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Polymorphism in high density lipoprotein paraoxonase gene and risk of acute myocardial infarction in men: prospective nested case-control studyCommentary: Causality—the Achilles' heel of observational studiesCommentary: How high density lipoprotein protects against heart disease

BMJ 1999; 319 doi: https://doi.org/10.1136/bmj.319.7208.487 (Published 21 August 1999) Cite this as: BMJ 1999;319:487
  1. Jukka T Salonen (jukka.salonen{at}uku.fi), professor of epidemiologya,
  2. Riikka Malin, associate research fellowb,
  3. Tomi-Pekka Tuomainen, research fellowa,
  4. Kristiina Nyyssönen, clinical biochemista,
  5. Timo A Lakka, senior research fellowa,
  6. Terho Lehtimäki, senior research fellowb
  1. a Research Institute of Public Health and Department of Public Health and General Practice, University of Kuopio, PO Box 1627, 70211 Kuopio, Kuopio, Finland
  2. b Department of Clinical Chemistry, Tampere University Hospital, and Research Laboratory for Atherosclerosis Genetics, University of Tampere Medical School, Tampere, Finland
  3. Clinical Trials and Evaluation Unit, Royal Brompton Hospital, London SW3 6NP
  4. BMJ
  1. Correspondence to: Professor Salonen
  • Accepted 26 November 1998

Increased lipid peroxidation is associated with accelerated progression of atherosclerosis.1 Paraoxonase (paraoxonase/arylesterase) is an antioxidative enzyme in high density lipoproteins, which protect against coronary disease2 3 It eliminates organophosphorus pesticides but also the products of lipid peroxidation2 4 The mutation at position 54 of the paraoxonase gene in which methionine is substituted by leucine (Met54Leu) has an effect on paraoxonase, increasing its activity; people who have the methionine allele show decreased paraoxonase activity.4 Only a few studies have looked at the association of the Met54Leu polymorphism with coronary disease,2 5 and the findings are inconclusive. Thus we carried out a prospective study of the role of this polymorphism on the risk of acute myocardial infarction in healthy men from eastern Finland.

Participants, methods, and results

Our prospective nested case-control study was carried out among participants in the Kuopio ischaemic heart disease risk factor study We examined 2682 (83%) of 3235 invited men aged 42, 48, 54 or 60 during 1984-9. Blood samples were collected and risk factors assessed at baseline. A DNA sample was available for this study for 1137 men who were free of coronary disease. We registered and verified all myocardial infarctions—definite or possible—between the baseline examinations and the end of 1995.3 The mean follow up time was 8.5 years, and in patients who had had multiple infarctions we considered only the first.

The cases were all 55 men (among the 1137) who had had an infarction by 1995. The controls were drawn from the remaining members of the same cohort. Two controls for each case (110 men) were matched according to the most important non-genetic risk factors for myocardial infarction in the Kuopio ischaemic heart disease risk factor study cohort. For the 165 men, paraoxonase genotypes were determined by using a polymerase chain reaction method with Hsp92II enzyme digestion.4 We used logistic regression modelling to analyse the association of paraoxonase genotypes with the risk of myocardial infarction.

Strongest risk factors for acute myocardial infarction in two multivariate logistic models

View this table:

Of the cases, 13 (24%) were homozygous for the M allele (MM), 22 (40%) were heterozygous (ML), and 20 (36%) did not carry it (LL). Of the controls, 11 (10%) had an MM genotype, 54 (49%) an ML, and 45 (41%) an LL. In a logistic model adjusted for the other strongest risk factors the odds ratio for the MM genotype was 3.38 (95% confidence interval 1.17 to 9.83; P=0.025) (table, model 1). An additional adjustment for serum concentration of the second subfraction of high density lipoprotein cholesterol (HDL2) attenuated this odds ratio to 3.07 (1.03 to 9.17; P=0.044; table, model 2). The odds ratio for the MM genotype remained unchanged after any other risk factor was forced into the model. The odds ratio was significantly (P<0.05) higher among the 59 smokers (40.8 (2.65 to 629.2); P=0.008) than the 106 non-smokers (1.55 (0.45 to 5.32); P=0.488).

Comment

In this prospective population based study, men who were MM homozygous for the Met54Leu polymorphism of paraoxonase had over a threefold risk of a first myocardial infarction compared with men who did not carry the M allele (LL). People with the M allele have reduced plasma concentrations and activities of paraoxonase.2

Men with the genotype conferring low activity of paraoxonase (MM) had about three times the risk of myocardial infarction than had men with the genotype conferring high activity (LL). The genotype at increased risk is common: a tenth of our healthy controls had it According to our data, close to a fifth of all infarctions in our healthy cohort of middle aged men would be attributable to this single genetic polymorphism through either direct causation or linkage to other genes Our findings suggest that paraoxonase has a protective role against coronary disease.

