R192 polymorphism and severity, progression and regression of coronary atherosclerosis, plasma lipid levels, clinical events and response to fluvastatin

Atherosclerosis 154 (2001) 633– 640 www.elsevier.com/locate/atherosclerosis

A prospective study of paraoxonase gene Q/R192 polymorphism and severity, progression and regression of coronary atherosclerosis, plasma lipid levels, clinical events and response to fluvastatin Sharon Turban a, Francisco Fuentes a, Laura Ferlic a, Ramon Brugada a, Antonio M. Gotto b, Christie M. Ballantyne a, Ali J. Marian a,* a

Department of Medicine, Sections of Cardiology and Atherosclerosis, Baylor College of Medicine, Houston, TX 77030, USA b Cornell Uni6ersity Weill Medical College of New York, NY 10021, USA Received 18 November 1999; received in revised form 2 February 2000; accepted 3 April 2000

Abstract Human serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme that is responsible for the protective effect of HDL against oxidation of low-density lipoprotein (LDL). PON1 has a Glu to Arg polymorphism at codon 192 (CGA“CAA) which is designated R/Q192. The R/Q192 polymorphism has been associated with coronary artery disease (CAD) in several, but not all, case-control studies. We prospectively studied the association of the Q/R192 genotypes with the severity, progression and regression of CAD, plasma lipid levels, clinical events and response to treatment with fluvastatin in a well-characterized cohort. Genotypes were determined by polymerase chain reaction (PCR) and restriction mapping with AlwI enzyme in 356 subjects in the Lipoprotein and Coronary Atherosclerosis Study (LCAS). Fasting plasma lipids were measured and quantitative coronary angiograms were obtained at baseline and 2.5 years following randomization to fluvastatin or placebo. A total of 177 (50%), 142 (40%) and 37 (10%) subjects had Q/Q, Q/R and R/R genotypes, respectively. Baseline and final plasma levels of HDL, LDL, triglyceride and other lipoproteins, lesion-specific minimum lumen diameters (MLD), mean MLD, number of coronary lesions and total occlusions at baseline and follow-up and clinical event rates were not significantly different among the genotypes. There was no genotype-treatment interaction with respect to plasma lipid levels and angiographic indices of CAD. The Q/R192 variants of PON1 are not associated with severity, progression or regression of coronary atherosclerosis, plasma lipid levels, clinical events, or response to treatment with fluvastatin. Thus, the Q/R192 polymorphism is not a major risk factor in susceptibility to CAD in the LCAS population. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Paraoxonase; Genetics; Coronary artery disease; Polymorphism

1. Introduction The predominant hypothesis of the pathogenesis of atherosclerosis proposes that the oxidation of LDL confers a large array of proatherogenic properties to LDL and is the fundamental step in the initiation and progression of atherosclerosis [1 – 3]. The mechanisms that promote or inhibit oxidative modification of LDL * Corresponding author. Present address: Section of Cardiology, Baylor College of Medicine, 1 Baylor Plaza, 543E, Houston, TX 77030, USA. Tel.: + 1-713-7987454; fax: + 1-713-7983147. E-mail address: [email protected] (A.J. Marian).

are likely to involve a variety of enzymatic and nonenzymatic pathways [1,2]. HDL is known to prevent oxidation of LDL [4,5], a function that may account, in part, for the protective effect of HDL against atherosclerosis. The ability of HDL to prevent oxidation of LDL has been attributed to several HDL-associated enzymes, namely paraoxonase (PON1) [6–8], platelet-activating factor acetyl hydrolase [9] and lecithin:cholesterol acyltransferase [10]. PON1 is a Ca2 + -dependent esterase that can hydrolyze many synthetic substrates including organophosphates. Recent data implicating PON1 in the metabolism of phospholipid peroxides [4–8,11]

