURPOSE: The adipocytokines adiponectin and leptin have been suggested as risk factors for cardiovascular disease, including stroke, acting through atherosclerosis. However, studies have provided conflicting results in underpowered cohorts with some suggesting that the leptin:adiponectin ratio is a better predictor of risk. We examined these associations with carotid intima-media thickness (IMT), a marker of early atherosclerosis and arterial remodeling and an independent predictor of stroke. We also examined association between genetic variants in the leptin and adiponectin genes and IMT. METHODS: Adiponectin and leptin levels were determined in 990 individuals from the community Carotid Atherosclerosis Progression Study. Five variants in the gene encoding adiponectin and 7 in the gene encoding leptin were genotyped and their effects on circulating levels assessed. Both were then correlated with IMT. RESULTS: Adiponectin levels negatively correlated with IMT (-0.079, P=0.013). There was no correlation between leptin levels or leptin:adiponectin ratio and IMT. Two variants in the ADIPOQ gene encoding adiponectin were associated with altered adiponectin levels, 1 of which (rs266729) was associated with IMT. There was also an interaction with body mass index (P=0.019) with the association being present in obese subjects (P=0.02). CONCLUSIONS: Our results support a causal role for adiponectin in early carotid IMT and suggest it may act through interaction with obesity. In contrast, we found no evidence of a role for leptin and no evidence that leptin:adiponectin ratio is a better predictor of risk that adiponectin levels alone.

Physical activity attenuates the influence of FTO variants on obesity risk: a meta-analysis of 218,166 adults and 19,268 children.

BACKGROUND: The FTO gene harbors the strongest known susceptibility locus for obesity. While many individual studies have suggested that physical activity (PA) may attenuate the effect of FTO on obesity risk, other studies have not been able to confirm this interaction. To confirm or refute unambiguously whether PA attenuates the association of FTO with obesity risk, we meta-analyzed data from 45 studies of adults (n = 218,166) and nine studies of children and adolescents (n = 19,268). METHODS AND FINDINGS: All studies identified to have data on the FTO rs9939609 variant (or any proxy [r(2)>0.8]) and PA were invited to participate, regardless of ethnicity or age of the participants. PA was standardized by categorizing it into a dichotomous variable (physically inactive versus active) in each study. Overall, 25% of adults and 13% of children were categorized as inactive. Interaction analyses were performed within each study by including the FTO×PA interaction term in an additive model, adjusting for age and sex. Subsequently, random effects meta-analysis was used to pool the interaction terms. In adults, the minor (A-) allele of rs9939609 increased the odds of obesity by 1.23-fold/allele (95% CI 1.20-1.26), but PA attenuated this effect (p(interaction)  = 0.001). More specifically, the minor allele of rs9939609 increased the odds of obesity less in the physically active group (odds ratio  = 1.22/allele, 95% CI 1.19-1.25) than in the inactive group (odds ratio  = 1.30/allele, 95% CI 1.24-1.36). No such interaction was found in children and adolescents. CONCLUSIONS: The association of the FTO risk allele with the odds of obesity is attenuated by 27% in physically active adults, highlighting the importance of PA in particular in those genetically predisposed to obesity.

Variation in genes coding for AMP-activated protein kinase (AMPK) and breast cancer risk in the European Prospective Investigation on Cancer (EPIC).

AMP-activated protein kinase (AMPK) is an energy sensing/signalling intracellular protein which is activated by an increase in the cellular AMP:ATP ratio after ATP depletion. Once activated, AMPK inhibits fatty acid synthesis and the Akt-mTOR pathway, and activates the p53-p21 axis. All these molecular mechanisms are thought to play a key role in breast carcinogenesis. We investigated the genetic variability of four genes encoding AMPK (PRKAA1, PRKAA2, PRKAB1 and PRKAB2). Using a tagging approach and selecting SNPs we covered all the common genetic variation of these genes. We tested association of tagging SNPs in our four candidate genes with breast cancer (BC) risk in a study of 1340 BC cases and 2536 controls nested into the European Prospective Investigation into Cancer and Nutrition (EPIC). Given the relevance of AMPK on fatty acid synthesis and the importance of body fatness as a BC risk factor, we tested association of SNPs and body-mass index as well. We observed no statistically significant association between the SNPs in the PRKAs genes and BC risk and BMI after correction for multiple testing.

