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Optimal lipid intake in relation to risk of cardiovascular disease in individuals with susceptible genotypes

Project Code: N02027

30/12/2005

MRC Dunn Nutrition Unit
Bingham, S ; Bowman, R; Wu, K; Joosens, A; Norat, T;
University of Cambridge EPIC Norfolk
Shakya-Shrestha, S; Welch, A; Luben, R

Aim

Risk of coronary heart disease (CHD) depends on both inherited susceptibility and environmental factors, including diet. The aim of this project was to examine the interrelationship between dietary lipid intake, intermediate cardiovascular disease risk factors (serum cholesterol fractions, fibrinogen, factor V11 and blood pressure) and single nucleotide polymorphisms in genes controlling these factors, andsusceptibility to CHD controlling for other aspects of lifestyle such as smoking and exercise. The effect of specific fatty acids, rather than total and types of fats, was also investigated using a new data base and two methods for assessing intake.

Approaches

This work utilised data from the Norfolk arm of the European Prospective Investigation of Cancer (EPIC), a study of 25,000 men and women aged 45 – 75 years who were healthy when their diet, blood lipids and other factors were assessed on recruitment in 1992. Three approaches were followed (i) DNA was extracted from blood collected at a second health check in 1997 and genotyped for polymorphisms in four genes with a newly established Pyrosequencing technique. Cross sectional analyses relating dietary lipids and urinary electrolytes to CVD risk factors were carried out on 12,828 individuals to determine possible interactions according to genotype, controlling for other lifestyle factors such as smoking (ii) Of the individuals who were free of CHD on recruitment, since 1992, 1,107 developed CHD. 2,205 controls who remained healthy matching for sex, date of birth and date of health
check were selected and genotype, diet and lifestyle factors compared between the case and control group to determine effects of diet and lifestyle factors on risk of CHD. (iii) Existing dietary data bases which are used to calculate intake of total and types of fat cannot be used to assess effects of separate fatty acids. Of the saturated fats for example, myristic acid is thought to be one of the most effective fatty acids in raising low density lipoprotein. The published RSC dietary data base was therefore extended to include individual fatty acids and the results applied to information on food consumption obtained at recruitment from a 7 day dietary diary and a food frequency questionnaire.

Key Findings

In the 12,828 individuals for whom blood for genotyping was available, people who ate more saturated fat and less polyunsaturated fat and dietary fibre had higher blood lipids than others, as expected. There were significant positive regression coefficients between serum LDL and total cholesterol (P < 0.001) and dietary saturated fat and inverse associations with plasma LDL and polyunsaturated fat. Inverse associations were found as expected between dietary fibre and plasma LDL and total cholesterol, and positive associations with HDL cholesterol, but unexpectedly inverse associations were also shown between plasma triglyceride and dietary fibre (P < 0.001). Adjusting for other lifestyle factors such as smoking, body weight, exercise levels did not affect these relationships.

Blood lipids varied as expected by genotype, for example low density lipoprotein choiesterol was higher in individuals carrying the Apolipoprotein E ε4 allele than those carrying either the ε2 allele or the ε3 allele (p<0.001) (Mahey and Rall 2000). Using the FFQ and original food data base, regression coefficients between diet and serum lipids were of a greater magnitude in individuals carrying the Apolipoprotein E ε4 allele compared with individuals carrying the ε3 allele; for example for dietary saturated fat, % energy, and plasma LDL cholesterol the β coefficients for ε3 and ε4 were 0.014 (P = 0.003) and 0.023 (P = 0.003). However only a significant interaction between ε2/ε4 genotype (β = 0.319, P = 0.011) and ε3/ε3 genotype (β = 0.108, P < 0.001), (Z = 2.38, P < 0.05) was detected. In individuals carrying the ε2 allele there were no significant associations between serum lipids and dietary
constituents.

There were no significant effects of factor VII genotype on blood lipids in 11,930 individuals studied. In the 4548 individuals for whom plasma factor VII, genotype and blood lipids were available, factor VII levels showed the expected variation with higher levels in the wild type CC (116+/- 36.7%), 99.1 (+/- 32.6%) in CT and 80.0 (+/-26%) in TT individuals. Factor VII blood levels were significantly related to plasma triglyceride and HDL cholesterol levels (p<0.001) and these associations remained in the CC, CT, and CT+TT combined genotypes. However there were no effects of diet on factor VII levels (β coefficients all less than 0.001), and thus no effects according to Factor VII genotype.

