Relationship of HDL and coronary heart disease to a common amino acid polymorphism in the cholesteryl ester transfer protein in men with and without hypertriglyceridemia
1998; Elsevier BV; Volume: 39; Issue: 5 Linguagem: Inglês
10.1016/s0022-2275(20)33876-1
ISSN1539-7262
AutoresCan Bruce, Dan S. Sharp, Alan R. Tall,
Tópico(s)Lipoproteins and Cardiovascular Health
ResumoPlasma triglyceride (TG) levels are inversely related to HDL-cholesterol levels and subjects with high TG and low HDL cholesterol have increased coronary heart disease (CHD) risk. Plasma cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from HDL to TG-rich lipoproteins. In this study we determined the relationship between a common CETP amino acid polymorphism (I405V) and CETP and HDL levels and CHD prevalence in 576 men of Japanese ancestry in the Honolulu Heart Program cohort. This conservative substitution was associated with altered plasma CETP concentration (1.95 ± 0.54, 1.91 ± 0.57, and 1.77 ± 0.57 μg/ml for the II, IV and VV genotypes, respectively). The distribution of plasma CETP concentrations among the VV, but not II, men appeared bimodal (P < 0.01), suggesting the presence of a functionally significant CETP gene mutation(s) in a subset of V alleles. HDL-C levels were higher in VV than IV for II men (55.4 ± 17.4, 51.3 ± 16.6, 51.1 ± 17.0 mg/dl, P < 0.04). However, the increase in HDL was only significant in VV men with plasma TG > 165 mg/dl. Although CHD prevalence was not significantly different among the three genotypes in this population, in the subpopulation with high plasma TG, CHD prevalence appeared higher among VV than IV or II subjects (38% vs. 27% vs. 18%, P < 0.05 for an interaction of genotype and plasma TG levels). In fresh plasma from a separate group of normolipidemic subjects, the V/I polymorphism was not associated with any change in plasma CETP specific activity. The data suggest that a widespread and common CETP gene mutation(s) in linkage disequilibrium with 405V causes low CETP. Among hypertriglyceridemic men this is associated with higher HDL and possibly with increased CHD.—Bruce, C., D. S. Sharp, and A. R. Tall. Relationship of HDL and coronary heart disease to a common amino acid polymorphism in the cholesteryl ester transfer protein in men with and without hypertriglyceridemia. J. Lipid Res. 1998. 39: 1071–1078. Plasma triglyceride (TG) levels are inversely related to HDL-cholesterol levels and subjects with high TG and low HDL cholesterol have increased coronary heart disease (CHD) risk. Plasma cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from HDL to TG-rich lipoproteins. In this study we determined the relationship between a common CETP amino acid polymorphism (I405V) and CETP and HDL levels and CHD prevalence in 576 men of Japanese ancestry in the Honolulu Heart Program cohort. This conservative substitution was associated with altered plasma CETP concentration (1.95 ± 0.54, 1.91 ± 0.57, and 1.77 ± 0.57 μg/ml for the II, IV and VV genotypes, respectively). The distribution of plasma CETP concentrations among the VV, but not II, men appeared bimodal (P < 0.01), suggesting the presence of a functionally significant CETP gene mutation(s) in a subset of V alleles. HDL-C levels were higher in VV than IV for II men (55.4 ± 17.4, 51.3 ± 16.6, 51.1 ± 17.0 mg/dl, P < 0.04). However, the increase in HDL was only significant in VV men with plasma TG > 165 mg/dl. Although CHD prevalence was not significantly different among the three genotypes in this population, in the subpopulation with high plasma TG, CHD prevalence appeared higher among VV than IV or II subjects (38% vs. 27% vs. 18%, P < 0.05 for an interaction of genotype and plasma TG levels). In fresh plasma from a separate group of normolipidemic subjects, the V/I polymorphism was not associated with any change in plasma CETP specific activity. The data suggest that a widespread and common CETP gene mutation(s) in linkage disequilibrium with 405V causes low CETP. Among hypertriglyceridemic men this is associated with higher HDL and possibly with increased CHD.—Bruce, C., D. S. Sharp, and A. R. Tall. Relationship of HDL and coronary heart disease to a common amino acid polymorphism in the cholesteryl ester transfer protein in men with and without hypertriglyceridemia. J. Lipid Res. 1998. 