Haplotypes of the porcine peroxisome proliferator-activated receptor delta gene are associated with backfat thickness.

BACKGROUND: Peroxisome proliferator-activated receptor delta belongs to the nuclear receptor superfamily of ligand-inducible transcription factors. It is a key regulator of lipid metabolism. The peroxisome proliferator-activated receptor delta gene (PPARD) has been assigned to a region on porcine chromosome 7, which harbours a quantitative trait locus for backfat. Thus, PPARD is considered a functional and positional candidate gene for backfat thickness. The purpose of this study was to test this candidate gene hypothesis in a cross of breeds that were highly divergent in lipid deposition characteristics. RESULTS: Screening for genetic variation in porcine PPARD revealed only silent mutations. Nevertheless, significant associations between PPARD haplotypes and backfat thickness were observed in the F2 generation of the Mangalitsa x Piétrain cross as well as a commercial German Landrace population. Haplotype 5 is associated with increased backfat in F2 Mangalitsa x Piétrain pigs, whereas haplotype 4 is associated with lower backfat thickness in the German Landrace population. Haplotype 4 and 5 carry the same alleles at all but one SNP. Interestingly, the opposite effects of PPARD haplotypes 4 and 5 on backfat thickness are reflected by opposite effects of these two haplotypes on PPAR-delta mRNA levels. Haplotype 4 significantly increases PPAR-delta mRNA levels, whereas haplotype 5 decreases mRNA levels of PPAR-delta. CONCLUSION: This study provides evidence for an association between PPARD and backfat thickness. The association is substantiated by mRNA quantification. Further studies are required to clarify, whether the observed associations are caused by PPARD or are the result of linkage disequilibrium with a causal variant in a neighbouring gene.

Gene-lifestyle interaction on risk of type 2 diabetes: A systematic review.

The pathophysiological influence of gene-lifestyle interactions on the risk to develop type 2 diabetes (T2D) is currently under intensive research. This systematic review summarizes the evidence for gene-lifestyle interactions regarding T2D incidence. MEDLINE, EMBASE, and Web of Science were systematically searched until 31 January 2019 to identify publication with (a) prospective study design; (b) T2D incidence; (c) gene-diet, gene-physical activity, and gene-weight loss intervention interaction; and (d) population who are healthy or prediabetic. Of 66 eligible publications, 28 reported significant interactions. A variety of different genetic variants and dietary factors were studied. Variants at TCF7L2 were most frequently investigated and showed interactions with fiber and whole grain on T2D incidence. Further gene-diet interactions were reported for, eg, a western dietary pattern with a T2D-GRS, fat and carbohydrate with IRS1 rs2943641, and heme iron with variants of HFE. Physical activity showed interaction with HNF1B, IRS1, PPARγ, ADRA2B, SLC2A2, and ABCC8 variants and weight loss interventions with ENPP1, PPARγ, ADIPOR2, ADRA2B, TNFα, and LIPC variants. However, most findings represent single study findings obtained in European ethnicities. Although some interactions have been reported, their conclusiveness is still low, as most findings were not yet replicated across multiple study populations.

Insulin-Like Growth Factor Binding Protein 2 (IGFBP-2) and the Risk of Developing Type 2 Diabetes.

Recent studies suggest that insulin-like growth factor binding protein 2 (IGFBP-2) may protect against type 2 diabetes, but population-based human studies are scarce. We aimed to investigate the prospective association of circulating IGFBP-2 concentrations and of differential methylation in the IGFBP-2 gene with type 2 diabetes risk.

Associations of autozygosity with a broad range of human phenotypes.

In many species, the offspring of related parents suffer reduced reproductive success, a phenomenon known as inbreeding depression. In humans, the importance of this effect has remained unclear, partly because reproduction between close relatives is both rare and frequently associated with confounding social factors. Here, using genomic inbreeding coefficients (FROH) for >1.4 million individuals, we show that FROH is significantly associated (p < 0.0005) with apparently deleterious changes in 32 out of 100 traits analysed. These changes are associated with runs of homozygosity (ROH), but not with common variant homozygosity, suggesting that genetic variants associated with inbreeding depression are predominantly rare. The effect on fertility is striking: FROH equivalent to the offspring of first cousins is associated with a 55% decrease [95% CI 44-66%] in the odds of having children. Finally, the effects of FROH are confirmed within full-sibling pairs, where the variation in FROH is independent of all environmental confounding.

