Attempt to replicate published genetic associations in a large, well-defined osteoarthritis case-control population (the GOAL study).

OBJECTIVE: Published studies have tested over 90 genes for association with osteoarthritis (OA), but few positives reported have been independently replicated. Using a new case-control study, our aim was to attempt the replication of findings from 12 genes reported to have significant genetic association with OA and to further examine the role of genetic variation in six of these genes. METHODS: A case-control study was undertaken in Nottingham, UK. Hospital-referred index cases with symptomatic, radiographic OA (ROA) of the knee (n=1040) or hip (n=1004) were recruited. Asymptomatic controls (n=1123) were recruited from intravenous urography waiting lists and screened for radiographic hip and knee OA. Sixty-eight polymorphisms were genotyped in IL1A, IL1B, IL1RN, IL4R, IL6, COL2A1, ADAM12, ASPN, IGF1, TGFB1, ESR1 and VDR. Statistical analysis compared allele or genotype frequencies of these polymorphisms in all asymptomatic controls and the subset of controls without ROA vs all OA, knee OA and hip OA. The analyses were adjusted for age, gender and body mass index. RESULTS: We were unable to replicate any of the published genetic associations investigated. Our extended exploratory analyses identified some associations between polymorphisms in TGFB1, IGF1 and IL1RN and OA; but the strength of evidence varied with the control group used. CONCLUSION: Lack of replication is common and could be due to differences in study design, phenotype, populations examined or the occurrence of false positives in the initial study. Variants within TGFB1, IGF1 and IL1RN could have a role in OA susceptibility; however, replication of these findings is required in an independent study.

Contribution of apolipoprotein C-III gene variants to determination of triglyceride levels and interaction with smoking in middle-aged men.

Variation within and around the apolipoprotein C-III (APOC3) gene has been associated with elevated triglyceride (Tg) levels and cardiovascular disease. The associations of 4 polymorphic variants in the APOC3 gene (3238C>G in the 3' untranslated region [SST:I], 1100C>T in exon 3, -482C>T in the insulin-responsive element, and -2854T>G in the APOC3-A4 intergenic region) with plasma Tg and cholesterol levels and their interaction with smoking have been investigated in the Second Northwick Park Heart Study (NPHSII), a large cohort of healthy men (n=2745). Analyzing the variants separately showed that 3238G, 1100T, and -482T alleles were all associated with raised Tg levels. For the 3238C>G and -482C>T sites, the Tg-raising effect appeared to depend on smoking status (test for interaction, P:=0.042 and P:=0.009, respectively), but for the 1100C>T site, the effect was constant irrespective of smoking status (test for interaction, P:=0.27). The -2854T>G site was not associated with effects on Tg levels in this sample. Because all of the variants showed significant allelic association, regression modeling was used to quantify the relative size of each effect and to assess whether the effects of the separate variants were independent. The 1100C>T variant had an independent effect on Tg levels that was not influenced by smoking status (increase of 8.2% in Tg with each T1100 allele), whereas the -482C>T variant had a separate effect that was dependent on smoking (increase of 13.7% in Tg for each -482T allele in current smokers, 8.6% in exsmokers, and -7.4% in those who never smoked). The 3238C>G variant did not show a separate independent effect on Tg concentration. Thus, by use of the regression model, it was possible to estimate how mean Tg levels would vary in groups of individuals with respect to APOC3 genotype and smoking information. Analysis in this large group of healthy men has allowed the identification of a statistically robust APOC3 genotype-smoking interaction, which now warrants further molecular study.

Epidemiological and genetic associations of activated factor XII concentration with factor VII activity, fibrinopeptide A concentration, and risk of coronary heart disease in men.

BACKGROUND: The relations of plasma activated factor XII (FXIIa) concentration and a common polymorphism (C46T) of the factor XII gene with hemostatic status and risk of coronary heart disease (CHD) were examined by prospective surveillance. METHODS AND RESULTS: Genotyping for the C46T variant was performed in 2624 men 50 to 61 years of age who were free of CHD at baseline. The genotype distribution was as follows: CC, 56.7%; CT; 36.9%; and TT, 6.6%. Plasma FXIIa was measured by ELISA on 1745 samples collected 1 year after baseline; median levels were (ng/mL) CC, 2.0; CT, 1.4; and TT, 0.8 (P:<0.0001). Respective values for plasma fibrinopeptide A (FPA, nmol/L) were 1.52, 1.35, and 1.15 (P:<0.0001); for factor VII coagulant activity (FVIIc, % standard), 114.5, 116.2, and 109.3 (P:=0.02). Group differences in FVIIc were unchanged by adjustment for body mass index and serum triglycerides. Whereas CHD incidence did not differ significantly by genotype, rates (per 1000 person-years) by thirds of FXIIa distribution were for <1.5 ng/mL, 7. 2; for 1.5 to 2.0 ng/mL, 7.2; and for >2.0 ng/mL, 13.6. Respective hazard ratios with the low third as reference group were 1.01 and 1. 96 (P:=0.007), which were essentially unchanged after allowance for genotype, blood lipids, blood pressure, body mass index, FVIIc, and FPA. CONCLUSIONS: The C46T polymorphism is a determinant of FXIIa, FPA, and possibly FVIIc, suggesting that FXII influences the activity state of the coagulation pathway and FPA cleavage from fibrinogen in vivo. Plasma FXIIa is increased in middle-aged men at high risk of CHD.