Acknowledgments

We thank Terhi Nissinen for DNA extractions; Kari Seppänen for lipoprotein analyses; and Kimmo Ronkainen for data management and matching.

Contributors: JTS is the guarantor of the study. JTS initiated and organised the Kuopio ischaemic heart disease risk factor study; JTS, TL, and T-PT planned the study concerning paraoxonase genotypes; RM and TL did the genotypings; KN is responsible for the chemical measurements; TAL classified the heart attacks; and JTS analysed the data. All authors participated in writing the manuscript.

Footnotes

  • Funding The Kuopio ischaemic heart disease risk factor study was supported by grant HL44199 from the National Heart, Lung, and Blood Institute to George A Kaplan and by grants from the Academy of Finland (to JTS), the Finnish Ministry of Education (JTS, TAL), the Emil Aaltonen Foundation (TL), and the Finnish Foundation of Cardiovascular Research (RM, TL).

  • Competing interests None declared.

References

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Commentary: Causality—the Achilles' heel of observational studies

  1. Marcus D Flather, director (m.flather{at}rbh.nthames.nhs.uk)
  1. a Research Institute of Public Health and Department of Public Health and General Practice, University of Kuopio, PO Box 1627, 70211 Kuopio, Kuopio, Finland
  2. b Department of Clinical Chemistry, Tampere University Hospital, and Research Laboratory for Atherosclerosis Genetics, University of Tampere Medical School, Tampere, Finland
  3. Clinical Trials and Evaluation Unit, Royal Brompton Hospital, London SW3 6NP
  4. BMJ

    Salonen et al use a nested case-control design to correlate risk of myocardial infarction for different paraoxonase genotypes. The method identified incident cases and matched them to controls in a ratio of 1 to 2. This approach uses a subset of subjects from a larger cohort to address a particular hypothesis.

    Case-control studies are one of the weakest methods to infer a causal association between a risk factor and a disease. On the other hand, they are simple to perform and useful for generating hypotheses Problems of bias arise because of difficulty in matching cases to controls and the presence of multiple known and unknown confounding variables. Apparently significant results can arise by chance because of multiple testing, and some statistical approaches can be tipped in favour of significance in borderline cases.

    In this study associations between genotype and risk of myocardial infarction were comparatively weak (lower limit of 95% confidence interval 1.17 (P=0.025) in the unadjusted analysis and 1.03 (P=0.044) in the adjusted model), which does not give great confidence in the causality of the findings. The main problem is that the number of subjects in the analysis is small. The hypothesis is intriguing and deserves a fair appraisal.

    The correct next step would be to replicate these findings in a similar cohort study that had obtained DNA samples. Collaboration among groups with well documented cohorts would provide a major advance in the assessment of promising new markers of risk in both epidemiological and interventional studies after the publication of the main results of individual studies. This approach would help to reinforce or refute observations of borderline significance.

    The importance of an association between a particular genotype and risk of disease allows people at high risk to be counselled appropriately and modifiable risk factors to be treated more aggressively. In certain circumstances, correcting a protein deficiency with supplements may be helpful. Genetic manipulation, although intuitively attractive, is associated with practical, technical, and ethical problems but may be a treatment of the future. Most cases of myocardial infarction are unlikely to be due to a single gene defect. Nevertheless, the report by Salonen et al helps to increase understanding of genotype and disease and to identify promising areas of research.

    Commentary: How high density lipoprotein protects against heart disease

    1. Abi Berger, science correspondent
    1. a Research Institute of Public Health and Department of Public Health and General Practice, University of Kuopio, PO Box 1627, 70211 Kuopio, Kuopio, Finland
    2. b Department of Clinical Chemistry, Tampere University Hospital, and Research Laboratory for Atherosclerosis Genetics, University of Tampere Medical School, Tampere, Finland
    3. Clinical Trials and Evaluation Unit, Royal Brompton Hospital, London SW3 6NP
    4. BMJ

      High density lipoprotein protects against cardiovascular disease. Protection is conferred in at least two ways The first way is by transporting cholesterol back from organs such as arteries to the liver, thus protecting the arteries from further atheromatous plaque formation. The second way is by acting as an antioxidant.1 Lipid peroxidation encourages atheroma formation, so that anything which limits lipid peroxidation should reduce the risk of cardiovascular events. The component of high density lipoprotein that is most likely to provide antioxidant activity is paraoxonase.

      In this study Salonen et al examined the paraoxonase gene of 165 men One genetic mutation appeared quite commonly. In this mutation methionine is substituted by leucine at one particular position in the enzyme's protein structure, increasing the enzyme's antioxidant activity. Men who did not carry this mutation (that is, those who were homozygous for the M allele) had three times the risk of myocardial infarction of those who did.

      These observations suggest that paraoxonase is more active when the mutation is present and less active when it is not and that paroxonase might be protective against cardiovascular disease.

      References

      1. 1.
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