39: 1071–1078. Cholesteryl ester transfer protein (CETP) plays an important role in the transport of cholesterol from tissues to the liver by mediating the transfer of cholesteryl esters from plasma high density lipoproteins (HDL) to triglyceride (TG)-rich lipoproteins (1Tall A. Plasma lipid transfer proteins.Annu. Rev. Biochem. 1995; 64: 235-257Google Scholar, 2Bruce C. Tall A.R. Cholesteryl ester transfer protein, reverse cholesterol transport and atherosclerosis.Curr. Opin. Lipidol. 1995; 6: 306-311Google Scholar). Because the transfer of the cholesteryl esters occurs as part of an exchange reaction with TG, the transfer rate is dependent on very low density lipoprotein TG, HDL-cholesterol (HDL-C), and CETP concentrations. Mann et al. (3Mann C.J. Yen F.T. Grant A.M. Bihain B.E. Mechanism of plasma cholesteryl ester transfer in hypertriglyceridemia.J. Clin. Invest. 1991; 88: 2059-2066Google Scholar) have suggested that in normolipidemic individuals CETP is in excess, so that cholesteryl ester transfer rates are determined by plasma triglyceride concentrations, while in hypertriglyceridemic patients, because CETP is at limiting concentrations, transfer rates are determined by the concentration of plasma CETP. Individuals deficient in CETP have increased HDL levels. Although epidemiological studies have shown high HDL levels to be protective against atherosclerosis (4Rhoads G.G. Gulbrandsen C.L. Kagan A. Serum lipoproteins and coronary heart disease in a population study of Hawaii Japanese men.N. Engl. J. Med. 1976; 294: 293-298Google Scholar, 5Miller N.E. Thelle D.S. Forde O.H. Mjos O.D. The Tromso Heart Study: high density lipoprotein and coronary heart disease: a prospective case-control study.Lancet. 1977; 1: 965-968Google Scholar, 6Gordon T. Castelli W.P. Hjortland M.C. Kannel W.B. Dawber T.R. High density lipoprotein as a protective factor against coronary heart disease: the Framingham Heart Study.Am. J. Med. 1977; 62: 707-714Google Scholar), a recent study of Japanese-American men in the Honolulu Heart Program has shown that CETP deficiency is associated with increased prevalence of coronary heart disease (CHD), despite the resulting elevated HDL levels (7Zhong S. Sharp D.S. Grove J.S. Bruce C. Yano K. Curb J.D. Tall A.R. Increased coronary heart disease in Japanese-American men with mutation in the cholesteryl ester transfer protein gene despite increased HDL levels.J. Clin. Invest. 1996; 97: 2917-2923Google Scholar). The difference in disease prevalence was found to be most marked in men with intermediate HDL levels (40–60 mg/dl), and men with HDL cholesterol >60 mg/dl with or without CETP deficiency had a low CHD prevalence. These results have suggested the importance of the dynamics of the cholesterol transport in plasma in addition to the lipoprotein profile as contributing to CHD risk. Several human CETP mutations have been identified that lead to decreased activity and/or expression. The two most common CETP mutations known to date, the D442G (8Takahashi K. Jiang X-C. Sakai N. Yamashita S. Hirano K. Bujo H. Yamazaki H. Kusunoki J. Miura T. Kussie P. Matsuzawa Y. Saito Y. Tall A. A missense mutation in the cholesteryl ester transfer protein gene with possible dominant effects on plasma high density lipoproteins.J. Clin Invest. 1993; 92: 2060-2064Google Scholar) and the intron 14 splicing (9Brown M.L. Inazu A. Hesler C.B. Abellon L.B. Mann C. Whitlock M.E. Marcel Y.L. Milne R.W. Koizumi J. Mabuchi H. Takeda R. Tall A.R. Molecular basis of lipid transfer protein deficiency in a family with increased high-density lipoproteins.Nature (London). 1989; 342: 448-451Google Scholar) mutations affect about 7 and 2% of the general Japanese population, respectively (10Inazu A. Jiang X-C. Haraki T. Yagi K. Kamon N. Koizumi J. Mabuchi H. Takeda R. Takata K. Moriyama Y. Doi M. Tall A.R. Genetic cholesteryl ester transfer protein deficiency caused by two prevalent mutations as a major determinant of increased levels of high density lipoprotein cholesterol.J. Clin. Invest. 