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Polymorphism in APOB Associated with Increased Low-Density Lipoprotein Levels in Both Genders in the General Population

Departments of Clinical Biochemistry (M.B., A.T.-H.), Medicine B (P.G.), and Vascular Surgery (H.S.), Rigshospitalet, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark; Department of Clinical Biochemistry, Herlev University Hospital (B.G.N.), DK-2730 Herlev, Denmark; Copenhagen City Heart Study, Bispebjerg University Hospital (B.G.N., J.S.J., A.T.-H.), DK-2400 Copenhagen, Denmark; and Department of Cardiology, Gentofte University Hospital (J.S.J.), 2900 Hellerup, Denmark

Address all correspondence and requests for reprints to: Dr. Anne Tybjærg-Hansen, Department of Clinical Biochemistry, KB3011, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. E-mail: at-h{at}rh.dk.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Rare mutations in APOB cause hypercholesterolemia. Whether common polymorphisms in APOB have similar effects remains controversial.

Objective: We tested the hypothesis that the APOB 7673C>T polymorphism (T2488T) is associated with variation in low-density lipoprotein (LDL) levels and with risk of ischemic heart disease (IHD), ischemic cerebrovascular disease (ICVD), and total mortality in the general population.

Design: The design was a cohort study with 22-yr follow-up (166,232 person years) and two case-control studies, The Copenhagen City Heart Study.

Settings: The study was performed within the Danish general population and at a university hospital.

Participants: The study was comprised of 9185 individuals from the general population, 2157 patients with IHD, and 378 patients with ICVD.

Main Outcome Measures: The main outcome measures were lipids, lipoproteins, apolipoproteins (apo), IHD, ICVD, and total mortality.

Results: Genotype was associated with increases in total cholesterol (women/men), LDL cholesterol, and apoB of 0.20/0.26 mmol/liter (3.3%/4.4%), 0.22/0.28 mmol/liter (5.9%/7.8%), and 5.0/5.6 mg/dl (5.9%/6.7%) in TT vs. CC homozygotes, respectively. These results were consistent over time. Despite this, the 7673C>T polymorphism was not associated with risk of IHD, ICVD, or total mortality prospectively or in case-control studies.

Conclusion: The APOB 7673C>T polymorphism is associated with moderate increases in total cholesterol, LDL cholesterol, and apoB in both genders in the general population, but not with risk of IHD, ICVD, or total mortality.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
RARE MUTATIONS IN the genes encoding the low-density lipoprotein (LDL) receptor and its ligand apolipoprotein B (apoB) may cause severely elevated LDL cholesterol levels and premature ischemic heart disease (IHD) (1, 2). However, polymorphisms in these two genes have never convincingly been demonstrated to affect LDL cholesterol levels or risk of IHD. Because it is well known that polymorphisms in another ligand for the LDL receptor, apoE, cause moderate elevations in LDL levels and increased risk of IHD (3), it is likely that variations in APOB have similar effects.

One of the most studied polymorphisms in APOB is due to the substitution of thymine (T) for cytosine (C) at nucleotide 7673 (4, 5), also known as the XbaI or T2488T polymorphism (6, 7, 8). TT homozygotes have been reported to have higher plasma cholesterol levels than CC homozygotes, although results are conflicting (6, 9, 10). Furthermore, the 7673C>T polymorphism has been associated with risk of IHD (11), although two recent meta-analyses reported conflicting results. Boekholdt et al. (12) found a decreased risk of IHD in TT vs. CC homozygotes (odds ratio, 0.80; 95% confidence interval, 0.66–0.96; n = 1906; P = 0.02), whereas Chiodini et al. (13) found an increased risk in TT individuals compared with CC and CT individuals (odds ratio, 1.19; 95% confidence interval, 1.01–1.39; n = 5874; P = 0.03).

