Interaction between Polymorphism of rs on CETP and. Environmental Factors are Associated with Low HDL-C Levels. in Chinese

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1 Interaction between Polymorphism of rs on CETP and Environmental Factors are Associated with Low HDL-C Levels in Chinese LIU Xiaoli,HE Shan 1,ZHOU Jichang 1,WANG Jun 1, SUN Xiufa 2,XU Jian 1 (1.Shenzhen center for chronic disease control, Shenzhen, China;2. Department of Nutrition and food hygiene, Huazhonguniversity of science and technology, Wuhan, China.) Abstract:Conflict results were reported about the association between multiple single nucleotide polymorphisms (SNPs) and low plasma high-density lipoprotein cholesterol (HDL-C) level in different populations. We conducted this study to find these potential associations in Chinese. Method: Twelve candidate SNPs in HDL-C metabolic genes were genotyped by PCR-RFLP method in 3809 Shenzhen citizens from a cross-sectional study. Plasma HDL-associated lipoproteins, enzymes and lipid parameters of each subject were also tested. Results:The genotype AA in rs of cholesteryl ester transfer protein (CETP) intron was associated with low HDL-C level in Chinese (OR adj =0.78 for AA vs. GG, P<0.05) and the HDL-C level of homozygous mutation carriers was significantly higher than that of homozygous wild-type carriers (1.28±0.31mmol/L vs. 1.20±0.16mmol/L). Multifactor dimensionality reduction analysis found that the male subjects with non-alcohol consumes, smoke and contained genotype GG in rs was subjects with highest risk for abnormally reduced HDL-C level (OR=7.32, P=0.012, compared with contrary subjects).conclusion:homozygous mutant of rs was associated with low HDL-C level in Chinese. Gene-environmental interaction was found between rs708272, sex, smoking and alcohol consumption, and associated with low HDL-C level. Keywords:high-density lipoprotein cholesterol, single nucleotide polymorphisms, Chinese 1 INTRODUCTION High-density lipoprotein (HDL) is groups of lipoprotein with the smallest size. Abnormally reduced HDL-C levels are generally believed as a risk factor for atherosclerosis and CHD [1].HDL-C level lower than 1.03mmol/L (40mg/dL) was considered as low HDL-C, and risk for heart disease increased under low HDL-C [2]. There were multiple apolipoproteins and enzymes involved in the metabolism of HDL-C. Among them the apolipoprotein A1 and A2 (ApoA1 and ApoA2) are the major structural components of HDL [3]. Main enzymes involved in the HDL-C metabolism include ATP-binding cassette transporter A1 (ABCA1), which regulates the cholesterol translation through cell membrane; cholesteryl ester transfer protein (CETP), which mediates the exchange of cholesterol ester between HDL and other kinds of lipoprotein; lecithin-cholesterol acyltransferase (LCAT), which converts free cholesterol into cholesteryl ester; and hepatic lipase (LIPC), which transfers cholesterol from HDL to liver. Previous studies showed that multiple single nucleotide polymorphisms (SNPs) on gene and promoters of those apolipoproteins and enzymes were probably related with HDL-C level. However for Author Biography: Liu Xiaoli, Shenzhen center for chronic disease control. 1

2 most of the SNPs, contradictory results were reported. Although differences in sample sizes and control for confounding factors may contribute to the conflict results, the ethnic heterogeneity of SNPs cannot be ignored. Ethnic difference in allele frequencies and risk of SNPs were widely reported [4], which indicated that results from a regional study for special population may not suitable for other populations. Therefore, in this study we try to find out the SNPs which may associate with abnormally reduced HDL-C level in Chinese. We were especially concerned the SNPs widely reported to be related with HDL-C levels but not studied or showed conflict results in Chinese. As environmental factors may affect HDL-C levels, we also analyzed the SNP-environmental interactions. 