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ApoC-III gene polymorphisms and risk of coronary artery disease

2002; Elsevier BV; Volume: 43; Issue: 9 Linguagem: Inglês

10.1194/jlr.m200145-jlr200

1539-7262

Oliviero Olivieri, Chiara Stranieri, Antonella Bassi, Barbara Zaia, Domenico Girelli, Francesca Pizzolo, Elisabetta Trabetti, Suzanne Cheng, Michael Grow, Pier Franco Pignatti, Roberto Corrocher,

Lipoproteins and Cardiovascular Health

Several polymorphisms in the apolipoprotein C-III (apoC-III) gene have been associated with hypertriglyceridemia, but the link with coronary artery disease risk is still controversial. In particular, apoC-III promoter sequence variants in the insulin responsive element (IRE), constitutively resistant to downregulation by insulin, have never been investigated in this connection. We studied a total of 800 patients, 549 of whom had angiographically documented coronary atherosclerosis, whereas 251 had normal coronary arteriograms. We measured plasma lipids, insulin, apoA-I, apoB, and apoC-III and assessed three polymorphisms in the apoC-III gene, namely, T-455C in the IRE promoter region, C1100T in exon 3, and Sst1 polymorphic site (S1/S2) in the 3′ untranslated region. Each variant influenced triglyceride levels, but only the T-455C (in homozygosity) and S2 alleles influenced apoC-III levels. In coronary artery disease (CAD) patients, 18.6% were homozygous for the −455C variant compared with only 9.2% in CAD-free group (P < 0.001).In logistic regression models, homozygosity for −455C variant was associated with a significantly increased risk of CAD (OR = 2.5 and 2.18 for unadjusted and adjusted models, respectively) suggesting that it represents an independent genetic susceptibility factor for CAD. Several polymorphisms in the apolipoprotein C-III (apoC-III) gene have been associated with hypertriglyceridemia, but the link with coronary artery disease risk is still controversial. In particular, apoC-III promoter sequence variants in the insulin responsive element (IRE), constitutively resistant to downregulation by insulin, have never been investigated in this connection. We studied a total of 800 patients, 549 of whom had angiographically documented coronary atherosclerosis, whereas 251 had normal coronary arteriograms. We measured plasma lipids, insulin, apoA-I, apoB, and apoC-III and assessed three polymorphisms in the apoC-III gene, namely, T-455C in the IRE promoter region, C1100T in exon 3, and Sst1 polymorphic site (S1/S2) in the 3′ untranslated region. Each variant influenced triglyceride levels, but only the T-455C (in homozygosity) and S2 alleles influenced apoC-III levels. In coronary artery disease (CAD) patients, 18.6% were homozygous for the −455C variant compared with only 9.2% in CAD-free group (P < 0.001). In logistic regression models, homozygosity for −455C variant was associated with a significantly increased risk of CAD (OR = 2.5 and 2.18 for unadjusted and adjusted models, respectively) suggesting that it represents an independent genetic susceptibility factor for CAD. Investigators have long disputed whether elevated serum triglyceride (TG) levels are an independent risk factor for coronary artery disease (CAD) (1Bloomfield Rubins H. The trouble with triglycerides.Arch. Intern. Med. 2000; 160: 1903-1904Google Scholar). A major reason for this controversy stems from the heterogeneity of factors measured by triglyceride (TG) determination (TGs are carried in virtually all plasma lipoproteins) and from the high variance and collinearity of TGs with other recognized risk factors (1Bloomfield Rubins H. The trouble with triglycerides.Arch. Intern. Med. 2000; 160: 1903-1904Google Scholar, 2Hodis H.N. Triglyceride-rich lipoprotein remnant particles and risk of atherosclerosis (Editorial).Circulation. 1999; 99: 2852-2854Google Scholar). Alternative, and more reliable, markers of TG metabolism have therefore been proposed (1Bloomfield Rubins H. The trouble with triglycerides.Arch. Intern. Med. 2000; 160: 1903-1904Google Scholar, 2Hodis H.N. Triglyceride-rich lipoprotein remnant particles and risk of atherosclerosis (Editorial).Circulation. 