To determine a possible association of the 7673C>T polymorphism in APOB with variation in lipid and lipoprotein levels and with risk of IHD and ischemic cerebrovascular disease (ICVD) as well as with total mortality, we genotyped 9185 individuals from the Danish general population, 2157 patients with IHD, and 378 patients with ICVD. 1) Variations in lipids, lipoproteins, and apoB as a function of genotype were determined in the general population cohort. 2) Very low-density lipoprotein (VLDL) and LDL metabolism as a function of genotype was examined in human turnover studies in vivo (n = 6 and 5). 3) The plasma apoE/VLDL-apoB ratio was determined in the general population cohort to detect genotype-dependent changes in the ability to clear VLDL via apoE. 4) Finally, the risk of IHD, ICVD, and total mortality was examined prospectively in the general population cohort, and the morbidity results were verified in two large case-control studies.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Participants

The Copenhagen City Heart Study is a prospective study of the Danish general population initiated in 1976–1978 with follow-up examinations in 1981–1983, 1991–1994, and 2001–2003 (14, 15). An almost equal number of women (55%) and men were stratified into 5-yr age groups from 20–80 yr and above. In the present study, 9185 individuals from the third examination, 1991–1994, were genotyped for the 7673C>T polymorphism. Information on diagnosis of IHD (n = 1104), myocardial infarction (MI; n = 573), ICVD (n = 417), and ischemic stroke (n = 330; World Health Organization; International Classification of Diseases; 8th edition: codes 410–414 and 432–435; 10th edition: I20-I25 and I63-I64) was collected and verified until December 31, 1999 by reviewing all hospital admissions and diagnoses entered in the Danish National Hospital Discharge Register, all causes of death entered in the Danish National Register of Causes of Death, and medical records from hospitals and general practitioners. IHD was determined on the basis of previous myocardial infarction or characteristic symptoms of stable angina pectoris based on location, character, and duration of pain and the relation of pain to exercise (16). A diagnosis of MI required the presence of at least two of the following criteria: characteristic chest pain, elevated cardiac enzymes, and electrocardiographic changes indicative of MI.

A second population comprised 2157 patients identified in 1991–2002 among patients referred for coronary angiography because of angina pectoris. Experienced cardiologists determined whether cases had IHD based on characteristic symptoms of stable angina pectoris as described above (16) plus at least one of the following: stenosis on coronary angiography, previous MI, or a positive exercise electrocardiography test.

A third population comprised 378 patients with ICVD identified among patients referred from 1994–1999 for ultrasonic evaluation of the carotid arteries because of focal neurological symptoms suggestive of ICVD (ischemic stroke, transient ischemic attack, or amaurosis fugax). Cases with ICVD were diagnosed by experienced vascular surgeons and neurologists on the basis of 50% or greater stenosis of the carotid artery on the symptomatic or most stenotic side and either sudden onset of focal neurological symptoms lasting 24 h or more (ischemic stroke), less than 24 h (transient ischemic attack), or transient monocular blindness (amaurosis fugax). Patients with cerebral hemorrhage were excluded on the basis of a computerized tomography scan.

Studies were approved by institutional review boards and Danish ethical committees and were conducted according to the Declaration of Helsinki. Permission to carry out the human in vivo turnover studies was obtained as an addendum. Participants gave written informed consent. More than 99% were white and of Danish descent.

Analyses

Genotyping was performed by PCR, followed by digestion with XbaI. Colorimetric and turbidimetric assays were used to measure plasma levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and apoB (2). VLDL and LDL cholesterol levels were calculated, respectively, as triglycerides x 0.45, and total cholesterol as HDL cholesterol – (triglycerides x 0.45) (all in millimoles per liter). ApoE was measured by nephelometry (Behring, Dudingen, Switzerland).

To calculate the number of plasma apoE particles per VLDL-apoB particle, plasma apoE was divided by VLDL-apoB, estimated as 12% of the total apoB (1) (both in millimoles per liter).

VLDL and LDL turnover studies

In vivo VLDL and LDL turnover studies were performed in humans to estimate the fractional catabolic rate (FCR) of VLDL and LDL from APOB 7673 CC homozygotes vs. VLDL and LDL from TT homozygotes.

