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Department of Clinical Biochemistry (P.R.K., B.G.N.), Herlev Hospital, Copenhagen University Hospital, DK-2730 Copenhagen, Denmark; Department of Clinical Biochemistry (A.T.-H.), Rigshospitalet, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark; The Copenhagen City Heart Study (B.G.N., A.T.-H.), Bispebjerg Hospital, Copenhagen University Hospital, DK-2400 Copenhagen, Denmark; and Department of Internal Medicine (R.S.), Nordsjællands Hospital-Hillerød, Faculty of Health Sciences, University of Copenhagen, Copenhagen, DK-2200 Denmark
Address all correspondence and requests for reprints to: Børge G. Nordestgaard, M.D., D.MSc., Professor and Chief Physician, Department of Clinical Biochemistry, Herlev Hospital, Copenhagen University Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark. E-mail: brno{at}heh.regionh.dk.
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Abstract |
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Objective: We tested the hypothesis that apolipoprotein(a) pentanucleotide repeat genotype predicts elevated lipoprotein(a) levels and risk of myocardial infarction (MI) and ischemic heart disease (IHD) in the general population.
Design: We used a cohort study of the Danish general population, The Copenhagen City Heart Study, including 10,276 individuals of which 860 and 1,781 developed MI and IHD, respectively, during up to 31 yr of follow-up, and a case-control study including 1,814 IHD patients and 5,076 controls. Follow-up was 100% complete.
Results: Allele frequencies were 0.0018, 0.0018, 0.6750, 0.1596, 0.1465, 0.0146, and 0.0004 for 6, 7, 8, 9, 10, 11, and 12 repeats, respectively. Mean lipoprotein(a) levels were 40, 36, and 27 mg/dl for individuals with 14–15, 16, and 17–22 repeats (sum of repeats on both alleles), respectively (trend, P < 0.001). Cumulative incidence of MI and IHD was increased for individuals with 14–15 vs. at least 16 repeats (log rank, P < 0.001 and P = 0.002). Multifactorially adjusted hazard ratios for 14–15 and 17–22 vs. 16 repeats were 3.1 (95% confidence interval, 1.6–5.8) and 1.0 (0.9–1.2) for MI and 2.2 (1.3–3.6) and 1.0 (0.9–1.1) for IHD. In the case-control study, multifactorially adjusted odds ratios for 14–15 and 17–22 vs. 16 repeats were 2.9 (1.1–7.8) and 0.9 (0.8–1.0) for MI and 2.5 (1.0–6.0) and 0.9 (0.8–1.0) for IHD.
Conclusions: Apolipoprotein(a) 14–15 pentanucleotide repeats predict elevated levels of lipoprotein(a) and a 3- and 2-fold increased risk of MI and IHD in the general population.
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Introduction |
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Levels of lipoprotein(a) vary up to a 1000-fold between individuals, and levels are predominantly genetically determined by the apolipoprotein(a) gene on chromosome 6 (1). The so-called kringle IV type 2 (KIV-2) size polymorphism responsible for the different isoforms of apolipoprotein(a) is estimated to determine up to 45% of the interindividual variation in lipoprotein(a) levels. However, a variable number of repeats of the pentanucleotide TTTTA in the promoter region of the apolipoprotein(a) gene has also been shown to correlate with lipoprotein(a) concentrations (10, 11, 12, 13, 14, 15). Between 4 and 12 pentanucleotide repeats have previously been described, and some studies have demonstrated an inverse relationship between number of repeats and lipoprotein(a) plasma concentration (10, 12, 15). One previous case-control study found that pentanucleotide repeat genotype predicts risk of MI in women (12), but the importance of this polymorphism for risk of MI and IHD in individuals in the general population is presently unknown.
