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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:1723-1729.)
© 1998 American Heart Association, Inc.

Original Contributions

Common Variants in the Promoter of the Hepatic Lipase Gene Are Associated With Lower Levels of Hepatic Lipase Activity, Buoyant LDL, and Higher HDL₂ Cholesterol

Alberto Zambon; Samir S. Deeb; John E. Hokanson; B. Greg Brown; ; John D. Brunzell

From the Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (A.Z., J.E.H., J.D.B.), the Division of Medical Genetics, Department of Genetics (S.S.D.), and the Division of Cardiology, Department of Medicine (B.G.B.), University of Washington, Seattle.

Correspondence to Dr John D. Brunzell, University of Washington, Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, 1959 NE Pacific St, Box 356178, Seattle, WA 98195-6178.

	Abstract

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Abstract—Increased hepatic lipase (HL) activity is associated with small, dense, low density lipoprotein (LDL) and low high density lipoprotein₂ (HDL₂) cholesterol (-C) levels. A polymorphism in the promoter region of the HL gene (LIPC) is associated with HDL-C levels. To test whether this association is mediated by differences in HL activity between different LIPC promoter genotypes, the LIPC promoter polymorphism at position -250 (GA), HL activity, LDL buoyancy, and HDL-C levels were studied in white normolipidemic men and men with coronary artery disease (CAD). The less common A allele (frequency=0.21 and 0.25 in normal and CAD subjects, respectively) was associated with lower HL activity (P<0.005 by ANOVA) and buoyant LDL particles (P0.01) in both groups. Normal and CAD subjects heterozygous for the A allele had lower HL activity (by 24% and 29%, respectively) and significantly more buoyant LDL particles. Homozygosity for this allele (AA) was associated with an even lower HL activity in normal (-26%) and CAD (-46%) subjects. The A allele was associated with higher HDL₂-C in CAD patients (P=0.007); heterozygotes and homozygotes for the A allele had a 92% and a 140% higher HDL₂-C level (P<0.01) than did GG individuals. In a small number of normolipidemic subjects, the same trend in HDL₂-C was seen. In a univariate analysis, the LIPC genotype accounted for 20% to 32% of the variance in HL levels among normal subjects and CAD patients, respectively. After adjustment for HL, the association between LIPC genotype and LDL buoyancy was no longer significant, suggesting that the effect of LIPC genotype on LDL buoyancy is mediated by its effects on HL activity. The LIPC A allele was more frequent in Japanese-Americans and African-Americans than in whites. In summary, these results suggest that variants in the LIPC promoter may significantly contribute to the variance in levels of HL activity and consequently, to the prevalence of the atherogenic small, dense, LDL particles and low HDL₂-C levels.

Key Words: LDL • HDL • triglycerides • cardiovascular disease • lipoprotein lipase

	Introduction

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Hepatic lipase (HL) is a glycoprotein that catalyzes the hydrolysis of triacylglycerols and phospholipids.^{1 2 3} Hepatic lipase is synthesized and secreted by the liver and is bound to the surface of sinusoidal endothelial cells and the external surfaces of microvilli of parenchymal cells in the space of Disse.⁴ The human HL gene (LIPC), located on chromosome 15q21, comprises 9 exons and 8 introns, spans >30 kb of DNA, and encodes a protein of 449 amino acids, with a signal peptide of 23 amino acids.^{5 6}

