(Circulation. 2000;101:2393.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
A Common Mutation in Lipoprotein Lipase Confers a 2-Fold Increase in Risk of Ischemic Cerebrovascular Disease in Women but Not in Men
From the Department of Clinical Biochemistry, Herlev and Glostrup University Hospitals (H.H.W., B.G.N., A.T.-H.), the Copenhagen City Heart Study, Bispebjerg University Hospital (B.G.N., P.S., A.T.-H.), and the Department of Vascular Surgery, Gentofte University Hospital (H.S.), Copenhagen, Denmark.
Correspondence to Anne Tybjærg-Hansen, Department of Clinical Biochemistry, Rigshospitalet 4111, National University Hospital, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. E-mail ath{at}image.dk
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Abstract |
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Background—We previously showed that the common Asn291Ser substitution in lipoprotein lipase is associated with elevated plasma triglyceride levels and a 2-fold increase in the risk of ischemic heart disease in women but not men. In this study, we tested the hypothesis that this substitution is also associated with an increased risk of ischemic cerebrovascular disease (ICVD).
Methods and Results—We compared 260 patients who had nonfatal
ICVD and carotid stenosis 
50% with 1560 age-matched controls
and also compared 205 Copenhagen City Heart Study cases who had
nonfatal ICVD with 1210 age-matched controls. All subjects were white
and from Denmark. Overall, no significant difference was observed
between carrier frequencies among those with and without ICVD; however,
sex interacted with genotype in predicting ICVD in the ICVD and
carotid stenosis cases (P=0.02). In Copenhagen
City Heart Study cases, sex was not significant
(P=0.18). Odds ratios for ICVD in female mutation
carriers were 2.9 (95% confidence interval [CI], 1.3 to 6.4) and 1.9
(95% CI, 0.8 to 4.6) in ICVD plus carotid stenosis cases and
Copenhagen City Heart Study cases, respectively. Equivalent values in
male mutation carriers were 0.8 (95% CI, 0.3 to 1.8) and 0.8 (95% CI,
0.3 to 2.0), respectively. These results were similar in
analyses that also allowed for other conventional
cardiovascular risk factors.
Conclusions—These results suggest that the Asn291Ser substitution in lipoprotein lipase is not associated with nonfatal ICVD in men but that it possibly confers a 2-fold risk in women.
Key Words: genetics • polymorphism (genetic) • triglycerides • cholesterol • cerebral ischemia
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The risk of ischemic heart disease1 and ischemic cerebrovascular disease (ICVD)2 increases with increasing plasma triglyceride levels. Lipoprotein lipase hydrolyses triglycerides contained in the core of chylomicrons and very-low-density lipoproteins.3 Thus, an impaired function of lipoprotein lipase may be associated with an increased risk of cardiovascular disease.
We recently showed that the common Asn291Ser substitution in
lipoprotein lipase in the heterozygous state (heterozygote frequency is
5% in the general population) is associated with an increase in
plasma triglycerides and a 2-fold increase in the risk of
ischemic heart disease in women but not men.4 In
the present study, we tested the hypothesis that this substitution
is also associated with an increased risk of nonfatal ICVD.
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Methods |
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A general population sample, the Copenhagen City Heart Study, which consisted of 9215 individuals, was collected from 1991 to 1994 and studied cross-sectionally.4 5 A total of 83 women and 122 men who had previous sudden onset of focal neurological symptoms due to ICVD were identified by an experienced neurologist5 who was unaware of the results of genotyping. ICVD was defined as ischemic stroke (focal neurological symptoms lasting >24 hours when hemorrhages were excluded on computed tomography scan; n=131), a transient ischemic attack (symptoms lasting <24 hours; n=66), or amaurosis fugax (transient blindness in 1 eye only; n=8). Patients with hemorrhagic stroke (n=10) or other nonischemic cerebral events (n=32), patients using cholesterol-lowering drugs (n=79), individuals among the 9215 subjects with plasma triglyceride levels
10
mmol/L (n=35; none of whom had ICVD), and participants with missing
information (n=203) were excluded from the analysis, which left
205 ICVD patients and 8651 controls. From this pool, age- and
sex-matched controls were drawn for all analyses.