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have raised significant interest in the possible role of this enzyme in atherosclerosis. In vitro and in vivo studies suggest that PON1 is the primary enzyme responsible for the antioxidant activity of HDL [4– 8,11,12]. Purified human PON1 decreases generation and accumulation of lipoperoxides in LDL [6]. In addition, PON1, by destroying oxidized phospholipids, reduces the ability of oxidized LDL to induce monocyte binding and transmigration and thus, inflammation in the vessel wall [8]. Furthermore, targeted deletion of the paraoxonase gene in the mouse led to an increased sensitivity of HDL and LDL to oxidation and enhanced susceptibility to atherosclerosis when placed on an atherogenic diet [12]. Collectively, these data strongly suggest a significant role for PON1 in oxidative modification of LDL and atherogenesis, particularly in the presence of an atherogenic diet. The human paraoxonase/acetylesterase (HUMPONA) gene is a member of a multigene family that also includes PON2 and PON3. While no mature proteins for PON2 and PON3 have been identified, the HUMPONA gene, located on chromosome 7q21-22, codes for a 44-kD protein comprised of 355 amino acids [13]. The enzymatic activity of PON1 against paraoxon varies 10- to 40-fold among individuals [14]. The molecular basis of this genetic variation is the presence of a R/Q polymorphism at amino acid position 192 (Q/R192) [15,16]. Individuals with the Q/Q genotype have lower enzymatic activity against paraoxon than those with the R/R genotype [15,16]. Regarding lipid peroxidation, preliminary studies suggest that the R/R isoform is less effective in hydrolyzing lipid peroxides than the Q/Q isoform [17,18]. Since PON1 is involved in oxidation of LDL — a fundamental step in the initiation and progression of atherosclerosis — the association of the Q/R192 polymorphism with CAD and myocardial infarction (MI) has been investigated in several case-control studies [19 – 29]. While some studies report a higher frequency of the R allele in patients with CAD and/or MI [19 – 24], others have found no association [25 – 29]. The conflicting results in part reflect the nature of the retrospective case-control polymorphism association studies, which are subject to a high rate of spurious association [30]. Similarly, the association of the Q/ R192 polymorphism with plasma lipoproteins remains unsettled [20,25,26,31,32]. While in genetic isolates an association between the Q/R192 variants and plasma lipoproteins has been reported [31,32], the majority of the studies in other populations show no association [25,26]. We analyzed the association of the Q/R192 polymorphism with the severity, progression and regression of CAD, as determined by serial quantitative coronary angiography, as well as plasma levels of lipids and clinical events in a prospective study of a well-characterized cohort. In view of the presence of a significant

inter-individual variability in the response of plasma lipids and angiographic indices of atherosclerosis to treatment with fluvastatin [33], we also determined possible genotype-treatment interactions (pharmacogenetics) between the Q/R192 genotypes and the response of phenotypic variables to treatment with fluvastatin.

2. Methods

2.1. Study population All subjects in the LCAS provided informed consent and the study was approved by the institutional review board. The study population was comprised of 429 subjects (349 male and 80 female). Design and results of LCAS have been published [33,34]. In brief, patients 35–75 years of age, who had at least one coronary lesion causing 30–75% diameter stenosis and LDL cholesterol of 115 –190 mg/dl despite diet, were randomized to fluvastatin (40 mg daily) or placebo. Total cholesterol, LDL, HDL, triglyceride, lipoprotein(a) and apolipoprotein levels were measured in all subjects at baseline and throughout the study. Quantitative coronary angiography was performed at baseline and 2.5 years after randomization and was available in 340 subjects. The primary end point was within-subject per-lesion change in the MLD of qualifying lesions, defined by MLD ] 25% of the reference lumen diameter at baseline and MLD ] 0.8 mm less than the reference lumen diameter at either baseline or followup. Subjects were also categorized as having definite progression, definite regression or mixed angiographic change. Definite progression was defined as ] 1 qualifying lesion with MLD decrease ]0.4 mm, including new total occlusions and no qualifying lesion with MLD increase ] 0.4 mm. Definite regression was defined as ] 1 qualifying lesion with MLD increase ] 0.4 mm, no qualifying lesion with MLD decrease ] 0.4 mm and no new total occlusion. Subjects that showed neither definite progression nor definite regression were classified as having mixed change. Clinical events monitored were definite or probable MI, unstable angina requiring hospitalization, percutaneous transluminal coronary angioplasty, coronary artery bypass grafting and death of any cause.