HL has emerged as a key player in the metabolism of both LDL and HDL. Plasma HDL cholesterol (HDL-C) levels are inversely correlated with HL activity⁷ ; specifically, HL promotes the conversion of large, buoyant HDL₂ to small, dense HDL₃ by modulating the phospholipid content of these particles.^{8 9} Epidemiological studies in humans have indicated that a low level of plasma HDL-C is one of the major risk factors for coronary artery disease (CAD).^{10 11} The plasma HDL concentration is modulated by environmental factors such as obesity,¹² cigarette smoking,¹³ and a sedentary lifestyle.¹⁴ A strong contribution by genetic factors has also been suggested by family^{15 16} and twin^{17 18} studies. It has been estimated that between 40% and 60% of the interindividual variation in HDL-C levels is accounted for by genetic variability.¹⁷ These studies have generated intense interest in identifying specific genetic polymorphisms that may influence HDL-C levels. The first evidence for involvement of the LIPC locus in influencing HDL-C levels was provided by Cohen et al.¹⁹ Their results suggested that in normolipidemic subjects, allelic variation at the LIPC locus accounted for 25% of the interindividual variation in plasma HDL-C levels. These findings were subsequently confirmed by the same group in a much larger population.²⁰ In addition, they observed 4 polymorphism in the 5'-flanking region of LIPC: GA at position -250, CT at -514, TC at -710, and AG at -763, with respect to the transcription start site.²⁰ These 4 polymorphisms were observed to be in complete linkage disequilibrium in the white population studied and in a subsequent African-American population²¹ and were defined a single haplotype. Association studies indicated that the presence of the T allele at position -514 was associated with elevated plasma HDL-C and apo A-I levels in men but not in women.²⁰ The authors speculated that the T allele at -514 is either directly or indirectly (in linkage disequilibrium with another mutation) associated with lower HL activity.

Low HL activity is also associated with more buoyant, less atherogenic LDL particles.^{22 23 24 25} HL catalyzes the hydrolysis of phospholipids in the IDL and large LDL to form smaller, denser LDL particles. The prevalence of small, dense LDL is associated with an increased risk of premature CAD^{26 27 28 29 30} and is a common trait in the general population.³¹ Complex segregation analysis indicated that a major gene influences small, dense LDL, with an allele frequency of 0.19 to 0.32.^{32 33} A study by Nishina et al³⁴ has suggested a positive linkage (log of the odds score >4.0) to a locus at or near the LDL receptor on chromosome 19. A recent report from the same group, however, shows no sequence variants in the LDL receptor gene in these families.³⁵ One study suggested multigenic control of LDL size, including the cholesteryl ester transfer protein and manganese superoxide dismutase loci, as well as the LDL receptor locus,³⁶ although the role of these loci in determining LDL size variability remains to be established. Recent evidence has shown that HL activity is an important modulator of LDL density in both normal subjects and patients with CAD.²²

Since the present study was completed, 2 groups reported an association between LIPC and HL activity. The LIPC rare allele was shown to be associated with decreased HL activity and modest elevations in HDL-C levels in Dutch patients with CAD.³⁷ LIPC has also been reported to account for the decrease in HL activity^{21 37} and the elevation in HDL-C levels seen in African-American males.²¹ We confirm these findings in the current study. However, the changes in HDL-C have been demonstrated to be confined to HDL₂-C. Importantly, the rare allele was associated with more buoyant LDL particles.

	Methods

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Study Subjects
Sixty-eight healthy, unrelated white subjects were studied: 40 women and 28 men aged 33±9 years (mean±SD) with no known lipid disorders or other major diseases affecting lipid metabolism and who were not taking any lipid-altering medication, including ß-blockers or estrogens. In addition, 60 unrelated dyslipidemic men with elevated apo B levels, who had originally participated in a clinical intervention trial,³⁸ aged 62 years at entry and with CAD diagnosed by coronary angiography, were also included in the study. Both the normolipidemic³¹ and CAD ³⁸ subjects have been characterized previously. For comparison of ethnic groups, 31 healthy Japanese-Americans and 56 healthy African-Americans were evaluated. When studied, patients with CAD were not taking any lipid-lowering medication and were free from diabetes, liver, thyroid, or kidney disease. Five CAD patients were taking ß-blockers at the time of the study. All subjects read and signed a consent form approved by the institutional review board.