We tested the hypotheses that apolipoprotein(a) pentanucleotide repeat genotype predicts 1) elevated levels of lipoprotein(a), and 2) increased risk of MI and IHD in the general population. For these purposes, we genotyped 10,276 individuals randomly drawn from the Danish general population, The Copenhagen City Heart Study, of which 860 and 1,781 developed MI and IHD during up to 31 yr of follow-up. To retest the second hypothesis, we also genotyped 1814 patients with IHD from The Copenhagen Ischemic Heart Disease Study and matched them to 5076 controls from The Copenhagen City Heart Study.
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Subjects and Methods |
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Both studies were approved by Herlev Hospital and Danish ethical committees and conducted according to the Declaration of Helsinki. Participants gave written informed consent. All participants were white and of Danish descent.
The Copenhagen City Heart Study A prospective cardiovascular study of the Danish general population was initiated in 1976–1978, with follow-up examinations in 1981–1983, 1991–1994, and 2001–2003 (16, 17). Participants were randomly selected within age and sex strata from the national Civil Registration System to represent the adult population of Copenhagen. At each of the follow-up examinations, the original cohort was supplemented with individuals in the younger age groups. All four examinations included a self-administered questionnaire, a physical examination, and blood samples. At the 1991–1994 and 2001–2003 examinations, participants gave blood for DNA analysis and had lipoprotein(a) levels determined shortly after sampling. In the present study, we included 10,276 individuals with no history of IHD before study entry and for whom blood for DNA analysis and at least one lipoprotein(a) measurement were available.
Hypertension was defined as use of antihypertensive medication, a systolic blood pressure of at least 140 mm Hg, and/or a diastolic blood pressure of at least 90 mm Hg. Diabetes mellitus was defined as self-reported disease, use of insulin or oral hypoglycemic drugs, and/or a nonfasting plasma glucose greater than 11 mmol/liter. Smokers were active smokers. Body mass index was weight in kilograms divided by height in meters squared.
We followed all individuals from study entry until the occurrence of IHD (including MI), death, or July 9, 2007, whichever came first. Maximum follow-up time was 31 yr. Follow-up was 100% complete. Information on diagnosis of MI and IHD (World Health Organization; International Classification of Diseases, 8th edition codes 410 and 410–414, respectively, and 10th edition codes I21-I22 and I20-I25, respectively) was collected and verified by reviewing hospital admissions and diagnoses entered in the national Danish Patient Registry, causes of death entered in the national Danish Causes of Death Registry, and medical records from hospitals and general practitioners. IHD was defined as the occurrence of MI or characteristic symptoms of angina pectoris based on location, character and duration of pain, and the relation of pain to exercise. A diagnosis of MI required the presence of at least two of the following criteria: characteristic chest pain, elevated cardiac enzymes, or electrocardiographic changes indicative of MI.
The Copenhagen Ischemic Heart Disease Study A case-control study comprising 1814 patients from the greater Copenhagen area referred for coronary angiography to Copenhagen University Hospital was conducted during the period 1991 through 2004. All patients had documented IHD based on characteristic symptoms of stable angina pectoris, plus at least one of the following: stenosis/atherosclerosis on coronary angiography, a previous MI, or a positive bicycle exercise electrocardiographic test. A diagnosis of MI was based on the same criteria as in the general population sample. Lipoprotein(a) measurements were available on 844 patients. Controls consisted of 5076 participants in The Copenhagen City Heart Study without IHD.
Laboratory analysis
The pentanucleotide repeat genotype was determined by PCR, as described by others (10), followed by fragment length analysis on the MegaBACE 500 capillary sequenator (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Rare genotypes were confirmed by sequencing, and in addition random samples of more common genotypes were also confirmed by sequencing. No discrepancy between the two methods of genotyping was found.