Circulating Fetuin-A and Risk of Type 2 Diabetes: A Mendelian Randomization Analysis.

Fetuin-A, a hepatic-origin protein, is strongly positively associated with risk of type 2 diabetes in human observational studies, but it is unknown whether this association is causal. We aimed to study the potential causal relation of circulating fetuin-A to risk of type 2 diabetes in a Mendelian randomization study with single nucleotide polymorphisms located in the fetuin-A-encoding AHSG gene. We used data from eight European countries of the European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct case-cohort study including 10,020 incident cases. Plasma fetuin-A concentration was measured in a subset of 965 subcohort participants and 654 case subjects. A genetic score of the AHSG single nucleotide polymorphisms was strongly associated with fetuin-A (28% explained variation). Using the genetic score as instrumental variable of fetuin-A, we observed no significant association of a 50 µg/mL higher fetuin-A concentration with diabetes risk (hazard ratio 1.02 [95% CI 0.97, 1.07]). Combining our results with those from the DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) consortium (12,171 case subjects) also did not suggest a clear significant relation of fetuin-A with diabetes risk. In conclusion, although there is mechanistic evidence for an effect of fetuin-A on insulin sensitivity and secretion, this study does not support a strong, relevant relationship between circulating fetuin-A and diabetes risk in the general population.

Interaction of Dietary and Genetic Factors Influencing Body Iron Status and Risk of Type 2 Diabetes Within the EPIC-InterAct Study.

OBJECTIVE: Meat intake has been consistently shown to be positively associated with incident type 2 diabetes. Part of that association may be mediated by body iron status, which is influenced by genetic factors. We aimed to test for interactions of genetic and dietary factors influencing body iron status in relation to the risk of incident type 2 diabetes. RESEARCH DESIGN AND METHODS: The case-cohort comprised 9,347 case subjects and 12,301 subcohort participants from eight European countries. Single nucleotide polymorphisms (SNPs) were selected from genome-wide association studies on iron status biomarkers and candidate gene studies. A ferritin-related gene score was constructed. Multiplicative and additive interactions of heme iron and SNPs as well as the gene score were evaluated using Cox proportional hazards regression. RESULTS: Higher heme iron intake (per 1 SD) was associated with higher ferritin levels (ß = 0.113 [95% CI 0.082; 0.144]), but not with transferrin (-0.019 [-0.043; 0.006]) or transferrin saturation (0.016 [-0.006; 0.037]). Five SNPs located in four genes (rs1799945 [HFE H63D], rs1800562 [HFE C282Y], rs236918 [PCK7], rs744653 [SLC40A1], and rs855791 [TMPRSS6 V736A]) were associated with ferritin. We did not detect an interaction of heme iron and the gene score on the risk of diabetes in the overall study population (Padd = 0.16, Pmult = 0.21) but did detect a trend toward a negative interaction in men (Padd = 0.04, Pmult = 0.03). CONCLUSIONS: We found no convincing evidence that the interplay of dietary and genetic factors related to body iron status associates with type 2 diabetes risk above the level expected from the sum or product of the two individual exposures.

Refining the accuracy of validated target identification through coding variant fine-mapping in type 2 diabetes.

We aggregated coding variant data for 81,412 type 2 diabetes cases and 370,832 controls of diverse ancestry, identifying 40 coding variant association signals (P < 2.2 × 10-7); of these, 16 map outside known risk-associated loci. We make two important observations. First, only five of these signals are driven by low-frequency variants: even for these, effect sizes are modest (odds ratio ≤1.29). Second, when we used large-scale genome-wide association data to fine-map the associated variants in their regional context, accounting for the global enrichment of complex trait associations in coding sequence, compelling evidence for coding variant causality was obtained for only 16 signals. At 13 others, the associated coding variants clearly represent 'false leads' with potential to generate erroneous mechanistic inference. Coding variant associations offer a direct route to biological insight for complex diseases and identification of validated therapeutic targets; however, appropriate mechanistic inference requires careful specification of their causal contribution to disease predisposition.

Genetic variants including markers from the exome chip and metabolite traits of type 2 diabetes.

