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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:560-568.)
© 1997 American Heart Association, Inc.

Articles

Heterogeneity at the CETP Gene Locus

Influence on Plasma CETP Concentrations and HDL Cholesterol Levels

Jan Albert Kuivenhoven; Peter de Knijff; Jolanda M.A. Boer; Harold A. Smalheer; Gert-Jan Botma; Jaap C. Seidell; John J.P. Kastelein; ; P. Haydn Pritchard

From the Department of Vascular Medicine, Academic Medical Center, University of Amsterdam (J.A.K., H.A.S., G.-J.B., J.J.P.K.); Department of Human Genetics, Sylvius Laboratory, Leiden University (P.de K.); and Department of Chronic Diseases and Environmental Epidemiology, National Institute of Public Health and Environmental Protection, Bilthoven (J.M.A.B, J.C.S.), The Netherlands; and the Atherosclerosis Specialty Laboratory, Department of Pathology and Laboratory Medicine, St Paul's Hospital and University of British Columbia, Canada (P.H.P.).

Correspondence to J.A. Kuivenhoven, Academic Medical Center, Department of Vascular Medicine (G1-114), Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail kuif{at}wnet.bos.nl.

	Abstract

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Abstract This study was designed to investigate the association(s) between heterogeneity at the cholesteryl ester transfer protein (CETP) gene locus, CETP plasma concentrations, and HDL cholesterol levels. Healthy men with the lowest, median, and highest deciles of HDL cholesterol were selected from a large population database. We accounted for factors that are known to influence HDL cholesterol levels, such as smoking, exercise, body mass index, alcohol consumption, and blood pressure. Plasma CETP concentrations were measured, and we determined the allele frequency distribution of six CETP DNA polymorphisms. The group with low HDL cholesterol exhibited a significant increase in CETP concentration compared with both the median and high HDL cholesterol groups, whereas CETP concentrations did not differ among the groups with median and high HDL cholesterol. The allele frequency distributions of the TaqIB (intron 1), Msp I (intron 8), and Rsa I (exon 14) polymorphisms differed significantly between the groups with low and high HDL cholesterol. Further analysis revealed that the Msp I polymorphism had a 1.5-fold larger impact on CETP concentration than the TaqIB polymorphism and a fivefold larger impact than the Rsa I polymorphism. In conclusion, we demonstrated that heterogeneity at the CETP gene locus is correlated with CETP plasma concentrations and HDL cholesterol levels. More specifically, our data indicate the presence of a strong association between common variants of the CETP gene, high plasma CETP concentrations, and consequently hypoalphalipoproteinemia in healthy white men.

Key Words: restriction fragment length polymorphism • cholesteryl ester transfer protein • high-density lipoprotein • coronary artery disease • haplotyping

	Introduction

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The strong inverse correlation between plasma HDL cholesterol levels and the incidence of CAD^{1 2 3} provides the rationale for a widespread interest in both environmental and genetic factors that regulate plasma HDL cholesterol levels. HDL plays a central role in the transport of cholesterol from peripheral tissues (including coronary arteries) to the liver. This process of reverse cholesterol transport^{4 5 6} is believed to explain the major antiatherogenic mode of action of this lipoprotein fraction. Part of this process involves the esterification of free cholesterol within the HDL fraction and its subsequent transfer by CETP to TG-rich lipoproteins.⁷ To date, our knowledge concerning the physiological role of CETP in lipoprotein metabolism is incomplete. Variations in the efficiency of the removal of excess cholesterol from peripheral tissues or the cholesteryl ester transfer between lipoprotein classes could be of vital importance in atherogenesis. Evidence is accumulating that increased CETP activity actually may be a risk factor for atherosclerosis. First, CETP activity is reported to be inversely associated with HDL cholesterol.^{8 9 10 11 12} Furthermore, CETP activity is increased in some hyperlipidemic conditions,^{13 14} and it was shown that patients with established CAD have increased transfer of cholesteryl ester from HDL to TG-rich (atherogenic) lipoproteins.¹⁵ Second, CETP mass levels are increased in certain metabolic states,¹⁶ and CETP mass is reported to be positively associated with carotid artery wall thickening.¹⁷ Finally, recent studies with transgenic mice overexpressing the human CETP gene indicate that these mammals develop an atherogenic lipoprotein profile as a result of enhanced CETP activity.^{18 19} In addition, Marotti et al²⁰ showed that transgenic mice overexpressing the simian CETP gene develop severe atherosclerosis on a high-fat, high-cholesterol diet. By contrast, overexpression of CETP has recently been shown to inhibit the development of early atherosclerotic lesions in transgenic mice that overexpressed apo C-III.²¹ This finding argues that enhanced CETP activity might be beneficial in specific hyperlipidemias.