2 MATERIALS AND METHODS 3815 subjects were collected by Shenzhen Center for Chronic Disease Control in annual nutritional survey of Shenzhen, China. Shenzhen is the biggest immigration city in China where more than 80% citizens came from different areas of China. Therefore to Chinese, the population of Shenzhen was a more representative sample compared with population of other Chinese cities. All subjects were randomly selected in different households from 73 communities in Shenzhen to minimize the possibility that subjects came from same family. Included subjects were citizens of Shenzhen, aged years, without CHD and CHD history, and were not taking medicines treating dyslipidemia, obesity and diabetes, or other medicines which may significantly change the HDL-C levels. All subjects accepted a questionnaire survey to provide their basic information. Fasting blood samples were collected and separated as blood cells and plasma. Plasma lipid parameters were measured by enzymatic colorimetric method, and then all subjects were divided into two groups as cases (with low HDL-C) and controls (with normal HDL-C) by their HDL-C level. The cutoff points to define low HDL-C were plasma HDL-C levels<1.03mmol/l [2]. The Restriction Fragment Length Polymorphism (RFLP) methods were used for SNPs typing. Twelve SNPs on five genes and promoters were included in the analysis. They were rs670 and rs5069 on ApoA1, rs5922, rs5923, and rs5924 on LCAT, rs , rs , rs , and rs on LIPC, rs , and rs on ABCA1, and rs on CETP. In there, primers and digest strategy were designed for each SNP. Linkage disequilibrium may be existed between SNPs in same chromosome; therefore, frequencies and odd ratios of each haplotype were calculated. To found the potential effects of SNP on the correspond enzymes or apolipoprotein, a subgroup (155cases and 399 controls) was selected randomly from whole subjects and then ApoA1, ApoA2, LCAT, LIPC and CETP levels in their plasma samples were measured by enzyme linked immune sorbent assay (ELISA) method. All data from our study were organized by Excel and output as suitable formats to fit different softwares. The Hardy-Weinberg Equilibrium (HWE) test for each SNP was performed by the Chi-square test, and the distribution of SNP genotype among cases and controls were counted and then the odds ratio (OR) of each SNP in recessive model, dominant model and additive model were calculated in a logistic analysis which adjusted for age, sex, body mass index (BMI), smoking and drinking, and daily energy intake. The age were divided into four groups as 15-30, 31-45, and 61-80years, and BMI were divided into four groups as smaller than 18.5, , and larger than 28 [5]. Differences of the plasma HDL-C and enzyme parameters in different SNP genotypes were tested by two-way analysis of variance. In all analysis, P<0.05 were considered statistically significant. 3 RESULTS Six subjects (all in controls) was excluded due to failed to extract DNA samples or missed data on plasma 2

3 HDL-C level, and at last 864 cases with low HDL-C were compared with 2945 controls in our study. The demographic and basic characteristics of study population samples were showed in Table 1. The age between cases and controls were not significantly different. Ratios of smoker were difference between cases and controls but not for that of alcohol consumer. The TG level was higher in cases. Surprisingly, the LDL-C and TC level were lower in cases compared with controls. However the TC/HDL-C was higher in cases than in control, as expected. Drawn from all subjects, characteristics of subjects of the subgroup which measured enzyme parameters were highly consistent with that of all subjects. However, ratio of smoker in cases of subgroup was significantly higher than that of all subjects. As the main compound of HDL, the level of ApoA1 were highly correlated with HDL-C level (r 2 =0.84, P<0.05) and significantly lower in cases. However as another compound of HDL, the level of ApoA2 was not showed same correlation with HDL (r 2 =0.14, P=0.53). In cases, the CETP level was significantly higher than that in controls. No difference was found about LCAT and LIPC level between cases and controls in this subgroup. 3