Participants in the turnover studies were selected from the general population cohort and comprised individuals who were either CC or TT homozygotes, APOE 33 homozygotes, and homozygous for the common alleles of the following nonsynonymous polymorphisms in APOB suspected of affecting cholesterol levels: P2712L, R3611Q, E4154K, and N4311S, i.e. they only differed at the 7673C>T site. APOB R3500Q/W, R3531C, and the three most common (45%) LDLR mutations in Danes (W23X, W66G, and W556S) (17) were excluded. All participants in turnover studies were screened for HIV, hepatitis B virus, and hepatitis C virus, and all were found to be negative.

LDL (or VLDL) from CC and TT homozygotes was isolated by ultracentrifugation and iodinated as previously described at a density of 1.019–1.050 g/ml to exclude lipoprotein(a) (or density <1.006 g/ml) (18, 19). Sixteen to 18 h after labeling of LDL or VLDL, a preparation containing a mixture of differently labeled LDL (or VLDL) from a pair of CC and TT homozygotes was injected simultaneously into the same recipients of either the CC or TT genotype (six pairs of donors, 12 recipients, 11 turnover studies, because one failed). This design with simultaneous injection of differently labeled CC-LDL and TT-LDL (or CC-VLDL and TT-VLDL) excludes variation due to differences between recipients, because the two types of LDL (or VLDL) are metabolized by the exact same pathways in the same recipient. We have recently shown for other genetic variants in APOB that even small differences in FCR can be determined using this method (20). After iv injection, 10-ml blood samples were collected regularly for the following 8 d. When labeled LDL (or VLDL) was injected, radioactivity was counted in total plasma after precipitation with trichloroacetic acid (18) [or in apoB in lipoprotein fractions after precipitation with isopropanol (21)].

FCR of LDL after injection of labeled LDL was calculated by the method of Matthews (22) using SAAM II software (23). The production rate (PR; millimoles per kilogram per day) was calculated by multiplying FCR (pools per day) by the distribution volume (milliliters per kilogram) and the LDL (or VLDL) cholesterol level (millimoles per liter).

Prospective study

Prospective studies were conducted using IHD, MI, ICVD, ischemic stroke, any ischemic event, or death as end points (incident events: IHD, n = 952; MI, n = 480; ICVD, n = 407; ischemic stroke, n = 321; any ischemic event, n = 1,141; death, n = 1,314). The cohort was comprised of participants in the Copenhagen City Heart Study who attended the 1991–1994 examination and gave blood for DNA analysis (n = 9,185). All end points were recorded in the follow-up period 1976–2000. The median follow-up time was 22 yr (166,232 person years). Individuals diagnosed with either end point before entry into the study were excluded (IHD, n = 152; MI, n = 93; ICVD, n = 10; ischemic stroke, n = 9; any ischemic event, n = 162). Follow-up was 100% complete.

Case-control studies

To retest with maximal statistical power whether genotype was associated with risk of ischemic events, all prevalent cases from the Copenhagen City Heart Study were combined with independent case populations of patients with IHD (952 + 152 + 2157 = 3261), ICVD (407 + 10 + 378 = 795), or any ischemic event (1402 + 2157 + 378 = 3937). All cases were compared with unmatched controls from the general population free from IHD, ICVD, or both (IHD, n = 8081; ICVD, n = 8769; IHD or ICVD, n = 7783).

Statistical analysis

Data were analyzed using SPSS (24) and STATA (25). P < 0.05 on a two-sided test was considered significant. Pearson’s ² test and Student’s t test were used in two-group comparisons, and one-way ANOVA was used in three-group comparisons. The percent phenotypic variance was estimated as previously described (26).

Cox proportional hazards regression models with age as a covariate or multifactorially adjusted for age, total cholesterol, triglycerides, HDL cholesterol, body mass index, hypertension, diabetes, hypothyroidism, smoking status, and menopausal status and use of hormonal replacement therapy for women were used to estimate hazard ratios for end points by genotype. In addition, Cox regression was performed with age as the time scale (left truncation), which means that differences in age were automatically adjusted.

