| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Clinical Biochemistry (M.B., A.T.-H.), Rigshospitalet, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark; Department of Clinical Biochemistry (M.B., B.G.N.), Herlev University Hospital, DK-2730 Herlev, Denmark; The Copenhagen City Heart Study, Bispebjerg University Hospital (A.T.-H., B.G.N., J.S.J.), DK-2400 Copenhagen, Denmark; and Department of Cardiology (J.S.J.), Gentofte University Hospital, University of Copenhagen, DK-2900 Gentofte, Denmark
Address all correspondence and requests for reprints to: Anne Tybjærg-Hansen, M.D., D.M.Sc., Chief Physician and Associate Professor, 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 IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Objective: We tested the hypothesis that the APOB T71I, A591V, P2712L, R3611Q, E4154K, and N4311S polymorphisms associate with risk of ischemic stroke in the general population and performed in vivo human LDL turnover studies of E4154K heterozygotes vs. K4154K homozygotes.
Design: This was a prospective study (the Copenhagen City Heart Study) with 23-yr, 100% complete follow-up.
Setting: The study was conducted with a Danish general population.
Participants: A total of 9157 women and men aged 20–80+ yr participated in the study.
Main Outcome Measures: Risk of ischemic cerebrovascular disease and ischemic stroke, apolipoprotein B and LDL levels, and LDL fractional catabolic rate were measured. The hypothesis was formulated before genotyping.
Results: APOB K4154K homozygotes had an age-adjusted hazard ratio of 0.4 (95% confidence interval 0.2–0.9) for ischemic cerebrovascular disease and 0.2 (0.1–0.7) for ischemic stroke relative to E4154E homozygotes. Corresponding multifactorially adjusted hazard ratios were 0.5 (0.2–1.0) and 0.2 (0.1–0.8). Furthermore, E4154K heterozygotes and K4154K homozygotes had lower levels of apolipoprotein B and LDL cholesterol, compared with E4154E homozygotes. Finally, E4154K heterozygotes had an increased fractional catabolic rate of LDL relative to E4154E homozygotes. None of the other polymorphisms studied influenced risk of ischemic stroke.
Conclusions: APOB K4154K homozygosity predicts a 3- to 5-fold reduction in risk of ischemic cerebrovascular disease and ischemic stroke. This may be explained by lower plasma levels of apolipoprotein B and LDL cholesterol caused by an increased catabolism of LDL particles, although another yet-unknown mechanism is also possible.
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
A total of 123 genetic variants have been reported in the APOB gene: one approximately 2000 bp upstream, one in the 5' untranslated region (UTR), 60 nonsynonymous coding, 21 synonymous coding, 39 intronic, and one variant in the 3'UTR (http://www.hapmap.org/index.html.en, http://www.ensembl.org/index.html). To study APOB polymorphisms with potential functional importance, we selected six nonsynonymous polymorphisms for genotyping: T71I and A591V located in domains directly interacting with the microsomal triglyceride transfer protein and the protein disulfide isomerase during lipidation (7, 8); P2712L located in the first of three proline rich domains suggested to be involved in structural changes of apolipoprotein B during the conversion of very low-density lipoprotein (VLDL) to LDL (9); R3611Q located close to the LDL receptor binding region (10); and finally, E4154K and N4311S, both in the carboxyl terminal tail of apolipoprotein B, thought to regulate binding of apolipoprotein B to the LDL receptor (10, 11). The T71I, A591V, and E4154K polymorphisms are three of the seven Tag single-nucleotide polymorphisms covering the APOB gene and represent as such variation in the 5'UTR, exons 1–14 and 21–29 of the 29 exons in the APOB gene (http://www.hapmap.org/index.html.en).