Variation in the PPARalpha gene is associated with altered function in vitro and plasma lipid concentrations in Type II diabetic subjects.

AIMS/HYPOTHESIS: Peroxisome proliferator activated receptor alpha (PPARalpha) regulates genes involved in lipid metabolism, haemostasis and inflammation, in response to fatty acids and fibrates, making it a candidate gene for risk of dyslipidaemia, atherosclerosis and coronary artery disease. Plasma non-esterified fatty acids are increased in subjects with Type II (non-insulin-dependent) diabetes mellitus, suggesting that PPARalpha could link Type II diabetes and dyslipidaemia, and affect response to fibrates. This has been investigated in association studies in healthy and diabetic subjects and in vitro studies. METHODS: The human PPARalpha gene was isolated and screened for variation by single strand conformation polymorphism analysis. Genotypes were determined for 129 Type II diabetic subjects and 2508 healthy men. The association with plasma lipid concentrations was examined. The function of the V162 variant was examined in co-transfection assays. RESULTS: We identified two polymorphisms, one in intron 3 and a missense mutation, leucine 162 to valine, in the DNA binding domain. In Type II diabetic patients, V162 allele carriers had higher total cholesterol, HDL cholesterol and apoAI whereas intron 3 rare allele carriers had higher apoAI concentrations. By contrast, no effect was observed in healthy rare allele carriers. In vitro, the V162 variant showed greater transactivation of a reporter gene construct. CONCLUSION/INTERPRETATION: Naturally occurring variation alters PPARalpha function, influencing plasma lipid concentrations in Type II diabetic patients but not healthy people. This demonstrates that PPARalpha is a link between diabetes and dyslipidaemia, and so could influence the risk of coronary artery disease, the greatest cause of morbidity and mortality in Type II diabetes.

Substitution of asparagine for aspartic acid at residue 9 (D9N) of lipoprotein lipase markedly augments risk of ischaemic heart disease in male smokers.

Genetic variants of lipoprotein lipase (LPL), a key enzyme in the hydrolysis of triglyceride (TG)-rich particles, may contribute to ischaemic heart disease (IHD) risk. We have examined the risk of IHD in carriers of two common LPL variants, asparagine substitution for aspartic acid at residue 9 (D9N) and serine for asparagine at residue 291 (N291S) in 2708 middle-aged healthy European men, followed for over 6 years. The carrier frequencies were 2.6% for N9, and 3.9% for S291. Both variants were associated with higher plasma TG at baseline of 9% and 14%, respectively. At baseline, 28% of men were current smokers and smoking was unrelated to genotype. Associations between LPL variants and disease outcome, according to smoking status, were assessed by Cox's proportional hazards analysis. S291 carriers showed no increased risk of IHD compared to non-carriers, while there was strong evidence of interaction between D9N genotype and smoking status (P = 0.0003) in determining the risk of IHD. In 2248 non-carriers of N9, smoking increased the risk of an IHD event by 1.6 (95% CI: 1.1-2.4%) times. Among 58 N9 carriers, no IHD events occurred in 42 who were non-smokers, whereas five events were reported in 16 who smoked. The combined effect of smoking and N9 allele was to increase the risk of an IHD event by 10.4 (95% CI: 4.7-22.8%) times compared with D9 non-smokers. These findings could not be explained by confounding effects of baseline TG. Carriers of N9 appear to be especially vulnerable to the adverse effects of cigarette smoking on IHD risk, but this susceptibility is unrelated to the influence of this variant on plasma TG levels.

Associations of IGF2 ApaI RFLP and INS VNTR class I allele size with obesity.

Body mass index (BMI) is an established epidemiological predictor of coronary disease, diabetes and hypertension. In a previous study of 2560 healthy British Caucasoid males aged 50-61 years (Northwick Park Heart Study II; NPHSII), we showed that IGF2 ApaI AA homozygotes display a mean body weight 3.3 kg lower than GG homozygotes (P = 0.0002) independent of height. Two RFLPs in the insulin (INS) gene, +1127/PstI shown previously and -23/HphI in this study, both of which are in strong linkage disequilibrium with class I/III alleles of the INS 5' variable number tandem repeat (VNTR), are not associated with weight or BMI. The IGF2 ApaI polymorphism therefore appears to mark an effect independent of INS VNTR class I vs class III. We now show by regression that there is a positive correlation of BMI with INS VNTR class I allele size, with an average 0.33% (95% CI = 0.13%, 0.50%) increase in BMI per extra tandem repeat (P < 0.0001) representing variation of 4.8% over the allele size range. However, an alternative interpretation is of 'step' rather than 'slope', the small class I subclass allele group (mode 669 bp) being lighter than the large subclass group (mode 814 bp). This small effect would not be evident as an association between INS VNTR class I/I1 genotype and BMI. The IGF2 ApaI association and INS VNTR class I subclass regression association account for at least 1.1% of population BMI variance. Neither, both, or a third site may be aetiological.