1999; 99: 2852-2854Google Scholar). Perhaps the most important among them is apolipoprotein C-III (apoC-III), a 79 amino acid protein synthesized in the liver and in the intestine, which is an essential constituent of circulating particles rich in triacylglycerol, i.e., chylomicrons and VLDL (3Jong M.C. Hofker M.H. Havekes L.M. Role of apo Cs in lipoprotein metabolism. Functional differences between apoC1, apoC2, and apoC3.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 472-484Google Scholar). The results of large clinical studies have indicated that apoC-III levels are a better predictor of risk for the development and progression of CAD than the traditionally measured TG levels (4Blankenhorn D.H. Alaupovic P. Wickham E. Chin H.P. Azen S.P. Prediction of angiographic change in native human coronary arteries and aortocoronary bypass grafts. Lipid and nonlipid factors.Circulation. 1990; 81: 470-476Google Scholar, 5Hodis H.N. Mack W.J. Azen S.P. Alaupovic P. Pogoda J.M. La Bree L. Hemphill L.C. Kramsch D.M. Blankenhorn D.H. Triglyceride and cholesterol-rich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin.Circulation. 1994; 90: 42-49Google Scholar, 6Mack W.J. Krauss R.M. Hodis H.N. Lipoprotein subclasses in the monitored atherosclerosis regression study (MARS). Treatment effects and relation to coronary angiographic progression.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 697-704Google Scholar, 7Luc G. Fievet C. Arveiler D. Evans A.E. Bard J.M. Cambien F. Fruchart J.C. Ducimetiere P. Apolipoproteins et al. Apolipoproteins C–III and E in apo B- and non-apo B-containing lipoproteins in two populations at contrasting risk for myocardial infarction: the ECTIM study.J. Lipid Res. 1996; 37: 508-517Google Scholar, 8Thompson G.R. Angiographic evidence for the role of triglyceride-rich lipoproteins in progression of coronary artery disease.Eur. Heart J. 1998; 19: H31-H36Google Scholar, 9Sacks F.M. Alaupovic P. Moye L.A. Cole T.G. Sussex B. Stampfer M.J. Pfeffer M.J. Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the cholesterol and recurrent events (CARE) trial.Circulation. 2000; 102: 1886-1892Google Scholar). ApoC-III inhibits the hydrolysis of TG-rich particles by lipoprotein lipase and their apoE–mediated hepatic uptake (10McConathy W.J. Gesquiere J.C. Bass H. Tartar A. Fruchart J.C. Wang C.S. Inhibition of lipoprotein lipase activity by synthetic peptides of apolipoprotein C–III.J. Lipid Res. 1992; 33: 995-1003Google Scholar, 11Ginsberg H.N. Le N.A. Goldberg I.J. Gibson J.C. Rubinstein A. Wang-Iverson P. Norum N. Brown W.V. Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CIII and AI. Evidence that apolipoprotein CIII inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo.J. Clin. Invest. 1986; 78: 1287-1295Google Scholar). In vitro and transgenic animal studies have demonstrated that overexpression of apoC-III causes delayed clearance of TG-rich lipoproteins from plasma, resulting in overt hypertriglyceridemia (3Jong M.C. Hofker M.H. Havekes L.M. Role of apo Cs in lipoprotein metabolism. Functional differences between apoC1, apoC2, and apoC3.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 472-484Google Scholar). The human apoC-III gene has been mapped on chromosome 11 and several variant alleles have been investigated as possible genetic markers of hypertriglyceridemia, an atherosclerosis-related "intermediate phenotype" (12Talmud P.J. Humphries S.E. Apolipoprotein C–III gene variation and dyslipidemia.Curr. Opin. Lipidol. 1997; 8: 154-158Google Scholar). Biallelic polymorphisms have been described in the 5′ promoter region (five polymorphisms in strong linkage disequilibrium with one another: T-455C, C-482T, T-625 deletion, G-630A, C-641A) (13Dammeman M. Sandkuijl L.A. Halaas J.L. Chung W. Breslow J.L. An apolipoprotein CIII aplotype protective against hypertriglyceridemia is specified by promoter and 3′ untranslated region polymorphisms.Proc. Natl. Acad. Sci. USA. 1993; 90: 4562-4566Google Scholar), in exon 3 (C1100T) and in the 3′ untranslated region (the so called Sstl polymorphic site S1/S2) (12Talmud P.J. Humphries S.E. Apolipoprotein C–III gene variation and dyslipidemia.Curr. Opin. Lipidol. 