4 To simplify the explanations, we defined the homozygous wildtype as W, the homozygous mutation as M, and the heterozygote as H. All frequencies in controls for all SNPs passed the HWE test. The frequencies of each genotype of each SNP in both cases and controls and the odds ratios calculated in different analysis model and adjusted for age, sex, BMI, smoking and drinking, and daily energy intake were showed in Table 2. The mutations of two SNPs (rs5923 and rs5924) were not found in our studies and other ten SNPs were included in the statistical analysis, therefore, the threshold of Bonferroni adjusted P-value would be (0.05/10). Rs on CETP was found associated with low HDL-C level. The frequencies of genotype AA (homozygous mutation) were Table 2. The frequencies of each genotype of each SNP in cases and controls, and the odds ratios calculated in different analysis model. Genes and Promoters SNP controls Cases OR* in additive model (95%CI) OR* in dominant model OR* in recessive model W H M W H M H vs. W M vs. W (95%CI) (95%CI) ABCA1 rs ( ) 1.06 ( ) 0.95 ( ) 1.15 ( ) ABCA1 rs ( ) 0.94 ( ) 0.97 ( ) 1.04 ( ) ApoA1 rs ( ) 0.87 ( ) 0.93 ( ) 0.88 ( ) ApoA1 rs ( ) a 0.64 ( ) 0.80 ( ) a 0.63 ( ) CETP rs ( ) 0.78 ( ) ab 0.92 ( ) 0.83 ( ) ab LCAT rs ( ) N/A 1.12 ( ) N/A LCAT rs c N/A N/A N/A N/A LCAT rs c N/A N/A N/A N/A LIPC rs ( ) 0.93 ( ) 1.03 ( ) 0.96 ( ) LIPC rs ( ) 0.93 ( ) 1.03 ( ) 0.96 ( ) LIPC rs ( ) 0.95 ( ) 0.99 ( ) 0.93 ( ) LIPC rs ( ) 1.01 ( ) 0.97 ( ) 1.02 ( ) W, wildtype; H, homozygous; M, homozygous. *,Odds ratios were calculated in logistic analysis which adjusted for age, sex, BMI, smoking and drinking, and daily energy intake. a, statistical significant before Bonferroni correction; b, statistical significant after Bonferroni correction; c, genotyping ceased after no mutation was found in 1000 controls. significantly lower in cases compared than that in controls. The odds ratio for low HDL-C in genotype AA was 0.78 (vs. GG, P=0.0043) and 0.83 (vs. GG+GA, P=0.0046). This result also significance even after Bonferroni correction. In multiplicative model, the frequency of allele A was lower in cases compared with that in control but this result cannot pass Bonferroni correction (P=0.0094). For rs5069 on ApoA1, we found significant difference of frequencies in additive model (TT vs. CC), dominant model (TT+TC vs. CC) and multiplicative model (T vs. C). However, the significance disappeared after Bonferroni correction. We did not found SNPs on ABCA1, LCAT and LIPC were associated with low HDL-C. Strong linkage disequilibrium was found between four SNPs on promoter of LIPC (r 2 >0.839, D > for any two of them), and the frequencies of each haplotype were not different between cases and controls. In the subgroup with enzymes measured, the plasma levels of enzymes and HDL-C divided by genotypes of each SNP were showed in Table 3. Significantly higher HDL-C level was found in genotype AA of rs compared with that in genotype TT. Although CETP level tends to raise along with the increase of number of A copies of rs708272, this tendency was not significant. No other notable results were found in this subgroup. 4

5 Table 3 Levels of Enzymes and HDL-C Divided by Genotypes of Each SNP Genotype W H M rs670 ApoA1/(mmol L -1 ) 1.35± ± ±0.24 HDL-C/(mmol L -1 ) 1.22± ± ±0.16 rs5069 ApoA1/(mmol L -1 ) 1.35± ± ±0.19 HDL-C/(mmol L -1 ) 1.22± ± ±0.11 rs CETP/(µg ml -1 ) 2.41± ± ±0.82 HDL-C/(mmol L -1 ) 1.20± ± ±0.31 a rs5922 LCAT/(µg ml -1 ) 12.07± ±5.13 N/A HDL-C/(mmol L -1 ) 1.23± ±0.35 N/A rs LIPC/(ng ml -1 ) 29.83± ± ±6.35 HDL-C/(mmol L -1 ) 1.23± ± ±0.34 rs LIPC/(ng ml -1 ) 29.84± ± ±6.35 HDL-C/(mmol L -1 ) 1.23± ± ±0.34 rs LIPC/(ng ml -1 ) 30.13± ± ±6.36 HDL-C/(mmol L -1 ) 1.23± ± ±0.34 rs LIPC/(ng ml -1 ) 30.12± ± ±6.35 HDL-C/(mmol L -1 ) 1.23± ± ±0.34 All values are expressed as mean±standard deviation; W, wildtype; H, homozygous; M, homozygous ; a, statistical significant. The results of MDR analysis were shown in Table 4. The BMI was the best single factor model, and model included with sex and smoke was the best two-factors model. However prediction accuracy of those models were not statistically significant (P=0.244, 0.105, respectively). The model included with sex, smoke and alcohol consumption was the best three-factors model (prediction accuracy=70.91%, P=0.0276). The model included with sex, smoke, alcohol consumption and rs was the best four-factors model. This model had the highest cross-validation consistency (100%) and the highest prediction accuracy (73.44%). The odds ratios for low HDL-C of male subjects with non-alcohol consumption, smoke and contained genotype GG in rs was 7.32, compared with the female subjects that with alcohol consumption, non-smoke and contained genotype AA in rs The prediction accuracy was statistically significant (P=0.012).In dendrogram of MDR analysis the sex, smoke, alcohol consumption and rs were on the same branch. Red and orange line in the dendrogram suggested that interaction existed between factors. The strongest interaction exists between sex and smoke, and the second strongest interaction found between them and rs (Fig.1). 5

6 Table 4 Summary of Multifactor Dimensionality Reduction Analysis No. of factors Factor(s) in the best models CV consistency Prediction Accuracy 1 BMI 78/ Sex Smoke 98/ Sex Smoke Alcohol 100/ a 4 Sex Smoke Alcohol rs / a a, statistical significant. Figure 1. Interaction dendogram of the factors in multifactor dimensionality reduction analysis. The color of the line representing a continuum from synergy to redundancy. 4 DISCUSSION This study analyzed some potential SNPs which may associate with low HDL-C. For twelve SNPs on five genes and promoters, genotype AA of rs on CETP was found to be a protective factors for low HDL-C and the OR were 0.78 (vs. GG) and 0.83 (vs. GG+GA). The HDL-C levels of subjects with genotype AA were significantly higher than that in subjects with genotype GG. Rs was one factor in the best four-factors model in MDR analysis which other three factors were sex, smoke status and alcohol consumption, and showed strong interactions with sex and smoke. Although multiple studies reported the association between SNPs in LIPC promoter and HDL-C level, our study suggested a negative result. For rs , the most concerned SNP on LIPC promoter as an example, a meta-analysis suggested a positive association between it and HDL levels and this associations are largely unaffected by gender, ethnicity and risk category [6]. On the other hand, studies in Canada and Iran did not found the association [7,8]. A study in United States suggested the relationship between HDL-C and LIPC polymorphisms modified by dietary fat [9]. Our study found a slight increased HDL-C level in subjects with genotype TT, but the association was not significant probably due to confounding factors such as sample sizes, ethnic heterogeneity, and ignorance of dietary factor and so on. Also, our study did not find relationships between SNPs on ABCA1. Previous studies showed conflict results on those relationships. Several studies carried out in worldwide did not find altered HDL-C levels in different genotype of rs [10,11] ; however, a meta-analysis suggested that rs was associated with a higher HDL-C level only in Asians [12]. Specifically, nine of ten articles included 6

