Hepatic Lipase Gene Polymorphisms Influence Plasma HDL Levels
Results From Finnish EARS Participants
Abstract Hepatic lipase (HL), a triglyceride lipase found in liver, adrenals, testes, and ovaries, takes part in the uptake, remodeling, and function of lipoproteins including HDL, as well as VLDL and chylomicrons. In the present study, the genotype distribution of five HL polymorphisms (−C480T, V133V, T202T, L334F, T457T) and their association to plasma lipid values were investigated. The study participants included 92 students with paternal history of myocardial infarction before the age of 55 and 194 matched control subjects, ie, the Finnish participants of the European Atherosclerosis Research Study (EARS). The allele T of the HL polymorphism −C480T showed an association with elevated HDL, apoA-I, and LpA-I values (ANOVA P<.01). No difference in genotype distribution was observed in the offspring with and without paternal history of myocardial infarction.
- hepatic lipase
- high-density lipoproteins
- European Atherosclerosis Research Study
- Received December 20, 1996.
- Accepted December 3, 1997.
Human HL (triacylglycerol lipase, EC 126.96.36.199)1 has a dual role in lipid metabolism; it is involved in chylomicron remnant catabolism and also in the metabolism of HDL. HL may change the presentation of apoE in chylomicron remnants, a process that may lead to their increased uptake by hepatocytes.2 It also influences HDL interconversion, a process essential for cholesterol metabolism.3 4 5 In addition to its presence in the liver, it is found in steroidogenic organs such as adrenals, ovaries, and testes.6 Of the two triglyceride lipases, HL and lipoprotein lipase, lipoprotein lipase has a central role in hydrolyzing lipoprotein triglycerides for metabolic use in extrahepatic tissues, whereas HL seems to take part in remodeling lipoproteins.7 Studies on HL enzyme activity in humans and in transgenic animals, show that HL deficiency in most cases leads to triglyceride enrichment in LDL and HDL lipoprotein fractions, presence of circulating β-VLDL, and abnormal catabolism of chylomicrons.8 9 10 11 12 13 That relation to lipid values holds also conversely; ie, high HL activity is associated with low concentration of HDL-C.14 15
Several mutations and gene polymorphisms of HL have been described. Polymorphisms causing an amino acid substitution include V73M, N193S, S267F, T383M,8 9 L334F,11 and R186H.12 Of those, S267F, L334F, T383M, and R186H have been found to lead to decreased postheparin plasma HL activity.9 11 12 Polymorphisms that do not cause amino acid substitutions have been reported in codons V133V, T202T,8 T457T,16 G175G,17 and T344T.11 Also, a C-to-T substitution in nucleotide −480 in the promoter region of the HL gene, leading to decreased postheparin plasma HL activity,18 and a mutation causing a splice site mutation in intron 1 of the HL gene13 have been described.
The relation of HL to premature atherosclerosis is not clear. The abnormal lipid profile resulting from the rare cases of total or almost total HL deficiency can certainly lead to premature atherosclerosis.19 In the development of atherosclerosis, the conditions before the clinical manifestation of the disease are important. One way to evaluate them is to compare offspring of fathers with premature atherosclerosis with matched control subjects. EARS is a multicenter collaborative project sponsored by the European Community with that goal20 (the collaborating centers of the EARS Group 1994 are listed in the “Appendix”). The theoretical strength to infer genotype associations from studies comparing matched offspring has been discussed in a previous study.21
In the present study, the Finnish subjects from EARS were genotyped for five HL polymorphic loci (−C480T, V133V, T202T, L334F, T457T). The associations of genotypes with clinical and biochemical phenotypes were studied.
