Circulating Apolipoprotein E Concentration and Cardiovascular Disease Risk: Meta-analysis of Results from Three Studies.

BACKGROUND: The association of APOE genotype with circulating apolipoprotein E (ApoE) concentration and cardiovascular disease (CVD) risk is well established. However, the relationship of circulating ApoE concentration and CVD has received little attention. METHODS AND FINDINGS: To address this, we measured circulating ApoE concentration in 9,587 individuals (with 1,413 CVD events) from three studies with incident CVD events: two population-based studies, the English Longitudinal Study of Ageing (ELSA) and the men-only Northwick Park Heart Study II (NPHSII), and a nested sub-study of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT). We examined the association of circulating ApoE with cardiovascular risk factors in the two population-based studies (ELSA and NPHSII) and the relationship between ApoE concentration and coronary heart disease and stroke in all three studies. Analyses were carried out within study, and, where appropriate, pooled effect estimates were derived using meta-analysis. In the population-based samples, circulating ApoE was associated with systolic blood pressure (correlation coefficient 0.08, p < 0.001, in both ELSA and NPHSII), total cholesterol (correlation coefficient 0.46 and 0.34 in ELSA and NPHSII, respectively; both p < 0.001), low-density lipoprotein cholesterol (correlation coefficient 0.30 and 0.14, respectively; both p < 0.001), high-density lipoprotein (correlation coefficient 0.16 and -0.14, respectively; both p < 0.001), and triglycerides (correlation coefficient 0.43 and 0.46, respectivly; both p < 0.001). In NPHSII, ApoE concentration was additionally associated with apolipoprotein B (correlation coefficient 0.13, p = 0.001) and lipoprotein(a) (correlation coefficient -0.11, p < 0.001). In the pooled analysis of ASCOT, ELSA, and NPHSII, there was no association of ApoE with CVD events; the odds ratio (OR) for CVD events per 1-standard-deviation higher ApoE concentration was 1.02 (95% CI 0.96, 1.09). After adjustment for cardiovascular risk factors, the OR for CVD per 1-standard-deviation higher ApoE concentration was 0.97 (95% CI 0.82, 1.15). Limitations of these analyses include a polyclonal method of ApoE measurement, rather than isoform-specific measurement, a moderate sample size (although larger than any other study to our knowledge and with a long lag between ApoE measures), and CVD events that may attenuate an effect. CONCLUSIONS: In the largest study to date on this question, we found no evidence of an association of circulating ApoE concentration with CVD events. The established association of APOE genotype with CVD events may be explained by isoform-specific functions as well as other mechanisms, rather than circulating concentrations of ApoE.

FVII, FVIIa, and downstream markers of extrinsic pathway activation differ by EPCR Ser219Gly variant in healthy men.

OBJECTIVE: The purpose of this study was to determine the effect of a variant in EPCR (Ser219Gly), previously shown to affect EPCR shedding, on plasma FVII, FVIIa, and downstream markers of activated coagulation. METHODS AND RESULTS: Statistical analysis was undertaken in approximately 2000 healthy middle aged men (NPHSII). Higher soluble EPCR levels were confirmed for Gly allele carriers (P<0.0001). Significantly higher levels of FVII, FVIIa, and downstream markers of activated coagulation in the extrinsic pathway (FIX activation pep [FIXpep]; FX activation pep [FXpep]), and prothrombin F1+2 (F1+2) were identified in baseline samples, in Gly carriers compared to Ser/Ser (P

Healthy individuals carrying the PCSK9 p.R46L variant and familial hypercholesterolemia patients carrying PCSK9 p.D374Y exhibit lower plasma concentrations of PCSK9.