In participants attending the 1991–1994 examination of The Copenhagen City Heart Study, lipoprotein(a) total mass was measured with a well-characterized in-house turbidimetric assay using a Technicon Axon autoanalyzer (Miles Inc., Diagnostics Division, Tarrytown, NY), rabbit antihuman lipoprotein(a) polyclonal antibodies (Q023, Dako A/S, Glostrup, Denmark), and a human serum lipoprotein(a) calibrator (Dako A/S) (9). At the 2001–2003 examination, lipoprotein(a) was measured again, using a sensitive immunoturbidimetric assay (DiaSys Diagnostic Systems, Holzheim, Germany). For the 4609 individuals with a lipoprotein(a) measurement in both 1991–1994 and 2001–2003, we observed a minimal bias between the two measurements of 1.6 mg/dl and an r2 value of 0.81 (P < 0.001) when comparing the two measurements using linear regression.
Enzymatic assays were used on fresh samples to measure plasma levels of total cholesterol at all four examinations and plasma levels of triglycerides at the examinations in 1976–1978, 1991–1994, and 2001–2003.
The KIV-2 size polymorphism was genotyped by real-time PCR analysis using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) (details available from the authors). Genotyping resulted in an estimate of the total number (sum of repeats on both alleles) of KIV-2 repeats. The single-copy gene albumin was used to normalize for different concentrations of DNA in different samples. Results for the KIV-2 size polymorphism will be reported elsewhere; the number of KIV-2 repeats was inversely associated with plasma lipoprotein(a) levels as reported by others (10).
Statistical analysis
We used STATA statistical software package version 9.2. A two-sided P < 0.05 was considered significant. 
2 analysis was used to test for departures from Hardy-Weinberg equilibrium of distribution of pentanucleotide repeat genotypes. One-way ANOVA was used to estimate the contribution of the pentanucleotide repeat genotype and of the KIV-2 size polymorphism to the variation in lipoprotein(a) levels. Before this analysis, lipoprotein(a) levels were square root-transformed, as done by others (11), due to skewness of the distribution. For further analysis of the association between apolipoprotein(a) pentanucleotide repeat genotype and lipoprotein(a) levels and risk of MI and IHD, genotypes were divided into groups based on the sum of repeats on both alleles. Cuzick nonparametric test for trend was used to test for differences in lipoprotein(a) levels between different pentanucleotide repeat genotypes. Mann-Whitney test was used to test for differences in KIV-2 repeat number between different pentanucleotide repeat genotypes. Cumulative incidences of MI and IHD were plotted using Kaplan-Meier curves, and differences between apolipoprotein(a) pentanucleotide genotypes were examined using log-rank tests. Cox proportional hazards regression was used to estimate hazard ratios with 95% confidence intervals. We analyzed age-at-event using left truncation (delayed entry) and age as time scale. Thus, age is automatically adjusted for, and we take into account that a period of ignorance exists before an individual enters into the study, a period in which the individual has been subjected to the effects of elevated lipoprotein(a) levels or a specific pentanucleotide repeat genotype. Data from the 1976–1978, 1981–1983, 1991–1994, and 2001–2003 examinations were used as time-dependent covariates for multifactorial adjustments. Therefore, covariate values change over the years of follow-up, depending on availability of data from more recent examinations. Total cholesterol was, depending on an available lipoprotein(a) measurement from the same examination, adjusted for the lipoprotein(a) contribution, according to compositional data where cholesterol accounts for approximately 30% of total lipoprotein(a) mass (9). Triglyceride values were logarithmically transformed due to skewness of the distribution. Interaction of pentanucleotide repeat genotype with other covariates was evaluated by comparing models with and without interaction terms using maximum likelihood ratio tests. Proportionality of hazards over time for different genotypes was assessed by plotting –ln[–ln(survival)] vs. ln(analysis time). No violations of the proportional hazard assumption were detected. Based on the second lipoprotein(a) measurement in 2001–2003, hazard ratios for increased lipoprotein(a) levels were corrected for regression dilution bias using a nonparametric method (18).
Conditional logistic regression was used to estimate odds ratios with 95% confidence intervals. Controls were matched to cases based on gender and 5-yr age groups. Risk factor status for controls was based on information from the 2001–2003 examination.