A detailed description of the design of EARS has been outlined previously.20 Briefly, the participants from 14 centers in 11 European countries were university students aged between 18 and 26 years representing both sexes. The study has been approved by review committees of collaborating centers, and the subjects have given informed consent. The students were chosen as study population on the basis of internal comparability; although they do not represent the general population of the same age group, they have roughly similar lifestyles and are exposed to similar environmental factors. Initially, a total of 286 students representing two centers, Helsinki and Oulu, participated in EARS from Finland. The index group consisted of 92 offspring of fathers who had had a documented premature acute MI before the age of 55 years, and the control group consisted of 194 individuals who were selected from the student register (two control subjects of the same sex with closest birth date to the index case). All subjects were examined, and blood samples were taken within a 5-month period from October 1990 to February 1991.
The sample handling and analyzing methods were previously described.20 22 In short, venous blood was collected after an overnight fast into tubes with an anticoagulant (potassium EDTA), centrifuged at 4°C, and 11 1-mL aliquots were snap-frozen on dry ice within 30 minutes of sample collection. The samples were sent through the EARS center in Nancy, France, to other participating laboratories, where the actual biochemical analyses were done. Plasma total cholesterol, total triglycerides, and HDL-C were measured according to the Lipid Research Clinic’s Manual of Laboratory Operations standardized according to the Centers for Disease Control and Prevention, Atlanta, Ga. The LDL cholesterol was calculated by using Friedewald’s formula. The apoA-I and apoB concentrations were measured by immunonephelometry. ApoA-II was measured by immunoturbidometry. ApoE, apoA-IV, and Lp(a) were measured by enzyme-linked immunosorbent assay. The apoA-I–containing lipoprotein (LpA-I) particles were measured by rocket immunoelectrophoresis. Blood glucose was measured after protein removal by the glucose dehydrogenase method and insulin level by radioimmunoassay.
The genomic DNA from white blood cells was isolated using the “salting-out” procedure.23 The mutations and polymorphisms at nucleotide position −480 and at amino acid codons 133, 202, 334, and 457 were determined. The L334F polymorphism was originally described by our group.11 The V133V, T202T, and T457T are the three most common neutral polymorphisms of HL.8 16 −C480T is the most common HL polymorphism reported to have an effect on HL activity.18 In the solid-phase minisequencing method,24 variable nucleotides were identified by a single nucleotide primer extension reaction catalyzed by DNA polymerase from a polymerase chain reaction product on a solid support. Three different primers were used to study each polymorphism; each DNA-fragment, containing a nucleotide to be tested, was first amplified by polymerase chain reaction using a pair of primers, and then the product was analyzed by a detection primer required in minisequencing. These primers are listed in Table 1⇓.
The database containing results obtained from various centers (including Oulu and Helsinki) is stored in Paris on an IMB Risc 6000 computer. Statistical analyses were performed with the SAS statistical software (SAS Institute Inc). Only statistically significant differences (P<.01) are reported. Genotypic effect was tested by ANOVA. Genotype-phenotype association analyses were adjusted for age, paternal history of MI, sex, and recruitment center. Triglyceride, insulin, and apoE levels were log transformed to remove positive skewness. Allele frequencies were estimated by gene counting. The nonrandom distribution of allele frequencies between case and control groups was tested with a χ2 test. The haplotype frequencies were estimated from genotypes by using the computer program MYRIAD.25 The linkage disequilibrium coefficient was calculated from control genotypes by log-linear analysis.26 Two-way ANOVA with HDL and triglycerides as dependent variables were performed to test whether the phenotypic variation associated with −C480T genotypes was independent of the four other polymorphisms.
Clinical and Biochemical Characteristics According to Genotypes
Five different HL polymorphisms were studied to detect their possible effect on phenotype. The measured characteristics of lipid metabolism according to genotype classes are shown in Table 2⇓⇓. Presence of the T allele in locus −C480T led to increase in HDL-C (P<.007), apoA-I (P<.01), and LpA-I (P<.005) levels. Small differences, which showed a statistical tendency in the range of P>.01 and P<.05, were found with the genotypes in loci V133V (Table 2⇓). Triglycerides (P=.02) and glucose (P<.05) were higher, and HDL-C (P=.03), apoA-I (P=.03), and LpA-I (P=.02) were lower in individuals having nucleotide T at codon 133. The combined effects of −C480T and the other loci on HDL-C and triglyceride levels are presented in Table 3⇓. The −C480T polymorphism remained significantly associated with HDL-C regardless of other polymorphisms jointly considered. Also, the lowering effect on HDL and the raising effect on triglycerides observed with the T allele at codon 133 proved to be significant when adjusted for the −C480T polymorphism.