2.2. Genotyping Laboratory personnel who had no knowledge of the angiographic and clinical data, performed the genotyping. DNA was extracted by the salting-out technique [36] and genotyping was performed by PCR and restriction mapping with Alw1 restriction endonuclease as published [19]. The genotype of each individual was determined by gel electrophoresis of the PCR product

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digested with Alw1 enzyme. Each genotype was read by two individuals independently and if in conflict, genotyping was repeated. The amplified PCR product was 99 bp. Individuals with the Q/Q genotype were identified by the presence of one product of 99 bp and those with R/R by the presence of two products of 69 and 30 bp. Heterozygous individuals were identified by the presence of all three products of 99, 69 and 30 bp.

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or Fisher’s exact test. To determine the association of the genotypes with response to fluvastatin treatment, mean changes in plasma lipid levels and MLD among the genotypes were compared using analysis of variance. Statistical analysis was performed using STATA, version 5.0 (Stata Corporation, College Station, TX).

3. Results

2.3. Statistical analysis 3.1. Genotypes Continuous variables were expressed as mean9 S.D., except for the lesion-specific MLD, which was expressed as mean9S.E. Differences among the genotypes were compared by analysis of variance. Variables that were unsuited for analysis of variance — due to inequalities of variance — were analyzed by Kruskal – Wallis test. Distribution of the categorical variables among genotypes was compared using Pearson  2-test

In 54 subjects, no DNA sample was available and in another 19 the PCR reaction did not work. Genotyping was completed in 356 subjects: 177 (50%) had Q/Q, 142 (40%) had Q/R and 37 (10%) had R/R genotypes. The frequencies of the Q and R alleles were 0.70 and 0.30, respectively. The distribution of genotypes followed the Hardy –Weinberg equilibrium.

3.2. Demographic data

Table 1 Baseline characteristics of subjects according to HUMPONA Q/R192 genotypesa HUMPONA Q/R192 genotypes

N =356 Age (years) Male (%) White Height (m) Weight (kg) BMI (kg/m2) Waist/hip ratio Previous MI Diabetes mellitus Systolic BP (mmHg) Diastolic BP (mmHg) Smoker No. of qualifying lesions No. of total occlusions ]1 Qualifying lesion ]1 Total occlusion Mean MLD (mm) Lesion-specific MLD (mm)

Q/Q

Q/R

R/R

177 (50%) 58.5 9 7.8 151 (85) 166 (94) 1.74 9 0.08 83.4 9 15.7 27.5 9 4.4 0.91 9 0.07 75 (42) 4 (2) 122.99 14.1

142 (40%) 59.0 97.7 117 (82) 124 (87) 1.73 90.08 85.59 15.3 28.59 4.6 0.91 9 0.07 59 (42) 9 (6) 125.3915.9

37 (10%) 59.3 9 8.1 27 (73) 30 (81) 1.72 9 0.09 86.0 9 14.2 29.09 3.9 0.89 9 0.07 14 (38) 1 (3) 127.69 16.8

76.7 9 8.9

77.1 9 9.3

77.19 8.6

40 (23) 2.68 9 1.8

22 (15) 2.7192.1

9 (24) 2.81 9 2.15

0.35 9 0.62

0.34 9 0.66

0.199 0.46

154 (87)

116 (82)

32 (86)

49 (28)

37 (26)

6 (16)

1.679 0.40

1.68 90.43

1.63 9 0.37

1.69 9 0.03

1.67 90.04

1.59 9 0.07

Values are presented as either mean 9 S.D. or n (%). BMI, body mass index; BP, blood pressure; MI, myocardial infarction; MLD, minimum lumen diameter; Q, glutamine; R, arginine. a

The baseline characteristics of subjects according to HUMPONA genotypes are shown in Table 1. There was no significant association between the genotypes and the demographic variables presented in Table 1. The mean number of qualifying lesions and total occlusions, the number of subjects with ]1 qualifying lesions or total occlusions and the mean MLD at baseline, did not differ significantly among the genotypes.