Diabetes-associated metabolites may aid the identification of new risk variants for type 2 diabetes. Using targeted metabolomics within a subsample of the German EPIC-Potsdam study (n = 2500), we tested previously published SNPs for their association with diabetes-associated metabolites and conducted an additional exploratory analysis using data from the exome chip including replication within 2,692 individuals from the German KORA F4 study. We identified a total of 16 loci associated with diabetes-related metabolite traits, including one novel association between rs499974 (MOGAT2) and a diacyl-phosphatidylcholine ratio (PC aa C40:5/PC aa C38:5). Gene-based tests on all exome chip variants revealed associations between GFRAL and PC aa C42:1/PC aa C42:0, BIN1 and SM (OH) C22:2/SM C18:0 and TFRC and SM (OH) C22:2/SM C16:1). Selecting variants for gene-based tests based on functional annotation identified one additional association between OR51Q1 and hexoses. Among single genetic variants consistently associated with diabetes-related metabolites, two (rs174550 (FADS1), rs3204953 (REV3L)) were significantly associated with type 2 diabetes in large-scale meta-analysis for type 2 diabetes. In conclusion, we identified a novel metabolite locus in single variant analyses and four genes within gene-based tests and confirmed two previously known mGWAS loci which might be relevant for the risk of type 2 diabetes.

Determining the binding affinities of phenolic compounds to proteins by quenching of the intrinsic tryptophan fluorescence.

The noncovalent binding of selected phenolic compounds (chlorogenic-, ferulic-, gallic acid, quercetin, rutin, and isoquercetin) to proteins (HSA, BSA, soy glycinin, and lysozyme) was studied by an indirect method applying the quenching of intrinsic tryptophan fluorescence. From the data obtained, the binding constants were calculated by nonlinear regression (one site binding; y = Bx/k + x). It has been reported that tannins inhibit human salivary amylase and that these complexes may reduce the development of cariogenic plaques. Further, amylase contains two tryptophan residues in its active site. Therefore, in a second part of the study involving 31 human subjects, evidence was sought for noncovalent interactions between the phenols of green tea and saliva proteins as measured by the fluorescence intensity. Amylase activity was determined before and after the addition of green tea to saliva of 31 subjects. Forty percent of the subjects showed an increase in amylase activity contrary to studies reporting only a decrease in activity. The interactions of tannin with amylase result in a decrease of its activity. It still remains to be elucidated why amylase does not react uniformly under conditions of applying green tea to saliva. Further, in terms of using phenols as caries inhibitors this finding should be of importance.

Metabolically healthy obesity and cardiovascular events: A systematic review and meta-analysis.

AIMS: Previous studies have provided inconsistent results about the cardiovascular risks for participants with metabolically healthy obesity (MHO). These uncertainties might partly reflect the lack of a uniform definition of MHO. We conducted a systematic review and meta-analysis to examine whether there is a suitable approach that identifies obese participants who are not at an increased risk of cardiovascular events compared with healthy normal-weight participants. METHODS AND RESULTS: Twenty-two prospective studies were eligible for the meta-analysis. Using random-effect models, pooled relative risks (RRs) were calculated for the combined effects of obesity with the presence or absence of metabolic syndrome, insulin resistance, hypertension, diabetes, hyperlipidaemia and any of these metabolic factors. Participants with MHO defined by the absence of metabolic syndrome were at increased risk for cardiovascular events compared with healthy normal-weight participants (pooled RR 1.45, 95% confidence interval (CI) 1.20-1.70), but had lower risks than unhealthy normal-weight (RR 2.07, 95% CI 1.62-2.65) and obese (RR 2.31, 95% CI 1.99-2.69) participants. The risk associated with participants who had MHO was particularly high over the long term. Similar risk estimates were observed when MHO was defined by other approaches. CONCLUSIONS: None of the approaches clearly identified an obese subgroup not at increased risk of cardiovascular events compared with normal-weight healthy participants. A benign obese phenotype might be defined by strict definitions, but insufficient studies exist to support this. More research is needed to better define MHO.

Association of Multiple Biomarkers of Iron Metabolism and Type 2 Diabetes: The EPIC-InterAct Study.