7 in the Asian s subgroup of the meta-analysis were about studies on Chinese, and eight of them did not provide significantly results. Therefore, the association between rs and HDL-C level may not exist in Chinese. Conflicted results also reported for rs [13,14], that may due to ethnic heterogeneity and need further study. Potential association was found between rs5069 on ApoA1 and low HDL-C level. The rs5069 located in intron1 of ApoA1, which may be relevant to the regulation of ApoA1 gene expression. Since ApoA1 is the main structural compound of HDL, the rs5069 may associate with HDL-C level. This SNP was first reported to have association with HDL-C level in Caucasians [15]. A study on non-hispanic whites found higher HDL-C level in females with genotype TT than in those with genotype CC [16]. However, our study did not found this association in Chinese, consistent with previous studies on Asians and Africans [17,18]. The MAF of rs5069 were varying from ethnic. It was much higher in Japanese (0.44) [17] and African (0.40) [18 ] than in white (0.08) [19] and Chinese (0.12) [20]. In our study the MAF of controls and cases was and Ethnic difference may be a reason that significant result was not found. Another reason was the sample size. Set power in 80% and significance level in 5%, to found a significantly result, a study must include more than 2940 cases and 8819 controls (calculated by an online sample size estimator, available in The potential mechanism for the effect of rs5069 on HDL-C level was unclear; a natural hypothesis was rs5069 changed ApoA1 level. However, conflict results were found between rs5069 and ApoA1 level [21,22]. In our study the ApoA1 levels in different genotype of rs5069 were not significantly difference. It must be noticed that increased ApoA1 level may not be good for health, since a decreased HDL-C/ApoA1 ratio strongly predicted CHD [23]. The CETP mediates the exchange of cholesterol ester between HDL and other kinds of lipoprotein, and high level CETP induced the decrease of the HDL-C level. The rs also located in an intron region. The frequencies of rs and its effect on HDL-C level differed from studies. A study on Korean found homozygous wildtype of rs was associated with significantly lower HDL-C levels in females and non-smoking males [24], while another study on American males found that homozygous mutation was associated with higher HDL-C level and therefore the lower risk of CHD [25]. However, this association were not found in Saudis and African Americans [26,27]. Those results indicated that associations between rs and HDL-C levels could be ethnic specific. The rs may altered HDL-C level by alter CETP level/activity [28]. On the other hand, a haplotype analysis suggested that rs was only a tagger of SNP C-629A of CETP, and the latter SNP independently affected HDL-C level. In our study, the rs on CETP was found to be associated with abnormally reduced HDL-C level. The homozygous mutation (AA) was found to be a protective factor for low HDL-C level in additive model and recessive model. Considering the Bonferroni correction was the most conservative method to control the family wise error rate, this results was believable. The HDL-C level of genotype AA was significantly higher than that of genotype GG, however, the CETP level was not altered significantly, indicated that rs may not regulate HDL-C level through altering CETP level. Therefore, the potential mechanisms need further study. Besides SNPs, sex, age, BMI, diet, smoke, alcohol consumption and some other environmental factors were believed to be involve in the HDL-C metabolism [29,30]. In our analysis, the best model identified from ten SNPs and eight potential environmental factors was a four-factors model which included sex, smoke, alcohol consumption and rs In this model, the combination of male, non-alcohol consumption, smoke and containing genotype GG in rs was associated with abnormally reduced HDL-C level. A relatively strong interaction was found between sex, smoke and rs Although high-order interaction was hard to explain, the interaction between rs and smoke or between rs and alcohol consumption were reported to be associated with HDL-C levels [31]. However, the nature of interactions between rs and smoke, drink and sex was not clear. It must be noticed that even being able to increase HDL-C level, genotype AA of rs may not be a 7