After an overnight fast, blood specimens were collected in 0.1% EDTA for DNA isolation, lipoprotein density gradient distribution, and plasma lipid measurements. An intravenous heparin bolus of 60 IU/kg was then administered, and after 10 minutes, blood was collected into iced lithium-heparin tubes for measurement of HL activity. Blood samples were immediately processed by centrifugation at 4°C and stored at -70°C for later determination of HL activity.²²

Lipid and Lipoprotein Determinations
LDL, HDL, HDL₂, and HDL₃ cholesterol and plasma triglycerides were measured at the Northwest Lipid Research Laboratories as previously described.^{31 38} HDL-C was measured in the supernatant after precipitation of apo B–containing lipoproteins with dextran sulfate.³⁹ A second precipitation with high-molecular-weight dextran sulfate was performed on the supernatant containing HDL to separate the HDL₂ and HDL₃ subspecies.³⁹

Density Gradient Ultracentrifugation
Nonequilibrium density gradient ultracentrifugation was used to study LDL density.⁴⁰ By layering 2 mL of plasma adjusted to a density of 1.080 g/mL (final volume, 5 mL) underneath 12 mL of a 1.006 g/mL NaCl solution, a discontinuous salt gradient was produced in a Sorvall TV-865B vertical rotor (DuPont). Samples were centrifuged at 65 000 rpm for 90 minutes (total t²=2.36x10¹¹) at 5°C; the tubes were then fractionated from the bottom at a flow rate of 1.7 mL/min, and 38 fractions were collected. Total cholesterol was measured in each fraction. The relative flotation rate (Rf), which characterizes LDL peak buoyancy, was obtained by dividing the fraction number containing the LDL-C peak by the total number of fractions collected. The LDL Rf number determined by single vertical-spin density gradient ultracentrifugation was found to be highly reproducible.²²

Postheparin Plasma Lipase Activity
Total lipolytic activity was measured in postheparin plasma as described previously.⁴¹ Substrate containing tri[1-¹⁴C]oleate and lecithin was incubated with aliquots of postheparin plasma for 60 minutes at 37°C, and the liberated free fatty acid radioactivity was extracted and counted. Lipase activity is expressed as nanomoles of fatty acid released per minute per milliliter of plasma nmol · min^-1 · mL^-1. Lipoprotein lipase (LPL) activity was selectively eliminated by incubation with a specific monoclonal antibody (5D2) against LPL. HL activity was defined as the remaining activity detected in the postheparin sample after incubation with the antibody.⁴² For each assay, a bovine milk LPL standard and a human postheparin plasma standard were included to adjust for interassay variation.

DNA Analysis
The genotype at position -250 of the LIPC promoter was determined by polymerase chain reaction amplification with the use of the following primer pairs: forward, 5'-CCTACCCCGACCTTTGGCAG-3'; reverse, 5'-GGGGTCCAGGCTTTCTTGG-3'. Amplification was carried out in a 10-µL reaction with an initial denaturation at 94°C for 2 minutes, followed by 35 cycles of amplification at 92°C for 15 seconds, annealing at 64°C for 30 seconds, and extension at 72°C for 30 seconds, with a final extension at 72°C for 7 minutes. Five microliters of the polymerase chain reaction product was digested with the restriction enzyme DraI, followed by electrophoresis on a 1% agarose gel.

Statistical Analysis
Values presented in the tables and figures are expressed as mean±SEM. Comparisons among groups were performed by ANOVA with Tukey's test for all pairwise comparisons between different LIPC genotypes. In normolipidemic subjects, although HL activity was lower in women than in men, the relationship between LIPC genotype and HL activity was similar between men and women (see Results). Therefore, all quantitative variables (HL activity, LDL-Rf, LDL-C, HDL-C, and triglycerides) were adjusted for sex.⁴³ Values were all adjusted to male levels for comparison with the CAD subjects, who were all men. Significance level was set at P<0.05.

