Allelic Variants of the Human Scavenger Receptor Class B Type 1 and Paraoxonase 1 on Coronary Heart Disease
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动脉硬化血栓血管生物学 2005年第4期
From the Research Unit (F.R.-E., J.C.R.-P., Y.H.-T., A.M.-R., A.M., A.C.), Nephrology and Cardiology Services, Hospital Universitario de Gran Canaria Dr. Negrín, Las Palmas de Gran Canaria, Spain; and Hypertension and Vascular Disease Center (C.M.F.). Wake Forest University School of Medicine, Winston-Salem, NC.
Correspondence to José C. Rodríguez-Pérez, MD, PhD, Research Unit, Nephrology, Hospital Universitario de Gran Canaria Dr Negrín, 35010 Las Palmas de Gran Canaria, Spain. E-mail jrodperd@gobiernodecanarias.org
Abstract
Objective— The antioxidant properties of high-density lipoprotein (HDL) have been attributed to paraoxonase (PON) enzyme activity. Human scavenger receptor class B type 1 (SR-BI; CD36 and lysosomal integral membrane protein-II analogous-1 [CLA-1]) plays a central role in HDL-mediated native and oxidized cholesteryl ester uptake. We tested for a significant contribution of common variant of these genes to coronary heart disease (CHD) risk and hypothesized that genetic-mediated PON activity and CLA-1/SR-BI receptor functional properties jointly reduce plasma oxidation status.
Methods and Results— We studied 304 cases and 315 controls. Polymorphisms were analyzed by polymerase chain reaction-restriction fragment analysis. CLA-1/SR-BI-relative expression levels and mRNA stability were analyzed by the comparative threshold cycle method. There was a significant difference in the male genotype distribution of the CLA-1/SR-BI exon 8 (C8/T8) variant between groups with an odds ratio of 1.7 (95% CI, 1.16 to 2.51). This significant risk was restricted to those subject carriers of Arg (R) and Leu (L) allele of the PON1 192 and 55 variants and was confirmed in multiple logistic regression analysis. CLA-1/SR-BI mRNA expression levels differed according to CLA-1/SR-BI genotypes.
Conclusions— These data suggest a plausible genetic interaction between the CLA-1 exon 8 gene polymorphism and the risk of CHD in males.
HDL antioxidant properties have been attributed to the genetic-determined paraoxonase activity. Human scavenger receptor class B type 1 plays a central role in HDL-mediated cholesteryl ester hydroperoxides uptake. We tested for a significant contribution of these genes to coronary disease and analyzed the paraoxonase activity and CLA-1/SR-BI receptor functional properties.
Key Words: paraoxonase ? arylesterase ? scavenger receptor class B type 1 ? polymorphism ? CD36 and lysosomal integral membrane protein-II analogous-1
Introduction
Plasma levels of high-density lipoprotein (HDL) cholesterol are inversely related to coronary heart disease (CHD) risk.1 Nascent HDL removes cholesterol from peripheral tissues by selective uptake.2 This selective uptake has remained elusive until identification of the mouse scavenger receptor class B type 1 (SR-BI)3 and its human homologue CD36 and lysosomal integral membrane protein-II analogous 1 (CLA-1).4 The atheroprotective role of SR-BI has been well established in engineered mice.5 Several studies demonstrated that CLA-1/SR-BI plays an important role in the bidirectional flux of free cholesterol (FC) and HDL-cholesteryl ester (CE) uptake.6 Interestingly, in vitro studies have shown a preferential SR-BI-mediated selective uptake of CE hydroperoxides (CEOOHs) compared with unoxidized CE.7 Several polymorphic variants have been described in the human CLA-1/SR-BI gene.8 A CT transition located at cDNA 1050 base position on exon 8 was associated in healthy women with lower low-density lipoprotein (LDL) concentrations and was found linked with a C to T variant at intron 5 of the gene. A glycine to serine substitution in exon 1 of the gene was described and associated with different HDL cholesterol concentrations in healthy men.8
It is known that HDL exerts other antiatherogenic properties such as preventing the oxidative modification of LDL.9,10 These HDL antioxidant properties have been attributed to paraoxonase 1 (PON1)11,12 and platelet-activating factor acetyl hydrolase (PAF-AH)13 enzyme activities. PON is a serum esterase entirely complexed to HDL, whereas most of the PAF-AH enzyme is located on the LDLs.14 PON has also been identified as a homocysteine thiolactonase15 and possesses PAF-AH-like activity.16 Although a recent study has shown that PON1 had no phospholipase A2 activity,17 conflicting results have been reported.18 Therefore, the protecting role of PON is the subject of considerable debate.19–21
There are allelic variants in the human PON1 gene, a glutamine (Q allele) for arginine (R allele) at codon 192 and a methionine (M allele) to leucine (L allele) at codon 55, that have been studied and associated with susceptibility to developing vascular disease.22,23 The R alloenzyme displays higher activity against paraoxon, whereas the Q alloenzyme displays low activity. Mackness et al24 showed that the protective effect of HDL from individuals with the PON RR genotype against LDL oxidation was lower than that from subjects with the PON1 QQ genotype. Similar results were obtained by Aviram et al25 using purified PON Q and R forms. The Met55Leu substitution modulates activity through an effect on PON1 concentration. Arylesterase activity lies on the same protein, correlated with the 55 variant, and is considered an index of protein concentration.22
Other studies have proved that the HDL isolated from QQ/MM homozygous subjects have lowest activity toward paraoxon22,26 and greatest protective capacity toward LDL oxidation in vitro.26
Therefore, CLA-1 receptor plays a central role in FC and HDL-CE uptake but a preferential selective uptake of CEOOHs regarding unoxidized CE as described.6,7 PON seems to prevent oxidation of LDL and HDL by hydrolyzing lipid hydroperoxides (LOOHs).11,12 These findings prompted us to investigate the role of CLA-1 and the PON1 gene variants in CHD and whether polymorphism-related effects could explain changes in plasma LOOH concentrations and lipid profile.