A second study population consisted of 97 women and 163 men who
had a sudden onset of focal neurological symptoms due to ICVD. These
patients were referred by neurologists for carotid ultrasound
examination on an outpatient basis at the National University Hospital
in Copenhagen from 1994 to 1996 because of their neurological symptoms.
These patients were consecutively included in the present study if
they fulfilled the entry criteria of a carotid artery stenosis
50% on the symptomatic or most stenotic side;
thus, neurological symptoms most likely were referable to large vessel
atherothrombotic disease. At least 1 computed tomography scan was
performed to exclude hemorrhage. These 260 patients either had
ischemic stroke (n=157), transient ischemic attack
(n=75), or amaurosis fugax (n=28). Furthermore, they were all residents
of the same geographical area as the individuals from the Copenhagen
City Heart Study, and none had plasma triglyceride levels
10 mmol/L.
In both study populations, <1% were non-whites, and >98% were of Danish descent. Diabetes mellitus and hypertension were diagnosed as described previously.5
We compared the 260 patients with ICVD and carotid artery
stenosis 50% with 1560 age- and sex-matched controls (6
controls per case when possible); all controls were sampled from the
general population and did not have ICVD. Furthermore, we compared the
205 individuals from the Copenhagen City Heart Study who had ICVD with
1210 age- and sex-matched controls. The study was approved by
the ethical committee of the City of Copenhagen and
Frederiksberg.
Screening for the Asn291Ser substitution, collection of basic characteristics, and biochemical measurements were performed as described previously.4 5 Blood was drawn in the nonfasting state among participants of the Copenhagen City Heart Study but in the fasting state among the hospital-based ICVD patients.
We used Student’s t test, ANOVA, Pearson’s
2 test, and logistic regression
analyses, as previously described, for statistical
analyses.4 5 Data were analyzed for
each sex, both separately and combined, after tests for interaction
between sex and genotype. Logistic regression analyses
were performed as bifactorial analyses allowing for age only
and as multifactorial backward stepwise analyses allowing for
age and other cardiovascular risk factors. The models
did not allow for matching. After age and other significant predictors
had entered the model, genotype was forced into the model.
Homogeneity of the association of genotype and conventional
cardiovascular risk factors in the prediction of ICVD
were tested by the introduction, one at a time, of all possible
2-factor interaction terms between genotype and conventional
cardiovascular risk factors in the logistic regression
analysis. The likelihood ratio test determined
significance.
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Results |
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Carrier frequencies and characteristics are shown in Table 1
. Genotype frequencies in
the general population (including 6 homozygous individuals) were not
different from values predicted by the Hardy-Weinberg equilibrium. The
analyses described below did not include 35 individuals with
triglyceride levels 10 mmol/L; however, the
inclusion of these individuals had no major influence on study
outcomes.
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Lipids and Lipoproteins
Genotype and sex interacted on triglyceride
(P=0.03) and high-density lipoprotein (HDL)
cholesterol (P=0.03) levels (Table 2). The Asn291Ser substitution was
significantly associated with increased plasma triglyceride
levels in women (P<0.001) but not men (P=0.98).
HDL cholesterol was reduced 2.5 times more in female
carriers than in male carriers.
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Risk of Ischemic Cerebrovascular Disease
Genotype and sex also interacted in predicting ICVD in
cases with ICVD plus carotid stenosis (P=0.02); in
Copenhagen City Heart Study cases, this interaction was not significant
(P=0.18). No interaction occurred between genotype
and other covariates entered in the multifactorial logistic regression
models predicting ICVD (data not shown).
On logistic regression analyses allowing for age only,
Asn291Ser heterozygosity was associated with a 2- to 3-fold increase in
the risk of ICVD in women (Table 3); in
Copenhagen City Heart Study cases, this was not significant. In men,
genotype did not predict ICVD.
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On multifactorial stepwise logistic regression analysis, odds
ratios for ICVD associated with Asn291Ser heterozygosity were similar
to those seen in the bifactorial analyses (Tables 3
and 4). In women, the odds ratio was 2 to 3,
whereas in men, the odds ratio was close to 1.