Human CETP is a plasma glycoprotein of M_r 74 000 that mediates the transfer of cholesteryl esters, phospholipids, and TGs between plasma lipoproteins.²² The gene spans 25 000 base pairs and is composed of 16 exons.²³ Gene defects that result in a complete loss of CETP activity are one cause of hyperalphalipoproteinemia,^{24 25} which was originally described to be associated with an increased lifespan.²⁴ However, Hirano et al²⁶ recently reported atherosclerotic disease in heterozygotes for CETP deficiency with low hepatic lipase activities.

Several DNA polymorphisms of the CETP gene have been described.^{27 28 29} A variant identified by the restriction enzyme Taq I located in intron 1 (TaqIB) has been reported to be associated with plasma CETP activity,^{9 10} apo A-I,³⁰ and HDL cholesterol^{9 10 30 31 32 33} as well as with HDL subfractions.³⁴ The effects of the TaqIB RFLP on HDL cholesterol levels were seen in both diseased³¹ and healthy subjects.^{9 30 31 32} Several investigators have concluded that the association between the TaqIB RFLP and HDL cholesterol most likely reflects a true biological phenomenon.^{9 30} Furthermore, a recent study showed that a polymorphic missense mutation in exon 14 of the CETP gene was associated with HDL cholesterol levels in the general population.³⁵

To date, the majority of single RFLP association studies have described an association between variation at the CETP gene locus and HDL cholesterol levels.^{9 10 30 31 32 33 34 35} In addition, Bu et al³⁶ confirmed the presence of such an association in a quantitative sib-pair linkage analysis. Interestingly, the genotype effect was also reported to be sex-specific in another sib-pair linkage analysis study.³⁷ This finding has also been confirmed by a RFLP association study by Kauma et al.³³ By contrast, other reports describe the absence of a relationship between the CETP gene and HDL cholesterol. Cohen et al³⁸ showed that none of the variation in plasma HDL cholesterol could be accounted for by allelic variation in CETP when they compared sibs of 73 nuclear families. The different findings of the sib-pair linkage analyses by Bu et al³⁶ and Cohen et al³⁸ might be related to the fact that the former studied normotriglyceridemic white families and the latter studied white families with CAD. This might illustrate the effect of different selection procedures. Furthermore, Mitchell et al³² and Tenkanen et al³⁹ reported the absence of such effects in Italian and Finnish subjects, respectively, which indicates that the association between the CETP gene variants and lipid phenotypes might be influenced by differences in genetic background.

To investigate the genetic control of low HDL cholesterol levels in the general population, we selected subjects from a large representative sample of healthy Dutch men with upper, middle, and lower deciles of HDL cholesterol. The present study describes the association between six CETP gene restriction site polymorphisms, plasma CETP concentrations, and plasma HDL cholesterol levels.

	Methods

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Subjects and Materials
The subjects were selected from more than 17 000 male participants of the Monitoring Project on Cardiovascular Disease Risk Factors that has been implemented in the Netherlands from 1987 through 1991.⁴⁰ In short, in this project risk factors for CAD were evaluated in a random population sample of both men and women from three Dutch towns. The height, weight, and blood pressure of each subject was measured. BMI was defined as weight (kg)/height² (m). Three 10-mL nonfasting blood samples were obtained in EDTA-coated vacuum tubes. After centrifugation for 10 minutes at 1000 rpm, plasma was stored at -20°C in 1.5-mL aliquots. From each tube, the buffy coat and the remaining red blood cells were stored at -20°C in 4-mL tubes. One sample of 1.5 mL of EDTA plasma was used directly for determination of total and HDL cholesterol.