1997; 8: 154-158Google Scholar). In particular, over the last decade, this latter polymorphism, which is also the one most extensively studied, has consistently been reported to be associated with hypertriglyceridemia (14Ordovas J.M. Civeira F. Genest Jr., J. Craig S. Robbins A.H. Meade T. Pocovi M. Frossard P.M. Masharani U. Wilson P.W. Salem D.N. Ward R.H. Schaefer E.J. Restriction fragment length polymorphisms of the apolipoprotein A-I, C–III, A-IV gene locus. Relationships with lipids, apolipoproteins, and premature coronary artery disease.Atherosclerosis. 1991; 87: 75-86Google Scholar, 15Surgucho P. Page G. Patsch W. Boerwinkle E. Polimorphic markers in apolipoprotein C–III gene flanking regions and hypertriglyceridemia.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 941-947Google Scholar). Despite the expected implications in terms of cardiovascular morbidity, the evidence of an association between S2 allelic variant and risk of CAD is still controversial (14Ordovas J.M. Civeira F. Genest Jr., J. Craig S. Robbins A.H. Meade T. Pocovi M. Frossard P.M. Masharani U. Wilson P.W. Salem D.N. Ward R.H. Schaefer E.J. Restriction fragment length polymorphisms of the apolipoprotein A-I, C–III, A-IV gene locus. Relationships with lipids, apolipoproteins, and premature coronary artery disease.Atherosclerosis. 1991; 87: 75-86Google Scholar, 15Surgucho P. Page G. Patsch W. Boerwinkle E. Polimorphic markers in apolipoprotein C–III gene flanking regions and hypertriglyceridemia.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 941-947Google Scholar, 16Kee F. Amouyel P. Fumeron F. Arveiler D. Cambou J.P. Evans A. Cambien F. Fruchart J.C. Ducimetiere P. Dallongeville J. Lack of association between genetic variations of apo AI-CIII-AIV gene cluster and myocardial infarction in a sample of European male: ECTIM study.Atherosclerosis. 1999; 145: 187-195Google Scholar, 17Russo G. Meigs J.B. Cupples L.A. Demissie S. Otvos J.D. Wilson P.W. Lahoz C. Cucinotta D. Couture P. Mallory T. Schaefer E.J. Ordovas J.M. Association of the Sst-I polymorphism at the APOC3 gene locus with variation in lipids levels, lipoprotein subclass profiles and coronary artery disease risk: the Framingham offspring study.Atherosclerosis. 2001; 158: 173-181Google Scholar). Less information is available regarding the possible coronary risk linked to the other polymorphic variants (18Peacock R.E. Hamsten A. Johansson J. Nilsson-Ehle P. Humphries S.E. Association of genotypes at the apolipoprotein AI-CIII-AIV, apolipoprotein B and lipoprotein lipase gene loci with coronary atherosclerosis and high density lipoprotein subclasses.Clin. Genet. 1994; 46: 273-282Google Scholar, 19Hegele R.A. Small genetic effect in complex diseases: a review of regulatory sequence variants in dyslipoproteinemia and atherosclerosis.Clin. Biochem. 1997; 30: 183-188Google Scholar). This relatively limited interest may be particularly surprising if one considers the expression studies concerning the apoC-III promoter sequence variants in the insulin responsive element (IRE) and their interaction with insulin (20Chen M. Breslow J.L. Li W. Leff T. Transcriptional regulation of the apo C–III gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels.J. Lipid Res. 1994; 35: 1918-1924Google Scholar). In animals and in cultured cells, the apoC-III gene is transcriptionally downregulated by insulin (20Chen M. Breslow J.L. Li W. Leff T. Transcriptional regulation of the apo C–III gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels.J. Lipid Res. 1994; 35: 1918-1924Google Scholar). In 1995, Li et al. showed that, unlike the wild-type promoter, the promoter containing variants at positions –455 and –482 remains constitutively active over a 108-fold range of insulin concentrations inasmuch as polymorphic sequences have a reduced affinity for the nuclear transcription factors mediating the insulin response (21Li W.W. Dammeman M. Smith J.D. Metzger S. Breslow J.L. Leff T. Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia.J. Clin. Invest. 1995; 96: 2601-2605Google Scholar). The variation in the promoter of the apoC-III gene is the first reported example of a genetic polymorphism in an insulin responsive element and of "insulin resistance" at the gene level (21Li W.W. Dammeman M. Smith J.D. Metzger S. Breslow J.L. Leff T. Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia.J. Clin. Invest. 1995; 96: 2601-2605Google Scholar). Such considerations prompt speculation as to the possible links between these genetic variants and hypothetical related "intermediate" phenotypes, characterized by increased synthesis of apoC-III- and TG-rich lipoproteins, which in turn may imply an increased risk of CAD. In the light of all these elements, we designed a large case-control study in patients with or without angiographically documented CAD to evaluate: i) a possible association between apoC-III gene polymorphisms and circulating levels of apoC-III and/or plasma lipids, and ii) whether the distribution of these polymorphisms was in turn associated with an increased risk of CAD. The criteria for selection of the study population have already been described in detail elsewhere (22Girelli D. Russo C. Ferraresi P. Olivieri O. Pinotti M. Friso S. Manzato F. Mazzucco A. Bernardi F. Corrocher R. Polymorphisms in the factor VII gene and the risk of myocardial infarction in patients with coronary artery disease.N. Engl. J. Med. 2000; 343: 774-780Google Scholar). In brief, we studied a total of 800 unrelated adult patients of both sexes who were recruited from those referred to the Institute of Cardiovascular Surgery or to the Cardiovascular-Hypertension Unit of the Department of Internal Medicine of the University of Verona in Italy. Of these patients, 549 had angiographically documented, severe, often multivessel coronary atherosclerosis and were candidates for coronary-artery bypass grafting. The disease severity was evaluated by counting the number of major epicardial coronary arteries (left anterior descending, circumflex, and right) affected with ⩾1 significant stenosis (⩾50%). As a control group, we considered 251 subjects with angiographically documented normal coronary arteries (CAD-free), examined for reasons other than possible coronary artery disease (in 90% of cases valvular heart disease; the remaining cases were studied for miscellany conditions including atypical chest pain of uncertain origin, congenital heart disease, etc.). The controls were required to have no stenosis in angiogram, no history of atherosclerosis, nor evidence of atherosclerosis in other vascular beds. Since the primary aim of our selection was to provide an objective and clear-cut definition of the atherosclerotic phenotype, subjects with nonsignificant coronary stenosis (<50%) were not included in the study. The angiograms were assessed by two cardiologists unaware that the patients were to be included in the study. All the study participants came from northern Italy and had similar socioeconomic and ethnic backgrounds. At the time of blood sampling, a complete clinical and pharmacological history, including the presence or absence of cardiovascular risk factors such as smoking, hypertension, and diabetes mellitus, was obtained from the patients. Patients who were taking statins or fibrates (n = 266) were excluded from the genotype-phenotype correlation studies because of the documented lowering effects of these drugs on apoC-III and lipids levels (5Hodis H.N. Mack W.J. Azen S.P. Alaupovic P. Pogoda J.M. La Bree L. Hemphill L.C. Kramsch D.M. Blankenhorn D.H. Triglyceride and cholesterol-rich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin.Circulation. 1994; 90: 42-49Google Scholar, 9Sacks F.M. Alaupovic P. Moye L.A. Cole T.G. Sussex B. Stampfer M.J. Pfeffer M.J. Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the cholesterol and recurrent events (CARE) trial.Circulation. 