We tested the hypothesis that the APOB T71I, A591V, P2712L, R3611Q, E4154K, and N3411S polymorphisms predicted risk of ischemic cerebrovascular disease and ischemic stroke in the general population. For this purpose we genotyped 9157 individuals from the Danish general population, The Copenhagen City Heart Study, of which 550 developed ischemic cerebrovascular disease and 441 ischemic stroke during 23 yr follow-up. Because K4154K homozygosity predicted decreased risk of ischemic cerebrovascular disease and ischemic stroke and decreased apolipoprotein B and LDL cholesterol levels in plasma, we performed in vivo turnover studies of iodinated LDL in E4154K heterozygotes vs. E4154E homozygotes to explore the mechanism behind these findings. These human in vivo turnover studies were carried out in a design with simultaneous injection of differently labeled E4154K and E4154E LDL, a design that excludes all variation due to differences between recipients because the two types of LDL are metabolized by the exact same pathways in the same recipient. Thus, even small differences in metabolism due to differences in LDL particles can be detected accurately.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The Copenhagen City Heart Study is a prospective cardiovascular population study of individuals randomly selected on the basis of the Danish Central Population Register to reflect the adult general population. The participants, age stratified within 10-yr age groups from 20 to 80 yr and above were examined in 1976–1978, 1981–1983, and 1991–1994. More than 99% were of Danish descent. All three examinations included a self-administered questionnaire, a physical examination, and blood samples. Individuals who participated in this study were those who gave blood for DNA extraction.
Identification of possible ischemic cerebrovascular disease events including ischemic stroke (World Health Organization International Classification of Diseases, 8th and 10th revisions: codes 431–438 and I61-I69) was obtained from the Danish National Patient Register and the Danish National Causes of Death Register, which contain data on all hospital admissions and all causes of death in Denmark, respectively. Experienced neurologists reviewed all potential cases of ischemic cerebrovascular disease, i.e. amaurosis fugax (transient blindness on one eye only), transient ischemic attack (focal neurological symptoms lasting < 24 h), or ischemic stroke. Ischemic stroke was an acute disturbance of focal or global cerebral function with symptoms lasting 24 h or longer or leading to death with presumably no other reason than of vascular origin. To distinguish among infarction, intracerebral hemorrhages, and subarachnoid hemorrhages, computed tomography or magnetic resonance imaging scan, autopsy, spinal fluid examination, or surgical description was necessary.
A total of 9157 participants from The Copenhagen City Heart Study were followed up from study entry until the occurrence of ischemic cerebrovascular disease, ischemic stroke, death, or beginning of 2002, whichever came first. During a median of 23 yr follow-up, 550 individuals were recorded with ischemic cerebrovascular disease (ischemic stroke, n = 441; amaurosis fugax, n = 7; transient ischemic attack, n = 102) and 8607 were free of ischemic cerebrovascular disease. Eleven individuals were excluded because of ischemic cerebrovascular disease before study entry.
The study was approved by institutional review boards and a Danish ethical committee (no. 100.2039/91 and KF 01-302/97, Copenhagen and Frederiksberg committee) and was conducted according to the Declaration of Helsinki. Permission to carry out the human in vivo turnover studies was obtained as an addendum to 100.2039/91 and KF 01-302/97. Participants gave written informed consent.
Cardiovascular risk factors
Hypertension was systolic blood pressure 140 mm Hg or greater, diastolic blood pressure 90 mm Hg or greater, or use of antihypertensive medication. Body mass index was weight in kilograms divided by height in meters squared. Smokers were current smokers. Diabetes mellitus was self-reported disease, use of insulin, use of antidiabetic drugs, or nonfasting plasma glucose above 11 mmol/liter. Information on atrial fibrillation was obtained from the Danish National Patient Register or presence of atrial fibrillation on an electrocardiogram at one of the examinations. Colorimetric and turbidimetric assays were used to measure plasma levels of total cholesterol, apolipoprotein B, high-density lipoprotein (HDL) cholesterol, and triglycerides. LDL cholesterol was calculated as total cholesterol – HDL cholesterol – (triglycerides x 0.45) (all in millimoles per liter).
Genotype determination
Genotyping was carried out using TaqMan assays (Applied Biosystems, Inc., Foster City, CA) for the T71I (c>t; rs17246849) and A591V (c>t; rs17240681) polymorphisms and by PCR followed by digestion with BfaI (P2712L; c>t; rs17240903), MspI (R3611Q; g>a; rs17247291), EcoRI (E4154K; g>a; rs1042031), and Eco57I (N4311S; a>g; rs17240958), respectively. Primers, TaqMan probes, and PCR conditions are available from the authors on request.
LDL turnover studies
Selection of participants. Fifty-two in vivo LDL turnover studies were performed in 26 humans to estimate fractional catabolic rate of LDL for eight E4154K heterozygotes vs. eight E4154E homozygotes and for 18 E4154E homozygotes vs. 18 E4154E homozygotes.