BACKGROUND: We measured plasma PCSK9 concentrations in healthy men with a PCSK9 (proprotein convertase subtilisin/kexin type 9) loss-of-function variant (p.R46L), in statin-treated patients with a clinical diagnosis of familial hypercholesterolemia (FH) and carrying a PCSK9 gain-of-function mutation (p.D374Y), and in statin-treated patients with FH due to different genetic causes. METHODS: PCSK9 was measured with a previously described ELISA. RESULTS: In 81 healthy middle-aged Caucasian men, the PCSK9 concentration was significantly associated with the concentrations of total cholesterol (r = 0.42; P < 0.0001), LDL cholesterol (r = 0.34; P = 0.01), and triglycerides (r = 0.25; P = 0.02). In p.R46L carriers, mean (SD) concentrations of PCSK9 were 15% lower than in RR individuals [65.5 microg/L (21.6 microg/L) vs 77.5 microg/L (18.2 microg/L); P = 0.03]. In patients with the p.D374Y variant (n = 7), the mean PCSK9 concentration was significantly lower than in the combined group of patients with an LDLR (low density lipoprotein receptor) mutation (n = 25), an APOB [apolipoprotein B (including Ag(x) antigen)] variant encoding p.R3527Q (n = 6), or no detectable mutation (n = 14) [96.4 microg/L (42.5 microg/L) vs 151.6 microg/L (69.6 microg/L); P = 0.02]. Two of the 14 patients with no mutation had PCSK9 concentrations below the mean for p.D374Y carriers; sequencing of the PCSK9 gene and promoter revealed no mutations. Among 409 FH patients, we identified 6 carriers of the promoter variant -287G>A (1.5%), a frequency similar to that (1.0%) previously reported for 2772 healthy men in the UK. In neither group was the -287G>A variant associated with differences in lipid traits. CONCLUSIONS: The loss-of-function p.R46L variant is associated with the expected lower concentrations of circulating PCSK9; the gain-of-function p.D374Y mutation is also associated with lower concentrations, presumably because of the higher affinity of this variant for the LDL receptor and its more rapid clearance. In treated FH patients, a low plasma PCSK9 concentration does not appear to be a useful screening tool for identifying novel PCSK9 mutations.

Interaction between the uncoupling protein 2 -866G>A gene variant and cigarette smoking to increase oxidative stress in subjects with diabetes.

BACKGROUND AND AIMS: Increased oxidative stress is associated with coronary heart disease (CHD). The mitochondrial uncoupling protein-2 (UCP2) negatively regulates reactive oxygen species generation. We have observed that a common variant (-866G>A) in the promoter region of UCP2 is associated with increased CHD risk in healthy men and increased oxidative stress in diabetic men with CHD. The aim of the current study was to test the hypothesis that this variant might interact with smoking (an environmental stress) to influence plasma markers of oxidative stress. METHODS AND RESULTS: Amongst 453 Caucasian diabetic men there was a significant interaction (p=0.001) between genotype and smoking in determining plasma Total AntiOxidant Status (TAOS). Current smokers with the -866AA genotype had the lowest TAOS (indicating higher oxidative stress) of all subjects (AA vs. GG: 32.00+/-17.4% vs. 45.8+/-12.6%, p=0.04). In a sub-sample of 20 subjects (10 GG, 10 AA) matched for baseline characteristics, plasma markers of oxidative stress in current smokers were significantly higher in AA compared to GG subjects (TAOS 36.8+/-9.5% vs. 51.4+/-9.5%, p=0.04; F(2)-isoprostanes 1133.6+/-701.2 pg ml(-1) vs. 500.8+/-64.7 pg ml(-1), p=0.04). CONCLUSIONS: This study demonstrates an interaction between the UCP2 -866G>A variant and smoking to increase oxidative stress in vivo.

The S447X variant of lipoprotein lipase gene is associated with metabolic syndrome and lipid levels among Turks.

