Luong, Le Anh; (2002) Genetic variations in the interleukin-6 (IL-6) gene: Implication in coronary heart disease (CHD). Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Interleukin-6 (IL-6) is an inflammatory cytokine that plays an important role in the pathogenesis of coronary heart disease (CHD) such as atherosclerosis. Single stranded conformational polymorphism (SSCP) was used to screen for genetic variations in the 5'- untranslated and coding regions of the human IL-6 gene. Three novel variants were identified, a wobble position in codon F201 (TTT>TTC), -627C>A, and -572G>C (frequency of rare allele=0.04, 0.01 and 0.05 respectively), plus also the reported - 597G>A, the AnTn tract (-392 to -373) and the -174G>C promoter variants. The -174G>C and -597G>A variants were in strong allelic association with each other (delta=0.94). Functional properties of the IL-6 -174G>C and -572G>C variants were examined in hepatocyte cell lines (HepG2 and HuH7) using a dual luciferase reporter assay system. The -174C variant was associated with higher relative promoter strength than the -174G variant over a 24 hours time course period after dexamethasone (DEX) repression or IL-1β plus DEX induction in both HepG2 and HuH7 cells. The -572C variant also gave higher promoter activity than the -572G variant in response to the same stimulus and time periods in these cells. When the glucocorticoid response element (GRE) at position -557 to -552 was destroyed by mutation, the -572G promoter construct in HuH7 cells no longer showed repression by DEX and had higher induction from IL-1β plus DEX compared to the wild-type -572G variant. No promoter activity difference was found for the -572C GRE knockout compared to the -572C wild type variant. This suggests that the impact of the -572G>C variant is not acting via GRE-related mechanisms. The IL-6 -174G>C and -572G>C promoter polymorphisms were examined in the second Northwick Park Heart Study (NPSHII) of ~3000 prospective healthy middle-aged men from the UK (frequency of rare allele 0.43 95% CI=0.42-0.44 and 0.05 95% CI=0.04- 0.06 respectively). The -174C allele was not associated with fibrinogen levels, but was significantly associated with higher systolic blood pressure in smokers and non-smokers, an effect which was greater in men in the top two tertiles for body mass index (>24.68kg/m²). Compared to those with the GG genotype, men carrying the -174C allele had a relative risk of CHD of 1.54 (95% CI=1.0-2.23, p=0.048) and this effect was greatest in smokers (RR=2.66, 95% CI=1.64-4.32). These effects remained statistically significant even after adjusting for classical risk factors including systolic blood pressure (p=0.04). The -572C allele was not associated with a significant effect on blood pressure, fibrinogen or relative risk for CHD. To confirm the finding of the NPHSII study, the same two polymorphisms were examined in MI survivors and healthy aged-matched controls from four European centres (HIFMECH study). A significant gradient of declining allele frequency was seen in the controls (p=<0.005) and the cases (p=0.032) for the -174C allele, but not for the -572C allele. There were no significant differences in genotype distribution or allele frequency between cases and controls in the North or the South. Significantly higher plasma concentrations for IL-6, fibrinogen and CRP were seen in MI survivors compared to the healthy controls in the North and South of Europe (p=<0.00005). Smoking was associated with greater risk in the South of Europe (OR=3.60, 95% CI=2.34-5.52) compared to the North (OR=2.30, 95% CI=1.48-3.55), but the North-South interaction was not significant. The -174G>C genotype was not significantly associated with levels of IL-6, fibrinogen, CRP or blood pressure in MI survivors or healthy control men from the North or South of Europe. Carriers for the -572C allele who were smokers had significantly higher systolic (5% higher, p=0.004) and diastolic blood pressure (3.4% higher, p=0.03) compared to -572GG genotype, an effect not seen in the nonsmokers. The lack of confirmation of the CHD risk associated with the functional -174C allele in the HIFMECH study and the novel associations seen with the - 572C allele may be a result of the case-control design, but demonstrates that care must be taken in extrapolating from single studies, and that further work to demonstrate functionality in vitro and in vivo is required.

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