Materials and Methods
Methods
Participants were selected from the PROCAGENE case-control study.27 For details on subjects, laboratory procedures; CLA-1/SR-BI and PON1 genotyping, cell isolation and culture; CLA-1/SR-BI expression levels; and mRNA stability studies, please see the online supplement, available at http://atvb.ahajournals.org (file I).
Statistical Analysis
The SPSS statistical software version 11.0 was used for data analysis. Haplotype frequency estimation was evaluated by using Arlequin version 2.000 software.28
Results
Main Characteristics of Population Studied
The clinical characteristics are depicted in Table 1. A total of 304 cases (mean age 56±10 years; 22% females) and 315 randomly selected age- and gender-matched community controls (mean age 54.5±11 years; 26% females) were included. Patients showed a significant increase in plasma Lp(a) levels (P<0.0001), plasma CE content, and ester ratio (P<0.0001), whereas controls showed higher levels of HDL cholesterol (P<0.001) and arylesterase activity (P<0.001). However, values for diastolic blood pressure, total cholesterol, triglycerides, and LDL cholesterol were lower in cases than in controls.
TABLE 1. Main Characteristics of the Cases and Controls
We tested for significant correlations between enzyme activities and plasma lipid profile in controls because most of our study patients (58.2%) were pharmacologically treated before recruitment. There was a significant correlation between plasma arylesterase and PON activities and HDL levels (=0.199; P<0.001; n=308 and =0.252; P<0.001; n=303). PAF-AH activity correlated with LDL values (=0.568; P<0.001; n=309) and HDL values (=–0.217; P<0.001; n=311). There was a significant correlation between plasma levels of LOOH and HDL concentrations (=–0.156; P=0.006; n=305) and PON and arylesterase activities (=–0.178; P=0.002; n=300 and =–0.201; P=0.001; n=293).
We performed logistic regression analyses with the main studied variables without inclusion of the PON, arylesterase, and PAF-AH activities. The same analysis was performed excluding LDL and HDL cholesterol values and including PON, arylesterase, and PAF-AH activities Those significant variables in the first analysis remained significant in the second analysis. A marked protecting value was obtained for HDL cholesterol concentrations instead of arylesterase activity. We estimated that an increase from 0.9 to 1.25 mmol/L in HDL cholesterol causes a 16.7% reduction of coronary event.
Genotype Distribution
The genotype distribution of CLA-1–analyzed polymorphisms did not differ from that expected in Hardy-Weinberg equilibrium (HWE). Genotype distribution of the CLA-1 exon 8 variant was statistically different among patients and controls. Those CLA-1 C8C8 homozygote subjects had a significant CHD risk with an odds ratio (OR) of 1.47 (95% CI, 1.05 to 2.07). There were no differences in the genotype distribution of CLA-1 intron 5 and exon 1 variants between cases and controls. The exon 8 and intron 5 variants were in linkage disequilibrium (P<0.001).8 Inferred haplotypes are depicted in Table 2. We observed gender-dependent differences regarding genotype distribution for the exon 8 variant, with a significant CHD risk for men C8C8 homozygotes with an OR of 1.705 (95% CI, 1.16 to 2.51) but not in women with an OR of 0.806 (95% CI, 0.38 to 1.69).
TABLE 2. Haplotypes Frequency Estimation
In multiple logistic regression analysis, we obtained an OR of 2.245 (95% CI, 1.32 to 4.09) for those male C8C8 homozygous (Table 3).
TABLE 3. Multivariate Models for Total Population and Men
Genotype distribution of the PON1 Gln192Arg polymorphism was in HWE. A significant deviation from HWE for the PON1 Met55Leu genotypes was observed, probably because an excess of heterozygotes was obtained. Both variants were in linkage disequilibrium (P<000.1). No association between PON1 polymorphisms and CHD was found. The genotype distribution of subjects who were carriers of PON1 R and L alleles versus the remaining genotype combinations among cases and controls did not differ in the total population or after gender stratification.
