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(Circulation. 1997;95:2628-2635.)
© 1997 American Heart Association, Inc.

Articles

Genetic Variant Showing a Positive Interaction With ß-Blocking Agents With a Beneficial Influence on Lipoprotein Lipase Activity, HDL Cholesterol, and Triglyceride Levels in Coronary Artery Disease Patients

The Ser⁴⁴⁷-Stop Substitution in the Lipoprotein Lipase Gene

Björn E. Groenemeijer, MD; Michael D. Hallman, PhD; Paul W.A. Reymer, BSc; Eric Gagné, BSc; Jan Albert Kuivenhoven, MSc; Taco Bruin, PhD; Hans Jansen, PhD; Kong I. Lie, PhD; Albert V.G. Bruschke, PhD; Eric Boerwinkle, PhD; Michael R. Hayden, PhD; John J.P. Kastelein, PhD; on behalf of the REGRESS Study Group

the Department of Vascular Medicine (B.E.G., P.W.A.R., J.A.K., T.B., J.J.P.K.) and Department of Cardiology (K.I.L.), Academic Medical Center, Amsterdam, Netherlands; the Human Genetics Center (M.D.H., E.B.), University of Texas Health Science Center, Houston; the Department of Medical Genetics (E.G., M.R.H.), University of British Columbia, Vancouver, Canada; the Department of Biochemistry (H.J.), University of Rotterdam, Netherlands; and the Department of Cardiology (A.V.G.B.), University Hospital, Leiden, Netherlands.

Correspondence to John J.P. Kastelein, MD, PhD, Department of Vascular Medicine (G1-114), Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail lansberg{at}uva.amc.nl

	Abstract

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Background Lipoprotein lipase (LPL) is the rate-limiting enzyme in the lipolysis of triglyceride-rich lipoproteins, and the gene coding for LPL is therefore a candidate gene in atherogenesis. We previously demonstrated that two amino acid substitutions in LPL, the Asn²⁹¹-Ser and the Asp⁹-Asn, are associated with elevated triglycerides and lower HDL cholesterol and are present with greater frequency in coronary artery disease (CAD) patients than in normolipidemic control subjects. Conversely, a third frequent mutation in this gene, the Ser⁴⁴⁷-Stop, is reported by some investigators to underlie higher HDL cholesterol levels and would represent a beneficial genetic variant in lipoprotein metabolism. We therefore sought conclusive evidence for these allegations by investigating the effects of the LPL Ser⁴⁴⁷-Stop mutation on LPL and hepatic lipase (HL) activity, HDL cholesterol, and triglycerides in a large group of CAD patients (n=820) with normal to mildly elevated total and LDL cholesterol levels.

Methods and Results Carriers of the Ser⁴⁴⁷-Stop allele (heterozygotes and homozygotes) had significantly higher postheparin LPL activity (P=.034), normal postheparin HL activity (P=.453), higher HDL cholesterol levels (P=.013), and lower triglyceride levels (P=.044) than noncarriers. The influence of the Ser⁴⁴⁷-Stop allele on LPL activity was pronounced in patients using ß-blockers (P=.042) and not significant in those not using them (P=.881), suggesting a gene-environment interaction between the Ser⁴⁴⁷-Stop mutation and ß-blockers.

Conclusions We conclude that the LPL Ser⁴⁴⁷-Stop mutation has a significant positive effect on LPL activity and HDL cholesterol and triglyceride levels and that certain subgroups of CAD patients carrying the Ser⁴⁴⁷-Stop mutation will have less adverse metabolic effects when placed on ß-blockers. The LPL Ser⁴⁴⁷-Stop mutation therefore should have a protective effect against the development of atherosclerosis and subsequent CAD.