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When only ischemic stroke (excluding transient ischemic attack and amaurosis fugax) was considered, the odds ratios for ICVD in women carrying the Asn291Ser substitution were 4.1 (95% confidence intervals [CI], 1.6 to 10.8) and 1.7 (95% CI, 0.6 to 5.1) in cases with ICVD plus carotid stenosis and Copenhagen City Heart Study cases, respectively. The equivalent odds ratios for men were 1.1 (95% CI, 0.4 to 2.9) and 0.6 (95% CI, 0.2 to 2.1), respectively.
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Discussion |
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Overall, we found no significant difference in Asn291Ser carrier frequencies between subjects with and without ICVD; no effect was observed among men, either in cases with ICVD plus carotid stenosis or in Copenhagen City Heart Study cases. Furthermore, no significant effect existed in Copenhagen City Heart Study female cases. In contrast, a roughly 2-fold risk of ICVD was observed in female Asn291Ser carriers among cases with ICVD plus carotid stenosis (n=10 carriers). It was also only in this study group, and not among Copenhagen City Heart Study cases, that significant sex interaction was seen.
We recently showed that the common Asn291Ser substitution in
lipoprotein lipase was associated with a significant increase in plasma
triglyceride levels and a 2-fold increase in the risk of
ischemic heart disease in women but not men.4 The
present data indicate that the Asn291Ser substitution may also be
associated with a 2-fold increase in risk of ICVD in women but not men.
These sex-specific effects are supported by the following points. (1)
Triglyceride level is a better predictor of the risk of
cardiovascular disease in women than in
men.1 (2) Triglyceride levels were
significantly increased in female but not male carriers in the
Copenhagen City Heart Study in 2 separate measurements taken in 1991 to
1994 and 1976 to 1978.4 (3) HDL cholesterol
was 2.5 times more reduced in female carriers than in male
carriers.4 (4) Odds ratios for ischemic heart
disease4 and ICVD in carriers were 2.0 in women and
1.0 in men.
Lipoprotein lipase degrades the triglycerides
contained in chylomicrons, and very-low-density lipoproteins thus
converting these particles into smaller remnants.3 The
Asn291Ser substitution does not block the formation of remnants
completely; it only reduces enzyme activity by 40%.6
Thus, this substitution probably increases the time between the
secretion of intact chylomicrons and very-low-density lipoproteins
until the chylomicrons and very-low-density lipoproteins have been
sufficiently depleted of triglycerides to be removed from
plasma. In other words, at a given time point, more remnants of
different sizes will be present in plasma than if the enzyme was
fully functional, and such remnants may be atherogenic7 ;
importantly, it is the cholesterol content in these
triglyceride-rich lipoproteins rather than the
triglyceride content that is thought to be atherogenic.
Dysfunctional lipoprotein lipase also leads to the reduced
production of HDL particles,3 which may also be
atherogenic.
Because the inclusion of plasma triglycerides and HDL cholesterol in the logistic regression analysis did not completely abolish the association between the mutation and the risk of ICVD, it could be hypothesized that the atherogenicity of a malfunctioning lipoprotein lipase may be due not only to a direct effect of triglyceride-rich lipoprotein and low HDL levels, but also to a pathological effect on the metabolic processes involved in their formation. This could be due to (1) deposition of surface lipid in the vessel wall, (2) local production of smaller remnant lipoproteins, and (3) lipolysis alteration of endothelial barrier function.8 However, it is perhaps more likely that a single measurement of triglyceride or HDL levels does not accurately reflect the lifelong impact of the mutation on these levels.