Genotype-Genotype Interaction
We analyzed the genotype-genotype interaction, considering separately those male R allele carriers versus those QQ homozygotes and evaluating the genotype distribution of the CLA-1 exon 8 C1050T polymorphism and, conversely, the genotype distribution of the PON1 (Gln192Arg) variant, considering separately those CC homozygotes and T allele carriers of the exon 8 variant between cases and controls. Thus, the CHD risk associated to C8C8 homozygotes of the CLA-1 gene was significantly different in those R allele carriers of the PON1 Gln192Arg gene polymorphism (12=6.078; P=0.014) with an OR of 1.948 (95% CI, 1.143 to 3.32). A similar analysis was performed stratifying the PON1 Met55Leu as Leu allele carriers and MM homozygotes and evaluating the C1050T CLA-1 gene distribution and conversely evaluating the Met55Leu gene distribution according to C8C8 homozygotes and T8 allele carriers of the exon 8 variant. We observed a significant difference in the genotype distribution of the CLA-1 exon 8 variant in those L allele carriers (12=5.705; P=0.017) with an OR of 1.657 (95% CI, 1.093 to 2.511) but not in those MM homozygotes. On the other hand, no differences were observed between PON1 Met55Leu genotypes and CHD in either those T8 allele carriers or C8C8 homozygotes. Because of the linkage disequilibrium between the PON1 gene variants, we analyzed the genotype distribution of the CLA-1 exon 8 C1050T gene variant, stratifying according to PON1 genotypes dichotomized as those R and L allele carriers and the remaining possible PON1 genetic combinations between groups. The CHD risk associated with the C8C8 genotype was 2.061 (95% CI, 1.198 to 3.545) and confined to those R and L allele carriers of the PON1 variants, whereas no statistical difference was obtained for those non-R, non-L allele carriers. A trend was observed only in the genotype distribution of those R and L allele carriers in the subgroup of C8C8 homozygotes for the C1050T gene polymorphism of the CLA-1 gene but not in those non-R non-L allele carriers. For more detailed data, see supplemental files II and III (available online at http://atvb.ahajournals.org).
Genotype-Phenotype Associations
Genotype-phenotype associations were evaluated in controls. Genotypes of the CLA-1 studied variants showed no differences when evaluated in relation to lipid profiles. We found significant differences regarding basal and after-copper sulfate treatment in LOOH levels for those C5C5 versus T5 allele carriers of the intron 5 variant (P=0.008 and P=0.011), with lower levels for those C5C5 homozygote subjects. In addition, there was a graduation in plasma LOOH values according to haplotypes (Figure 1).
Figure 1. LOOH concentration values according to CLA-1/SR-BI haplotypes. Subscript indicates the corresponding allele of the intron 5 or exon 8 variants. Values are means±SD. *P=0.007; **P=0.038 for the Mann-Whitney U test comparing LOOH concentrations of the indicated haplotype vs C5C8/T5C8 haplotype.
There were significant differences in PON and arylesterase activities according to PON1 Gln192Arg and Met55Leu polymorphisms. No differences regarding basal and after-copper sulfate treatment of LOOH levels between RRLL and QQMM subjects were observed. In addition, there were no differences according to PON1 haplotypes in plasma PAF-AH values or LOOH concentration values.
CLA-1/SR-BI Expression Levels and mRNA Stability
We analyzed the relative amount of CLA-1/SR-BI mRNA levels in peripheral blood mononuclear cells (PBMCs) isolated from subjects genotyped previously as T8T8/C5C5, C8T8/C5T5 and C8C8/C5C5 of exon 8 and intron 5 variants, respectively. CLA-1/SR-BI- and GAPDH-amplified products showed similar linearity and efficiency. Both parameters were assessed using standard curves generated by increasing amounts of total RNA ranging from 0.06 to 1 μg (Figure 2).
Figure 2. CT validation experiment. Plot of logarithm of total RNA amount vs CT. Slope and statistical value are assessed using LightCycler software. Data are means±SD of an experiment replicated 3x.
Relative quantitation results are depicted in Figure 3. As shown, there was a significant difference in CLA-1/SR-BI basal expression levels between exon 8 C8C8 and T8T8 homozygous (3.3-fold). A similar difference was observed between exon 8 C8T8 heterozygous and T8T8 (5.9-fold) homozygous. The CLA-1/SR-BI mRNA stability was analyzed in cultured monocytes/macrophages. Experiments were performed in previously genotyped T8T8C5C5 and C8C8C5T5 cells. Total RNA was analyzed in control versus treated cells by relative quantitation. At the time point indicated, the relative expression of CLA-1/SR-BI mRNA did not reach statistical significance, but a trend was observed (P=0.06).
Figure 3. Relative quantification results using the CT method. Different RNA concentrations were used. Data are means±SD of T8T8C5C5 (n=5); C8C8C5T5 (n=5). C8T8C5T5 (n=5) samples replicated 2x.
Discussion
We describe for the first time that the CLA-1 exon 8 (C1050T) gene polymorphism contributes per se to CHD risk in our male population.