BACKGROUND: We evaluated the relationship of the lipoprotein lipase (LPL) S447X variant with serum lipid levels and the metabolic syndrome (MS) in the Turkish Adult Risk Factor (TARF) study. This is the first study examining this LPL variant in the Turkish population. METHODS: The sample comprised 1586 Turkish adults. Genotyping was performed using the Taqman allelic discrimination assay. RESULTS: The X447 allele frequency was 0.11 (95% CI: 0.10-0.12). X447 allele carriers had significantly higher levels of HDL-C, LDL-C and total cholesterol; and lower fasting glucose, when compared with the SS genotype in females. In men, no significant association with any parameters was seen. The genotypic impact of the S447X variant on lipid levels appears to be modulated by environmental factors, such as cigarette smoking in women. Logistic regression analysis demonstrated a significantly reduced likelihood for metabolic syndrome in female X447 allele carriers (p=0.04), after adjustment for age, cigarette smoking, alcohol usage and physical activity grade. CONCLUSIONS: In especially Turkish women, compared to non-carriers, carriers of the LPL X447 allele have higher levels of HDL-C, LDL-C and total cholesterol, and show a degree of protection against developing the metabolic syndrome.

A common variant in the glutathione S transferase gene is associated with elevated markers of inflammation and lipid peroxidation in subjects with diabetes mellitus.

INTRODUCTION: Glutathione S transferases (GST) are enzymes responsible for the metabolism of numerous xenobiotics and play a major cellular antioxidant role. Our aim was firstly, to examine the association between the GST M1/GST mu-1 (GSTM1) and GST T1/GST theta-1 (GSTT1) gene variants with markers of oxidative stress and inflammation in diabetic patients, and secondly to examine the association and potential interaction between these variants and cigarette smoking. METHODS: Seven hundred and seventy-three Caucasian subjects with diabetes and 2592 Caucasian non-diabetic subjects were successfully genotyped. Plasma total antioxidant status, C-reactive protein (CRP), oxidized-LDL (Ox-LDL) and LDL-mean/peak particle diameter were recorded in the diabetes sample. RESULTS: No association was seen between genotype and cardiovascular disease (CVD) risk. In the diabetic subjects, GSTT1-1 compared to GSTT1-0 subjects had significantly higher CRP (p=0.001), Ox-LDL (p=0.004) and smaller LDL particles (p=0.01). In subjects without CVD, there was a significant interaction between the GSTT1-1 variant and smoking in determining Ox-LDL (p=0.04). Furthermore, CVD risk was higher in smokers compared to non-smokers with GSTT1-1. No significant associations were observed by GSTM1. Within the non-diabetic sample, no association was observed between genotype and prospective coronary heart disease (CHD) risk. Of note, the frequency of the GSTT1-1 variant was significantly lower in the diabetes subjects compared to the non-diabetic sample (p=0.01). CONCLUSIONS: This study demonstrates an association between the GSTT1-1 variant and markers of inflammation and lipid peroxidation. Furthermore this variant interacts with smoking to increase lipid peroxidation.

A common variant in the ACE gene is associated with peripheral neuropathy in women with type 2 diabetes mellitus.

AIMS: The D allele of the ACE I/D gene variant is associated with higher tissue and serum ACE activity. Previously, studies have suggested an association between the D allele with the microvascular complications of diabetes. The aim of this study was to explore the impact of this genotype in relation to clinically manifest peripheral neuropathy (PN) in a cohort of subjects with type 2 diabetes mellitus (type 2 DM). METHODS: Five hundred and seventy-two Caucasian subjects (230 females, 342 males) with type 2 DM were recruited from the diabetes clinic at University College London Hospitals NHS Trust. Clinically manifest PN was determined from a standardized clinical examination. RESULTS: The ACE I/D genotype distribution was in Hardy-Weinberg equilibrium. In the whole group, no significant association was seen between genotype and PN; however, when stratified by sex, the D allele was associated with PN in females but not in males. The odds ratio (OR) for PN in the D allele carriers compared to those homozygous for the I allele was significantly higher in females [OR 2.93 (1.09-7.63), P=.027] but not in males [OR 1.2 (0.61-2.36), P=.60]. CONCLUSIONS: The presence of the D allele is associated with increased risk of peripheral neuropathy in females but not in male subjects with type 2 DM, suggesting a role for the renin-angiotensin system in the development of PN.

Effect of six type II diabetes susceptibility loci and an FTO variant on obesity in Pakistani subjects.