In cross sectional analyses, sodium potassium ratio was positively associated with increased blood pressure (b = 3.7 (2.02 – 5.38) p<0.001). However, in individuals with the wild type A the regression coefficient was almost double that of the population as a whole (b = 6.64 (3.92 – 9.36) p <0.001) and in those with the G variant associations failed to reach significance (p 0.085). Polyunsaturated fat was inversely associated with plasma fibrinogen levels, although there was no interaction according to genotype.

1104 individuals who did not report heart attack or stroke on recruitment in 1992 later developed CHD. Their data were compared with 2208 people who remained healthy and also free of heart attack or stroke on recruitment, matched for age, sex and date of recruitment. Taking dietary and other factors into account, the Apolipoprotein E polymorphisms were not associated with risk of CHD. After adjusting for dietary and other lifestyle factors, the odds ratio (OR) for ε2 and ε4 compared to the ε3 were as follows (OR = 1.19, P = 0.287, 95% confidence interval (C.I.) = 0.87, 1.63, OR = 0.93, P = 0.513, 95% C.I. = 0.73, 1.17), respectively.
Further genotyping is needed to confirm this lack of effect in a cohort wide analysis.

The G variant of the polymorphism for angiotensinogen was protective against CHD risk in men (0.76 (0.58 – 0.98), excluding those who were prescribed drugs for hypertension at baseline. However, there were no effects in women. There were strong positive associations with the intake of sodium to potassium ratio and CHD risk especially using data derived from the food diary (OR 1.58 (1.06 – 2.35) p 0.0076 and a highly significant interaction for the A wild type (OR 3.18 (1.72 – 5.89) versus G variant (OR 1.02 (0.36 – 2.88) for the top quintile of Na/K ratio for the diary, p interaction 0.007.

Plasma fibrinogen levels were strongly associated with increased CVD risk, especially in smokers (OR 4.43 (1.26 – 15.63) p trend 0.001 for the top versus lower quintile of fibrinogen. However there were no associations with cardiovascular disease and the mutation (G455-A) in the promoter region of the beta fibrinogen gene.

Despite the expected relation between blood lipids and dietary factors found in the cross sectional study, in the case control study there were highly significant (p <0.001) inverse associations between dietary saturated fat and CHD risk and positive associations with polyunsaturated fats and dietary fibre (p<0.001). After excluding cases and controls receiving medication for high blood pressure at recruitment, and those who reported eating a modified diet at recruitment, these associations became non significant. The unexpected inverse association with saturated fat and risk of CHD is therefore probably due to a change in diet close to the diagnosis of CHD. Further studies are ongoing with longer follow up time to
assess this possibility.

To examine effects of specific fatty acids on blood lipids, in house data entry and processing programmes were used. The 10,464 food items on these entry programmes are mapped to 2480 foods with published RSC food codes or recipes, which were in-filled with data on specific fatty acids. Nine sources of published compilations on 25 fatty acid fractions including eight specific saturated fatty acids, four monounsaturated fatty acids and nine polyunsaturated fatty acids were used for this process. As before, there were significant inverse associations with total and LDL cholesterol and polyunsaturated fat, mainly due to n-6 fats and linoleic acid. There were weaker positive associations between saturated fat but much stronger with stearic acid and total cholesterol but not LDL cholesterol. Myristic acid was not significantly related to total or LDL cholesterol. A positive association was found between plasma triglycerides and palmitic and stearic acid, and an inverse association with n-3 polyunsaturated fatty acids.

In conclusion, this is the largest definitive study relating diet, genes, risk markers and CHD risk ever conducted. This data set is very large and, in the interests of time, analyses reported here in this report are preliminary. Full analyses, for example in relation to other dietary variables, will be reported as full publications in the scientific press as soon as possible.

Nevertheless, the results reported here showed that regression coefficients relating dietary fat to blood lipids and odds ratios for risk markers of CHD were of different magnitude when assessed according to ApoE genotype, but findings were all in the same direction, thus supporting public health recommendations to reduce saturated fat intake in the population as a whole. Effects were unchanged when controlling for other lifestyle factors and there were no added effects of specific fatty acids. There were no associations between plasma factor VII and dietary lipids, and an inverse association between fibrinogen and polyunsaturated fatty acids. There were no effects according to genotype on CHD risk.

The only genotype, which had a significant effect, was the polymorphism of the angiotensinogen gene, which was not associated with increased risk of high blood pressure with increasing sodium intake. Thus the overall population effect was diluted and those with the wild type were at greater risk from high salt intakes than was apparent in the population as a whole. As the wild type is the most common form, these findings support the current FSA policy of reducing salt intake in the population as a whole.

Summary Table of Main Findings
(ND = not determined)

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