Key Words: genetics • lipoproteins • coronary disease

	Introduction

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Coronary artery disease is the main cause of death in most industrialized nations. In the vast majority of cases, atherosclerosis, a process influenced by both environmental and genetic factors, underlies the development of CAD. Disorders of lipoprotein metabolism such as elevated LDL cholesterol and low HDL cholesterol are considered important risk factors in the pathogenesis of atherosclerosis.^{1 2}

LPL is the rate-limiting enzyme in the lipolysis of plasma triglyceride-rich lipoproteins and is bound to glycosaminoglycans on the surface of the endothelium in muscle and adipose tissue. The hydrolytic function of LPL is essential for the processing of triglyceride-rich chylomicrons and VLDL to remnant particles and for the transfer of phospholipids and apolipoproteins to HDL.^{3 4} Furthermore, LPL plays a key role in the removal of lipoproteins from the circulation.⁵

A number of polymorphisms in the LPL gene^{6 7 8 9 10} have been identified and reported to underlie variation in plasma lipoprotein levels or to be associated with severity of CAD. For example, the (+) allele of the HindIII polymorphism at the LPL locus was found to be associated predominantly with elevated triglyceride levels and to a varying extent with low HDL cholesterol levels,⁹ and the (+) allele of the Pvu II polymorphism was significantly associated with CAD severity and type II diabetes in CAD patients.¹⁰ Moreover, many structural defects in this gene have been identified at the molecular level,¹¹ and the heterozygous state of LPL deficiency has been associated with low HDL cholesterol levels^{12 13} and with recognized lipid disorders such as familial combined hyperlipidemia^{14 15 16} and familial dysbetalipoproteinemia.¹⁷

We recently demonstrated that 10% of our CAD patients were heterozygous for either the LPL Asp⁹-Asn (unpublished data, 1996) or the LPL Asn²⁹¹-Ser¹⁸ mutation. These mutations were associated with significantly lower postheparin LPL activity, higher triglyceride levels, and lower HDL cholesterol than in normolipidemic matched control subjects. We subsequently sought to determine the frequency, lipid phenotype, and LPL activity associated with a third common mutation in the LPL gene, which was first described by Hata et al,¹⁹ ie, the C-to-G substitution (Ser⁴⁴⁷Stop) in the LPL gene, resulting in a truncation of the C-terminal end of the LPL protein by two amino acids. This mutation was reported to be present at a lower frequency in hypertriglyceridemic subjects, and it was suggested that this mutation might be protective against hypertriglyceridemia.²⁰ However, no significant effects of the Ser⁴⁴⁷-Stop mutation on HDL cholesterol levels could be demonstrated. Other investigators did not identify associations of the Ser⁴⁴⁷-Stop mutation with plasma lipid levels, concluding that it is a neutral polymorphism.²¹

With regard to this mutation, no studies have been reported to date in any substantial sample of subjects having angiographically defined CAD. We report here the effects of the Ser⁴⁴⁷-Stop mutation on LPL and HL activity, plasma lipid levels, and the interaction with ß-blocking agents in a large group of male patients with angiographically proven coronary atherosclerosis.

	Methods

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Subjects
The subjects included in this study have been described in detail elsewhere.²² Briefly, a total of 820 male CAD subjects taking part in the placebo-controlled Regression Growth Evaluation Statin Study (REGRESS) were genotyped for the Ser⁴⁴⁷-Stop mutation in the LPL gene. After a washout period of 6 weeks for bile acid sequestrants and 12 weeks for HMG-CoA reductase inhibitors, patients who were symptomatic for coronary heart disease were included and treated with the HMG-CoA reductase inhibitor pravastatin or placebo. Lipoprotein levels and postheparin LPL and HL activity were determined at the start of the study. Each subject had to have at least one coronary artery with a stenosis of >50% and a qualifying baseline blood total cholesterol level of 4 to 8 mmol/L (155 to 310 mg/dL) and triglycerides <4 mmol/L (<350 mg/dL). Patients >70 years old at entry and those who were unable or refused to undergo a repeat coronary arteriography, as well as patients with obvious life-threatening illness, were excluded from the study. The REGRESS study was conducted under the auspices of the Interuniversity Cardiology Institute of the Netherlands.