OBJECTIVE: Observational studies show an association between ferritin and type 2 diabetes (T2D), suggesting a role of high iron stores in T2D development. However, ferritin is influenced by factors other than iron stores, which is less the case for other biomarkers of iron metabolism. We investigated associations of ferritin, transferrin saturation (TSAT), serum iron, and transferrin with T2D incidence to clarify the role of iron in the pathogenesis of T2D. RESEARCH DESIGN AND METHODS: The European Prospective Investigation into Cancer and Nutrition-InterAct study includes 12,403 incident T2D cases and a representative subcohort of 16,154 individuals from a European cohort with 3.99 million person-years of follow-up. We studied the prospective association of ferritin, TSAT, serum iron, and transferrin with incident T2D in 11,052 cases and a random subcohort of 15,182 individuals and assessed whether these associations differed by subgroups of the population. RESULTS: Higher levels of ferritin and transferrin were associated with a higher risk of T2D (hazard ratio [HR] [95% CI] in men and women, respectively: 1.07 [1.01-1.12] and 1.12 [1.05-1.19] per 100 µg/L higher ferritin level; 1.11 [1.00-1.24] and 1.22 [1.12-1.33] per 0.5 g/L higher transferrin level) after adjustment for age, center, BMI, physical activity, smoking status, education, hs-CRP, alanine aminotransferase, and γ-glutamyl transferase. Elevated TSAT (≥45% vs. <45%) was associated with a lower risk of T2D in women (0.68 [0.54-0.86]) but was not statistically significantly associated in men (0.90 [0.75-1.08]). Serum iron was not associated with T2D. The association of ferritin with T2D was stronger among leaner individuals (Pinteraction < 0.01). CONCLUSIONS: The pattern of association of TSAT and transferrin with T2D suggests that the underlying relationship between iron stores and T2D is more complex than the simple link suggested by the association of ferritin with T2D.

Binding of selected phenolic compounds to proteins.

In the context of this study, the noncovalent binding of selected phenolic compounds (chlorogenic, ferulic, and gallic acids, quercetin, rutin, and isoquercetin) to different proteins (human serum albumin, bovine serum albumin, soy glycinin, and lysozyme) was studied with direct (Hummel-Dreyer/size exclusion chromatography) and/or indirect methods (fluorescence absorbance properties of the binding components). In the latter case, the measurement of the phenol binding was achieved by exploiting the intrinsic fluorescence emission properties of quercetin as a probe. From the data obtained, the binding constants and the number of binding sites were calculated. The binding parameters were influenced by different factors, where, e.g., increasing temperature and ionic strength as well as decreasing pH cause a diminished binding. The structures of the proteins as determined by circular dichroism indicate changes in the tertiary structure with the secondary structure remaining intact.

Characterization of metabolically unhealthy normal-weight individuals: Risk factors and their associations with type 2 diabetes.

OBJECTIVE: A proportion of type 2 diabetes cases arise from normal-weight individuals who can therefore be considered to be "metabolically unhealthy normal-weight" (MUH-NW). It remains unclear which factors account for this access risk. Our aims were to identify risk factors for type 2 diabetes in normal-weight individuals and to compare the strengths of their associations with type 2 diabetes to that observed in overweight and obese participants. METHODS: A case-cohort, including 2027 sub-cohort participants and 706 incident type 2 cases, was designed within the population-based European Prospective Investigation into Cancer and Nutrition Potsdam study. Adjusted means and relative frequencies of anthropometric, lifestyle and biochemical risk factors were calculated in groups stratified by BMI and incident diabetes status. Cox regressions were applied to evaluate associations between these variables and diabetes risk stratified by BMI category. RESULTS: MUH-NW individuals were characterized by known diabetes risk factors, e.g. they were significantly more likely to be male, former smokers, hypertensive, and less physically active compared to normal-weight individuals without incident diabetes. Higher waist circumference (women: 75.5 vs. 73.1cm; men: 88.0 vs. 85.1cm), higher HbA1c (6.1 vs. 5.3%), higher triglycerides (1.47 vs. 1.11 mmol/l), and higher levels of high sensitive C-reactive protein (0.81 vs. 0.51 mg/l) as well as lower levels of HDL-cholesterol (1.28 vs. 1.49 mmol/l) and adiponectin (6.32 vs. 8.25 µg/ml) characterized this phenotype. Stronger associations with diabetes among normal-weight participants compared to overweight and obese (p for interaction<0.05) were observed for height, waist circumference, former smoking, and hypertension. CONCLUSIONS: Normal-weight individuals who develop diabetes have higher levels of diabetes risk factors, however, frequently still among the normal range. Still, hypertension, elevated HbA1c and lifestyle risk factors might be useful indicators of risk.