Previous studies have reported different lipoprotein profiles and lipoprotein particle size associated with the 3 CLA-1/SR-BI-analyzed variants.8,29 Acton et al8 reported significant differences in LDL cholesterol and body mass index (BMI) according to the exon 8 and intron 5 variants in women. Our analysis did not reveal any sex-related difference in BMI and lipid profiles according to the CLA-1/SR-BI variants, probably because we analyzed an older population. Acton et al8 reported that the association with BMI was more evident in premenopausal women, a finding that suggests hormonal regulation of the CLA-1/SR-BI gene.
We also found a significant difference in LOOH content according to CLA-1 genotypes and haplotypes. Because there is no amino acid change because of C8 to T8 substitution, it is possible that the C1050T polymorphism could constitute a marker of other functional polymorphisms. This possibility was also discussed by Acton et al.8 These authors sequenced the entering CLA-1 coding region in 3 individuals but did not find a functional mutation, and hypothesized that other genetic variants located at the 12q24 region could be linked with the phenotypic changes associated with polymorphisms. New variants have been characterized recently in the promoter region of the human CLA-1 gene.30 An interesting 11-bp (–140 to –150) insertion/deletion promoter variant was described. Hsu et al30 showed that this variant significantly influenced CLA-1 transcriptional gene activity. From our total population, 200 subjects were selected at random and genotyped for the 11-bp insertion/deletion. We found that the frequency of the deleted variant was low but similar to the 0.02 described previously. However, our analyses did not reveal any linkage with the exon 8 or intron 5 polymorphisms (data not shown). A larger population will be necessary to rule out this possibility. In addition, several other possibilities remain unexplored, including changes in the structure and stability of CLA-1 mRNA. Thus, we found in isolated PBMCs a significant difference in basal CLA-1/SR-BI mRNA expression levels according to CLA-1 genotypes. Those T8T8 and C5C5 homozygous subjects showed higher CLA-1/SR-BI mRNA expression levels than those C8 and T5 allele carriers. It was difficult for us to detect which of the linked studied variants was the main determinant of this difference because among all studied donors from which PBMCs were isolated, no T5T5 homozygous subjects of the intron 5 variant were found. Our findings concur with previous studies showing an atheroprotective role for CLA-1/SR-BI and could partially explain the CHD risk associated with the C1050T gene polymorphism. Total plasma LOOH content also differed according to CLA-1 genotypes, and higher levels were found in those T8T8 homozygous subjects. Thus, it seems that those subjects with higher CLA-1/SR-BI mRNA levels also presented high plasma levels of total plasma LOOHs. There are several reasons that could help to explain this paradox. First, we used a bulky method for detecting total plasma LOOHs. Also, there is considerable disagreement regarding LOOH levels and lipoprotein subfraction location, and it is difficult to explain this finding without measuring LOOHs in isolated lipoprotein subfraction.31,32 Although CLA-1/SR-BI receptor has a broad substrate range, it shares a highly efficient selective uptake of CEOOHs regardless of CE. In our study, its activity was indirectly evaluated. It is known that human plasma lipoproteins are heterogenous in their CE and phospholipid content, and these molecules contain a large proportion of peroxidizable fatty acid. Finally, there are other determinants of total plasma LOOHs, including the genetic-determined PON enzyme activity.
In our study, there was no association of PON1 genotypes with CHD. However, we found that the CHD risk associated with C8C8 homozygosis of CLA-1 (C1050T) polymorphism was confined to the subset of individuals who were also carriers of R and L alleles of the PON1 variants. Conversely, the CHD risk associated with the PON1 gene variants was statistically different only in the subset of individual C8C8 homozygotes for the CLA-1 polymorphism.
Several studies have suggested that HDL-associated PON activity protects against atherosclerosis in part by inhibiting the oxidative modification of LDL.11,12,14,24,26 However, PON1 variants have been inconsistently associated with CHD risk.21,33,34 Thus, it seems that gene environment or gene-gene interactions modulate the CHD risk associated with the PON1 polymorphisms. Previously, the PON1 Met55Leu variant has been associated with reduced HDL-associated PAF-AH activity.18 We did not obtain significant associations of the studied PON1 gene variant with plasma total LOOHs or PAF-AH enzyme activity. Most PAF-AH activity lies on the LDL particle, but HDL, to a lesser degree (<20%), also expresses PAF-AH activity.13 Again, it is difficult to evaluate the biological meaning of PAF-AH measurements in serum samples without lipoprotein particle isolation. Nevertheless, we did not detect changes in PAF-AH activity not related to variations in LDL cholesterol values.
In summary, it has been demonstrated that CLA-1/SR-BI mediates the selective uptake of HDL-CE.1,6 Furthermore, HDL containing oxidized CE may transfer it to the liver in an antiatherogenic pathway, and some studies showed a preferential CLA-1/SR-BI-selective uptake of oxidized CE regarding native CE.7 PON1 gene variants have been associated with different degrees of protection against lipid peroxidation.11 Thus, it is plausible that functional differences in CLA-1/SR-BI basal mRNA expression and activity linked to those reported differences associated with the PON1 gene-studied variants could explain the genetic interaction described here.