The aim of the current study was to analyze the effect of six type II diabetes GWAS loci rs3923113 (GRB14), rs16861329 (ST6GAL1), rs1802295 (VPS26A), rs7178572 (HMG20A), rs2028299 (AP3S2) and rs4812829 (HNF4A), and an FTO polymorphism (rs9939609) on obesity. The probable mechanism of action of these SNPs was analyzed by studying their association with various biochemical and anthropometric parameters. A total of 475 subjects (obese=250, controls=225) were genotyped by TaqMan assay and their lipid profile was determined. Allele/genotype frequencies and an unweighted/weighted gene score were calculated. The effect of the gene score on anthropometric and biochemical parameters was analyzed. The minor allele frequencies of all variants were comparable to that reported in the original studies and were associated with obesity in these Pakistani subjects. Subjects with 9 risk alleles differ from those with <3 and overall there is no significant effect (P-value for trend 0.26). None of the SNPs were associated with any of the serum lipid traits. We are the first to report the association of these T2D SNPs with obesity. In the Pakistani population the reported effect of six SNPs for obesity is similar to that reported for T2D and having a combination of risk alleles on obesity can be considerable. The mechanism of this effect is unclear, but appears not to be mediated by changing serum lipid chemistry.

Comparison of coronary heart disease genetic assessment with conventional cardiovascular risk assessment in primary care: reflections on a feasibility study.

AIM: This study assesses the feasibility of collecting genetic samples and self-reported outcome measures after cardiovascular risk assessment, and presenting the genetic test results to participants. BACKGROUND: Coronary heart disease (CHD) genetic tests are increasingly available through direct-to-consumer marketing, but their potential clinical impact on cardiovascular risk assessment is unclear. METHODS: Observational study in 10 British general practices in Central England. A total of 320 individuals, who had completed conventional cardiovascular risk assessment, were offered CHD genetic test, with follow-up outcome questionnaire at eight months for lifestyle change and State-Trait Anxiety. FINDINGS: A total of 119 (37%) participants returned genetic test specimens, with over a third reporting family history of CHD in a specified relative; 79 (66.4%) were categorized above-average risk on conventional cardiovascular risk assessment, 65 of whom (82.3%) were only average risk on genetic assessment. The dietary fat questionnaire was poorly completed while study participation was not associated with increased anxiety (mean increase in anxiety score=2.1; 95% CI -0.1-4.3; P=0.06). CONCLUSION: As a feasibility study, over a third of individuals offered genetic testing in primary care, as part of CVD risk assessment, took up the offer. Although intervention did not appear to increase anxiety, this needs further evaluation. To improve generalizability and effect size, future studies should actively engage individuals from wider socio-economic backgrounds who may not have already contemplated lifestyle change. The current research suggests general practitioners will face the clinical challenge of patients presenting with direct-to-consumer genetic results that are inconsistent with conventional cardiovascular risk assessment.

Postprandial variations in fibrinolytic activity in middle-aged men are modulated by plasminogen activator inhibitor I 4G-675/5G genotype but not by the fat content of a meal.

BACKGROUND: Decreased fibrinolytic activity is associated with an increased risk of ischemic heart disease and elevated plasma triacylglycerol concentrations. OBJECTIVE: The goal was to determine whether plasminogen activator inhibitor 1 (PAI-1) and fibrinolytic activities are influenced by 1) dietary fat intake from a test meal and 2) the PAI-1 4G allele. DESIGN: A parallel randomized controlled trial was used to compare the effect on fibrinolytic activity, measured as dilute clot lysis time, of high-oleate or high-palmitate test meals (both containing 50 g fat; both n = 18) with that of a low-fat test meal (15 g fat; n = 15) in men aged > 52 y. In a second study, postprandial changes in PAI-1 activity were measured in 32 men in response to a high-oleate meal containing 50 g fat. The results from both studies were analyzed according to PAI-1 4G-675/5G genotype. RESULTS: Fasting dilute clot lysis time was positively associated with body mass index (r = 0.326, P = 0.02) and was shortened postprandially (P < 0.00001) independent of the fat content of the meal. Fasting PAI-1 activity was higher in those carrying the 4G allele and was correlated with fasting plasma triacylglycerol concentrations (r = 0.48, P = 0.008) and factor VII coagulant activity (r = 0.46, P = 0.012) after adjustments for age, body mass index, and genotype. Plasma PAI-1 activity decreased significantly after a meal but was not associated with postprandial changes in plasma triacylglycerols after a high-fat meal. The postprandial increase in plasma triacylglycerols was higher in subjects carrying the 4G allele. CONCLUSION: Fibrinolytic activity is not lower after meals rich in palmitate or oleate than after a low-fat meal.