1994; 94: 1872-1882Google Scholar). Recently, other less common mutations have also been described (11Arai T. Yamashita S. Sakai N. Hirano K. Okada S. Ishigami M. Maruyama T. Yamane M. Kobayashi H. Nozaki S. Funahashi T. Kameda-Takemura K. Nakajima N. Matsuzawa Y. A novel nonsense mutation (G181X) in the human cholesteryl ester transfer protein gene in Japanese hyperalphalipoproteinemic subjects.J Lipid Res. 1996; 37: 2145-2154Google Scholar, 12Sakai N. Santamarina-Fojo S. Yamashita S. Matsuzawa Y. Brewer Jr., H.B. Exon 10 skipping caused by intron 10 splice donor site mutation in cholesteryl ester transfer protein gene results in abnormal downstream splice site selection.J Lipid Res. 1996; 37: 2065-2073Google Scholar, 13Gotoda T. Kinoshita M. Shimano H. Harada K. Shimada M. Ohsuga J. Teramoto T. Yazaki Y. Yamada N. Cholesteryl ester transfer protein deficiency caused by a nonsense mutation detected in the patient's macrophage mRNA.Biochem. Biophys. Res. Commun. 1993; 194: 519-524Google Scholar). No common CETP gene mutations have been described in European populations. The contribution of the CETP locus to HDL-C in Europeans has been assessed by sib-pair analysis, and an association between the CETP gene and HDL levels has been shown in one population having many individuals with heart disease (14Cohen J.C. Wang Z. Grundy S.M. Stoesz M.R. Guerra R. Variation at the hepatic lipase and apolipoprotein AI/CII/AIV loci is a major cause of genetically determined variation in plasma HDL cholesterol levels.J. Clin. Invest. 1994; 94: 2377-2384Google Scholar), while a larger study on healthy individuals showed no such association (15Bu X. Warden C.H. Xia Y.R. De Meester C. Puppione D.L. Teruya S. Lokensgard B. Daneshmand S. Brown J. Gray R.J. Rotter J.I. Lusis A.J. Linkage analysis of the genetic determinants of high density lipoprotein concentrations and composition: evidence for involvement of the apolipoprotein A-II and cholesteryl ester transfer protein loci.Hum. Genet. 1994; 93: 639-648Google Scholar). Position 405 of CETP is polymorphic, being either an isoleucine or a valine, due to an A-to-G substitution in exon 14 of the CETP gene (16Agellon L.B. Quinet E.M. Gillette T.G. Drayna D.T. Brown M.L. Tall A.R. Organization of the human cholesteryl ester transfer protein gene.Biochemistry. 1990; 29: 1372-1376Google Scholar). In a preliminary abstract report, the CETP I405V polymorphism was reported to be significantly associated with HDL-C concentration in two European populations (17Funke H. Wiebusch H. Fuer L. Muntoni S. Schulte H. Assmann G. Identification of mutations in the cholesterol ester transfer protein in Europeans with elevated high density lipoprotein cholesterol.Circulation. 1994; 90 (Abstr.): I-241Google Scholar). This association suggested to us that this polymorphism may be associated with variations in the plasma CETP concentration, and if so, possibly with CHD status as well. We undertook to examine whether this association was also present in a population of men of Japanese ancestry living in Hawaii (18Trombold J.C. Moellering Jr., R.C. Kagan A. Epidemiological aspects of coronary heart disease and cerebrovascular disease: the Honolulu Heart Program.Hawaii Med J. 1966; 25: 231-234Google Scholar). The Honolulu Heart Program is a prospective epidemiologic investigation of heart disease and stroke in a population-based cohort of Japanese-American men who were living in the island of Oahu in 1965 (18Trombold J.C. Moellering Jr., R.C. Kagan A. Epidemiological aspects of coronary heart disease and cerebrovascular disease: the Honolulu Heart Program.Hawaii Med J. 1966; 25: 231-234Google Scholar). The men genotyped in this study are a subgroup of the men reported previously (7Zhong S. Sharp D.S. Grove J.S. Bruce C. Yano K. Curb J.D. Tall A.R. Increased coronary heart disease in Japanese-American men with mutation in the cholesteryl ester transfer protein gene despite increased HDL levels.J. Clin. Invest. 