2000; 102: 1886-1892Google Scholar, 23Packard C.J. Overview of fenofibrate.Eur. Heart J. 1998; 19: A62-A65Google Scholar). The study was approved by our institutional review boards. Either written or oral informed consent was obtained from all the patients after a full explanation of the study. Samples of venous blood were drawn from each subject after an overnight fast within 10 days from the angiographic procedures. Serum lipids and the other common biochemical parameters were determined as previously described (22Girelli D. Russo C. Ferraresi P. Olivieri O. Pinotti M. Friso S. Manzato F. Mazzucco A. Bernardi F. Corrocher R. Polymorphisms in the factor VII gene and the risk of myocardial infarction in patients with coronary artery disease.N. Engl. J. Med. 2000; 343: 774-780Google Scholar). Insulin was measured by an immunometric sandwich assay (Immulite 2000 Insulin) from Diagnostic Products Corporation, Los Angeles, CA; intra- and interassay CVs of the method were 30 mg/dl, the sample was diluted manually. Imprecision was assessed on three pools of control sera with low, medium and high concentrations of apoC-III; intraassay CV was 1.84, 2.02, and 1.98% and interassay CV 4.4, 3.4, and 2.29% for low, medium, and high concentration, respectively. Mutation analysis (as well as routine biochemical analysis) was conducted as a study that was blinded as to whether the sample came from CAD or a CAD-free subject. Three polymorphic variants mapping on the promoter (T-455C), on exon 3 in the coding region (C1100T), and on the 3′ untranslated region (S1/S2), were evaluated (Fig. 1). Genomic DNA was prepared from whole blood samples by phenol-chloroform extraction and was then used according to a recently described multilocus genotyping assay protocol (24Cheng S. Grow M.A. Pallaud C. Klitz W. Erlich H.A. Visvikis S. Chen J.J. Pullinger C.R. Malloy M.J. Siest G. Kane J.P. A multilocus genotyping assay for candidate markers of cardiovascular disease risk.Genome Res. 1999; 9: 936-949Google Scholar). Briefly, each sample was amplified by two 33 cycle Multiplex Polymerase Chain Reactions (32 ng of genomic DNA each) and the PCR products were then hybridized to an array of immobilized oligonucleotide probes. The colorimetric detection was based upon streptavidin-horseradish peroxidase method. All computations were performed by using the SPSS 10.0 statistical package (SPSS Inc., Chicago, IL). Distributions of continuous variables in groups were expressed as means ± SD. Logarithmic transformation was performed for skewed variables, i.e., apoC-III and TG, and the statistical differences concerning these parameters were also computed on the corresponding log-transformed values, although, for the sake of simplicity and clarity, crude data are reported in the Results. Statistical significance of differences in quantitative variables was assessed by Student's t-test, and it was also tested by one-way ANOVA adjusted for age and sex (General Linear Model procedure). Qualitative data were analyzed using the chi-square test. Pearson coefficient was used for correlation between variables. In each of the two patient groups (with or without CAD), genotype frequencies were compared by chi-square analysis with the values predicted on the basis of Hardy-Weinberg equilibrium. Intragenic haplotypes for multiple markers within the apoC-III locus were estimated using the EH program (25Terwilliger J. Ott J. Handbook of human genetic linkage. John Hopkins University Press, Baltimore, MD1994: 188-198Google Scholar) and were used to detect pairwise linkage disequilibrium values (D′). Insulin and lipid variables were compared among patients with different polymorphisms by ANOVA, using the Tukey procedure for post hoc multivariate comparison of the means (ANOVA). For T-455C and C1100T polymorphisms, computation was also performed considering the less frequent allele as recessive (homozygous for the less frequent variant vs. all other patients). To assess the extent to which the various genotypes were associated with coronary artery disease, odds