Distribution of Genotypes and Haplotypes
The genotype and allele frequencies of the HL polymorphisms are shown in Table 4⇓. The index and control groups did not have different allele or genotype frequencies with any of the HL polymorphisms. The allele frequencies from the two recruitment centers did not differ either. The haplotypes constructed from HL polymorphisms are shown in Table 5⇓. The haplotype C-T-G-A-C, in the order of the position in the HL gene −480, 133, 202, 334, 457, was the most common in both groups (index group, 19%; control group, 24%). Alleles of the HL polymorphism 202 were in strong linkage disequilibrium with alleles at the loci L334F and T457T; linkage disequilibrium coefficients were −1.00 (P<.01) and −0.96 (P<.001), respectively. There was also a weaker linkage disequilibrium between HL −480 and HL 334 (0.54, P<.01).
The aggregation of coronary heart disease in families has been previously demonstrated in several studies, but to what extent and in which metabolic pathways the genetic factors are risk determinants need to be clarified.27 28 It is believed27 that no single familial risk factor will ever explain as much of the total risk of heart disease mortality as the conventional risk factors such as elevated lipids, smoking, and hypertension. The genetic variation of enzymes in metabolic pathways may explain part of the interindividual variation of risk for premature atherosclerosis also in nonfamilial cases, especially if the frequencies of risk alleles are high. On the other hand, if mutation increases the risk for premature atherosclerosis only moderately, it may be more efficient to study the effects of that particular genetic factor directly on the metabolic determinants. In the present study, both strategies were applied: a possible difference in genotype frequencies in the groups of students with or without paternal history of premature MI was tested, and the effects of genotypes on phenotypes were studied by ANOVA.
Although most of the clinical and biochemical characteristics were independent of genotype, HDL-C and apoA-I showed association to genotypes at the position of −480 of the HL gene. Individuals carrying the allele T of that polymorphism have elevated HDL-C levels. This promoter region polymorphism has previously been shown to be associated with lowered lipase activity.18 Of the other polymorphisms used in the present study, HL enzyme with phenylalanine at codon 334 has been reported in in vitro expression studies to have only about 30% of the enzymatic activity of the wild-type enzyme.11 In the present study, none of the 270 individuals was homozygous for the rare allele C at codon 334, and its possible allelic effect was too small to be statistically significant in heterozygous individuals. The locus V133V had effects on both triglyceride and HDL-C levels that were statistically significant after adjustment for the −C480T genotypes.
The mechanisms of how −C480T or V133V polymorphisms cause these effects on phenotype are not known. The effects are most probably due to interactions of several polymorphisms that may be in linkage disequilibrium with each other. Constructing haplotypes and testing linkage disequilibrium between the polymorphisms may also reveal the history of genetic variation. The most common haplotype C-T-G-A-C was found in about 20% of chromosomes. Polymorphism T202T was in linkage disequilibrium with L334F and T457T. The association of L334F and T457T to T202T can also be seen from haplotypes; the allele G in T202T is always associated to the A in L334F and C in T457T. This observation may refer to the history of these mutations, ie, T202T mutation C to G may have occurred on an ancestral chromosome that had the allele C at T457T. The next mutation may have been A to C at locus HL334 in a chromosome having allele C at 202.
Allele frequencies of the HL polymorphisms observed in the present study were similar to those reported earlier from the Netherlands, Finland, Japan, and Canada.8 11 16 18 When the possible association of the HL gene polymorphisms with genetic background of students according to paternal MI history was studied, the students whose fathers had had an MI did not have different allele frequencies of HL polymorphisms compared with their fellow students. Over 46% of individuals in both study groups carried the C-to-T substitution at the position −480 in the HL gene, but the present study did not give any evidence that the presence of it would associate to the genetic background of individuals susceptible to premature atherosclerosis. The fact that the −C480T mutation was associated with HDL-C level, a recognized risk factor for coronary heart disease, may warrant other interpretations and further studies on the effects of HL polymorphisms on HDL particle metabolism and on the development of atherosclerosis.