1996; 97: 2917-2923Google Scholar) and consist of all men with the low (<30 mg/dl) and high (≥75 mg/dl) HDL-C, and a random 14% of men with intermediate HDL levels. The HDL distribution of the sample was normal. Only those with either definite CHD or no CHD were studied; "possible" CHD cases were excluded. Definite CHD categorization required a) prior myocardial infarction detected by hospital surveillance, b) silent myocardial infarction from electrocardiogram data, c) acute coronary insufficiency, or angina pectoris resulting in surgical intervention, and d) temporal changes in electrocardiogram diagnostic of myocardial infarction. Men carrying the intron 14 splicing defect and the D442G mutations of CETP were excluded in the study of the relationship between the aa405 polymorphism and HDL-C levels and CHD (see Table 4, Fig. 1, 2, 3). To study the relationship of the intron 14 and D442G mutations to the aa405 polymorphism, a subset of the subjects carrying these mutations were genotyped for I405V (see Tables 1, 2, 3).TABLE 4.Relationship of the CETP aa405 polymorphism to CETP and HDL-cholesterolCETP aa405 GenotypeIIIVVVCETP (μg/ml)1.95 ± 0.541.91 ± 0.571.77 ± 0.57aaLess than II and IV values with P < 0.01 and < 0.02, respectively.HDL-C (mg/dl)51.15 ± 17.051.27 ± 16.655.38 ± 17.4bbMore than II and IV values with P < 0.04 and < 0.02, respectively.n126301149a aLess than II and IV values with P < 0.01 and < 0.02, respectively.b bMore than II and IV values with P < 0.04 and < 0.02, respectively. Open table in a new tab Fig. 2.The effect of the aa405 polymorphism on adjusted HDL-C levels is modulated by plasma TG levels. Subjects were segregated into the TG < 165 (n = 374) and TG ≥ 165 (n = 102) mg/dl groups. HDL-C levels between the II and VV subjects in the high-TG group were significantly different (P = 0.05 by unpaired t test).View Large Image Figure ViewerDownload (PPT)Fig. 3.The effect of diffferent TG cutoff points on the interaction between HDL levels and aa405 genotype.View Large Image Figure ViewerDownload (PPT)TABLE 1.Association of the intron 14 splicing mutation and the aa405 polymorphismaa405 PolymorphismInt 14 mutationIIIVVVTotal−/−126301149576+/−59014+/+0000Total1313100590Plus (+) indicates presence of the mutation. Open table in a new tab TABLE 2.Association of the D442G mutation and the aa405 polymorphismaa405 Polymorphismaa442IIIVVVTotalDD126301149576DG1824042GG4000Total148325149618 Open table in a new tab TABLE 3.Relationship of the MspI and aa405 polymorphisms on plasma CETP concentratinsaa405 PolymorphismbbNumber of subjects and their mean ± SD of plasma CETP (μg/ml).MspI PolymorphismaaM1 and M2 indicate the presence and absence of the Msp I cutting site, respectively.IIIVVVTotal13323883M2M2(2.03 ± 0.52)(1.71 ± 0.57)(1.57 ± 0.49)211013M2M1(2.10, 2.10)(1.73 ± 0.41)0000M1M1Total15433896a aM1 and M2 indicate the presence and absence of the Msp I cutting site, respectively.b bNumber of subjects and their mean ± SD of plasma CETP (μg/ml). Open table in a new tab Plus (+) indicates presence of the mutation. Genomic DNA used for genotyping was obtained from buffy coat. The A→G transversion at position 1394 of the CETP cDNA (corresponding to aa405 of the protein) was detected by using a pair of PCR primers (5′-ctcaccatgggcatttgattgcagagcagctccgactcc-3′ and 5′-aatgggaagctctgtcagcctcggccacccag-3′) designed to create a Tth111 I site in the amplification product when the 405V allele was present. For PCR, a 50 μl reaction mixture contained 1 unit of Taq polymerase, about 100 ng of genomic DNA, 50 pmol of each primer, and 10 μm each of dNTPs in a buffer containing 50 mm KCl, 10 mm Tris-HCl, pH 8.3, 1.5 mm MgCl2 and 0.01% gelatin. After 30 cycles (94°C 30 sec, 63°C 1 min, 74°C 1 min) using a Perkin-Elmer DNA Temperature Cycler, 15 μl of the reaction mixture was digested with 1 unit of Tth111 I at the conditions recommended by the manufacturer (New England Biolabs). The Msp I polymorphism was determined as described (19Kuivenhoven J.A. de Knijff P. Boer J.M.A. Smalheer H.A. Botma G. Seidell J.C. Kastelein J.J.P. Pritchard P.H. Heterogeneity at the CETP gene locus. Influence on plasma CETP concentrations and HDL cholesterol levels.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 560-568Google Scholar). The M1 and M2 alleles refer to the presence and absence of the Msp I cutting site. Plasma CETP concentration was determined by competitive radioimmunoassay (20Marcel Y.L. McPherson R. Hogue M. Czarneka H. Zawadski Z. Weech P. Whitlock M.E. Tall A.R. Milne R.W. Distribution and concentration of cholesteryl ester transfer protein in plasma of normolipidemic subjects.J. Clin. Invest. 1990; 85: 10-17Google Scholar). HDL cholesterol was measured as described previously (21Fried L.N. Borhani N.O. Enright P. The Cardiovascular Health Study: design and rationale.Ann. Epidemiol. 