The selection of subjects with low, median, or high HDL cholesterol from the Monitoring Project as well as the matching procedure is described in Table 1. Genomic DNA was extracted from buffy coats as described previously.⁴¹

View this table: [in this window] [in a new window]   Table 1. Selection and Matching of Subjects Investigated

Lipids, HDL Cholesterol, and CETP Mass Analysis
Plasma total cholesterol was determined enzymatically with a Boehringer test kit.⁴² HDL cholesterol was determined by the same method after precipitation of the apo B–containing lipoproteins with magnesium phosphotungstate.⁴³ Nonfasting plasma TG levels were determined enzymatically (Biomerieux). Plasma CETP concentration was measured by solid-phase competitive radioimmunoassay in the presence of 0.5% Triton⁴⁴ and a specific monoclonal antibody⁴⁵ as described previously.¹⁶

PCR-Based Detection of Polymorphisms of the CETP Gene
The Taq I polymorphisms (TaqIA and TaqIB) and Msp I polymorphism were originally described by Drayna and Lawn,²⁷ and the EcoNI RFLP was reported by Zuliani and Hobbs.²⁹ Our laboratory successfully mapped these DNA polymorphisms to their corresponding exons and introns, which enabled us to design PCR-based analyses. The BamHI RFLP analysis was performed as described by Inazu et al.²⁴ The sixth RFLP investigated, considered an A-to-G nucleotide substitution in exon 14, was recently reported by Funke and coworkers.³⁵ This base substitution did not result in the creation or disappearance of an enzyme restriction site. Therefore, we used a mismatch primer that introduced an Rsa I restriction site in the presence of the mutation. Target sequences were amplified with oligonucleotides as presented in Table 2. The sequence information was obtained from data supplied by Agellon et al.²³ The amplification reactions were carried out in 10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 0.1% wt/vol gelatin, 1.5 mmol/L MgCl₂, 1% Triton X-100, and 20 mg/dL BSA containing 0.1 to 0.5 µg genomic DNA and final concentrations of 100 to 200 µmol/L dNTPs and 0.5 µmol/L primers in a total volume of 50 µL. After initial denaturation (10 minutes, 95°C), 0.3 to 0.5 U thermostable DNA polymerase (Supertaq, HT Biotechnology) was added, followed by 30 amplification cycles at 95°C (1 minute), 60°C (1 minute), and 72°C (1 to 2 minutes), with a final extension step of 10 minutes at 72°C. Twenty percent of the PCR reaction product was used for digestion with 2 to 3 U of the respective endonucleases according to the instructions of the manufacturer (New England Biolabs) in a total volume of 20 µL for 2 hours at 37°C. After electrophoresis of the PCR product in 1% to 2% agarose containing ethidium bromide, DNA restriction fragments were visualized and analyzed on a transilluminator.

View this table: [in this window] [in a new window]   Table 2. Oligonucleotides Used for Amplification of Polymorphic Regions of CETP Gene, Estimated Length of PCR Products, and Estimated Length of Digestion Fragments

Statistical Analysis
All subjects selected for this study were categorized according to their HDL cholesterol levels. Means were compared between groups by ANOVA, and the Tukey test was performed to adjust for multiple comparisons. Allele frequencies were determined by direct counting, and ² analysis was used to test for Hardy-Weinberg equilibrium of all CETP gene variants. The Fisher exact test implemented in the statistical package StatXact-Turbo (CYTEL Software Corp) was used to estimate differences in genotype distributions and estimated haplotype distributions between the three HDL cholesterol classes. For the most promising RFLPs, ie, TaqIB, Msp I, and Rsa I, a three-marker haplotype was estimated in each of the three HDL cholesterol groups. Since all individuals were unrelated, E-M algorithms implemented in the program 3LOCUSA⁴⁶ were used to provide maximum-likelihood estimates of the three-marker haplotype frequencies and to quantify pairwise and global linkage disequilibria. These likelihood calculations, which were based on the observed Hardy-Weinberg equilibria, allowed linkage disequilibrium to differ between the three HDL cholesterol classes and assumed no interaction between any allele or haplotype by an individual.