	Results

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Association of LIPC -250 Polymorphism and HL Activity
Subjects in both white populations were divided according to their genotype at position -250 of the LIPC promoter. The observed genotypic frequencies were consistent with Hardy-Weinberg equilibrium in both the normolipidemic group (GG=44, GA=20, and AA=4; ²=0.14, P=0.71) and the CAD group (GG=32, GA=24, and AA=4; ²=0.03, P=0.85). The frequency of the A allele, 0.21 in normolipidemic subjects and 0.25 in CAD subjects, was not different between the 2 groups. The -250 A allele was found to occur at a much higher frequency in unselected, unrelated African-Americans (n=56) and Japanese-Americans (n=31) (Table 1). The 4 promoter variants were in strong, but not complete, linkage disequilibrium in these 2 additional populations. In the white population, complete linkage disequilibrium was found.

View this table: [in this window] [in a new window]   Table 1. LIPC Promoter Varients in Unrelated, Random American Populations

The distribution of HL activity by genotype was not different between normolipidemic and CAD subjects. Therefore, data from the 2 groups were combined (Figure 1). HL activity in both GG and GA genotypes was normally distributed. Mean HL activity was higher in subjects with the GG than in those with the GA or AA genotype: 285±9.5, 207±7.0, and 184±10.6 nmol · min^-1 · mL^-1 respectively, P<0.001 by ANOVA. The same relationship between HL activity and LIPC genotype was noted in the normolipidemic women: 202±19, 116±11, and 131±8 nmol · min^-1 · mL^-1, respectively, P<0.005 by ANOVA. In the whole group, only 2 subjects with the GA genotype had an HL activity value in the 300 to 349 nmol · min^-1 · mL^-1 range, and none was found with an HL value 350 nmol · min^-1 · mL^-1. The range of HL activity within the GG genotype (143 to 538 nmol · min^-1 · mL^-1) was remarkably wider that that of the GA genotype (124 to 364 nmol · min^-1 · mL^-1).

View larger version (18K): [in this window] [in a new window]   Figure 1. Frequency distribution of LIPC -250 polymorphism in normolipidemic and CAD white subjects combined (n=128).

Analysis of the association between polymorphism at the LIPC promoter region and HL activity in the 2 white populations revealed a significantly lower HL activity in carriers of the GA and AA genotypes in both normolipidemic (P=0.005 by ANOVA, Figure 2) and CAD (P<0.001, Figure 3) groups. The GA genotype was associated with a 24% and 29% lower HL activity compared with the GG genotype (P<0.05) in normal and CAD subjects, respectively. In addition, HL activity was even lower in subjects with the AA genotype in both groups (by 26% in normal subjects and by 46% in CAD patients). In a univariate analysis, the polymorphism in the promoter region of LIPC accounted for 20% and 32% of the variance in HL activity among normolipidemic and CAD subjects, respectively.

View larger version (20K): [in this window] [in a new window]   Figure 2. LIPC promoter polymorphism at position -250, HL activity (solid bars), and LDL buoyancy (open bars) in normolipidemic white subjects (n=68).

View larger version (19K): [in this window] [in a new window]   Figure 3. LIPC promoter polymorphism at position -250, HL activity (solid bars), and LDL buoyancy (open bars) in white patients with CAD (n=60).

Association of LIPC -250 Polymorphism and LDL Buoyancy
The polymorphism in the promoter of LIPC was associated with LDL buoyancy. LDL particles were significantly more buoyant in subjects with the GA and AA genotypes in both normolipidemic individuals (P=0.01, Figure 2) and patients with CAD (P=0.01, Figure 3). In multivariate analysis, after adjustment for HL activity levels, the association between LIPC genotype and LDL buoyancy was no longer significant.

Association of LIPC -250 Polymorphism and HDL₂-C Levels
No significant association was observed in either group between the LIPC promoter polymorphism and plasma triglyceride, LDL-C, or HDL-C levels (Table 2), although there was a trend toward higher HDL-C in GA and AA subjects compared with GG individuals in both groups. It is possible that the lack of a significant association between LIPC genotype and HDL-C level is due to the small number of subjects homozygous for the rare allele.