1991; 1 (for the CHS Investigators): 263-276Google Scholar). Activity assays were done as described (22Tall A.R. Granot E. Brocia R. Tabas I. Hesler C. Williams K. Denke M. Accelerated transfer of cholesteryl esters in dyslipidemic plasma. Role of cholesteryl ester transfer protein.J. Clin. Invest. 1987; 79: 1217-1225Google Scholar), essentially by incubating 1 μl of fresh plasma at 37°C for 4 h with HDL (5 μg protein) containing radiolabeled cholesteryl ester and low density lipoproteins (LDL) (100 μg protein) in a volume of 100 μl, then precipitating the LDL and counting the radioactivity left in the supernatant. Analyses were conducted using the SAS software program. Descriptive statistics included means, standard deviations, and frequencies statistics were done using PROC FREQ. For continuous variables, adjustments were made to 23.5 kg/m2 body mass index, 50 mg/dl HDL-C, 110 mg/dl calculated LDL-cholesterol, 78 years of age, 80 mm Hg diastolic blood pressure, and 110 mg/dl blood glucose. CHD prevalence was adjusted to reflect no alcohol consumption, no smoking, and no use of medication for hypertension by logistic regression methods (23Wilcosky T.C. Chanbless L.E. A comparison of direct adjustment and regression adjustment of epidemiologic measures.J. Chronic Dis. 1985; 38: 849-856Google Scholar). Measures of association included differences in adjusted means for continuous measures among allelic groups, and odds ratios for measures of frequency as the outcome variable. Statistical significance was set as two-sided type I error of 0.05. Measures of association included differences in adjusted means for continuous measures among allelic groups and odds ratios for measures of frequency as the outcome variable. Statistical significance was set as a two-sided type I error of 0.05. An adjustment with a weighing factor to account for the 14% sampling frame for the 30–75 mg/dl HDL-C range did not change magnitudes of association or descriptive statistics significantly and was not used in order to allow the calculation of P values in statistical tests. The test for bimodality was done using the NOCOM program (24Ott J. Detection of rare major genes in lipid levels.Hum. Genet. 1979; 51: 79-91Google Scholar), which estimates the means, variances, and proportions of a mixture of normal distributions for independent observations and calculates their likelihood values. Briefly, this program calculates the best fit to a given sample distribution after it is constrained to assume that this population consists of a mixture of two normal distributed populations or of a single one. The difference in the likelihood values for the two alternatives indicates whether the assumption of a second component in the population fits the distribution significantly better. The CETP aa405 polymorphism reported previously in European populations (17Funke H. Wiebusch H. Fuer L. Muntoni S. Schulte H. Assmann G. Identification of mutations in the cholesterol ester transfer protein in Europeans with elevated high density lipoprotein cholesterol.Circulation. 1994; 90 (Abstr.): I-241Google Scholar) was found to be similarly prevalent in the Japanese-American population of the Honolulu Heart Program cohort. Of 576 subjects genotyped who had neither the D442G mutation nor the intron 14 splicing mutation in the CETP gene, 126, 301, and 149 had the II, IV and VV genotypes, respectively, at the amino acid 405 (aa405) locus. The I and V alleles have gene frequencies of 0.46 and 0.54, respectively, while the genotype frequencies are consistent with the Hardy-Weinberg equilibrium. We characterized the CETP aa405 polymorphism also in men who did carry the D442G and the intron 14 splicing mutation. The relationships between the genotypes are shown in Tables 1, 2, 3. We also genotyped a subset of our population (96 men) for the Msp I restriction fragment polymorphism in intron 8 (23Wilcosky T.C. Chanbless L.E. A comparison of direct adjustment and regression adjustment of epidemiologic measures.J. Chronic Dis. 1985; 38: 849-856Google Scholar). Individuals who were heterozygous at any of these three loci did not have the VV genotype for the aa405 locus (Tables 1, 2, 3). The data suggest that the D442G and the