	Results

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Population Sample Characteristics and CETP Mass
Sufficient DNA was isolated from samples from 98 men with low plasma HDL cholesterol, 70 with median HDL cholesterol, and 73 with high HDL cholesterol. Table 3 summarizes the lifestyle parameters, clinical features, and lipids of the men included in this study. The three groups were matched for total cho-lesterol, age, BMI, systolic blood pressure, inactivity, and alcohol consumption. However, a tendency remained toward a lower BMI, higher physical activity, and higher alcohol consumption with increasing HDL cholesterol. Moreover, analysis of the lifestyle and clinical parameters revealed that subjects in the high-HDL group had significantly lower diastolic blood pressure than men with low plasma HDL cholesterol (P<.05).

View this table: [in this window] [in a new window]   Table 3. Clinical Features, Lifestyle Parameters, Lipids, and Lipoproteins of the Population Samples Investigated

With regard to lipid values, the statistically significant decrease in TG levels when both the groups with low and median HDL cholesterol and median and high HDL cholesterol (P<.05) are compared illustrates a clear inverse relation between plasma HDL cholesterol and TG levels.

The sample with low HDL cholesterol presented with highly significantly elevated CETP concentrations (P<.000001) compared with both the groups with median and high HDL cholesterol (Table 4).

View this table: [in this window] [in a new window]   Table 4. Plasma CETP Concentrations in Healthy Male Subjects According to HDL Cholesterol Percentiles

Distributions and Frequencies of CETP Polymorphisms
Using PCR-based DNA analysis, we determined the allele frequency distribution of six CETP RFLPs (ie, TaqIB, intron 1; Msp I, intron 8; BamHI, intron 9; EcoNI, intron 9; TaqIA, intron 10; and Rsa I, exon 14) in the three population samples (Table 5). For each of the three different population samples (analyzed separately as well as in the combined population), the observed frequencies were in Hardy-Weinberg equilibrium. The observed allele frequencies of the rare gene variants of the median HDL cholesterol group (data not shown) were similar to those reported for other white population samples.^{10 30 31 32 34 35 39} In addition, the allele frequencies did not differ between the subjects from the three Dutch towns (data not shown). Furthermore, the TaqIB, Msp I, and Rsa I RFLPs, encompassing a large region of the CETP gene (intron 1 up to exon 14), were in linkage disequilibrium in the groups with low and median HDL cholesterol (P<.01; corrected for multiple comparison) as well as for the whole group of subjects investigated (P<.01).

View this table: [in this window] [in a new window]   Table 5. Distributions of the CETP RFLPs in Healthy Male Subjects According to HDL Cholesterol Percentiles

We identified significant statistical differences between the allele frequency distribution of the TaqIB, Msp I, and Rsa I polymorphisms in the three HDL groups. First, the allele frequency distribution for the TaqIB polymorphism was significantly different (P<.003) between the groups with the lowest and highest deciles of HDL cholesterol. Second, the allele frequencies for the Msp I RFLP were statistically significantly different between both the groups with low and median HDL cholesterol (P<.027) and the groups with low and high HDL cholesterol (P<.000001). The presence of a TaqIB site in intron 1 (B1 allele) and the presence of an Msp I site in intron 8 (M1 allele) were both strongly associated with decreased plasma HDL cholesterol. Third, the presence of an Rsa I site in exon 14 (R1 allele) was associated with high HDL cholesterol levels (P<.034).

CETP Genotypes and CETP Mass
After pooling of the three groups investigated, we identified highly significant associations between the TaqIB, Msp I, and Rsa I genotypes and plasma CETP concentrations (P<.0001, P<.0001, and P=.015, respectively) (Table 6). However, since a pool of all subjects cannot be regarded as a random population sample, caution should be used in interpreting the statistical significance of this association. Further analysis revealed that variation at the TaqIB and Msp I loci explained 10.6% and 16.8%, respectively, of the variance of CETP concentrations, whereas the Rsa I genotype variation explained 3.6% of this variance.