Unconditional logistic regression models estimated odds ratios for end points in case-control studies. Bivariate interactions on end points between genotype and covariates listed in Table 1 were tested (age, total cholesterol, triglycerides, HDL cholesterol, body mass index, hypertension, diabetes, hypothyroidism, smoking status, and menopausal status and use of hormonal replacement therapy for women); none was significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

Lipids, lipoproteins, and apoB

The APOB 7673C>T genotype was associated with increases in plasma total cholesterol (women/men), LDL cholesterol, and apoB of 0.20/0.26 mmol/liter (3.3%/4.4%), 0.22/0.28 mmol/liter (5.9%/7.8%), and 5.0/5.6 mg/dl (5.9%/6.7%), for TT vs. CC homozygotes, respectively (Fig. 1). These findings were consistent in both genders and across different examinations spanning 10–25 yr. Results at the 2001–2003 examination were less significant, most likely due to a reduction in the number of subjects to about 50% from the 1991–1994 examination. Levels of triglycerides, VLDL cholesterol, and HDL cholesterol were not affected by genotype.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Lipids, lipoproteins, and apoB levels at four examinations, 1976–1978, 1981–1983, 1991–1994, and 2001–2003, of the Copenhagen City Heart Study as a function of APOB 7673C>T genotype. Bars represent the mean difference between the APOB 7673C>T CC and CT genotypes and the CC and TT genotypes, respectively. Mean values for the CC genotype are given below bars. *, P < 0.05; **, P 0.01; ***, P 0.001 (comparing CC with CT or TT genotype, by Student’s t test).

FCRs of LDL from CC and TT homozygotes were not significantly different when labeled LDL was injected simultaneously in the same individuals as recipients [FCR CC-LDL, 0.41 ± 0.04 (mean ± SEM); FCR TT-LDL, 0.44 ± 0.03 pools/d; P = 0.33; Fig. 2; see supplemental Fig. 1 and supplemental Table 1, published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org] or when labeled VLDL (converted to labeled LDL in vivo) was injected in similar experiments (FCR CC-LDL, 0.48 ± 0.05; FCR TT-LDL, 0.47 ± 0.07 pools/d; P = 0.21; Fig. 2). For all LDL turnover studies, combined FCR (n = 11) was 0.44 ± 0.03 pools/d for CC-LDL and 0.45 ± 0.03 for TT-LDL (n = 11; P = 0.62).

View larger version (24K): [in this window] [in a new window]   FIG. 2. In vivo human turnover studies of labeled VLDL and LDL directly comparing APOB 7673C>T CC and TT individuals. Circles depict compartments (q), arrows denote rate constants (k), the syringe shows where labeled VLDL or LDL was injected, and S1 and S2 indicate where samples were drawn. The VLDL turnover model comprises two VLDL compartments (q1 and q2) and two LDL compartments (q3 and q4; upper panel). The LDL turnover model comprises two LDL compartments (q1 and q2; lower panel). Values are the mean ± SE. P values were by paired Student’s t tests.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Number of plasma apoE particles per apoB particle in the VLDL fraction as a function of APOB 7673C>T genotype. Based on 4529 individuals from the 2001–2003 examination of the Copenhagen City Heart Study. By ANOVA, P = 0.001; by post hoc tests, *P = 0.01; ***P = 0.001 (Student’s t test).

The APOB 7673C>T polymorphism contributed 0.4–0.8%, 0.4–1.0%, and 0.7–0.9%, respectively, to the total phenotypic variance in total cholesterol, LDL cholesterol, and apoB levels in the general population (Table 2). For comparison, the rare APOB R3500Q mutation contributed 0.1–0.2%, 0.2–0.3%, and 0.2–0.3%, and the common APOE polymorphism contributed 2.3–3.7%, 4.1–5.8%, and 5.1–7.2%, respectively, to the total phenotypic variance in total cholesterol, LDL cholesterol, and apoB levels.