Participants were selected in gender matched pairs from the general population cohort and for each study comprised an individual who was E4154K heterozygous and an individual who was E4154E homozygous (or two E4154E homozygous individuals). All were noncarriers for four APOB mutations with known functional effects [R3500Q, R3531C, R3480P, and R3480W (12)], were APOE 
33 homozygotes (13), and were noncarriers for all of the three most common (45%) LDLR mutations in Danes (W23X, W66G, and W556S) (14). Furthermore, individuals selected for the E4154K turnover study in addition were homozygous for the most common allele at the five other polymorphisms studied. This strict selection was done to minimize interindividual variation due to other genetic variation but unfortunately resulted in a limited number of individuals able and willing to participate and none with the K4154K homozygous genotype. All participants in turnover studies were screened for human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, and all were found negative.
Isolation, iodination, and preparation of LDL for in vivo injection in humans
Three days before turnover studies in a pair of an E4154K heterozygote and an E4154E homozygote (or two E4154E homozygotes), 150 ml of blood was obtained from each of the two participants. LDL from the two individuals was isolated by ultracentrifugation as previously described, and at a density of 1.019–1.050 g/ml, to exclude lipoprotein (a) (15). For each pair of participants, LDL (5 mg of protein) was labeled with either 18.5 MBq 125I or 18.5 MBq 131I, or vice versa, using the iodine-monochloride method of McFarlane, as modified by Bilheimer et al. (16).
Study protocol for turnover studies
Sixteen to 18 h after labeling of LDL, a preparation containing a mixture of differently labeled LDL from a pair of an E4154K heterozygote and an E4154E homozygote (or two E4154E homozygotes) was injected simultaneously into the same recipients of either E4154K heterozygous or E4154E homozygous genotype (13 pairs of donors, 26 recipients, 52 turnover studies) (supplemental Fig. 1, left panel, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). This design with simultaneous injection of two differently labeled LDL species excludes variation due to differences between recipients because the two types of LDL are metabolized by the exact same pathways in the same recipient. Furthermore, the two recipients in a pair are injected with the same mixture of autologous and heterologous labeled LDL and thus act as each other’s controls. We have previously shown that even small differences in fractional catabolic rate can be determined using this method (12, 17). After iv injection, 10-ml blood samples were collected from both individuals in a pair at 10 and 20 min; at 1, 2, 4, 6, and 8 h; and daily for the following 8 d of the turnover study (supplemental Fig. 1, right panel). Radioactivity was counted in total plasma after precipitation with trichloroacetic acid (15). Fractional catabolic rate (pools per day) of LDL after injection of labeled LDL was calculated using the SAAM II software (18) (supplemental Fig. 2).
Statistical analysis
The statistical software package Stata/S.E. 9.2 (Stata Corp., College Station, TX) was used. A two-sided P < 0.05 was considered significant. Pearson’s 
2 test and Student’s t test were used in two-group comparisons and one-way ANOVA in three-group comparisons. Deviation from Hardy-Weinberg equilibrium was assessed using the 2 statistic.
Kaplan-Meier curves and log-rank tests compared cumulative incidences of ischemic cerebrovascular disease and ischemic stroke. Cox proportional hazards regression was used to estimate hazard ratios with 95% confidence intervals. The assumption of proportional hazards was tested using Schoenfeld residuals. Analyses were adjusted for age, using age as time scale or multifactorially adjusted for age, gender, total cholesterol, HDL cholesterol, triglycerides, body mass index, hypertension, diabetes mellitus, hypothyroidism, smoking, atrial fibrillation, use of lipid-lowering drugs, and for women also menopausal status and use of hormonal replacement therapy. For multifactorial adjustments, we used time-dependent covariates from the 1976–1978, 1981–1983, and 1991–1994 examinations.
Bivariate tests of interactions between the covariates mentioned above and the six different genotypes on risk of ischemic cerebrovascular disease and ischemic stroke were all nonsignificant. Bonferroni correction was used for multiple comparisons.