No APOEepsilon4 effect on coronary heart disease risk in a cohort with low smoking prevalence: the Whitehall II study.

Carriers of the APOEepsilon4 allele have consistently shown higher, and epsilon2 carriers have lower, plasma cholesterol and coronary heart disease (CHD) risk compared with epsilon3 homozygotes. An epsilon4:smoking interaction was observed in NPHSII, consistent with context dependency of the epsilon4 effect on CHD risk. In this study, APOE genotype was determined in 3787 male British civil servants followed for fatal and non-fatal myocardial infarction for median of 5.8 years, with 159 validated CHD events. APOE genotype was associated with expected effects on lipid traits (all P <0.0001). We tested the hypothesis that APOEepsilon4 was not a risk factor for CHD among non-smokers. In non-smokers, the odds ratio (OR) for epsilon2 and epsilon4 carriers were 0.51 (0.27, 0.97) and 0.70 (0.46, 1.08), respectively, compared with epsilon3 homozygotes. Thus epsilon2 carriers showed expected risk-protection, but despite 80% power to detect an OR in epsilon4 subjects of 1.71 (i.e. of magnitude increase reported in prospective studies), the epsilon4 non-smokers showed no increased risk compared with epsilon3 homozygotes. Smoking prevalence in this study was low (12.8%), but smokers had higher CHD risk which was of similar magnitude in risk in all genotypes [(OR 1.57 (1.03, 2.40)]. These data, therefore, provide strong corroborative evidence that there is no elevated risk of CHD in epsilon4 non-smokers, but failed to confirm the epsilon4:smoking interaction on risk. This supports the context dependency of the epsilon4 risk effect, but the low smoking incidence in the Whitehall men reduced our ability to examine a smoking:genotype interaction.

An interaction between the interleukin-6 -174G>C gene variant and urinary protein excretion influences plasma oxidative stress in subjects with type 2 diabetes.

BACKGROUND: Microalbuminuria and subsequent progression to proteinuria and nephropathy is associated with increased oxidative stress, increased inflammatory cytokines and increased cardiovascular (CVD) risk. The common functional IL-6 -174G>C gene variant is also associated with elevated levels of inflammatory cytokines and CVD risk. METHODS: The aim of this study was to examine the association between the IL-6 -174G>C gene variant with plasma total antioxidant status (TAOS) in 552 subjects with type 2 diabetes in relation to urinary protein excretion. RESULTS: In subjects free from CVD, there was a significant interaction between urinary protein excretion (normoalbuminuria/ microalbuminuria/proteinuria) and the -174C allele (compared to -174GG) in determining plasma TAOS (p value for interaction = 0.03). In the -174C allele carriers there was a significant association between plasma TAOS and urinary protein excretion: normalbuminuria v microalbuminuria v proteinuria: 44.30% +/- 11.32 vs. 39.74% +/- 14.83 vs. 37.93% +/- 16.42, ANOVA p = 0.025. In those with CVD, no interaction or association was observed with the -174C allele (p = 0.246). CONCLUSION: The IL-6 -174G>C gene variant is associated with differences in plasma oxidative stress in response to altered protein excretion in subjects with type 2 diabetes.

Association of vitamin D status with arterial blood pressure and hypertension risk: a mendelian randomisation study.