ratios with 95% confidence intervals were estimated by logistic-regression analysis. To provide separate odds ratios for each genotype, dummy variables were used, with wild-type genotype used as the reference group. Adjustment for the patients' conventional risk factors (age, gender, smoking status, presence of diabetes and hypertension, cholesterol, triglycerides, apoA-I and apoB) was done by including these covariates in a second set of logistic-regression models. The clinical and biochemical characteristics of the population studied are summarized in Table 1. CAD patients had more conventional cardiovascular risk factors and significantly higher levels of apoC-III than control patients. There were no statistical differences in insulin plasma levels between the two groups (Table 1). In the population as a whole, apoC-III was statistically correlated with total (R = 0.40, P < 0.001), LDL (R = 0.238, P < 0.001), and HDL cholesterol (HDL-C) (R = −0.08, P < 0.05) and, more strongly, with TG levels (R = 0.68, P < 0.001).TABLE 1Characteristics of the study patients and controlsaPlus-minus values are means ± SD.ParametersCAD Patients (n = 549)CAD-free (n = 251)P valueAge (years)60.4 ± 9.457.6 ± 12.6<0.01Male sex (%)81.866.9<0.001BMI (kg/height2)bAge- and sex-adjusted values.26.5 ± 3.325.3 ± 3.4<0.001CholesterolbAge- and sex-adjusted values.Total (mmol/l)5.83 ± 1.125.51 ± 1.050.001LDL (mmol/l)3. 88 ± 0.983.53 ± 0.93<0.001HDL (mmol/l)1.20 ± 0.321.42 ± 0.43<0.001Triglycerides (mmol/l)bAge- and sex-adjusted values.2.01 ± 1.131.50 ± 0.71<0.001ApoA-I (g/l)bAge- and sex-adjusted values.1.31 ± 0.241.42 ± 0.31<0.001ApoB (g/l)bAge- and sex-adjusted values.1.22 ± 0.301.06 ± 0.25<0.001ApoC-III (mg/dl)bAge- and sex-adjusted values.12.31 ± 4.410.7 ± 3.25<0.001Insulin (μIU/ml)bAge- and sex-adjusted values.14.64 ± 7.715.75 ± 12.2NSSmoking (%)69.741.4<0.001Hypertension (%)58.330.8<0.001Diabetes (%)21.913.3<0.01Patients taking lipid-lowering drugs266—Statistical significance for differences was tested by Student's unpaired t-test or by χ2 test when appropriate. P value was considered significant when <0.05.To convert the values for cholesterol to milligrams per deciliter, divide by 0.02586. To convert the values for triglycerides to milligrams per deciliter, divide by 0.01129.a Plus-minus values are means ± SD.b Age- and sex-adjusted values. Open table in a new tab Statistical significance for differences was tested by Student's unpaired t-test or by χ2 test when appropriate. P value was considered significant when <0.05. To convert the values for cholesterol to milligrams per deciliter, divide by 0.02586. To convert the values for triglycerides to milligrams per deciliter, divide by 0.01129. Genotype frequencies of the apoC-III polymorphic variants for CAD and CAD-free groups are described in Table 2. All three polymorphisms were in the Hardy-Weinberg equilibrium in both groups of patients. No homozygous individuals for S2 allele were found. The distribution of C1100T and S1/S2 polymorphisms was similar in CAD and CAD-free patients. In contrast, the frequency of −455C homozygous subjects in CAD group was significantly higher than that observed in individuals free of coronary artery disease (18.6 vs. 9.2%, P < 0.001; Table 2).TABLE 2ApoC-III genotypes in the study patients and controlsGenotype (% of Patients)CAD-free Patients (n = 251)CAD Patients (n = 549)Chi- SquareP valueT-455C−455 TT(%) 43.8 35.3−455 TC (%) 47 46.1−455 CC(%) 9.2 18.613.070.001C1100T1100 CC (%) 50.2 54.31100 CT (%) 43 37.31100 TT (%) 6.8 8.42.529NSS1/S2S1/S1 (%) 85.2 82.4S1/S2 (%) 14.8 17.60.95NS Open table in a new tab Strong linkage disequilibrium between S1/S2 and both T-455C and C1100T (D' = 0.98, D' = 0.97, respectively) was observed, but not between T-455C and C1100T polymorphisms (D' = 0.13) (Fig. 1). It should be noted that, out of a total of 125 homozygotes for −455 variant, there were 55 (44%) subjects with S1/S2 and 13 (10.4%) with 1100TT genotype. In order to estimate the impact of the polymorphisms on apoC-III and/or other lipid metabolism parameters, genotype-phenotype analysis was performed with data from the entire study population. Due to the well known lowering effect of statins and fibrates on apoC-III (5Hodis H.N. Mack W.J. Azen S.P. Alaupovic P. Pogoda J.M. La Bree L. Hemphill L.C. Kramsch D.M. Blankenhorn D.H. Triglyceride and cholesterol-rich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin.Circulation. 