During 22 yr of follow-up (166,232 person years), we observed the following number of incident events in the Copenhagen City Heart Study: IHD, 952; MI, 480; ICVD, 407; ischemic stroke, 321; any ischemic event, 1,141; and deaths 1,314. Hazard ratios as a function of genotype did not differ from 1.0 for any of the end points studied, for both genders combined, or for women and men separately (Table 3). When adjusting the analyses for covariates shown in Table 1 (total cholesterol, triglycerides, HDL cholesterol, body mass index, hypertension, diabetes, hypothyroidism, smoking status, and menopausal status and use of hormonal replacement therapy in women), the results were similar (data not shown). In case-control studies, we included the following number of prevalent cases: IHD, 3,261; ICVD, 795; and any ischemic event, 3,937. Odds ratios as a function of genotype did not differ from 1.0 for any of the end points studied, for both genders combined, or for women and men separately (data not shown).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Lipids, lipoproteins, and apoB

Previous results on the association of APOB 7673C>T with variation in lipids and lipoproteins have been somewhat conflicting; however, some studies have reported higher plasma cholesterol levels in TT vs. CC homozygotes (6, 9, 10, 27). In the present study we found moderate, but consistent, increases in total cholesterol, LDL cholesterol, and apoB in both genders at examinations spanning 10–25 yr. Increases in these variables reflected increased LDL levels, because plasma triglycerides, VLDL cholesterol, and HDL cholesterol levels were not affected by genotype.

VLDL and LDL metabolism

We did not find any difference in the FCR of LDL between CC and TT homozygotes, but we did find an increase in the PR of LDL in TT compared with CC homozygotes. These results are in agreement with human in vivo turnover studies performed by Korhonen et al. (28) and with in vitro studies of human fibroblasts (29). However, they are in conflict with two other studies (30, 31) that included more participants than ours, reporting decreased LDL FCR in TT homozygotes. The main differences between our study and the latter two studies (30, 31) are 1) we compared the LDL turnover of individuals from the general population matched for gender and a number of nonsynonymous polymorphisms in APOB suspected of affecting cholesterol metabolism as well as for APOE genotype; and 2) we injected differently labeled autologous and heterologous CC and TT LDL (or VLDL) simultaneously into the same recipients, a design that excludes interindividual variation and more accurately shows differences in metabolism of the lipoprotein studied.

The novel observation of a reduction in the plasma apoE/VLDL-apoB ratio in TT vs. CC homozygotes (6%) could cause a reduced ability to remove VLDL via apoE in TT homozygotes and thus potentially explain the higher LDL cholesterol levels in these individuals.

This observation might seem to be in conflict with the fact that we could not detect statistically significant higher PRs of LDL from VLDL in the VLDL turnover studies; however, it is difficult to argue that data on plasma apoE/VLDL-apoB ratios measured in 4529 individuals could be rendered irrelevant by results for VLDL PR in only five individuals. Furthermore, when all 11 turnover studies were combined, the LDL PR was increased in TT vs. CC homozygotes.

Risk of IHD and ICVD

Two recent and conflicting meta-analyses (12, 13) reported that TT vs. CC homozygotes had either reduced or slightly elevated risk of IHD. Our study was several times larger than all previous studies combined into these two meta-analyses, was carried out using a well-characterized homogenous sample from a general population known to be at high risk of ICVD, and clearly demonstrated that the 7673C>T polymorphism was not associated with risk of IHD, ICVD, or total mortality in either gender. Furthermore, we demonstrated this lack of association with IHD and ICVD in a prospective study with 22 yr of follow-up (with 98% power to detect a hazard ratio of 1.3), whereas all previous studies were case-control studies. Even when we combined all end points available to us in the largest possible case-control studies (with 99% power to detect an odds ratio of 1.2), TT vs. CC homozygotes did not have odds ratios different from 1.0. This lack of association with risk of IHD and ICVD in the present study, despite a clear association of genotype with an increase in LDL cholesterol levels, was most likely due to the modest contribution of this polymorphism to the overall variability in LDL cholesterol and apoB levels in the general population. In our study the difference in total cholesterol is approximately 0.2 mmol/liter between CC and TT homozygotes. When comparing with other cohort studies, it is clear that such a small difference in total cholesterol would not increase the risk of ICVD substantially when the mean total cholesterol is in the normal range (as in our study); however, in the upper quartile of the total cholesterol range, it seems that even small changes in total cholesterol might have an effect on the risk of cardiovascular events, at least in men; i.e. the risk of ischemic cardiovascular events increases exponentially with increasing levels of total cholesterol (32, 33, 34). If the TT genotype in itself had an effect on the risk of cardiovascular disease, i.e. reducing the risk and thus counteracting the risk normally associated with increased total cholesterol levels, this effect would probably be revealed when adjusting the Cox regression analysis for total cholesterol. This was not the case in our study.