BACKGROUND: Low plasma 25-hydroxyvitamin D (25[OH]D) concentration is associated with high arterial blood pressure and hypertension risk, but whether this association is causal is unknown. We used a mendelian randomisation approach to test whether 25(OH)D concentration is causally associated with blood pressure and hypertension risk. METHODS: In this mendelian randomisation study, we generated an allele score (25[OH]D synthesis score) based on variants of genes that affect 25(OH)D synthesis or substrate availability (CYP2R1 and DHCR7), which we used as a proxy for 25(OH)D concentration. We meta-analysed data for up to 108 173 individuals from 35 studies in the D-CarDia collaboration to investigate associations between the allele score and blood pressure measurements. We complemented these analyses with previously published summary statistics from the International Consortium on Blood Pressure (ICBP), the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, and the Global Blood Pressure Genetics (Global BPGen) consortium. FINDINGS: In phenotypic analyses (up to n=49 363), increased 25(OH)D concentration was associated with decreased systolic blood pressure (ß per 10% increase, -0·12 mm Hg, 95% CI -0·20 to -0·04; p=0·003) and reduced odds of hypertension (odds ratio [OR] 0·98, 95% CI 0·97-0·99; p=0·0003), but not with decreased diastolic blood pressure (ß per 10% increase, -0·02 mm Hg, -0·08 to 0·03; p=0·37). In meta-analyses in which we combined data from D-CarDia and the ICBP (n=146 581, after exclusion of overlapping studies), each 25(OH)D-increasing allele of the synthesis score was associated with a change of -0·10 mm Hg in systolic blood pressure (-0·21 to -0·0001; p=0·0498) and a change of -0·08 mm Hg in diastolic blood pressure (-0·15 to -0·02; p=0·01). When D-CarDia and consortia data for hypertension were meta-analysed together (n=142 255), the synthesis score was associated with a reduced odds of hypertension (OR per allele, 0·98, 0·96-0·99; p=0·001). In instrumental variable analysis, each 10% increase in genetically instrumented 25(OH)D concentration was associated with a change of -0·29 mm Hg in diastolic blood pressure (-0·52 to -0·07; p=0·01), a change of -0·37 mm Hg in systolic blood pressure (-0·73 to 0·003; p=0·052), and an 8·1% decreased odds of hypertension (OR 0·92, 0·87-0·97; p=0·002). INTERPRETATION: Increased plasma concentrations of 25(OH)D might reduce the risk of hypertension. This finding warrants further investigation in an independent, similarly powered study. FUNDING: British Heart Foundation, UK Medical Research Council, and Academy of Finland.

Antibodies to citrullinated peptides and risk of coronary heart disease.

OBJECTIVE: Increased cardiovascular risk has been associated with high levels of serum antibodies to citrullinated proteins (ACPA) in patients with rheumatoid arthritis (RA). Citrullination is part of many chronic inflammatory processes and we therefore investigated whether ACPA might be associated with coronary artery disease, in the absence of RA. METHODS: To maximize the potential predictive value of this retrospective study we included sera from a cohort of 3052 healthy male individuals, subsequently followed for the development of coronary artery disease, and documented for other disease risk factors. With each case event (myocardial infarction; n = 144), 2 matched controls were assigned. RESULTS: We found 10.4% of cases were ACPA positive compared to 3.8% of controls (odds ratio (95% CI) = 3.26 (1.36-7.80), p = 0.008), remaining significant after adjustment for classical risk factors including smoking and CRP (4.23 (1.22-14.61) p = 0.02). CONCLUSION: The genesis and fine specificity of ACPA in patients with atherosclerosis, in the absence of Rheumatoid arthritis, may prove worthy of further investigation.

Acute renal failure in a patient with paroxysmal cold hemoglobinuria.

Acute renal failure following auto-immune hemolysis is rare. We report a child with acute paroxysmal cold hemoglobinuria (PCH) complicated by renal failure. She was treated by peritoneal dialysis and red blood cell transfusion. After 2 weeks she had made a complete recovery with a normal blood count and renal profile, and the peritoneal dialysis catheter was removed. Extensive investigation, including an analysis of heme oxygenase-1 gene promoter region polymorphisms, failed to identify a cause for the renal failure in this patient.