Plasma Lipoprotein and Apolipoprotein Determination
Blood samples were collected at baseline from patients after an overnight fast. Total serum cholesterol, HDL cholesterol, and triglycerides were measured by standard techniques. Total cholesterol was determined with an enzymatic kit (Boehringer Mannheim) and calibrated with a serum calibrator. HDL cholesterol was measured after precipitation of apo B–containing lipoproteins with a 4% tungstate solution and centrifugation.²³ Triglycerides were analyzed enzymatically (Bayer/Technicon).²⁴ LDL cholesterol was calculated by use of the Friedewald formula.²⁵

LPL and HL Activity Measurements
At baseline, LPL and HL activities were determined in postheparin plasma after an overnight fast. Twenty minutes after an injection of heparin (50 IU/kg body wt IV), blood was taken from the other arm and collected in heparin-containing tubes. Plasma was separated by centrifugation of the samples at 3000 rpm for 10 minutes at 4°C and stored at -70°C. LPL and HL activities were determined after separation by an immunochemical method.²⁶ HL activity was determined as the salt-resistant lipase in the presence of 1 mol/L NaCl. LPL activity was determined after inhibition of HL with a goat antibody raised against HL purified from postheparin human plasma. For correction of the extraction of fatty acids, [¹⁴C³H]oleate was added to the substrate. To reduce interassay variability, pooled plasmas with high and low LPL and HL were included as a reference in each series of determinations.

PCR-Based Detection of the Ser⁴⁴⁷-Stop Mutation
DNA was extracted from leukocytes by a standard procedure and stored at 4°C. Oligonucleotides located in introns 8 and 9 of the LPL gene were used to amplify the region of interest (5' primer, 5'-TACACTAGCAATGTCTAGGTGA-3'; 3' primer, 5'-TCAGCTTTAGCCCAGAATGC-3'). PCR reactions were carried out in a 96-well programmable thermal controller apparatus (PTC-100-96) from MJ Research. Each amplification reaction contained 0.1 µg genomic DNA, 40 pmol of each primer, BSA, and 25 pmol dNTPs (Pharmacia) in a total volume of 50 µL. The reaction mixture was overlaid with 30 µL mineral oil and heated at 90°C for 5 minutes. Subsequently, 0.4 U Taq polymerase (Supertaq, Sphaero Q) was added along with 5 mL 10x buffer solution. The amplification strategy was denaturation for 1 minute at 94°C, annealing for 30 seconds at 60°C, and extension for 30 seconds at 72°C (n=30).

The LPL Ser⁴⁴⁷-Stop mutation can be detected by cutting the PCR product with the restriction endonuclease Mnl I. The PCR product of 488 bp contains two Mnl I restriction sites, of which one is polymorphic,²⁷ which reveals the Ser⁴⁴⁷-Stop mutation. Digestion of the PCR product with 10 U Mnl I results in three fragments of 290, 250, and 200 bp, respectively. Analysis was performed on 2% agarose gels stained with ethidium bromide. By chance, digestion of the same PCR product with the restriction enzyme BstNI revealed the localization of the BstNI polymorphism⁸ to be in intron 8. Digestion with BstNI and electrophoresis on 3% agarose showed the following polymorphic bands: 488, 320, and 170 bp (see Fig 1).

View larger version (64K): [in this window] [in a new window]   Figure 1. Lane 1, Undigested PCR product (488 bp). Lanes 2, 6, and 10, DNA molecular weight marker, a combination of -DNA cut with HindIII and fX-174-RF DNA cut with HaeIII. Lanes 3, 4, and 5, PCR product digested with Mnl I; lanes 7, 8, and 9, PCR product digested with BstNI. Homozygosity for absence (lanes 3 and 7), heterozygosity (lanes 4 and 8), and homozygosity for presence (lanes 5 and 9) of restriction site are shown.

Statistical Analysis
Wherever possible, both parametric and nonparametric analyses were conducted. In tests of differences among means, probability values are from the nonparametric Kruskal-Wallis test unless otherwise noted. Analyses were carried out both on unadjusted variables and on variables adjusted for the effects of age, (age²), BMI, (BMI²), alcohol use (yes [1 to 3 drinks per day]/no), and smoking (present/former/never) without materially affecting our conclusions. Results for the unadjusted values are reported here.

	Results

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Baseline characteristics of the CAD patients are shown in Table 1. The mean age was 56.1±8.1 years; mean BMI was 26.0±2.7 kg/m². Nearly half of the patients (48.4%) had a family history of CAD, most (87.8%) were either current or former smokers, and most (72.1%) drank alcohol at least occasionally. The majority (71.8%) were being treated with ß-adrenergic blocking agents. The mean total cholesterol level for the full sample was 6.04±0.86 mmol/L.