Acknowledgments
This work was supported by Fondo de Investigación Sanitaria grant FIS 01/0190 and Fundación Mapfre-Guauarteine. The authors gratefully acknowledge the help of T. Kosaka and the technical assistance Lidia Estupi?án-Quintana and Sandra García-Domínguez. Dr David Shea of the University of Las Palmas de Gran Canaria also provided editorial assistance in proofreading this manuscript.
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Correspondence to José C. Rodríguez-Pérez, MD, PhD, Research Unit, Nephrology, Hospital Universitario de Gran Canaria Dr Negrín, 35010 Las Palmas de Gran Canaria, Spain. E-mail jrodperd@gobiernodecanarias.org
Abstract
Objective— The antioxidant properties of high-density lipoprotein (HDL) have been attributed to paraoxonase (PON) enzyme activity. Human scavenger receptor class B type 1 (SR-BI; CD36 and lysosomal integral membrane protein-II analogous-1 [CLA-1]) plays a central role in HDL-mediated native and oxidized cholesteryl ester uptake. We tested for a significant contribution of common variant of these genes to coronary heart disease (CHD) risk and hypothesized that genetic-mediated PON activity and CLA-1/SR-BI receptor functional properties jointly reduce plasma oxidation status.
Methods and Results— We studied 304 cases and 315 controls. Polymorphisms were analyzed by polymerase chain reaction-restriction fragment analysis. CLA-1/SR-BI-relative expression levels and mRNA stability were analyzed by the comparative threshold cycle method. There was a significant difference in the male genotype distribution of the CLA-1/SR-BI exon 8 (C8/T8) variant between groups with an odds ratio of 1.7 (95% CI, 1.16 to 2.51). This significant risk was restricted to those subject carriers of Arg (R) and Leu (L) allele of the PON1 192 and 55 variants and was confirmed in multiple logistic regression analysis. CLA-1/SR-BI mRNA expression levels differed according to CLA-1/SR-BI genotypes.
Conclusions— These data suggest a plausible genetic interaction between the CLA-1 exon 8 gene polymorphism and the risk of CHD in males.
HDL antioxidant properties have been attributed to the genetic-determined paraoxonase activity. Human scavenger receptor class B type 1 plays a central role in HDL-mediated cholesteryl ester hydroperoxides uptake. We tested for a significant contribution of these genes to coronary disease and analyzed the paraoxonase activity and CLA-1/SR-BI receptor functional properties.
Key Words: paraoxonase ? arylesterase ? scavenger receptor class B type 1 ? polymorphism ? CD36 and lysosomal integral membrane protein-II analogous-1
Introduction
Plasma levels of high-density lipoprotein (HDL) cholesterol are inversely related to coronary heart disease (CHD) risk.1 Nascent HDL removes cholesterol from peripheral tissues by selective uptake.2 This selective uptake has remained elusive until identification of the mouse scavenger receptor class B type 1 (SR-BI)3 and its human homologue CD36 and lysosomal integral membrane protein-II analogous 1 (CLA-1).4 The atheroprotective role of SR-BI has been well established in engineered mice.5 Several studies demonstrated that CLA-1/SR-BI plays an important role in the bidirectional flux of free cholesterol (FC) and HDL-cholesteryl ester (CE) uptake.6 Interestingly, in vitro studies have shown a preferential SR-BI-mediated selective uptake of CE hydroperoxides (CEOOHs) compared with unoxidized CE.7 Several polymorphic variants have been described in the human CLA-1/SR-BI gene.8 A CT transition located at cDNA 1050 base position on exon 8 was associated in healthy women with lower low-density lipoprotein (LDL) concentrations and was found linked with a C to T variant at intron 5 of the gene. A glycine to serine substitution in exon 1 of the gene was described and associated with different HDL cholesterol concentrations in healthy men.8
It is known that HDL exerts other antiatherogenic properties such as preventing the oxidative modification of LDL.9,10 These HDL antioxidant properties have been attributed to paraoxonase 1 (PON1)11,12 and platelet-activating factor acetyl hydrolase (PAF-AH)13 enzyme activities. PON is a serum esterase entirely complexed to HDL, whereas most of the PAF-AH enzyme is located on the LDLs.14 PON has also been identified as a homocysteine thiolactonase15 and possesses PAF-AH-like activity.16 Although a recent study has shown that PON1 had no phospholipase A2 activity,17 conflicting results have been reported.18 Therefore, the protecting role of PON is the subject of considerable debate.19–21
There are allelic variants in the human PON1 gene, a glutamine (Q allele) for arginine (R allele) at codon 192 and a methionine (M allele) to leucine (L allele) at codon 55, that have been studied and associated with susceptibility to developing vascular disease.22,23 The R alloenzyme displays higher activity against paraoxon, whereas the Q alloenzyme displays low activity. Mackness et al24 showed that the protective effect of HDL from individuals with the PON RR genotype against LDL oxidation was lower than that from subjects with the PON1 QQ genotype. Similar results were obtained by Aviram et al25 using purified PON Q and R forms. The Met55Leu substitution modulates activity through an effect on PON1 concentration. Arylesterase activity lies on the same protein, correlated with the 55 variant, and is considered an index of protein concentration.22
Other studies have proved that the HDL isolated from QQ/MM homozygous subjects have lowest activity toward paraoxon22,26 and greatest protective capacity toward LDL oxidation in vitro.26
Therefore, CLA-1 receptor plays a central role in FC and HDL-CE uptake but a preferential selective uptake of CEOOHs regarding unoxidized CE as described.6,7 PON seems to prevent oxidation of LDL and HDL by hydrolyzing lipid hydroperoxides (LOOHs).11,12 These findings prompted us to investigate the role of CLA-1 and the PON1 gene variants in CHD and whether polymorphism-related effects could explain changes in plasma LOOH concentrations and lipid profile.