Three novel mutations in the apolipoprotein E gene in a sample of individuals with type 2 diabetes mellitus.

BACKGROUND: Apolipoprotein E (apoE) is found in association with triglyceride-rich lipoproteins and is the ligand for the removal of these particles from the plasma. Genetic variations in exon 4 lead to three common gene variants: E2, E3, and E4. METHODS: We performed apoE genotyping in 765 individuals with type 2 diabetes. RESULTS: We identified three new variant heteroduplex patterns. Sequencing of these variants revealed three novel mutations that were related to biochemical and clinical characteristics. One mutation produced a frameshift at amino acid position 166, which predicted termination of protein synthesis. This individual had a heteroduplex pattern and sequence of E3E3, which was associated with a change in the plasma isoelectric focusing pattern and a 70% lower plasma concentration of apoE compared with healthy individuals. The other mutations were both single base changes. A CGC>CAC change at amino acid position 150 predicted a substitution of Arg>His. This individual had a heteroduplex pattern and sequence of E2E2, which was not associated with major changes in plasma lipids or apoE concentration. The third individual had a CGC>CCC base change at amino acid position 114, which predicted an Arg>Pro change. This person had a heteroduplex pattern and sequence of E3E3, higher plasma total cholesterol, and moderately decreased plasma apoE. CONCLUSIONS: The frequency of new mutations in this sample (1 in 255) is higher than that of a healthy population (1 in 7900). Further screening for common apoE gene variants in individuals at risk for dyslipidemia may reveal abnormal heteroduplex patterns and uncover further mutations in this important lipid-regulating gene.

The D allele of the ACE I/D common gene variant is associated with Type 2 diabetes mellitus in Caucasian subjects.

The deletion D allele of the angiotensin-I converting enzyme (ACE) I/D gene variant is associated with higher ACE activity in Caucasians and previous studies in non-Caucasian samples have suggested an association between the D allele and type 2 diabetes (Type 2DM). The aim of this study was to compare the genotype distribution between Caucasian subjects with Type 2DM and non-diabetic Caucasian men. Genotype distribution was compared between 574 Caucasian subjects with Type 2DM, recruited from the UCL Diabetes and Cardiovascular Disease Study and 2413 non-diabetic Caucasian men, recruited from the second Northwick Park Heart Study. Within both samples, genotype distributions were in Hardy-Weinberg equilibrium. The genotype distributions in those with Type 2DM compared to the non-diabetic men (II/ID/DD) was 18%/50%/32% vs. 23%/49%/27%, p=0.004. In accordance with this, the frequency of the D allele was higher in those with Type 2DM (0.574 [0.55-0.60] vs. 0.519 [0.50-0.53], p=0.001). On combining the two samples, the odds ratio (OR) for Type 2DM was significantly higher in D allele carriers compared to II subjects (OR=1.55, p=0.02, after adjustment for age, sex, BMI, blood pressure, and lipids). In those with diabetes, there was a significant association between genotype and a family history of diabetes. The odds ratio for a family history of diabetes in DD compared to II subjects was 1.52 [0.89-2.60], p=0.03. This study clearly shows an association between the ACE I/D common gene variant and Type 2DM.

The APOA4 T347S variant is associated with reduced plasma TAOS in subjects with diabetes mellitus and cardiovascular disease.

Apolipoprotein A-IV (apoA-IV) has been postulated to be antiatherogenic. Transgenic APOA4/Apoe-/- mice are protected against atherosclerosis, with plasma apoA-IV displaying antioxidant activity in vitro. In humans, there is an inverse relationship between apoA-IV levels and risk of coronary heart disease (CHD). Furthermore, the APOA4 T347S rare allele has been associated with increased risk of CHD and reduced apoA-IV levels. Reduced total antioxidant status (TAOS) due to increased oxidative stress is implicated in the process of atherogenesis. Thus, this study aimed to examine the association between the APOA4 T347S variant and TAOS in diabetic patients with (n = 196) or without (n = 509) cardiovascular disease (CVD). A higher percentage of CVD patients were present in the lowest quartile of TAOS, compared with the rest (P = 0.04). Overall, there was no association between genotype and TAOS. However, in patients with CVD, homozygotes for the S347 allele had significantly lower TAOS compared with TT and TS subjects (31.2 +/- 9.89% and 42.5 +/- 13.04% TAOS, respectively; P = 0.0024), an effect that was not seen in the patients without CVD. This study offers direct support for an antioxidant capacity of apoA-IV, thus providing some explanation for the antiatherogenic role of apoA-IV and the higher CVD risk in S347 homozygotes.