3.3. Plasma le6els of lipids There were no significant differences between mean plasma HDL, total cholesterol, LDL, triglyceride, lipoprotein(a) and apolipoprotein levels at baseline and upon completion of the study, as shown in Table 2. Overall, genotypes were not associated with either baseline or final lipid levels and there was no interaction between response of plasma lipids to treatment with fluvastatin and the genotypes.

3.4. Progression and regression of coronary artery disease There was no association between Q/R192 genotypes and the development of new lesions or new total occlusions or change in mean MLD or lesion-specific MLD (Table 3). The numbers of subjects with definite progression, definite regression or mixed change did not differ significantly among the genotypes. Overall, there was no association between the genotypes and regression or progression of coronary atherosclerosis, whether defined as a categorical or continuous variable and regardless of the treatment group.

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Table 2 Paraoxonase Q/R192 genotypes and baseline and final plasma lipid levelsa Genotypes (N = 353)

Placebo

Fluvastatin

Q/Q (84)

Q/R (74)

R/R (14)

Q/Q (92)

Q/R (67)

R/R (22)

218.99 20.7 218.89 31.7 0.229 12.7

220.89 29.2 212.99 32.8 −3.09 −13.2

223.0 9 26.2 221.5 9 37.9 −0.879 10.4

222.7 9 25.8 184.4 9 29.6 −16.89 12.1

216.4 9 22.9 189.2 9 35.3 −12.49 13.8

230.6 9 20.1 190.3 9 27.1 −17.09 13.3

HDL BL FU %D

42.89 9.6 44.39 10.0 4.79 16.2

46.49 12.3 47.69 13.4 3.19 14.2

44.6 9 9.3 46.5 913.2 3.5 9 13.7

44.1 9 12.4 47.0 9 11.5 8.4 9 14.4

41.9 9 11.8 45.9 9 13.3 10.2 9 15.0

45.8 9 9.9 50.9 9 12.5 12.4 9 21.4

LDL BL FU %D

144.9 9 18.3 140.59 25.8 −2.49 16.6

143.7 9 22.5 132.49 27.2 −7.09 18.3

146.3 919.9 140.2 9 37.7 −5.09 18.0

145.2 9 20.4 106.4 9 25.5 −26.19 17.4

142.6 9 19.4 109.9 9 27.2 −22.69 17.7

152.8 919.2 108.4 925.5 −28.39 17.9

TG BL FU %D

157.0 9 55.8 168.69 77.5 11.19 40.9

155.5 9 52.9 165.6 9 77.0 8.19 40.4

159.6 9 46.6 173.3 9 71.5 11.3 9 39.4

167.2 9 60.9 155.0 9 65.0 −4.09 36.7

158.8 9 56.0 169.4 9 93.8 8.0 9 47.6

160.4 9 62.4 154.4 9 66.0 1.3 9 36.5

Apo A-I BL FU %D

131.79 23.0 134.29 25.6 2.79 15.3

138.09 31.1 136.9 9 34.9 −0.49 15.0

134.1 9 23.2 137.6 9 23.6 3.3 9 11.1

132.4 9 28.8 138.4 9 29.4 6.3 9 19.3

127.5 9 25.6 136.5 9 35.3 7.3 9 17.1

135.6 9 23.7 151.2 9 39.2 13.1 9 28.9

Apo B BL FU %D

132.99 18.8 140.09 24.5 6.49 19.2

131.99 24.0 132.59 25.2 2.39 20.9

133.8 9 22.8 141.5 9 27.3 7.3 9 19.8

136.7 9 20.8 112.9 9 20.7 −16.49 15.2

136.1 9 18.7 116.1 9 21.9 −14.09 15.0

145.8 9 17.8 117.1 9 22.8 −18.99 17.3

35.89 11.5 35.49 13.1 3.59 34.5

36.49 13.7 34.99 13.7 −0.79 32.0

35.6 9 13.4 34.5 9 13.7 0.2 9 30.7

39.6 9 10.9 36.0 9 12.1 −7.49 25.0

35.3 9 10.9 35.4 9 15.3 10.9 9 33.3

34.9 9 12.7 34.1 913.6 2.0 9 31.7

37.2 933.7 40.5939.8 −2.3932.7

34.3 9 33.9 37.39 45.1 −5.29 29.6

38.1 936.2 39.6 9 43.0 −7.59 38.5

34.5 9 33.7 39.4 9 44.8 −2.49 37.6

39.9 936.2 42.9 940.9 −0.5935.8

38.2 928.5 37.7 932.2 −10.4929.8

TC BL FU %D

Apo CIII BL FU %D Lp(a) BL FU %D

a All values are mg/dl and are represented as mean 9 S.D. Ap, apolipoprotein; BL, baseline; FU, final follow-up; HDL, high density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; Lp(a), lipoprotein(a); TC, total cholesterol; TG, triglyceride. The %D was calculated as follows: %D= ni = 1 (((FUi−BLi )/(BLi ))/n), where i is the number of subgroup patients and FUi and BLi are the values at follow-up and baseline, respectively.