View this table: [in this window] [in a new window]   Table 1.

The distribution of Ser⁴⁴⁷-Stop genotypes in the sample is shown in Table 2. In this sample, 149 subjects (18.37%) carried at least one copy of the Ser⁴⁴⁷-Stop allele; only 6 (0.74%) were homozygous for this allele. The observed genotype frequencies did not differ from those expected under Hardy-Weinberg equilibrium (²=0.33, 1 df, P=.566). The frequencies of the wild-type and the Ser⁴⁴⁷-Stop alleles were estimated to be 0.904 and 0.096, respectively. No significant differences in age, BMI, blood pressure, alcohol use, or smoking habits were found between carriers and noncarriers of the Ser⁴⁴⁷-Stop mutation (Table 3). However, carriers showed significantly higher levels of HDL cholesterol (0.96 versus 0.92 mmol/L, P=.007) and lower levels of triglycerides (1.67 versus 1.81 mmol/L, P=.044). HDL cholesterol levels also differed among genotypes (P=.013), with subjects homozygous for the presence of the Ser⁴⁴⁷-Stop mutation having the highest values (1.08±0.23 mmol/L) (data not shown) and subjects homozygous for absence of the Ser⁴⁴⁷-Stop mutation the lowest (0.92±0.23 mmol/L); the mean heterozygote value was 0.96±0.20 mmol/L. As shown in Fig 2, carrier frequency was significantly higher in higher quartiles of HDL cholesterol (P=.006).

View this table: [in this window] [in a new window]   Table 2.

View this table: [in this window] [in a new window]   Table 3.

View larger version (10K): [in this window] [in a new window]   Figure 2. Percentage of LPL447-Stop carriers in each HDL cholesterol quartile. Overall 2=12.388, 3 df, P=.006; highest vs lowest quartile, 2=12.268, 1 df, P<.001.

Subjects carrying the Ser⁴⁴⁷-Stop mutation showed significantly higher LPL (P=.034) but normal HL (P=.453) activity levels compared with those of noncarriers (Table 4). Average LPL activity in subjects with the Ser⁴⁴⁷-Stop allele was 12.0 nmol free fatty acid·min^-1·mL^-1 higher than in the noncarrier groups. Fig 3 shows carrier frequency by quartiles of LPL activity. Although the overall ² value of 4.698 was not significant (P=.195, 3 df), there was a significant linear trend toward higher carrier frequency in higher quartiles of LPL activity (²=4.539, 1 df, P=.033).

View this table: [in this window] [in a new window]   Table 4.

View larger version (10K): [in this window] [in a new window]   Figure 3. Percentage of LPL447-Stop carriers in each quartile of LPL activity. Overall 2=4.698, 3 df, P=.195; highest vs lowest quartile: 2=3.921, 1 df, P=.048.

Because of the previously noted effect of the Ser⁴⁴⁷-Stop mutation on HDL cholesterol concentrations and the accepted relationship of HDL cholesterol with both LPL activity and ß-blocker use, we carried out similar analyses to determine whether the effect of the Ser⁴⁴⁷-Stop mutation was modified by the use of ß-blockers. The results are shown in Fig 4. Frequencies of the Ser⁴⁴⁷-Stop mutation between ß-blocker users and nonusers did not differ significantly (²=1.398, 2 df, P=.497). By itself, ß-blocker use significantly affected HDL cholesterol (P<.01), LDL cholesterol (P=.05), and triglyceride (P<.01) levels. In subjects taking ß-blockers, the frequency of individuals carrying a Ser⁴⁴⁷-Stop mutant allele was significantly different among quartiles of the HDL cholesterol distribution (P=.003). The frequency of individuals in the lowest quartile carrying this mutation was 0.095; in the highest quartile, this number was 0.26. In those not taking ß-blockers, the frequency of individuals carrying a Ser⁴⁴⁷-Stop mutant allele was not significantly different among quartiles of the HDL distribution (P=.517). Results were similar for LPL activity. As shown in Fig 5, the association of the Ser⁴⁴⁷-Stop mutation with LPL activity differed according to ß-blocker use. Among those using ß-blockers, fewer individuals in the lowest quartile of LPL activity carried the mutation than those in the highest quartile (P=.006), whereas there was no significant difference in carrier frequency among quartiles in those not using ß-blockers (P=.530). The carrier frequency patterns across quartiles in Fig 5 strongly suggest interaction between ß-blocker use and the Ser⁴⁴⁷-Stop mutation. When a two-way ANOVA was carried out to determine whether ß-blocker use modified the effect of the Ser⁴⁴⁷-Stop mutation, the interaction term was statistically significant (P=.026). Table 5 shows mean LPL and HL activity and HDL cholesterol levels among those who used ß-blockers and those who did not, with dichotomized genotypes. The probability values are from the Kruskal-Wallis test; differences are the same when t tests are used. Another way of expressing the effects of ß-blocker use is shown in Table 6, which gives the odds that a subject in the lowest quartile of HDL cholesterol level or LPL activity does not carry the Ser⁴⁴⁷-Stop mutation relative to the odds for subjects in all higher quartiles. Only among those using ß-blockers is a subject with higher LPL activity or higher HDL cholesterol levels more likely to carry the Ser⁴⁴⁷-Stop mutation.