View this table: [in this window] [in a new window]   Table 6. TaqIB, Msp I, and Rsa I RFLPs and CETP Mass Regarding All Subjects Investigated Who Were Selected for Low, Median, and High HDL Cholesterol

Distributions of CETP Haplotypes
Table 7 illustrates maximum-likelihood estimates of the three-marker haplotypes in the CETP gene. All pairwise marker comparisons showed significant linkage disequilibrium in the low HDL cholesterol group (P<.05, corrected for multiple comparisons). In the median HDL cholesterol group, only the TaqIB–Msp I combination was in linkage disequilibrium. Finally, we identified complete equilibrium between the three markers in the high HDL cholesterol group. When the estimated haplotype distributions were compared between the three groups, we observed a marked difference between the groups with low and high HDL cholesterol, respectively (Table 7). This significant difference (P=.024) was primarily due to differences in the frequencies of two haplotypes: B1-M1-R2 and B2-M2-R1. This is in line with the differences in genotype distributions between the three HDL cholesterol groups and the associations between the genotypes, HDL cholesterol levels, and CETP mass (Tables 5 and 6). The haplotype that, according to our results, should associate with the lowest HDL cholesterol levels and highest CETP mass, B1-M1-R2, is indeed markedly overrepresented in the low HDL cholesterol group, whereas the opposite is true for the haplotype associated with the highest HDL cholesterol and lowest CETP mass (B2-M2-R1).

View this table: [in this window] [in a new window]   Table 7. CETP Haplotype Frequency Distribution in the Three HDL Cholesterol Classes

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Male subjects who present with premature CAD often have decreased HDL cholesterol levels as an isolated lipid disorder.⁴⁷ Since we are interested in the genetic contribution to atherosclerosis and elevated CETP activity is thought to be one metabolic cause of low HDL cholesterol, we studied the relationship(s) between CETP gene variants, plasma CETP concentrations, and plasma HDL cholesterol levels. The frequency of six RFLPs spread over the CETP gene was determined in healthy men with low, median, and high plasma HDL cholesterol. These groups were matched for lifestyle parameters and clinical features that affect HDL cholesterol levels. Consequently, differences in this lipoprotein fraction most likely reflect variation in the genetic constitution of our study participants. Interestingly, the samples, which were selected from a large database (17 138 subjects), could not be entirely matched for BMI, alcohol consumption, and inactivity, which indicates the strong influence of these factors on plasma HDL cholesterol levels in the general population.

CETP Concentration and HDL Cholesterol Levels
CETP concentration measurements revealed that subjects with low HDL cholesterol exhibited highly significantly elevated plasma CETP concentrations compared with both other samples. Since CETP concentrations correlate closely with CETP activity in normal plasma,¹⁶ this finding provides in vivo evidence that low HDL cholesterol may be associated with high CETP activity in healthy male subjects. These results are in agreement with findings by Tato et al,¹² who recently suggested that elevated CETP activity may be one significant factor related to low HDL cholesterol levels in a group of normolipidemic patients. However, in contrast to the findings of Tato et al,¹² who reported a bimodal distribution in CETP concentrations among the subjects with low HDL cholesterol, we identified a unimodal distribution of CETP concentrations (data not shown). These differences might be related to the fact that we did account for environmental factors that are known to affect HDL cholesterol levels, whereas the population sample studied by Tato et al included ß-blocker users (22%) and subjects with hypertension (46%). Especially the inclusion of smokers (38%) might underlie the differential findings. In this respect, Hannuksela et al¹⁰ recently showed that the association of TaqIB with plasma CETP activity was affected by smoking.

Furthermore, our data demonstrated that upper decile HDL cholesterol was not associated with low CETP concentrations. This may indicate the presence of other genetic factors that determine high HDL cholesterol levels in our population.