View this table: [in this window] [in a new window]   Table 2. LIPC Promoter Polymorphisms and Plasma Lipid Levels

In subjects with CAD, for whom HDL subclasses were analyzed separately, the LIPC promoter polymorphism was strongly associated with the HDL₂-C level (P=0.007), but not with HDL₃-C (Table 2). Heterozygous and homozygous individuals for the A allele had HDL₂-C levels that were 92% and 140% higher, respectively, than those of GG individuals. In 14 of 68 normolipidemic whites who had HDL subfractions measured, a similar trend was noted. Within each genotype, HDL₂ was lower in the CAD patients compared with those without CAD. In multivariate analysis, after adjustment for HL activity levels, the association between LIPC genotype and HDL₂-C was no longer significant.

	Discussion

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This study demonstrates that the GA substitution at position -250 of the LIPC promoter is associated with lower HL activity, more buoyant LDL particles, and higher HDL₂-C levels, with no effect on HDL₃-C.

Modest associations between LIPC promoter polymorphisms and total HDL-C levels have been detected in a number of population studies.^{19 20 36 37 44} Presumably, these changes in HDL-C are a reflection of changes in HDL₂-C. These findings have led to the hypothesis that LIPC promoter polymorphisms may be associated with lower HL activity values, which are known to be inversely related to HDL-C levels.^{7 8 9} Our study directly addressed this hypothesis and confirmed a strong association between LIPC promoter polymorphism and HL activity in normolipidemic subjects as well as in patients with CAD. Normolipidemic and CAD subjects with the GA genotype at position -250 had 24% and 29% lower HL activity, respectively, compared with homozygous GG individuals. Even lower HL activity (by 26% and 46%, normal subjects and CAD patients) was detected in individuals with the AA genotype. Variability in the LIPC promoter region accounted for 20% and 30% of the variance in HL activity among normal subjects and CAD patients, respectively. In this study population, HL activity was normally distributed among individuals with either the GG or GA genotype (Figure 1). However, the range of HL activity among individuals with the GG genotype (143 to 538 nmol · min^-1 · mL^-1) was wider then that of GA individuals (124 to 364 nmol · min^-1 · mL^-1), indicating a strong negative effect of the A allele on HL expression. Since completion of this study, similar findings were reported in Dutch³⁷ and African-American populations.²¹

In previous studies,²⁰ no functional mutations were detected in the coding region of LIPC that could account for the observed association between polymorphism in the LIPC promoter region and plasma HL activity. This suggests that 1 or more of the promoter variants may actually be responsible for diminished transcription of the gene, as has also been described in a recent preliminary report.⁴⁵ Our results are consistent with this notion. An alternative explanation is that the promoter polymorphism might be in linkage disequilibrium with other variants located either within LIPC itself or within an adjacent gene encoding a protein that regulates LIPC expression. The observation^{19 20 21 44 46} that the same LIPC haplotype was associated with HDL-C or HL activity in different populations makes this a less likely explanation.

Not all studies have detected an association or linkage between HDL-C and markers at the LIPC locus. Apparently conflicting results have been presented by Mahaney et al⁴⁷ in a Mexican-American population from the San Antonio Family Heart Study. Using model-based linkage analysis, these authors found evidence for a major locus effect on plasma HDL-C levels after adjustment for apo A-I levels but excluded the possibility of linkage between the observed major locus and the LIPC locus. A possible explanation for this discrepancy might be that the predominant locus affecting HDL-C levels in Mexican-Americans is different from that in the non-Hispanic white population. An alternative explanation for the apparent discrepancy of these findings may be provided by the results of the current study, showing that only HDL₂-C levels, which represent a fraction of total HDL-C, appear to be strongly associated with LIPC polymorphism, whereas the major HDL₃ component is not. It has been known that HL activity is primarily associated with HDL₂-C and not HDL₃-C.^{48 49 50} This suggests that HDL₂-C levels rather than total HDL-C, which in the current report was not significantly associated with LIPC promoter polymorphism, may represent a more sensitive parameter for testing the effect of LIPC promoter polymorphisms on HDL metabolism. Carriers of the A allele with CAD had 92% to 140% higher HDL₂-C levels compared with the GG individuals, whereas no differences in HDL₃-C were detected. A similar trend was seen in the normolipidemic subjects, though because of the small number of samples in which HDL₂ was measured, the differences were not statistically significant. It is also possible that the relationship between genotype and HDL₂ is true only for CAD patients. The association between LIPC polymorphisms and HDL₂-C levels was mediated by the effects of LIPC polymorphisms on HL activity. These findings and perhaps the study size may account for the different results of the current study compared with previous reports in which total HDL-C levels were measured.^{19 20 21 37}