View larger version (20K): [in this window] [in a new window]   Figure 4. Percentage of LPL447-Stop carriers in each HDL cholesterol quartile by ß-blocker use. ß-Blockers: overall 2=14.036, 3 df, P=.003; highest vs lowest quartile: 2=13.494, 1 df, P<.001. No ß-blockers: overall 2=2.276, 3 df, P=.517; highest vs lowest quartile: 2=0.632, 1 df, P=.427.

View larger version (21K): [in this window] [in a new window]   Figure 5. Percentage of LPL447-Stop carriers in each quartile of LPL activity by ß-blocker use. ß-Blockers: overall 2=8.177, 3 df, P=.042; highest vs lowest quartile: 2=7.562, 1 df, P=.006. No ß-blockers: overall 2=0.667, 3 df, P=.881; highest vs lowest quartile: 2=0.395, 1 df, P=.530.

View this table: [in this window] [in a new window]   Table 5.

View this table: [in this window] [in a new window]   Table 6.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
A number of candidate genes potentially involved in the development of CAD, such as the apo B-100 gene, the apo A-I–C-III–A-IV gene cluster, and the gene coding for apo E, have been studied extensively, but results concerning variation in these potential risk modulators on a population basis have been disappointing. One of the most appealing candidate genes that might explain at least part of the lipid and lipoprotein abnormalities seen in many CAD patients is the LPL gene. A functional mutation in this gene can result in lower LPL activity in plasma, thus affecting the catabolism of triglyceride-rich lipoproteins, the transfer of phospholipids and apolipoproteins to HDL,^{3 4} and the removal of lipoproteins from the circulation.⁵ The diminished clearance of these triglyceride-rich lipoproteins has been shown to induce higher triglycerides and lower HDL cholesterol levels in plasma.^{14 28} Large regression trials, such as the Cholesterol Lowering Atherosclerosis Study and the Monitoring on Atherosclerosis Regression Study, demonstrated that the apo C-III moiety of HDL particles, as a marker of inefficient lipolysis of triglyceride-rich lipoproteins, is one of the most powerful predictors for the progression of coronary atherosclerosis when LDL cholesterol levels are reduced.

The lipid abnormality most frequently found in patients with premature CAD consists of decreased HDL cholesterol levels.²⁹ We recently found two common mutations in the LPL gene, the Asp⁹-Asn and the Asn²⁹¹-Ser mutations, with the Asn²⁹¹-Ser being present at an increased frequency in a large group of male patients with familial combined hyperlipidemia.³⁰ We also established that those same mutations together were present in 10% of our sample of male CAD patients (5.2% Asn²⁹¹-Ser and 4.8% Asp⁹-Asn), with the Asp⁹-Asn being found at a significantly higher frequency than in the control group (4.8% in CAD patients versus 1.8% in control subjects) (unpublished data, 1996).

Both LPL mutations were associated with low HDL cholesterol and were more frequent among CAD patients with high triglyceride levels. We now report on a third frequently occurring mutation in the LPL gene. The allelic frequency of this mutation, the Ser⁴⁴⁷-Stop, is 0.096 in our cohort of CAD patients and is associated with a >10% increase in mean LPL activity, higher HDL cholesterol, and lower triglyceride levels.