Interestingly, relatively frequent mutations in the CETP gene identified in Japan are shown to be associated with hyperalphalipoproteinemia.^{48 49} We speculate, therefore, that the role of the CETP gene in determining high HDL cholesterol in Japanese and whites differs as a result of the absence of frequent functional CETP mutations in whites.

Variants of the CETP Gene and Plasma HDL Cholesterol Levels
The TaqIB and Msp I RFLPs, and to a lesser extent the Rsa I polymorphism, provided the most conclusive results. First, on its own, the B1 allele was strongly associated with low HDL cholesterol levels, which is in agreement with the results of a number of other studies.^{9 10 30 32} However, our findings strongly suggest that the B1 allele merely serves as a marker for specific CETP haplotypes, which will be discussed below. The lack of an association between the TaqIB RFLP and HDL cholesterol reported by some investigators might be due to the interference of factors such as ethnic origin^{32 39} and the fact that the associations were not determined in the context of the specific CETP haplotypes.

In this study, we compared three carefully selected groups that were fully matched for factors known to influence HDL cholesterol levels. Therefore, our findings strongly support the hypothesis that the B1 allele is indeed associated with hypoalphalipoproteinemia. In addition to the TaqIB RFLP, we also identified a highly significant association between the Msp I genotype and plasma HDL cholesterol levels. The M1 allele, like the B1 allele, was associated with low HDL cholesterol. Further studies are needed to investigate the potential of this DNA marker to predict HDL cholesterol levels. The question as to whether the Msp I RFLP is part of a haplotype that includes the TaqIB RFLP will be discussed below.

In contrast to the other CETP gene variants, the Rsa I RFLP affects the primary structure of the CETP protein (Ile⁴⁰⁵Val).³⁵ Funke and coworkers³⁵ showed that the R1 allele (denoted ⁴⁰⁵V) was associated with increased HDL cholesterol. In support of these findings, we identified 6%, 13%, and 20% homozygotes for the R1R1 genotype in samples with low, median, and high HDL cholesterol levels, respectively. However, the differences in allele frequency distributions between the three groups reached only borderline statistical significance, and the Rsa I RFLP locus explained only 3.6% of the variance of CETP concentrations in the total population sample. This indicates that a direct or indirect effect of this mutation on CETP will not have significant effects on HDL cholesterol levels in vivo.

The absence of associations between the TaqIA, EcoNI, and BamHI RFLPs and HDL cholesterol levels indicates the importance of studying DNA polymorphisms throughout a candidate gene while performing RFLP association studies.

CETP Gene Variants, CETP Mass, and HDL Cholesterol Levels
Several studies provide evidence for the presence of an association between the TaqIB RFLP and CETP activity^{9 10} or between this RFLP and plasma HDL cholesterol levels.^{9 10 30 31 32 34} However, the relationship between plasma CETP activity and HDL cholesterol levels has not been clarified. Freeman et al⁹ initially reported an inverse association between HDL and CETP activity in highly selected population samples for low and high HDL cholesterol levels. However, a different study³⁴ did not detect an association between the TaqIB genotype, plasma CETP activity, and HDL cholesterol. The lack of an association between CETP activity and HDL cholesterol has also been reported by other investigators.⁴⁴

The present study, however, provides strong evidence for a correlation between variants of the CETP gene, high CETP concentrations, and low HDL cholesterol levels. First, genetic variation at the loci for TaqIB and Msp I RFLPs explains a significant proportion of the variation in CETP concentrations (ie, 10.6% and 16.8%, respectively) in all subjects investigated, whereby the B1 and M1 alleles were associated with high CETP concentrations. Second, we demonstrated that these alleles are associated with low HDL cholesterol. When we assume a positive correlation between CETP concentration and CETP activity⁴⁴ and an inverse correlation of these parameters with HDL cholesterol levels, our findings are in agreement with the outcome of a study performed by Hannuksela et al.¹⁰ These investigators observed a relation between the TaqIB RFLP and CETP activity that was opposite to that between the RFLP and HDL cholesterol levels in nonalcoholic, nonsmoking male subjects. In an attempt to explain the association between the TaqIB and Msp I RFLPs and plasma HDL cholesterol levels, we suggest that these DNA markers are in linkage disequilibrium with genetic changes that affect the expression of the CETP gene. CETP mass is directly proportional to the cholesteryl ester transfer activity in normal plasma,¹⁶ and therefore, the observed differences in HDL cholesterol could result from differences in total enzyme activity.