Population attributable risk was calculated as [f(HR-1)]/[1+f(HR-1)], where f is the frequency of the genotype of interest in the general population and HR is the hazard ratio for ischemic stroke.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
. Rare allele frequencies of the APOB polymorphisms were 0.33 for T71I, 0.47 for A591V, 0.21 for P2712L, 0.09 for R3611Q, 0.17 for E4154K, and 0.21 for N4311S. All genotype distributions were in Hardy-Weinberg equilibrium (P > 0.05).
|
Age-adjusted and multifactorially adjusted hazard ratios for risk of ischemic cerebrovascular disease and ischemic stroke for all six genotypes are shown in Fig. 1. APOB K4154K homozygosity associated with a 3-fold reduced risk of ischemic cerebrovascular disease [age adjusted hazard ratio 0.4 (95% confidence interval 0.2–0.9)] and a 5-fold reduced risk of ischemic stroke [age adjusted hazard ratio 0.2 (0.1–0.7)], compared with E4154E homozygosity. Corresponding multifactorially adjusted hazard ratios were 0.5 (0.2–1.0) and 0.2 (0.1–0.8), respectively. Excluding individuals with diabetes mellitus from the analyses gave similar risk estimates.
|
). After correction for multiple comparisons for six polymorphisms, the findings for ischemic stroke remained significant. Based on a genotype frequency of 3.1% for the K4154K genotype and an age-adjusted hazard ratio for ischemic stroke of 0.2 (0.1–0.7), the population-attributable risk reduction for ischemic stroke was 2.6% (95% confidence interval 0.9–2.9%).
|
). In accordance with this, E4154K was only in weak linkage disequilibrium with the T71I (r2 = 0.08), A591V (r2 = 0.23), P2712L (r2 = 0.06), R3611Q (r2 = 0.02), and N4311S (r2 = 0.06) polymorphisms. There was no gender-genotype interaction for any of the six APOB polymorphisms on risk of ischemic cerebrovascular disease and ischemic stroke (all P > 0.05). In accordance with this, hazard ratios for risk of ischemic cerebrovascular disease and ischemic stroke were similar in men and women for all six polymorphisms (supplemental Table 1).
Apolipoprotein B and LDL cholesterol levels
E4154K and K4154K vs. E4154E genotype associated with a 2 and 5% reduction in levels of plasma apolipoprotein B and a 2 and 5% reduction in levels of LDL cholesterol, respectively (Fig. 3). Homozygosity for the rare vs. the common allele of the T71I, P2712L, R3611Q, or N4311S did not associate with reduced levels of plasma apolipoprotein B or LDL cholesterol; however, A591V and V591V vs. A591A genotypes associated with a 3 and 4% reduction in levels of apolipoprotein B and a 3 and 5% reduction in levels of LDL cholesterol, respectively (details to be reported elsewhere). The E4154K polymorphism did not influence levels of total cholesterol, VLDL cholesterol, HDL cholesterol, or triglycerides (data not shown). Apolipoprotein B level for the E4154E genotype corresponded to the 51st percentile, E4154K to the 48th percentile, and the K4154K genotype to the 45th percentile in the general population. Corresponding values for LDL cholesterol levels were the 51st, 49th, and 46th percentiles.
|
LDL fractional catabolic rate ratio was increased for E4154K heterozygotes vs. E4154E homozygotes (fractional catabolic rate: E4154K-LDL = 0.39 ± 0.04 (mean ± SE) pools/d vs. E4154E-LDL = 0.35 ± 0.04 pools/d; P = 0.04) and did not differ for E4154E homozygotes vs. E4154E homozygotes (fractional catabolic rate: E4154E-LDL = 0.40 ± 0.02 pools/d vs. E4154E-LDL = 0.40 ± 0.02 pools/d; P = 0.89) (Fig. 4) (supplemental Table 2).
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Elevated LDL cholesterol as a risk factor for ischemic stroke is not as well established as for ischemic heart disease (19, 20). However, two recent large prospective studies have shown association of increased levels of LDL cholesterol as well as apolipoprotein B with increased risk of ischemic stroke (1, 2). Furthermore, large double-blind intervention studies have shown that treatment with statins reducing LDL cholesterol and apolipoprotein B levels by 25–45% during 5 yr reduces the risk of ischemic stroke (21, 22). In this study, the E4154K polymorphism K4154K vs. E4154E genotype is associated with a lifelong reduction of apolipoprotein B and LDL cholesterol of 5%. Therefore, the most simple and straightforward explanation for the present findings is that the lower apolipoprotein B and LDL cholesterol levels, caused by increased fractional catabolic rate of LDL, leads to reduced risk of ischemic cerebrovascular disease and ischemic stroke in K4154K vs. E4154E homozygotes. The lack of risk reduction seen in E4154K heterozygotes vs. E4154E homozygotes could be due to the modest 2% reduction in levels of apolipoprotein B and LDL cholesterol in this group. However, because the A591V polymorphism also associates with reduced levels of apolipoprotein B and LDL cholesterol, but not with reduced risk of ischemic cerebrovascular disease and ischemic stroke, another yet-unknown mechanism may explain the reduced risk of ischemic cerebrovascular disease and ischemic stroke in K4154K vs. E4154E homozygotes.