3.5. Clinical e6ents

4. Discussion

Morbid or fatal cardiovascular events occurred in 53 patients (15%). The distribution of cardiovascular events was similar among the genotypes in the placebo and fluvastatin groups, as shown in Table 3. Similarly, the number of events in the subgroup with evaluable angiography did not differ significantly among the genotypes (Table 3).

The results of this prospective study show no significant association between the Q/R192 polymorphism in HUMPONA gene and the angiographic severity, progression or regression of coronary atherosclerosis, plasma levels of lipids, clinical events or the response to treatment with fluvastatin. There was also no association between Q/R192 genotypes and the baseline characteristics of subjects, including history of MI. Overall, these results suggest that the Q/R192 polymorphism is not a major risk factor for coronary atherosclerosis in the LCAS population. The association of the Q/R192 polymorphism with atherosclerosis and MI is controversial, largely because

3.6. Genotype-treatment interaction (pharmacogenetics) Q/R192 genotypes did not affect the response of plasma lipids, angiographic indices of CAD or clinical events to treatment with fluvastatin (Tables 2 and 3).

S. Turban et al. / Atherosclerosis 154 (2001) 633–640

of the conflicting results of retrospective case-control studies. While several show a positive association between the R allele and the risk for CAD or MI [19 –24], others show no association [25 – 29]. This dichotomy is due, in part, to the high rate of spurious association that is intrinsic to case-control polymorphism association studies [30], differences in ethnic background of the populations and differences in the criteria used for phenotypic definitions. The present study, unlike previous studies, is a prospectively designed study in a well-characterized cohort, which has undergone extensive phenotypic characterization. The severity, progression and regression of coronary atherosclerosis were assessed by serial quantitative coronary angiography and extensive lipid profiles were measured at baseline and on completion of the study. We analyzed the association of Q/R192 polymorphism with a large number of demographic, biochemical, angiographic and clinical variables and a total of 46 different P values were calculated. The results were concordant for plasma levels of lipids, angiographic indices of CAD and clinical events. The frequencies of Q/R192 alleles in the LCAS population are similar to those reported in previous studies of Caucasians [19 – 21,23,25,26,28,29] and follow the Hardy – Weinberg equilibrium. Collectively, these data further reduce the likelihood of a false result in the present study. LCAS was designed to test the effects of fluvastatin on angiographic indices of progression and regression of coronary atherosclerosis. Hence, the choice of end points, duration of the study and the inclusion criteria were determined prior to genetic analysis. Therefore, genetic studies are secondary data analyses. The criteria for inclusion in LCAS required the presence of 30–75% diameter stenosis in at least one major coronary artery. Therefore, the results of the present study do not exclude the possible association of the R/Q192 poly-