Another explanation might involve enhanced cholesteryl ester transfer and consequently low HDL cholesterol as the result of higher TG levels (the availability of higher concentrations of TG-rich acceptor lipoproteins)^{50 51} in the low-HDL group.

To further study the proposed effect of the CETP genotype on HDL cholesterol levels, we estimated the three-marker haplotype frequencies for the TaqIB, Msp I, and Rsa I RFLPs in the three HDL cholesterol classes as described by Long et al.⁴⁶ These three-marker haplotype distributions differed significantly between the groups with low and high HDL cholesterol. In line with our previous description of associations between the TaqIB, Msp I, and Rsa I genotypes, CETP mass, and HDL cholesterol, the B1-M1-R2 haplotype was overrepresented in the low HDL cholesterol group, whereas the B2-M2-R1 haplotype was overrepresented in the high HDL cholesterol group. To further investigate this finding, we selected for all subjects who were homozygous for either B1-M1-R2 or B2-M2-R1. The former (n=8) exhibited higher CETP concentrations and lower HDL cholesterol than did homozygotes for B2-M2-R1 (n=10) (CETP: 3.30±0.86 versus 2.14±0.65 µg/mL; HDL cholesterol: 0.95±0.25 versus 1.32 mmol/L). In addition, we have estimated the three marker haplotype distributions in CETP mass subgroups that were defined by tertiles of the distribution described in the present study (data not shown). In agreement with our other findings, the B2-M2-R1 haplotype was underrepresented and B1-M1-R2 overrepresented in the group with highest CETP concentrations. In addition, the linkage between the three markers was strongest in the group with highest CETP concentration, as was identified in the group with lowest HDL. Although not obtained from a random population sample, these data illustrate biological consistency between our findings and moreover support the notion that CETP gene variants affect plasma HDL cholesterol levels.

Conclusions
In conclusion, we have demonstrated that heterogeneity at the CETP gene locus is associated with changes in CETP plasma concentrations and HDL cholesterol levels. Specifically, we have shown that common CETP gene variants are associated with hypoalphalipoproteinemia in healthy men. The association of this phenotype with an increased risk for atherosclerosis indicates the need to identify the actual gene change(s) that cause the suggested differential expression of the CETP gene. Although the present study permits only speculation about the underlying mechanisms, our results indicate that the association between specific CETP gene variants and low HDL cholesterol levels is mediated by high plasma CETP concentrations. These findings would support the development of drug treatment of low HDL cholesterol by inhibitors of the CETP protein. However, Tato et al¹² recently reported less CAD in patients with low HDL and high CETP activity than in subjects with low HDL and normal CETP activity. In addition, others indicated that CETP activity might inhibit the progression of atherosclerotic lesions in hypertriglyceridemic mice.²¹ These findings clearly illustrate our incomplete knowledge of the exact role of CETP in atherosclerosis and the need of further in-depth investigations.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein BMI = body mass index CAD = coronary artery disease CETP = cholesteryl ester transfer protein PCR = polymerase chain reaction RFLP = restriction fragment length polymorphism TG = triglyceride

	Acknowledgments

 
This project was supported by a grant (89201) from the Dutch Heart Foundation. Dr J.J.P. Kastelein is a clinical investigator of the Dutch Heart Foundation. We would like to thank Paul Reymer and Björn Groenemeyer for providing us with the information on the location of the RFLPs studied. Furthermore, we wish to thank Anne Weenink for her assistance in carrying out the PCR analyses and Anneke Blokstra for performing the selection and matching procedure of the population samples. In addition, we are obliged to Dr R. McPherson for the determination of CETP mass.

Received December 12, 1995; accepted July 12, 1996.

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