The Asn291Ser substitution was only significantly related to ICVD in
the group of patients with carotid artery stenosis. Our
patients with ICVD and 50% carotid stenosis probably
represent the 20% of the general ICVD population with
atherosclerotic cerebrovascular disease,9 whereas the
individuals with ICVD identified in the general population probably
represent a more broad ICVD population. Thus, the promotion of
atherosclerosis through an effect of Asn291Ser on
plasma levels of triglycerides or HDL
cholesterol would potentially result in a higher risk of
ICVD in the subgroup of patients with verified carotid
atherosclerosis, thereby explaining the observed higher
risk in the ICVD plus carotid stenosis cases compared with
Copenhagen City Heart Study cases. This is supported by the findings in
the previous study on ischemic heart disease, which showed an
increase in the odds ratio for ischemic heart disease in women
who also had verified stenosis on coronary
angiography.4
As expected, hypertension and diabetes mellitus seemed to be the most important factors in the risk of ICVD.9 However, the models explaining ICVD risk in ICVD plus carotid stenosis cases and Copenhagen City Heart Study cases differed. In women, triglyceride levels were only important in the model for Copenhagen City Heart Study cases. This may be due to the fact that plasma triglyceride levels were measured in the fasting state in cases with ICVD plus carotid stenosis but in the nonfasting state in Copenhagen City Heart Study cases4 ; the results could therefore have underestimated the effect of plasma triglyceride levels on risk of ICVD and may help explain why a triglyceride-modulating mutation would still be associated with an increased risk of ICVD, despite the seemingly small-to-no difference between hospital-based ICVD cases and controls.
It is important to note, however, that in the present study, the risk association with genotype was unaffected by whether triglyceride levels were measured in the fasting or nonfasting state. The apparent protective effect in cases with ICVD plus carotid stenosis for subjects with a body mass index in the highest tertile may reflect the fact that stroke patients in a hospital environment generally have a lower body mass index, due to more advanced disease, than nonhospitalized individuals10 and that body mass index in this setting would be inversely related to severity of disease.
Finally, female smokers seem to have an increased ICVD risk, whereas
male smokers among cases with ICVD plus carotid stenosis
paradoxically seem to be protected; 89% of men in the general
population sample and 81% of men among patients with ICVD and 50%
carotid stenosis were current or former smokers. One
explanation for this finding could be an underreporting of smoking
among ICVD plus carotid stenosis cases. It is more likely,
however, that smoking among Danish men is so prevalent that the
importance of smoking as a risk factor for ICVD is difficult to
determine. Importantly, it would be erroneous to conclude that smoking
is a protective factor in men.
The misclassification of some ICVD cases cannot be entirely ruled
out; however, the patients with ICVD and 50% stenosis of the
relevant carotid artery were all first evaluated by a neurologist and
later by an experienced vascular surgeon, and participants in the
Copenhagen City Heart Study who had ICVD were all diagnosed by an
experienced neurologist who reviewed all hospital admissions and
diagnoses; in both cases, the evaluators were unaware of the results of
genotyping. Furthermore, confounding by another mutation in linkage
disequilibrium with the mutation causing the Asn291Ser substitution is
a possible, although unlikely, explanation of our results. In vivo
evidence suggests that this substitution reduces the activity of
lipoprotein lipase by 40%,6 which is sufficient to cause
the effects observed. Despite this, we cannot completely rule out the
possibility that some of our findings may be chance observations.
The differences in the effects of the Asn291Ser substitution that were observed among women and men are not easily explained. Possible explanations may be found in the differences in the hormonal regulation of metabolic processes in women and men. In men, low levels of testosterone are associated with low levels of lipoprotein lipase activity11 and, in a group of hypogonadal males, testosterone substitution increased the activity of lipoprotein lipase.12 In contrast, estrogen decreases lipoprotein lipase activity,13 possibly through a posttranscriptional modification of protein levels.14 Raised plasma triglyceride levels seem to be a better predictor of ischemic cardiovascular disease in women than men,1 which suggests that any genetic factor raising plasma triglyceride levels would also have a greater effect on the development of disease in women than in men. The consistency of the sex-specific effect of the Asn291Ser substitution on plasma lipid levels, the risk of ischemic heart disease,4 the risk of ICVD (as shown in the present study), and the strength of these associations suggest that these are not chance findings.
In conclusion, the Asn291Ser substitution in lipoprotein lipase, which
is present in 5% of Danes, is not associated with an increased
risk of ICVD in men, but it is possibly associated with a 2-fold risk
in women.
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Acknowledgments |
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Supported by The Danish Heart Foundation, the Danish Medical Research Council, and Chief Physician Johan Boserup and Lise Boserup’s Fund. We are grateful for the technical assistance of Pia T. Petersen and Anne-Merete Bengtsen.
Received August 30, 1999; revision received November 23, 1999; accepted December 22, 1999.
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