8 protective factor for atherosclerosis or CHD. Previous study found that enhanced HDL-C level caused by activity inhibition of CETP was not an effective way to prevent atherosclerosis or CHD [32]. Besides, the risk of myocardial infarction was not reduced in rs allele A carrier, even the HDL-C level was increased[33]. Although the CETP level was not associated with genotypes of rs708272, the relationship between rs and atherosclerosis or CHD should be estimated in caution. In conclusion, our study suggested rs in CETP was associated with abnormally reduced HDL-C level in Chinese, and the HDL-C level of genotype AA carrier was significantly higher than that of genotype GG carrier. Gene-environmental interaction was found between rs708272, sex, smoke and alcohol consumption and associated with abnormally reduced HDL-C level. Acknowledgments:This study was funded by the National Natural Science Foundation of China (No ), Science project of Shenzhen (No ), and National Key Technology Research and Development Program of China (2012BAI02B02). REFERENCES [1] Castelli WP, Garrison RJ, Wilson PW, et al. Incidence of coronary heart disease and lipoprotein cholesterol levels[j]. JAMA: the Journal of the American Medical Association,1986, 256(20): [2] National Cholesterol Education Program. Expert Panel on Detection E, Adults ToHBCi: Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (adult Treatment Panel III): Executive Summary: National Cholesterol Education Program, National Heart, Lung, and Blood Institute, National Institutes of Health[R] [3] Lilja HE, Soro A, Ylitalo K, et al. A candidate gene study in low HDL-cholesterol families provides evidence for the involvement of the APOA2 gene and the APOA1C3A4 gene cluster[j]. Atherosclerosis,2002, 164(1): [4] Bridges SL, Jenq G, Moran M, et al. Single-nucleotide polymorphisms in tumor necrosis factor receptor genes: Definition of novel haplotypes and racial/ethnic differences[j]. Arthritis & Rheumatism,2002, 46(8): [5] Zhou B. Predictive value for body mass index and waistline on related disease: a study on proper cutpoint of body mass index and waistline[j]. Chinese Journal of Epidemiology,2002, 23(1):5-10. [6] Isaacs A, Sayed-Tabatabaei FA, Njajou OT, et al. The 514C Thepatic lipase promoter region polymorphism and plasma lipids: a meta-analysis[j]. Journal of Clinical Endocrinology & Metabolism,2004, 89(8): [7] St-Pierre J, Miller-Felix I, Paradis M-È, et al. Visceral obesity attenuates the effect of the hepatic lipase 514C>T polymorphism on plasma HDL-cholesterol levels in French-Canadian men[j]. Molecular genetics and metabolism,2003, 78(1): [8] Daneshpour MS, Hedayati M, Azizi F. Hepatic lipase C-514T polymorphism and its association with high-density lipoprotein cholesterol level in Tehran[J]. European Journal of Cardiovascular Prevention & Rehabilitation,2006, 13(1): [9] Nettleton JA, Steffen LM, Ballantyne CM, et al. Associations between HDL-cholesterol and polymorphisms in hepatic lipase and lipoprotein lipase genes are modified by dietary fat intake in African American and White adults[j]. Atherosclerosis,2007, 194(2):e131-e140. [10] Evans D, Beil FU. The association of the R219K polymorphism in the ATP-binding cassette transporter 1 (ABCA1) gene with coronary heart disease and hyperlipidaemia[j]. Journal of molecular medicine,2003, 81(4): [11] Clee SM, Zwinderman AH, Engert JC, et al. Common genetic variation in ABCA1 is associated with altered lipoprotein levels and a modified risk for coronary artery disease[j]. Circulation,2001, 103(9): [12] Ma X Y, Liu J P, Song Z Y. Associations of the ATP-binding cassette transporter A1 R219K polymorphism with HDL-C level and coronary artery disease risk: a meta-analysis[j]. Atherosclerosis,2011, 215(2): [13] Harada T, Imai Y, Nojiri T, et al. A common Ile 823 Met variant of ATP-binding cassette transporter A1 gene alters high density lipoprotein cholesterol level in Japanese population[j]. Atherosclerosis,2003, 169(1): [14] Tan JH, Low PS, Tan YS, et al. ABCA1 gene polymorphisms and their associations with coronary artery disease and plasma lipids in males from three ethnic populations in Singapore[J]. Human genetics,2003, 113(2):

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