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Results |
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Of the 10,276 participants, 56% were women. Age at entry ranged from 20 to 87 yr with a mean of 45. During a follow-up of up to 31 yr, 1781 participants were diagnosed with IHD, including 860 with MI. Baseline characteristics of participants are displayed by apolipoprotein(a) pentanucleotide repeat genotype in Table 1
and by disease status in supplemental Table 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org).
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2 test, P = 0.79). The pentanucleotide repeat polymorphism was estimated to contribute 1.4% to the interindividual variance of lipoprotein(a) concentrations (ANOVA; F-value 8.2, df 18, P < 0.001), whereas the KIV-2 size polymorphism contributed 22% (ANOVA; F-value 43.1, df 65). Considering both alleles, the most frequent sum of repeats was 16, of which 99.8% were homozygous for the 8 repeat allele (supplemental Table 2). This group served as the reference group, representing 46% of the cohort. The number of repeats was inversely associated with lipoprotein(a) levels, and mean lipoprotein(a) levels were 40, 36, and 27 mg/dl for individuals with 14–15, 16, and 17–22 repeats (sum of repeats on both alleles), respectively (Fig. 1; Cuzick nonparametric test for trend of P < 0.001). This association was not explained by linkage disequilibrium with the KIV-2 size polymorphism genotype (supplemental Fig. 1). There was no association of the pentanucleotide repeat genotype with levels of total cholesterol or triglycerides (data not shown).
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; tests for trend ranged from P = 0.01 to P < 0.001). Multifactorially adjusted hazard ratios for MI and IHD were 1.7 (95% confidence interval, 1.4-.2.2) and 1.3 (1.1–1.6) for individuals in the top third compared with individuals in the bottom third of the lipoprotein(a) distribution.
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; log rank tests of P < 0.001 and P = 0.002). On Cox regression analysis, individuals with 14–15 vs. 16 repeats had multifactorially adjusted hazard ratios for MI and IHD of 3.1 (1.6–5.8) and 2.2 (1.3–3.6); in multifactorially adjusted models including adjustment for KIV-2 repeat number, corresponding hazard ratios were 3.0 (1.6–5.6) and 2.1 (1.3–3.6). However, a pentanucleotide repeat genotype of more than 16 vs. 16 or less repeats was not associated with reduced risk of MI and IHD (Fig. 4; tests for trend ranged from P = 0.29 to P = 0.97). The multifactorially adjusted hazard ratio for 17–22 vs. 16 repeats was 1.0 (0.9–1.2) for MI and 1.0 (0.9–1.1) for IHD, with similar results when KIV-2 repeat number was also adjusted for. Also, upon adjustment for lipoprotein(a) values, risk estimates were only slightly attenuated (Supplemental Fig. 2). Cox regression models analyzing risk according to number of repeats per allele confirmed that short alleles (6–7 repeats) associate with increased risk and that no decrease in risk is seen for the longer alleles (9–12 repeats), compared with 8 repeat alleles (Supplemental Fig. 3).
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Case-control study
Characteristics of cases and controls are displayed in supplemental Table 1. Among cases with IHD, number of repeats inversely associated with lipoprotein(a) levels (supplemental Fig. 4; Cuzick nonparametric test for trend of P < 0.001). On logistic regression analysis, individuals with 14–15 vs. 16 repeats had multifactorially adjusted odds ratios for MI and IHD of 2.9 (1.1–7.8) and 2.5 (1.0–6.0) (Fig. 5). For individuals with 14–15 through 16 and 17–22 repeats, a trend toward lower risk of MI and IHD was seen in some models (Fig. 5; tests for trend ranged from P < 0.01 to P = 0.16). The multifactorially adjusted odds ratios for MI and IHD for 17–22 vs. 16 repeats were 0.9 (0.8–1.0) and 0.9 (0.8–1.0). In models including adjustment for KIV-2 repeat number, results were similar.