Conflicting results have been published with regard to the effects of the Ser⁴⁴⁷-Stop mutation on LPL activity and lipoprotein levels, and its status as a functional mutation or neutral polymorphism in the LPL gene is still questioned. Explanations for these opposing results could lie in the fact that in most studies, patients and/or control populations were not well defined, LPL activity measurement was not standardized, or patient and control groups were not large enough to detect small but significant differences.

We recently showed, however, that in carefully selected and matched groups of healthy individuals with low (0.87 mmol/L), normal (1.09 to 1.15 mmol/L), and high (1.44 mmol/L) HDL cholesterol levels, the frequency of the Ser⁴⁴⁷-Stop allele increases strongly from individuals with the lowest to those with the highest HDL cholesterol levels.³¹

LPL Activity
The functionality of the Ser⁴⁴⁷-Stop mutation has also been assessed by in vitro expression studies by various groups. As in human studies, conflicting results have been reported. Some groups reported a similar³² or even lower³³ specific LPL activity for this truncated protein compared with wild-type LPL, whereas others assigned an almost double lipolytic activity for the Ser⁴⁴⁷-Stop mutation.³⁴

Until now, other groups have determined the effects of the Ser⁴⁴⁷-Stop mutation on LPL activity mainly by in vitro testing; in vivo studies have been conducted only in small groups of individuals. Studying a large cohort of CAD patients, we found that carriers who were either heterozygous or homozygous for presence of the Ser⁴⁴⁷-Stop mutation have an 10% higher mean LPL activity than patients who do not carry this premature stop codon. This difference was also found, although to a much greater extent, by Kozaki et al,³⁴ who found an almost twice as high overall and specific lipolytic activity of the truncated enzyme in a well-designed in vitro expression study. Conversely, Peacock et al²¹ found a trend, although not significant, toward lower LPL activity in a small group of carriers of the Ser⁴⁴⁷-Stop mutation. However, the low frequency of this genetic variation in the Swedish population and the consequently few individuals that were left for comparison could explain the negative results in this study. Kobayashi et al³³ reported normal LPL activity for the Ser⁴⁴⁷-Stop mutation in one patient with type I hyperlipidemia in which the Ser⁴⁴⁷-Stop was transfected and expressed in Cos-1 cells but detected lower specific activity (43% of wild-type LPL) with other substrates used, suggesting that this mutation could influence the lipid interface recognition of LPL with VLDL or chylomicrons. These conclusions were not supported by Faustinella et al,³² who found normal enzyme activity for the Ser⁴⁴⁷-Stop mutant and stated that this is a sequence polymorphism of no functional significance. The inconsistency of these in vitro findings might be due to methodological differences between laboratories and the fact that in vitro assessment of LPL expression and function appears not to mimic the in vivo situation completely.

The functional role of the C-terminal end of LPL has yet not been fully elucidated. Endothelial binding, interaction with lipoproteins, and modification of binding to triglyceride-rich lipoproteins like chylomicrons or to the LDL-related protein^{5 35} have been proposed, but no definite proof for any of these hypotheses has been provided to date.

HDL Cholesterol
With regard to lipid and lipoprotein levels, we have shown that within a large CAD population, Ser⁴⁴⁷-Stop allele carriers exhibit significantly higher HDL cholesterol levels than noncarriers. When our CAD sample is subdivided into quartiles, the presence of the Ser⁴⁴⁷-Stop mutation increases significantly from those in the lowest to those in the highest quartile of HDL cholesterol, pointing toward a positive association of this premature stop codon with levels of HDL cholesterol and thus subsequently with the development of CAD.