intron 14 mutations and the Msp I M2 allele are in linkage disequilibrium with the aa405 I allele (Fisher exact test P-value (two-sided) <0.001) (Tables TABLE 1., TABLE 2.). The CETP levels were not significantly different between the M2M1 and M2M2 individuals (Table 3). Based on the 96 people who were genotyped for both the aa405 and the Msp I polymorphism, we determined the haplotype frequencies of the M2-I, M1-I, M2-V, and M1-V alleles to be 0.30, 0.07, 0.63, and 0.00. These numbers are comparable to those reported for the Dutch population (in its middle HDL-C decile): 0.48, 0.13, 0.37, 0.02 (19Kuivenhoven J.A. de Knijff P. Boer J.M.A. Smalheer H.A. Botma G. Seidell J.C. Kastelein J.J.P. Pritchard P.H. Heterogeneity at the CETP gene locus. Influence on plasma CETP concentrations and HDL cholesterol levels.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 560-568Google Scholar), suggesting that the Msp I mutation in the two populations has the same origin. Measurement of plasma CETP concentration of 576 men in the Honolulu Heart Program cohort showed that the aa405 polymorphism is associated with differences in CETP levels. Men with the VV genotype had a lower CETP concentration (1.77 ± 0.57 mg/ml) than II men (1.95 ± 0.54 mg/ml; P < 0.01) while IV heterozygotes were intermediate (1.91 ± 0.57; P < 0.02 for IV vs. VV, P not significant for IV vs. II) (Table 4). As a recent study reported an Msp I polymorphism in the CETP gene to be a better predictor of plasma CETP levels than the aa405 polymorphism (19Kuivenhoven J.A. de Knijff P. Boer J.M.A. Smalheer H.A. Botma G. Seidell J.C. Kastelein J.J.P. Pritchard P.H. Heterogeneity at the CETP gene locus. Influence on plasma CETP concentrations and HDL cholesterol levels.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 560-568Google Scholar), we examined whether this was the case for the Japanese-American population of Hawaii. The CETP levels were not significantly different between the M2M1 and M2M2 individuals (Table 3c). The I405V substitution of CETP is a conservative substitution that is unlikely to alter function. Moreover, the specific activity of plasma CETP is identical in II and VV subjects (see below). This suggests that the V allele may be linkage disequilibrium with one or more mutations that determine plasma CETP levels. To evaluate this possibility, the frequency distribution of CETP levels among the three genotypes (II, IV, VV) was determined. Despite their different mean CETP values, their modes were essentially identical. The distribution of CETP values in subjects below the 50th percentile appeared strikingly different for the three genotypes. In the VV, IV, and II groups about 34%, 20%, and 15% of the subjects had a CETP concentration less than 1.5 μg/ml, respectively (Fig. 1). A bimodal distribution is suggested by a shoulder in the VV curve, while the II curve has the typical shape for a normal distribution. A test for bimodality of the distribution curve using the NOCOM program (24Ott J. Detection of rare major genes in lipid levels.Hum. Genet. 1979; 51: 79-91Google Scholar), indicated that for the VV population the distribution is more consistent with a 2-component mixture (1.03 ± 0.05 μg/ml, 8% proportion, and 1.83 ± 0.55 μg/ml, 92% proportion) than a one-component mixture (P < 0.01). Two subpopulations with 22% and 78% proportions, having means of 1.26 ± 0.29 and 1.91 ± 0.54 μg/ml, respectively, also provide a good fit to the observed distribution of the VV subjects, but their likelihood value does not quite reach significance. For the II population, the likelihood value for a two component mixture was not significantly better than a one component mixture with a mean CETP concentration of 1.95 ± 0.54 μg/ml. Similar to previous observations on European populations (17Funke H. Wiebusch H. Fuer L. Muntoni S. Schulte H. Assmann G. Identification of mutations in the cholesterol ester transfer protein in Europeans with elevated high density lipoprotein cholesterol.Circulation. 