A common functional variant in the interleukin-6 gene is associated with increased body mass index in subjects with type 2 diabetes mellitus.

Circulating levels of interleukin-6 (IL-6) are raised in insulin resistant states such as obesity, impaired glucose tolerance (IGT), and type 2 diabetes mellitus (T2DM). Growing evidence suggests that IL-6 is not only produced by fat cells but is also capable of inducing insulin resistance in these cells. The expected result of this in vivo, would be to increase adipose mass and subsequently body mass index (BMI). The IL-6 -174G > C common functional gene variant has consistently been associated with increased plasma IL-6, insulin resistance, and increased cardiovascular risk. We looked at the association between genotype and BMI in 571 Caucasian subjects with T2DM. There was a significant linear association between genotype and BMI: Median (interquartile range) GG 28.8 kg/m2 (26.0-31.6) vs GC; 29.4 kg/m2 (26.3-32.5) vs CC; 30.4 kg/m2 (26.1-33.0), p=0.05. When the group was divided by the median BMI (29.1 kg/m2), 62% of -174CC subjects were in the higher group compared to 38% in the lower group (p=0.008). By contrast, in 2,652 non-diabetic Caucasian men with a median BMI of 26.1 kg/m2, there was no difference in genotype distribution (p=0.288). The frequency of the -174C allele was lower in type 2 diabetes compared to the non-diabetic men (-174C allele frequency: 0.35[0.33-0.38] vs 0.43[0.42-0.45], p <0.00001; -174CC homozygotes: 12.3 vs 18.3%, respectively). The -174C allele is associated with higher BMI in type 2 diabetes, but not amongst healthy subjects. The increased cardiovascular risk associated with the -174C allele may account for the lower frequency of this allele in those with type 2 diabetes.

Cardiovascular risk in healthy men and markers of oxidative stress in diabetic men are associated with common variation in the gene for uncoupling protein 2.

BACKGROUND: Oxidative stress reduces total antioxidant status (TAOS) and is implicated in atherogenesis. Mitochondrial uncoupling protein 2 (UCP2) negatively regulates reactive oxygen species generation. The UCP2 gene demonstrates a common functional promoter variant (-866G>A). METHODS AND RESULTS: Amongst 465 diabetic men (age 61.7 +/- 13.3 years), an association of the UCP2-866A allele with significantly lower TAOS in those without CHD was even more pronounced in those with CHD (TAOS 30.1 +/- 16.1% vs. 41.6 +/- 12.4% for AA vs. GG; P=0.016). In a sample of 20 diabetic men selected for homozygosity for the UCP2-866G>A variant, matched for baseline characteristics, plasma markers of oxidative stress in those with CHD were significantly higher in AA genotype men (TAOS 31.7 +/- 7.3% vs. 52.6 +/- 6.3%; P=0.001 and F2-isoprostanes 220.6 +/- 37.2 pg ml(-1) vs. 109.9 +/- 51.1 pg ml(-1); P=0.005 for AA vs. GG). Amongst 2695 healthy men (age 56.1 +/- 3.5 years) prospectively studied for a median 10.2 years, AA homozygotes had a highly significant doubling in CHD risk after adjustment for established risk factors (HR 1.99 [1.37-2.90]; P=0.002). Risk associated with this genotype was substantially increased by the presence of other risk factors (obesity, hypertension and diabetes). CONCLUSIONS: This study provides the first in vivo evidence of a role for UCP2 in modifying oxidative stress and CHD risk in humans.