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Discussion |
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Lipoprotein(a) consists of what is essentially a low-density lipoprotein particle bound to a glycoprotein named apolipoprotein(a) (1). Lipoprotein(a) could influence risk of MI and IHD either through promotion of atherosclerosis or increased risk of thrombosis. Previous studies have implicated lipoprotein(a) in the process of atherogenesis including entry into the arterial intima and promotion of foam cell formation, smooth muscle cell proliferation, and plaque inflammation and instability (19, 20, 21, 22). Also, apolipoprotein(a) is structurally similar to plasminogen, and apolipoprotein(a) has been shown to interfere with the process of fibrinolysis (20). Levels of lipoprotein(a) vary up to 1000-fold between healthy individuals, with the variability largely determined by polymorphisms in the apolipoprotein(a) gene, mainly the KIV-2 size polymorphism and the pentanucleotide repeat promoter polymorphism (1). The latter has previously been estimated to account for up to 10% of the total variability in lipoprotein(a) concentrations in whites (1). However, luciferase reporter gene assays have revealed no difference in promoter activity between different pentanucleotide repeat alleles (23), indicating that the pentanucleotide repeat polymorphism may be in linkage disequilibrium with other genetic variation directly affecting levels of lipoprotein(a).
Studies of the association of elevated lipoprotein(a) levels with risk of IHD have yielded conflicting results, but a large metaanalysis from 2000 concluded that elevated levels of lipoprotein(a) associate with increased risk of IHD (2). Several subsequent studies have supported this conclusion (3, 4, 5, 6, 7, 8). We recently demonstrated a stepwise increase in risk of MI and IHD with increasing levels of lipoprotein(a), with no evidence of a threshold effect, and we showed that extreme levels above the 90th percentile predict a 3- to 4-fold increase in risk of MI in the general population (9). Whether this association is causal is, however, unclear.
An association between apolipoprotein(a) gene polymorphisms and both elevated lipoprotein(a) levels and risk of MI and IHD could indicate a causal relationship. Previous studies of the association between the apolipoprotein(a) pentanucleotide repeat polymorphism and risk of atherosclerosis and IHD are few, and results are not consistent (12, 13, 14). One study from 1996 including 96 patients and 64 controls evaluated the association of degree of atherosclerosis, based on coronary angiographic findings, with levels of lipoprotein(a), apolipoprotein(a) size, and pentanucleotide repeat genotype (14). An association with lipoprotein(a) levels and apolipoprotein(a) size was found, whereas only a borderline significant association with pentanucleotide repeat genotype was found. Furthermore, a case-control study from 1999 including 594 patients with MI vs. 682 controls demonstrated association of pentanucleotide repeat genotype with levels of lipoprotein(a), but pentanucleotide repeat genotype was not reported to predict risk of MI (13). Finally, a case-control study from 2003 including 834 individuals with MI vs. 1548 controls genotyped for the pentanucleotide repeat polymorphism and phenotyped for the KIV-2 size polymorphism, found that lipoprotein(a) levels were inversely related with both number of KIV-2 and pentanucleotide repeats. In addition, the odds of presenting with MI were elevated in individuals with small apolipoprotein(a) phenotypes. Finally, women, but not men, with a pentanucleotide repeat genotype of no more than 8 repeats on both alleles (equivalent to our 14–16 repeats) had an increased risk of MI (odds ratio, 1.5; P = 0.009), compared with individuals with other genotypes (12). In the present general population study, we demonstrate a clear inverse association between total number of pentanucleotide repeats and levels of lipoprotein(a), although no more than 1.4% of the interindividual variation in lipoprotein(a) levels was estimated to be determined by the pentanucleotide repeat polymorphism, an estimate somewhat lower than what has been reported previously (1, 11). Furthermore, we show that individuals with 14–15 repeats, and thus increased levels of lipoprotein(a), are at increased risk of MI and IHD. Because there was no difference in mean KIV-2 number between groups defined by 14–15 and 16 pentanucleotide repeats, and accordingly, risk estimates were not affected by adjustment for the KIV-2 size polymorphism genotype, these results cannot be explained by linkage disequilibrium with the KIV-2 size polymorphism. Upon adjustment for lipoprotein levels, risk estimates were only slightly attenuated, indicating that the pentanucleotide repeat genotype provides information above and beyond a plasma lipoprotein(a) measurement. This could be due to linkage disequilibrium with other variation in the coding regions of the apolipoprotein(a) gene resulting in different properties of different isoforms of lipoprotein(a). Our risk estimates are higher than those presented in previous studies, most likely because we, due to our large sample size, were able to focus on a relatively small group with an extreme genotype.