1994; 90 (Abstr.): I-241Google Scholar), there was an association between the polymorphism at aa405 and HDL levels (Table 4). When HDL-C levels were analyzed among the three aa405 polymorphism categories, the adjusted mean value of 55.4 mg/dl is about 10% higher in the VV group compared to the II and IV groups (P < 0.04 and < 0.02, respectively), while there appears to be no significant difference between the II and IV groups in HDL-C (Table 4). There was no correlation between the CETP concentration in the subjects analyzed and their HDL levels. Because the activity of plasma CETP is driven by hypertriglyceridemia (3Mann C.J. Yen F.T. Grant A.M. Bihain B.E. Mechanism of plasma cholesteryl ester transfer in hypertriglyceridemia.J. Clin. Invest. 1991; 88: 2059-2066Google Scholar, 25Hayek T. Azrolan N. Verdery R.B. Walsh A. Chajek-Shaul T. Agellon L.B. Tall A.R. Breslow J.L. Hypertriglyceridemia and cholesteryl ester transfer protein interact to dramatically alter high density lipoprotein levels, particle sizes and metabolism. Studies in transgenic mice.J. Clin. Invest. 1993; 92: 1143-1152Google Scholar), we next segregated the population by plasma total triglyceride values to see whether the relationships between the aa405 polymorphism and CETP were enhanced among men with high TG values. A cut-off of 165 mg/dl TG placed about one-third of our population in the "high TG" group. With this cut-off, the high-TG group had lower HDL levels than the low-TG group (Fig. 2). The TG ≥ 165 mg/dl group accounted for nearly all the effect of the aa405 polymorphism on HDL levels (P < 0.05), while the low-TG group's HDL values did not differ significantly among the II, IV, and VV men (Fig. 2). Cut-off points at higher TG levels produced similar results, i.e. the slope of the relationship between CETP genotype and HDL levels was steeper in more hypertriglyceridemic subjects (Fig. 3). There was no significant difference in CETP levels between the low- and high-TG groups: the mean (±SD) of plasma CETP concentration for the II, IV, and VV groups were, respectively, 1.94 (±0.56), 1.88 (±0.56), and 1.80 (±0.59) for the low-TG group, and 1.99 (±0.50), 1.98 (±0.59), and 1.68 (±0.51) for the high-TG group. VV men had significantly lower CETP levels than II men (P < 0.05) in both the low and high triglyceride groups. A logistic regression model predicting CHD status from HDL cholesterol was used to confirm that in the present population, the odds ratio for definite CHD for a 1 mg/dl increase in HDL cholesterol is 0.978, which is highly significant (P < 0.001) and consistent with previous data showing an overall inverse relationship between HDL levels and CHD (4Rhoads G.G. Gulbrandsen C.L. Kagan A. Serum lipoproteins and coronary heart disease in a population study of Hawaii Japanese men.N. Engl. J. Med. 1976; 294: 293-298Google Scholar). However, several statistical tests failed to show any overall relationship between the aa405 polymorphism and CHD status. Also, there was no significant difference in CHD prevalence among the three aa405 genotypes when tertiles of HDL-C values were analyzed individually. However, when the effect of the aa405 polymorphism on disease prevalence was examined for the low- and high-TG groups, the patterns of CHD prevalence showed different relationships with the aa405 genotype, i.e., amongst high TG subjects, disease prevalence tended to increase going from the II to IV to VV genotypes, while amongst low TG subjects it tended to decrease (chi-square = 6.010, df = 2, P = 0.05). In the hypertriglyceridemic group, CHD prevalence among VV men was found to be about twice that of II men (37% vs. 18%) (Fig. 4). In the group with TG < 165 mg/dl, there was an insignificant trend for lower CHD prevalence among VV men relative to IV and II men (20% vs. 30% and 29%, respectively). These findings suggest that hypertriglyceridemic VV subjects have increased CHD, while normotriglyceridemic VV subjects have a trend toward lower CHD prevalence. Although the aa405 polymorphism of CETP is a conservative amino acid substitution, the lower CETP levels in VV subjects could directly reflect this change. We reasoned that a functionally significant missense mutation causing low CETP levels would probably cause changes in specific activity. To assess this possibility, we collected fresh plasma from 22 healthy volunteers (reliable activity measurements necessitate fresh plasma), measured their plasma CETP concentration, performed an activity assay
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