1994; 90: 42-49Google Scholar, 9Sacks F.M. Alaupovic P. Moye L.A. Cole T.G. Sussex B. Stampfer M.J. Pfeffer M.J. Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the cholesterol and recurrent events (CARE) trial.Circulation. 2000; 102: 1886-1892Google Scholar, 23Packard C.J. Overview of fenofibrate.Eur. Heart J. 1998; 19: A62-A65Google Scholar), patients taking these drugs (n = 266, all CAD patients) were excluded from the analysis. Results for the levels of apoC-III, triglycerides, and insulin, according to each genotype in the remaining population (n = 534), are reported in Table 3. Different genotype groups of such population were similar for age and sex distribution (data not shown). Homozygous individuals for the promoter variant had increased levels of apoC-III as compared with carriers of other genotypes, but a statistical significance was evident only upon assuming recessive allelic transmission as the model (Table 3). Homozygotes for −455C had significantly higher triglyceride values than those observed in heterozygous and wild-type individuals (whether considered separately or as a single group, Table 3).TABLE 3Levels of apoC-III, TGs, and insulin in the study population, according to the ApoC-III genotypes (by ANOVA)ApoC-III GenotypesNo. of Patients (n = 534)ApoC-III (mg/dl)Triglycerides (mmol/l)Insulin (μIU/ml)−455TT20611.47 ± 3.91.79 ± 0.94aStatistically different from patients homozygous for the less common allele.15.7 ± 11−455TC25211.30 ± 3.71.73 ± 0.86aStatistically different from patients homozygous for the less common allele.14.6 ± 8.8−455CC7612.65 ± 5.12.12 ± 1.4014.8 ± 7.2−455TT and −455TC45811.38 ± 3.8aStatistically different from patients homozygous for the less common allele.1.76 ± 0.89aStatistically different from patients homozygous for the less common allele.15.1 ± 9.9−455CC7612.65 ± 5.12.12 ± 1.4014.8 ± 7.21100CC27411.3 ± 3.61.73 ± 0.85aStatistically different from patients homozygous for the less common allele.15.3 ± 10.61100CT21811.8 ± 4.51.85 ± 1.0514.9 ± 8.71100TT4212.1 ± 4.52.13 ± 1.4014.1 ± 5.71100CC and 1100CT49211.5 ± 4.01.79 ± 0.94aStatistically different from patients homozygous for the less common allele.15.1 ± 9.81100TT4212.1 ± 4.52.13 ± 1.4014.1 ± 5.7S1/S143811.3 ± 3.8bStatistically different from S1/S2 patients.1.75 ± 0.92bStatistically different from S1/S2 patients.14.9 ± 9.9S1/S29612.8 ± 4.82.10 ± 1.2215.6 ± 7.7a Statistically different from patients homozygous for the less common allele.b Statistically different from S1/S2 patients. Open table in a new tab C1100T polymorphism was significantly associated with plasma triglycerides (with the raising effect confined to the homozygotes), but this site was not associated with variations in apoC-III levels (Table 3). Different levels of apoC-III and triglycerides were also associated with the S1/S2 polymorphism, with higher values being observed in S2 carriers (Table 3). No substantial effect on fasting insulin values due to the different apoC-III gene polymorphisms was evident. Similarly, none of the other lipid variables analyzed (total, LDL, and HDL-C, apoA-I, apoB) showed any statistically relevant differences according to apoC-III genotype (data not shown). To estimate the disease risk associated with apoC-III genotypes, logistic regression analysis was performed. Crude and adjusted odds ratios for coronary disease in relation to each apoC-III genotype are shown in Table 4. The greatest risk was conferred by homozygosity for the −455C variant, which was associated with a more than 2-fold increased probability of disease. Adjustment for the main vascular risk factors produced no change in the result, indicating that this polymorp