ß-Blocker Use
The data show that there is an intriguing interaction between the Ser⁴⁴⁷-Stop mutation and the use of ß-blockers. We provide evidence for a significant interaction between ß-blockers and the Ser⁴⁴⁷-Stop mutation. An explanation for this effect cannot be given easily. Kihara et al³⁶ stated that different types of ß-blockers have different effects on human LPL in vitro, with propranolol showing the strongest and metoprolol a moderate inhibitory effect, and that nadolol at low concentrations may stimulate LPL activity. In other studies,^{37 38} metoprolol was found to have no significant effect on postheparin LPL activity. Day et al³⁹ found slower fractional triglyceride removal, with higher total and LDL cholesterol and lower HDL cholesterol in subjects after 3 months of ß-blocker use, suggesting LPL inhibition. The mechanism by which ß-blockers inhibit LPL is not yet clarified. Kubo and Hostetler⁴⁰ reported noncompetitive inhibition by ß-blockers of bovine LPL in vitro. Kihara et al³⁶ found similar effects for human LPL but at higher ß-blocker doses than required for bovine LPL. These in vitro studies used nonpharmacological doses of ß-blockers, but Kubo and Hostetler cite evidence that lipophilic ß-blockers, such as propranolol, may bind phospholipids on lipoproteins and on cell surface membranes to produce much higher effective concentrations of LPL activity than plasma activity would indicate. If so, assays of LPL activity in postheparin plasma may not be optimal for detecting ß-blocker effects. Since we assayed postheparin plasma as well, several factors, such as our large sample, the disease state of our subjects, and possibly differences in the duration of ß-blocker use and the types and dosages used, may be involved in the fact that in our study, ß-blocker use influenced LPL activity.

The effects of high doses of ß-blockers in inhibiting LPL activity in vitro cannot be due to altered LPL gene transcription and/or translation of LPL mRNA,^{41 42} but there could be an effect at the protein level. ß-Blockers tend to reduce HDL cholesterol and raise triglycerides. In Ser⁴⁴⁷-Stop allele carriers, this effect on HDL cholesterol may be partly inhibited and ß-blockers may be less effective in lowering the activity of the mutated LPL variant. In our subjects, it appears that Ser⁴⁴⁷-Stop carriers escape the negative effects of ß-blocker use. If this is correct, it is possible that either the loss of the two C-terminal amino acids of LPL interferes with the interactions of ß-blockers and LPL at the endothelial surface, possibly through altered binding, or that another mutation in disequilibrium with the Ser⁴⁴⁷-Stop allele results in such interference. Our findings, therefore, require confirmation in future studies.

Triglycerides
Triglyceride levels were significantly lower in carriers of the Ser⁴⁴⁷-Stop mutation than in noncarriers of this mutation. Generally, when triglyceride levels decrease, HDL cholesterol levels increase, reflecting the enhanced clearance of triglyceride-rich particles from the circulation and the increased synthesis of HDL particles, which would be beneficial. In our study, the effects on triglyceride levels are weaker than the favorable effects on HDL cholesterol, for which we have no explanation. The effects of the Ser⁴⁴⁷-Stop mutation on triglyceride levels should therefore preferably be investigated in a large sample of hypertriglyceridemic individuals and appropriate control subjects.

In summary, we identified a large number of carriers of the Ser⁴⁴⁷-Stop mutation in male CAD patients. Carriers of this mutation show significantly higher LPL activity and HDL cholesterol and lower triglyceride levels than noncarriers and a positive interaction with ß-blockers, suggesting a protective role of this mutation against CAD, especially in those using ß-blocking agents.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein BMI = body mass index CAD = coronary artery disease HL = hepatic lipase HMG-CoA = 3-hydroxy-3-methylglutaryl coenzyme A LPL = lipoprotein lipase PCR = polymerase chain reaction

	Acknowledgments

 
Dr Hayden is an Established Investigator of the British Columbia Children's Hospital and an investigator of the Canadian Genetic Disease Network. Dr Kastelein is a clinical investigator of the Dutch Heart Foundation. The REGRESS study was sponsored by Bristol-Myers Squibb Co, Princeton, NJ. The Lipid Reference Laboratory is an international member of the US National Cholesterol Reference Method Laboratory Network chaired by the Centers for Disease Control and Prevention (Atlanta, Ga).^{43 44} We are grateful to all the cardiologists and coworkers who made plasma and DNA samples from patients available to us.

	Footnotes

 
Guest editor for this article was Garret A. FitzGerald, MD, University of Pennsylvania, Philadelphia.

Received June 19, 1996; revision received December 4, 1996; accepted January 2, 1997.

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