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morphism with the initiation of coronary atherosclerosis. We note that there was also no significant association between development of new coronary lesions and the Q/R192 polymorphism in the LCAS population. It is also possible that the 2.5-year duration of the study was not sufficient and a longer time may be needed to detect a potential modest effect of the genotypes on progression or regression of coronary atherosclerosis. In addition, LCAS inclusion and exclusion criteria can also introduce a selection bias, which can confound the possible association of the genotypes with the baseline severity of coronary atherosclerosis. We also note that, compared to the previously published studies [20 –24], the LCAS population had a lower prevalence of diabetes mellitus and smoking, which can affect genotype-related risk in atherosclerosis. However, when analyzed, there were no significant interactions between smoking and the R/Q192 genotypes with regard to progression or regression of coronary atherosclerosis or clinical events in the LCAS population (data not shown). A total of 108 subjects in LCAS were also treated with cholestyramine. The distribution of the Q/R192 genotypes among those who were or were not treated with cholestyramine was not significantly different. In addition, there were also no significant interactions between the Q/R192 genotypes and treatment with cholestyramine with regards to plasma lipids or angiographic indices of coronary atherosclerosis (data not shown). In any study with null results, it is important to exclude the possibility of type II error. The sample size of 356 subjects provided an : 95% chance to detect a 20% difference in mean MLD and mean HDL level at baseline among the genotypes at a two-sided h value of 0.05. Similarly, the study had : 95% power to detect a 20% difference in the final MLD among the genotypes in the placebo or in the fluvastatin group. Since change

Table 3 Paraoxonase Q/R192 genotypes and progression or regression of coronary lesions and clinical eventsa Genotypes (N = 303)

Placebo Q/Q (73)

] 1 New lesion ] 1 New total occlusion Categorical angiographic change Progression (%) Regression (%) Mixed change (%) DMLD (mm) Lesion-specific DMLD (mm) Clinical e6ents All subjects (53/356) Angiographic subgroup (38/303) a

25 (34) 4 (5) 33 (45) 5(7) 35 (48) −0.139 0.27 −0.159 0.03 14 (17) 9 (12)

Fluvastatin Q/R (60) 21 (35) 4 (7) 18 (30) 6 (10) 36 (60) −0.089 0.22 −0.099 0.03 13 (18) 9 (15)

R/R (12) 4 (33) 2 (17) 6 (50) 0 (0) 6 (50) −0.1490.18 −0.1490.07 2 (14) 1 (8)

Q/Q (82) 25 (30) 4 (5) 30 (37) 11 (13) 41 (50) −0.069 0.27 −0.069 0.03 14 (15) 12 (15)

Q/R (56) 11 (20) 1 (2) 12 (21) 11 (20) 33 (59) −0.0090.24 −0.0290.03 9 (13) 6 (11)

Values for DMLD are presented as mean 9 S.D., for lesion-specific DMLD as mean 9S.E. and for all others as n (%).

R/R (20) 6 (30) 0 (0) 4 (20) 3 (15) 13 (65) −0.039 0.17 −0.049 0.05 1 (4) 1 (5)