Materials and Methods
Methods
Participants were selected from the PROCAGENE case-control study.27 For details on subjects, laboratory procedures; CLA-1/SR-BI and PON1 genotyping, cell isolation and culture; CLA-1/SR-BI expression levels; and mRNA stability studies, please see the online supplement, available at http://atvb.ahajournals.org (file I).
Statistical Analysis
The SPSS statistical software version 11.0 was used for data analysis. Haplotype frequency estimation was evaluated by using Arlequin version 2.000 software.28
Results
Main Characteristics of Population Studied
The clinical characteristics are depicted in Table 1. A total of 304 cases (mean age 56±10 years; 22% females) and 315 randomly selected age- and gender-matched community controls (mean age 54.5±11 years; 26% females) were included. Patients showed a significant increase in plasma Lp(a) levels (P<0.0001), plasma CE content, and ester ratio (P<0.0001), whereas controls showed higher levels of HDL cholesterol (P<0.001) and arylesterase activity (P<0.001). However, values for diastolic blood pressure, total cholesterol, triglycerides, and LDL cholesterol were lower in cases than in controls.
TABLE 1. Main Characteristics of the Cases and Controls
We tested for significant correlations between enzyme activities and plasma lipid profile in controls because most of our study patients (58.2%) were pharmacologically treated before recruitment. There was a significant correlation between plasma arylesterase and PON activities and HDL levels (=0.199; P<0.001; n=308 and =0.252; P<0.001; n=303). PAF-AH activity correlated with LDL values (=0.568; P<0.001; n=309) and HDL values (=–0.217; P<0.001; n=311). There was a significant correlation between plasma levels of LOOH and HDL concentrations (=–0.156; P=0.006; n=305) and PON and arylesterase activities (=–0.178; P=0.002; n=300 and =–0.201; P=0.001; n=293).
We performed logistic regression analyses with the main studied variables without inclusion of the PON, arylesterase, and PAF-AH activities. The same analysis was performed excluding LDL and HDL cholesterol values and including PON, arylesterase, and PAF-AH activities Those significant variables in the first analysis remained significant in the second analysis. A marked protecting value was obtained for HDL cholesterol concentrations instead of arylesterase activity. We estimated that an increase from 0.9 to 1.25 mmol/L in HDL cholesterol causes a 16.7% reduction of coronary event.
Genotype Distribution
The genotype distribution of CLA-1–analyzed polymorphisms did not differ from that expected in Hardy-Weinberg equilibrium (HWE). Genotype distribution of the CLA-1 exon 8 variant was statistically different among patients and controls. Those CLA-1 C8C8 homozygote subjects had a significant CHD risk with an odds ratio (OR) of 1.47 (95% CI, 1.05 to 2.07). There were no differences in the genotype distribution of CLA-1 intron 5 and exon 1 variants between cases and controls. The exon 8 and intron 5 variants were in linkage disequilibrium (P<0.001).8 Inferred haplotypes are depicted in Table 2. We observed gender-dependent differences regarding genotype distribution for the exon 8 variant, with a significant CHD risk for men C8C8 homozygotes with an OR of 1.705 (95% CI, 1.16 to 2.51) but not in women with an OR of 0.806 (95% CI, 0.38 to 1.69).
TABLE 2. Haplotypes Frequency Estimation
In multiple logistic regression analysis, we obtained an OR of 2.245 (95% CI, 1.32 to 4.09) for those male C8C8 homozygous (Table 3).
TABLE 3. Multivariate Models for Total Population and Men
Genotype distribution of the PON1 Gln192Arg polymorphism was in HWE. A significant deviation from HWE for the PON1 Met55Leu genotypes was observed, probably because an excess of heterozygotes was obtained. Both variants were in linkage disequilibrium (P<000.1). No association between PON1 polymorphisms and CHD was found. The genotype distribution of subjects who were carriers of PON1 R and L alleles versus the remaining genotype combinations among cases and controls did not differ in the total population or after gender stratification.