To the best of our knowledge, none of the APOB polymorphisms included in this study have previously been studied with respect to risk of ischemic cerebrovascular disease or ischemic stroke (English language MEDLINE search to January 2007). We previously demonstrated that the APOB T2488T polymorphism is not associated with risk of ischemic cerebrovascular disease or ischemic stroke (17).
In human in vivo turnover studies, we found an 11% increase in fractional catabolic rate of LDL in E4154K heterozygotes, compared with E4154E homozygotes, findings that are in accordance with two previous studies (23, 24). Several studies have shown functional importance of regions and domains in apolipoprotein B (25, 26, 27), and an amino acid change in a functional domain caused by a polymorphism could be a potential mechanism for the observed increase in fractional catabolic rate of LDL. Structural studies of apolipoprotein B by immunoelectron microscopy suggested that apolipoprotein B enwraps VLDL, intermediate-density lipoprotein, and LDL particles like a belt completing the encirclement by about amino acid residue 4050 and that the carboxy terminal end, including residue E4154K, forms a bow that crosses backward over the chain between residues 3000 and 3500 (11). A structural study speculated that the carboxy terminal sequence of apolipoprotein B could act as a regulator of LDL receptor binding and proposed that in VLDL particles the bow would inhibit binding of apolipoprotein B to the LDL receptor (11). In contrast, after lipolysis has transformed VLDL into intermediate-density lipoprotein and finally LDL, the carboxy terminal bow would move sufficiently to allow interaction of apolipoprotein B with the LDL receptor. Mutations resulting in truncations of apolipoprotein B are known to be able to enhance LDL receptor binding when located carboxy terminal to the LDL receptor binding site, indicating that the negative regulatory effect of the bow of apolipoprotein B can be abolished by removal of the carboxyl terminal tail (27, 28, 29). The E4154K polymorphism, located in the proximal part of the carboxy terminal bow, could potentially have impact on the movement of the bow and thus on LDL receptor binding after conversion of VLDL to LDL. This could explain the increased fractional catabolic rate of LDL in E4154K heterozygotes vs. E4154E homozygotes observed in the present study.
One limitation of the present study is that we studied only individuals of Northern European descent. Frequencies of the selected polymorphisms may differ between different ethnic groups, and thus results from the present study may not be applicable to other ethnic groups. It is unlikely that our results suffer from selection bias because we selected participants at random from the general population without knowledge of genotypes and development of ischemic cerebrovascular disease and ischemic stroke. Regarding cases, a diagnosis of ischemic cerebrovascular disease is less certain than a diagnosis of ischemic stroke, simply because ischemic stroke is a harder end point less subjectively assessed than a diagnosis of other ischemic cerebrovascular diseases like transient ischemic attack and amaurosis fugax. Misclassification of either controls or cases will tend to give more conservative risk estimates, and this might be the cause of the slightly more moderate risk estimates obtained for ischemic cerebrovascular disease, compared with those for ischemic stroke. Linkage disequilibrium with one of the five other polymorphisms studied is an unlikely explanation for the association observed between K4154K vs. E4154E and risk of ischemic cerebrovascular disease and ischemic stroke because linkage disequilibrium was only weak. According to the HapMap database the only polymorphisms in linkage disequilibrium with the E4154K polymorphism are two noncoding variants (rs12720828 and rs3749054) of unknown functional importance in introns 19 and 24, respectively (http://www.hapmap.org/index.html.en).
In summary, we show that K4154K homozygosity predicts a 3- to 5-fold reduction in risk of ischemic cerebrovascular disease and ischemic stroke. This may be explained by lower plasma levels of apolipoprotein B and LDL cholesterol, caused by an increased catabolism of LDL particles, although another yet-unknown mechanism is also possible.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Disclosure Statement: The authors have nothing to disclose.
First Published Online June 26, 2007
Abbreviations: HDL, High-density lipoprotein; LDL, low-density lipoprotein; UTR, untranslated region; VLDL, very low-density lipoprotein.
Received January 30, 2007.
Accepted June 14, 2007.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
32 women are protected while 43 and epsilon44 men are susceptible to ischemic heart disease: the Copenhagen City Heart Study. J Am Coll Cardiol 35:1192–1199
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