Epidemiological studies have suggested that both low HDL-C^{10 11} and the presence of small, dense LDL^{26 27 28 29 30} are associated with increased risk of CAD. These lipid abnormalities often coexist in the same subject as part of a multifaceted phenotype referred to as an atherogenic lipoprotein profile.⁵¹ In the current study, the lipid profile characterized by more buoyant LDL and higher HDL₂-C, associated with the presence of the A allele and lower HL activity, appears to bear less atherogenic potential than the lipid profile associated with the GG genotype (small, dense LDL and lower HDL₂-C). In addition, after adjustment for plasma HL levels, the association between LIPC promoter polymorphism and LDL density is no longer significant, suggesting that this association is mediated by the effect of LIPC promoter polymorphism on plasma HL activity. This observation is in agreement with previous studies demonstrating an important role of HL activity as a major player in determining LDL size and density,^{21 22 23 24} and it provides further evidence for genetic regulation of LDL subclass distribution. A number of other factors have also been shown to affect HL activity in association with changes in LDL size and density, HDL₂-C levels, and CAD risk. These include sex,⁵² intra-abdominal fat, and insulin resistance.^{53 54} In addition, intensive pharmacological intervention with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or niacin plus colestipol, resulted in a significantly lower HL activity, more buoyant LDL, higher HDL₂-C, and regression of CAD.⁵⁵ Therefore, plasma HL activity levels are modulated by a number of endogenous (estrogen levels, central adiposity) and exogenous (drugs) factors, in addition to the LIPC promoter polymorphisms (Figure 4).

View larger version (13K): [in this window] [in a new window]   Figure 4. Endogenous and exogenous factors affecting HL activity. - indicates negative (inhibitory) effect; +, positive (stimulatory) effect.

Because the less-common allele frequency of the HL promoter polymorphisms is much higher in African-Americans²¹ and Japanese-Americans compared with white Americans, the former 2 populations would offer the possibility of obtaining a large number of homozygotes for association studies. The presence of a common haplotype in all 3 of these populations indicates that these polymorphisms are derived from a common ancient origin.

In summary, the current study demonstrates that the LIPC promoter polymorphisms and plasma HL activity levels are strongly associated. This association is potentially clinically important, because the frequency of the A allele among whites is 0.2. In addition, the clinical relevance of this observation results from the association between LIPC promoter polymorphism and both LDL density and HDL₂-C levels, mediated by the potential effect of these polymorphisms on LIPC transcription. Because the polymorphism at -250 is in nearly complete linkage disequilibrium with the 3 other promoter polymorphisms, it is not known which of these potentially affects promoter activity. These findings further highlight the contribution of genetic factors to the regulation of both HDL and LDL subclass distribution. Finally, the strong association of LIPC genotype with large HDL₂ but not with smaller HDL₃ particles suggests that HDL₂ concentration may represent a better lipid measure to study the effect of LIPC on HDL metabolism. Functional analysis of the wild-type and of the different LIPC promoter variants is needed to investigate whether the effect on HL activity is accounted for by 1 or more of these LIPC promoter polymorphisms or is due to linkage disequilibrium between these polymorphisms and other as-yet-unidentified variants located within LIPC itself or within a gene encoding a protein regulating LIPC expression.

	Acknowledgments

 
The current study has been supported by National Institutes of Health (NIH) grant HL-30086. A portion of these studies was performed in the Clinical Research Center of the University of Washington (NIH RR 37). A.Z. is a recipient of an American Heart Association, Washington Affiliate, Fellowship.

Received March 3, 1998; accepted April 28, 1998.

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