Genotype-Genotype Interaction
We analyzed the genotype-genotype interaction, considering separately those male R allele carriers versus those QQ homozygotes and evaluating the genotype distribution of the CLA-1 exon 8 C1050T polymorphism and, conversely, the genotype distribution of the PON1 (Gln192Arg) variant, considering separately those CC homozygotes and T allele carriers of the exon 8 variant between cases and controls. Thus, the CHD risk associated to C8C8 homozygotes of the CLA-1 gene was significantly different in those R allele carriers of the PON1 Gln192Arg gene polymorphism (12=6.078; P=0.014) with an OR of 1.948 (95% CI, 1.143 to 3.32). A similar analysis was performed stratifying the PON1 Met55Leu as Leu allele carriers and MM homozygotes and evaluating the C1050T CLA-1 gene distribution and conversely evaluating the Met55Leu gene distribution according to C8C8 homozygotes and T8 allele carriers of the exon 8 variant. We observed a significant difference in the genotype distribution of the CLA-1 exon 8 variant in those L allele carriers (12=5.705; P=0.017) with an OR of 1.657 (95% CI, 1.093 to 2.511) but not in those MM homozygotes. On the other hand, no differences were observed between PON1 Met55Leu genotypes and CHD in either those T8 allele carriers or C8C8 homozygotes. Because of the linkage disequilibrium between the PON1 gene variants, we analyzed the genotype distribution of the CLA-1 exon 8 C1050T gene variant, stratifying according to PON1 genotypes dichotomized as those R and L allele carriers and the remaining possible PON1 genetic combinations between groups. The CHD risk associated with the C8C8 genotype was 2.061 (95% CI, 1.198 to 3.545) and confined to those R and L allele carriers of the PON1 variants, whereas no statistical difference was obtained for those non-R, non-L allele carriers. A trend was observed only in the genotype distribution of those R and L allele carriers in the subgroup of C8C8 homozygotes for the C1050T gene polymorphism of the CLA-1 gene but not in those non-R non-L allele carriers. For more detailed data, see supplemental files II and III (available online at http://atvb.ahajournals.org).
Genotype-Phenotype Associations
Genotype-phenotype associations were evaluated in controls. Genotypes of the CLA-1 studied variants showed no differences when evaluated in relation to lipid profiles. We found significant differences regarding basal and after-copper sulfate treatment in LOOH levels for those C5C5 versus T5 allele carriers of the intron 5 variant (P=0.008 and P=0.011), with lower levels for those C5C5 homozygote subjects. In addition, there was a graduation in plasma LOOH values according to haplotypes (Figure 1).
Figure 1. LOOH concentration values according to CLA-1/SR-BI haplotypes. Subscript indicates the corresponding allele of the intron 5 or exon 8 variants. Values are means±SD. *P=0.007; **P=0.038 for the Mann-Whitney U test comparing LOOH concentrations of the indicated haplotype vs C5C8/T5C8 haplotype.
There were significant differences in PON and arylesterase activities according to PON1 Gln192Arg and Met55Leu polymorphisms. No differences regarding basal and after-copper sulfate treatment of LOOH levels between RRLL and QQMM subjects were observed. In addition, there were no differences according to PON1 haplotypes in plasma PAF-AH values or LOOH concentration values.
CLA-1/SR-BI Expression Levels and mRNA Stability
We analyzed the relative amount of CLA-1/SR-BI mRNA levels in peripheral blood mononuclear cells (PBMCs) isolated from subjects genotyped previously as T8T8/C5C5, C8T8/C5T5 and C8C8/C5C5 of exon 8 and intron 5 variants, respectively. CLA-1/SR-BI- and GAPDH-amplified products showed similar linearity and efficiency. Both parameters were assessed using standard curves generated by increasing amounts of total RNA ranging from 0.06 to 1 μg (Figure 2).
Figure 2. CT validation experiment. Plot of logarithm of total RNA amount vs CT. Slope and statistical value are assessed using LightCycler software. Data are means±SD of an experiment replicated 3x.
Relative quantitation results are depicted in Figure 3. As shown, there was a significant difference in CLA-1/SR-BI basal expression levels between exon 8 C8C8 and T8T8 homozygous (3.3-fold). A similar difference was observed between exon 8 C8T8 heterozygous and T8T8 (5.9-fold) homozygous. The CLA-1/SR-BI mRNA stability was analyzed in cultured monocytes/macrophages. Experiments were performed in previously genotyped T8T8C5C5 and C8C8C5T5 cells. Total RNA was analyzed in control versus treated cells by relative quantitation. At the time point indicated, the relative expression of CLA-1/SR-BI mRNA did not reach statistical significance, but a trend was observed (P=0.06).
Figure 3. Relative quantification results using the CT method. Different RNA concentrations were used. Data are means±SD of T8T8C5C5 (n=5); C8C8C5T5 (n=5). C8T8C5T5 (n=5) samples replicated 2x.
Discussion
We describe for the first time that the CLA-1 exon 8 (C1050T) gene polymorphism contributes per se to CHD risk in our male population.
Previous studies have reported different lipoprotein profiles and lipoprotein particle size associated with the 3 CLA-1/SR-BI-analyzed variants.8,29 Acton et al8 reported significant differences in LDL cholesterol and body mass index (BMI) according to the exon 8 and intron 5 variants in women. Our analysis did not reveal any sex-related difference in BMI and lipid profiles according to the CLA-1/SR-BI variants, probably because we analyzed an older population. Acton et al8 reported that the association with BMI was more evident in premenopausal women, a finding that suggests hormonal regulation of the CLA-1/SR-BI gene.