Mechanism

The base change, nt 7673C>T, which creates the XbaI site, does not change the amino acid threonine at codon 2488. Thus, it is likely that there is additional sequence variation elsewhere in the APOB gene, in the promoter or a nonsynonymous variant in the coding region, in linkage disequilibrium with the 7673C>T polymorphism. The effect of this unknown variant is probably underestimated in our study due to a dilution of the associations by incomplete linkage disequilibrium between the 7673C>T polymorphism and this unknown variant. Like other groups, we have tried to identify this unknown variant by estimating the two-site linkage disequilibrium between the 7673C>T variant and other variants in the APOB gene. This analysis showed 32.0% linkage disequilibrium (D') with the T71I polymorphism, 40.6% with A591V, 99.9% with P2712L, 10.9% with R3611Q, 100.0% with E4154K, and 99.9% with N4311S. These results indicate that the rare allele at 7673C>T is tightly linked to a combined genotype extending further downstream in the gene, but does not point out with which specific candidate polymorphism in the APOB gene the 7673C>T variant is in linkage disequilibrium (our unpublished observations).

The mechanisms by which an APOB variation might affect plasma lipids, lipoprotein, and apoB levels are not completely understood. Variations in the APOB gene product could affect the interaction of apoB bearing lipoproteins with HDL in the cholesteryl ester transfer complex, the interaction with apoE or hepatic lipase, or the affinity of LDL particles for the LDL receptor. Our population-based findings of an association with the 7673C>T polymorphism confined to LDL does not favor a mechanism via interaction of apoB with HDL. Likewise, our in vivo turnover studies do not favor a mechanism via interaction between apoB and the LDL receptor. More likely, the association between genotype and LDL levels, LDL production rate, and plasma apoE/VLDL-apoB ratio could be explained by an influence on VLDL removal mediated via apoE and resulting in increased production of LDL from VLDL in TT homozygotes.

Limitations

Unfortunately we do not have data for fatty acid composition of the participant’s diet and are not able to look into this. However, because of Mendelian randomization (35), we would not expect to find a difference in the intake of fatty acids between genotypes; therefore, we do not believe that differences in diet would change the main study outcome on risk of ICVD.

Conclusion

The APOB 7673C>T polymorphism is associated with increased LDL cholesterol levels, but not with risk of IHD or ICVD or with total mortality. Thus, we demonstrate that common polymorphisms in APOB affect levels of lipids and lipoproteins in both genders in the general population.

	Acknowledgments

 
We thank Mette Refstrup, Hanne Damm, and Kurt Svarre Jensen for expert technical assistance.

	Footnotes

 
This work was supported by the Danish Heart Foundation, the Danish Medical Research Council, Chief Physician Johan Boserup and Lise Boserup’s Fund, Ingeborg and Leo Dannin’s Grant, and the Research Fund at Rigshospitalet, Copenhagen University Hospital.

First Published Online July 19, 2005

Abbreviations: apo, Apolipoprotein; FCR, fractional catabolic rate; HDL, high-density lipoprotein; ICVD, ischemic cerebrovascular disease; IHD, ischemic heart disease; LDL, low-density lipoprotein; MI, myocardial infarction; PR, production rate; VLDL, very low-density lipoprotein.

Received May 3, 2005.

Accepted July 7, 2005.

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