Selected Abbreviations and Acronyms
|EARS||=||European Atherosclerosis Research Study|
EARS project leader. J. Shepherd, Glasgow, UK.
EARS project management group. F. Cambien, Paris, France; D. De Backer, Ghent, Belgium; M.-M. Galteau, Nancy, France; A. St J. O’Reilly, Glasgow, UK; M. Rosseneu, Brugge, Belgium; L. Wilhelmsen, Göteborg, Sweden.
EC COMAC epidemiology liaison officer. T. Sorensen, Copenhagen, Denmark.
EARS Group, Collaborating Centers, and Their Associated Investigators
Austria. C. Sandholzer, C. Duba, H.-G. Kraft, H.-J. Menzel, Institute for Medical Biology and Genetics, University of Innsbruck; Recruitment Center and Laboratory.
Belgium. G. De Backer, S. De Henauw, D. De Bacquer, A. Bael, Department of Hygiene and Social Medicine, State University of Ghent; Recruitment Center. M. Rosseneu, C. Labeur, N. Vinaimont, Department of Clinical Chemistry, University Hospital St Jan, Brugge; Laboratory.
Denmark. C. Gerdes, O. Faergeman, Medical Department I, Aarhus Amtssygehus; Recruitment Center and Laboratory.
Finland. C. Ehnholm, National Public Health Institute, Helsinki; Recruitment Center and Laboratory. R. Elovainio, J. Peräsalo, The Finnish Student Health Service; Recruitment Center. A. Kesäniemi, Department of Internal Medicine, University of Oulu; Recruitment Center. P. Palomaa, The Finnish Student Health Service; Recruitment Center.
France. F. Gambien, L. Tiret, R. Agher, V. Nicaud, R. Rakotovao, INSERM U.258, Unité de Recherche d’Epidémiologie Cardiovasculaire, Hôpital Broussais, Paris; EARS Data Center and Recruitment Center. M.-M. Galteau, S.M. Visvikis, Centre de Médecine Préventive, Nancy; EARS Central Laboratory. J.C. Fruchart, J.M. Bard, P. Lebel, Service de Recherche sur les Lipoprotéines et l’Atherosclérose (SERLIA), INSERM U.325, Institut Pasteur, Lille; Laboratory. L. Bara: Laboratories de Thombose Expérimentale, Paris; Laboratory. C. Bady, J. Beylot, A. Lindousi, L. Tiret, UFR de Sante Publique, Bordeaux; Recruitment Center.
Germany. U. Beisiegel, A. Jorge, M. Papanikolaou, Medizinische Klinik Universitätskrankenhaus, Hamburg; Recruitment Center and Laboratory.
Italy. E. Farinaro, C. Cortese, M. Liguori, F. De Lorenzo, Institute of Internal Medicine and Metabolic Disease, University of Naples; Recruitment Center.
The Netherlands. L.M. Havekes, P. de Knijff, IVVO-TNO Health Research, Gaubius Institute, Leiden; Laboratory.
Spain. S. Sans, T. Puig. Programma CRONICAT, Hospital Sant Pau, Barcelona; Recruitment Center. J. Ribalta, J. Balanya, P.R. Turner, L. Masana, Unitat Recerca Lipids, Universitat Barcelona, Reus; Recruitment Center and Laboratory.
Sweden. L. Wilhelmsen, I. Wallin, S. Johansson, Department of Medicine, Ostra Hospital, University of Göteborg; Recruitment Center.
Switzerland. F. Gutzwiller, B. Marti, M. Knobloch, P. Anliker, Institute of Social and Preventive Medicine, University of Zürich; Recruitment Center.