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in the mean MLD in the fluvastatin group was B10% of the baseline mean MLD, we cannot exclude the presence of modest interactions between the Q/R192 genotypes and the response of MLD to treatment with fluvastatin. In addition, because the number of clinical events was relatively low (53/356) in the LCAS population, it can be inferred that the possible association of the Q/R192 genotypes with clinical events was not conclusively excluded in this study. Either a larger sample size or a longer duration of follow-up is necessary to resolve these issues conclusively. Furthermore, the enzymatic activities of PON1 alloenzymes could not be determined in this study. Overall, the main results of the LCAS showed that treatment with fluvastatin reduced mean LDL cholesterol by 24% and slowed progression of coronary lesions significantly [33]. In the LCAS population, the plasma level of HDL was a strong predictor of angiographic progression of coronary atherosclerosis and patients with low HDL received the greatest angiographic and clinical benefit [35]. The results of the present study suggest that the Q/R192 polymorphism is unlikely to play a major role in susceptibility to coronary atherosclerosis, but does not discount the possible role of PON1 in atherogenesis. The protective role of PON1 against oxidation of LDL has been extensively documented [6 – 8,11,12]. The null results of the present study may imply that PON1 activity against paraoxon — which is largely dependent on the Q/R192 polymorphism — is dissociated from its protective function in atherosclerosis. This possibility has been put forth by the results of a recent study, which showed that the ability of PON1 to protect LDL against oxidation was independent of its esterase activity and the Q/R192 polymorphism [37]. This finding, however, is in contrast to the results of two reports showing that PON1 alloenzymes confer variable ability to HDL to protect against accumulation of lipid peroxides in LDL [17,18]. Additional data suggest that different active sites are involved in paraoxonase/arylesterase activities and protection against LDL oxidation [38]. Accordingly, PON1 activity against LDL oxidation, although different between the two alloenzymes, requires the cysteine residue at position 283. Inhibition of the sulfhydryl group at this position blocks the arylesterase as well as antioxidant activities [38]. Collectively, these data suggest that the protective function of PON1 in atherosclerosis is mediated through a yet-tobe determined mechanism, which is at least partially independent of its polymorphic esterase activity. In view of the potential significance of PON1 in atherosclerosis, studies are needed to further elucidate the mechanism(s) by which PON1 protects against oxidative modification of LDL and atherogenesis. A second polymorphism in the HUMPONA gene in which leucine substitutes for methionine at position 55

(M/L55) has also been described [15,16]. The M/L55 polymorphism is in linkage disequilibrium with the Q/R192 polymorphism [19] and its functional significance is the subject of controversy. The original studies that described the M/L55 polymorphism showed that it had no effect on the enzymatic activity of PON1 against paraoxon [15,16]. However, in diabetic patients, the LL genotype has been associated with a higher plasma concentration of PON1 and greater paraoxonase activity than the LM or MM genotypes [39,40]. In addition, HDL from subjects with the combination of Q/Q and MM genotypes appears to be more effective in protecting against LDL oxidation [40]. However, a case-control association study found no significant association between the M/L55 polymorphism and CAD [41]. In conclusion, the results of this relatively large prospective study show that the Q/R192 genotypes of HUMPONA gene are not associated with the severity, progression or regression of coronary atherosclerosis, as determined by serial quantitative coronary angiography, or with plasma lipid levels or clinical events. In addition, the response of these variables to treatment with fluvastatin was not affected by the Q/R192 genotypes. Thus, the Q/R192 polymorphism is unlikely to be a major risk factor in the susceptibility to CAD in the LCAS population. Acknowledgements We wish to acknowledge Kerrie Jara for reading the manuscript and excellent suggestions. Funding for LCAS was provided by Novartis Pharmaceuticals Corporation Grant No. B351 and National Institutes of Health GCRC Grant No. 5M01RR00350. This work was also supported in part by a grant from Abercrombie Foundation. C.M. Ballantyne and A.J. Marian are recipients of Established Investigator Awards from the American Heart Association National Center, Dallas, TX, USA. References [1] Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem 1997;272:20963– 6. [2] Steinberg D. At last, direct evidence that lipoxygenases play a role in atherogenesis. J Clin Invest 1999;103:1487– 8. [3] Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. New Engl J Med 1989;320:915– 24. [4] Mackness MI, Durrington PN. HDL, its enzymes and its potential to influence lipid peroxidation. Atherosclerosis 1995;115:243– 53. [5] Parthasarathy S, Barnett J, Fong LG. High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein. Biochim Biophys Acta 1990;1044:275– 83.

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