Surprisingly, individuals with 17–22 vs. 16 repeats did not clearly associate with reduced risk of MI and IHD despite reduced lipoprotein(a) levels. However, individuals with more than 17 pentanucleotide repeats had decreased mean number of KIV-2 compared with individuals with 16 pentanucleotide repeats, equivalent to former findings of 10, 11, and 12 pentanucleotide repeat alleles associating with a low number of KIV-2 repeats (12). If small isoforms are particularly harmful, as previously indicated (5, 24), this might explain why increased numbers of pentanucleotide repeats did not clearly associate with decreased risk of MI and IHD. Nevertheless, after adjusting for the number of KIV-2 repeats in Cox regression analyses, risk estimates for MI and IHD for more than 16 vs. 16 pentanucleotide repeats still did not reach statistical significance. This, therefore, would seem to question a causal relationship between elevated levels of lipoprotein(a) and increased risk of MI and IHD, except for extreme levels of lipoprotein(a) (9).
Despite 43% of cases in the case-control study receiving lipid-lowering therapy known to modify IHD predictivity attributed to lipoprotein(a) (25), we obtained comparable risk estimates in the general population study and the case-control study. However, before medication start, cases may already have suffered from extensive atherosclerotic disease, not easily ameliorated by lipid-lowering therapy.
A study limitation is that results of the present study may not be applicable to other ethnic groups because levels of lipoprotein(a) and the impact of different apolipoprotein(a) gene polymorphisms on lipoprotein(a) levels vary between populations (1). It is unlikely that our results suffer from any major degree of selection bias because we selected participants at random from the general population and had 100% follow-up. However, we only included participants who gave blood for DNA analysis and therefore participated in the examination in either 1991–1994 or 2001–2003. One possible source of selection bias is that individuals with a high-risk genotype did not survive to participate in these examinations. This is unlikely though, because genotypes were in Hardy-Weinberg equilibrium and because the distribution of genotypes was similar to that reported earlier (10, 11, 13). We cannot rule out misclassification of a few cases or controls. Misclassification of either cases or controls will tend to give more conservative risk estimates, and this might be the cause of the somewhat more moderate risk estimates obtained for IHD compared with those for MI, simply because a diagnosis of IHD is less certain than a diagnosis of MI. A limitation of the case-control study is that risk factor status such as smoking, hypertension, and lipid parameters are affected by the lifestyle changes and medication often associated with case status. However, results were similar in age- and gender-adjusted models and multifactorially adjusted models (data not shown).
The present study finds elevated levels of lipoprotein(a) and low number of apolipoprotein(a) promoter pentanucleotide repeats associated with increased risk of MI and IHD in the general population. The association of 14–15 repeats with both elevated levels of lipoprotein(a) and increased risk of MI and IHD supports a causal role for lipoprotein(a) in atherosclerosis; however, the lack of clearly reduced risk of MI and IHD in those with 17–22 repeats, despite reduced lipoprotein(a) levels, raises questions about such a causal relationship.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online August 5, 2008
Abbreviations: IHD, Ischemic heart disease; KIV-2, kringle IV type 2; MI, myocardial infarction.
Received April 16, 2008.
Accepted July 30, 2008.
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References |
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This article has been cited by other articles:
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  P. R. Kamstrup, A. Tybjaerg-Hansen, R. Steffensen, and B. G. Nordestgaard Genetically Elevated Lipoprotein(a) and Increased Risk of Myocardial Infarction JAMA, June 10, 2009; 301(22): 2331 - 2339. [Abstract] [Full Text] [PDF]
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