United Kingdom. D. Stansbie, H. Denton, S. Blumridge, Department of Chemical Pathology, Bristol, Royal Infirmary; Recruitment Center. J. Shepherd, D. St J. O’Reilly, G.W. Tait, G.M. Hamilton, Institute of Biochemistry, Royal Infirmary, Glasgow; Recruitment Center and Laboratory. S. Humphries, P. Talmud, S. Ye, University College London, School of Medicine, Laboratory.
This study was supported by the Finnish Foundation for Cardiovascular Research and by EC Concerted Action MRH4-COMAC Epidemiology.
Shafi S, Brady SE, Bensadoun A, Havel RJ. Role of hepatic lipase in the uptake and processing of chylomicron remnants in rat liver. J Lipid Res. 1994;35:709-720.
Atherosclerosis X, Proceedings of the 10th International Symposium on Atherosclerosis. In: Woodford FP, Davignon J, Sniderman, eds. Amsterdam, Netherlands. Elsevier Science; 1995:731-735.
Hegele RA, Little JA, Vezina C, Maguire G, Tu L, Wolever T, Jenkins DJA, Connelly PW. Hepatic lipase deficiency: clinical, biochemical, and molecular genetic characteristics. Arterioscler Thromb. 1993;13:720-728.
Homanics GE, de Silva HV, Osada J, Zhang SH, Wong H, Borensztajn J, Maeda N. Mild dyslipidemia in mice following targeted inactivation of the hepatic lipase gene. J Biol Chem. 1995;270:2974-2980.
Knudsen P, Antikainen M, Ehnholm S, Uusi-Oukari M, Tenkanen H, Lahdenperä S, Kahri J, Tilly-Kiesi M, Bensadoun A, Taskinen M-R, Ehnholm C. A compound heterozygote for hepatic lipase gene mutations Leu334-Phe and Thr383-Met: correlation between hepatic lipase activity and phenotypic expression. J Lipid Res. 1996;37:825-834.
Knudsen P, Antikainen M, Uusi-Oukari M, Ehnholm S, Lahdenperä S, Bensadoun A, Funke H, Wiebusch H, Assmann G, Taskinen M-R, Ehnholm C. Heterozygous hepatic lipase deficiency, due to two missense mutations R186H and L334F, in the HL gene. Atherosclerosis. 1996;128:165-174.
Brand K, Dugi KA, Brunzell JD, Nevin DN, Santamarina-Fojo S. A novel A-G mutation in intron I of the hepatic lipase gene leads to alternative splicing resulting in enzyme deficiency. J Lipid Res. 1996;37:1213-1223.
Blades B, Vega GL, Grundy SM. Activities of lipoprotein lipase and hepatic triglyceride lipase in postheparin plasma of patients with low concentrations of HDL cholesterol. Arterioscler Thromb. 1993;13:1227-1235.
Kuusi T, Ehnholm C, Viikari J, Härkönen R, Vartiainen E, Puska P, Taskinen M-R. Postheparin plasma lipoprotein and hepatic lipase are determinants of hypo- and hyperalphalipoproteinemia. J Lipid Res. 1989;30:1117-1126.
Jansen H, Verhoeven AJM, Weeks L, Kastelein JJP, Halley DJJ, van den Ouweland A, Jukema JW, Seidell JC, Birkenhager JC. A common C to T substitution at position −480 of the hepatic lipase promoter associated with a lowered lipase activity in coronary artery in coronary artery disease patients. ATVB 1997. In press.
The EARS Group. The European Atherosclerosis Research Study (EARS): design and objectives. Int J Epidemiol. 1994;23:465-471.
Tiret L, de Knijff P, Menzel H-J, Ehnholm C, Nicaud V, Havekes LM, for the EARS group. ApoE polymorphism and predisposition to coronary heart disease in youths of different European populations: the EARS study. Arterioscler Thromb. 1994;14:1617-1624.
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215.
Perkins KA. Family history of coronary heart disease: is it an independent risk factor? Am J Epidemiol. 1989;124:182-194.