We also found a significant difference in LOOH content according to CLA-1 genotypes and haplotypes. Because there is no amino acid change because of C8 to T8 substitution, it is possible that the C1050T polymorphism could constitute a marker of other functional polymorphisms. This possibility was also discussed by Acton et al.8 These authors sequenced the entering CLA-1 coding region in 3 individuals but did not find a functional mutation, and hypothesized that other genetic variants located at the 12q24 region could be linked with the phenotypic changes associated with polymorphisms. New variants have been characterized recently in the promoter region of the human CLA-1 gene.30 An interesting 11-bp (–140 to –150) insertion/deletion promoter variant was described. Hsu et al30 showed that this variant significantly influenced CLA-1 transcriptional gene activity. From our total population, 200 subjects were selected at random and genotyped for the 11-bp insertion/deletion. We found that the frequency of the deleted variant was low but similar to the 0.02 described previously. However, our analyses did not reveal any linkage with the exon 8 or intron 5 polymorphisms (data not shown). A larger population will be necessary to rule out this possibility. In addition, several other possibilities remain unexplored, including changes in the structure and stability of CLA-1 mRNA. Thus, we found in isolated PBMCs a significant difference in basal CLA-1/SR-BI mRNA expression levels according to CLA-1 genotypes. Those T8T8 and C5C5 homozygous subjects showed higher CLA-1/SR-BI mRNA expression levels than those C8 and T5 allele carriers. It was difficult for us to detect which of the linked studied variants was the main determinant of this difference because among all studied donors from which PBMCs were isolated, no T5T5 homozygous subjects of the intron 5 variant were found. Our findings concur with previous studies showing an atheroprotective role for CLA-1/SR-BI and could partially explain the CHD risk associated with the C1050T gene polymorphism. Total plasma LOOH content also differed according to CLA-1 genotypes, and higher levels were found in those T8T8 homozygous subjects. Thus, it seems that those subjects with higher CLA-1/SR-BI mRNA levels also presented high plasma levels of total plasma LOOHs. There are several reasons that could help to explain this paradox. First, we used a bulky method for detecting total plasma LOOHs. Also, there is considerable disagreement regarding LOOH levels and lipoprotein subfraction location, and it is difficult to explain this finding without measuring LOOHs in isolated lipoprotein subfraction.31,32 Although CLA-1/SR-BI receptor has a broad substrate range, it shares a highly efficient selective uptake of CEOOHs regardless of CE. In our study, its activity was indirectly evaluated. It is known that human plasma lipoproteins are heterogenous in their CE and phospholipid content, and these molecules contain a large proportion of peroxidizable fatty acid. Finally, there are other determinants of total plasma LOOHs, including the genetic-determined PON enzyme activity.
In our study, there was no association of PON1 genotypes with CHD. However, we found that the CHD risk associated with C8C8 homozygosis of CLA-1 (C1050T) polymorphism was confined to the subset of individuals who were also carriers of R and L alleles of the PON1 variants. Conversely, the CHD risk associated with the PON1 gene variants was statistically different only in the subset of individual C8C8 homozygotes for the CLA-1 polymorphism.
Several studies have suggested that HDL-associated PON activity protects against atherosclerosis in part by inhibiting the oxidative modification of LDL.11,12,14,24,26 However, PON1 variants have been inconsistently associated with CHD risk.21,33,34 Thus, it seems that gene environment or gene-gene interactions modulate the CHD risk associated with the PON1 polymorphisms. Previously, the PON1 Met55Leu variant has been associated with reduced HDL-associated PAF-AH activity.18 We did not obtain significant associations of the studied PON1 gene variant with plasma total LOOHs or PAF-AH enzyme activity. Most PAF-AH activity lies on the LDL particle, but HDL, to a lesser degree (<20%), also expresses PAF-AH activity.13 Again, it is difficult to evaluate the biological meaning of PAF-AH measurements in serum samples without lipoprotein particle isolation. Nevertheless, we did not detect changes in PAF-AH activity not related to variations in LDL cholesterol values.
In summary, it has been demonstrated that CLA-1/SR-BI mediates the selective uptake of HDL-CE.1,6 Furthermore, HDL containing oxidized CE may transfer it to the liver in an antiatherogenic pathway, and some studies showed a preferential CLA-1/SR-BI-selective uptake of oxidized CE regarding native CE.7 PON1 gene variants have been associated with different degrees of protection against lipid peroxidation.11 Thus, it is plausible that functional differences in CLA-1/SR-BI basal mRNA expression and activity linked to those reported differences associated with the PON1 gene-studied variants could explain the genetic interaction described here.
Acknowledgments
This work was supported by Fondo de Investigación Sanitaria grant FIS 01/0190 and Fundación Mapfre-Guauarteine. The authors gratefully acknowledge the help of T. Kosaka and the technical assistance Lidia Estupi?án-Quintana and Sandra García-Domínguez. Dr David Shea of the University of Las Palmas de Gran Canaria also provided editorial assistance in proofreading this manuscript.
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