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(Stroke. 2002;33:1459.)
© 2002 American Heart Association, Inc.

Original Contributions

Paraoxonase 192 GlnArg Polymorphism

An Independent Risk Factor for Nonfatal Arterial Ischemic Stroke Among Young Adults

Barbara Voetsch, MD; Kelly S. Benke, AB; Benito P. Damasceno, MD; Lúcia H. Siqueira, BA Joseph Loscalzo, MD, PhD

From the Whitaker Cardiovascular Institute, Evans Department of Medicine (B.V., J.L.), and Genetics Program, Evans Department of Medicine (K.S.B.), Boston University School of Medicine, Boston, Mass; and Department of Neurology (B.V., B.P.D.) and Hematology and Hemotherapy Center, Department of Medicine (L.H.S.), State University of Campinas, Campinas, São Paulo, Brazil.

Correspondence to Joseph Loscalzo, MD, PhD, Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany St, W507, Boston, MA 02118. E-mail jloscalz{at}bu.edu

	Abstract

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Background and Purpose— The etiology of arterial ischemic stroke (AIS) in the young remains unknown in one third of patients. Serum paraoxonase (PON1) is an HDL-associated esterase that hydrolyzes products of lipid peroxidation and prevents the oxidation of LDL. Two common polymorphisms in the PON1 gene, the 192 Gln (Q) Arg (R) and 55 Leu (L) Met (M) substitutions, determine interindividual variation in PON1 activity. The association of these polymorphisms with the risk of AIS remains controversial.

Methods— We analyzed 118 patients (64 women) with a first nonfatal AIS occurring <45 years of age and 118 1:1 age (±2 years)- and sex-matched controls. The PON1 polymorphisms were determined by polymerase chain reaction amplification and restriction digestion.

Results— The prevalence of the PON1 192RR genotype (P=0.006) and the frequency of the R allele (P=0.010) were significantly increased among young AIS patients compared with controls. After adjustment for conventional vascular and prothrombotic risk factors, the 192RR genotype remained independently associated with a 4-fold increase in the risk of AIS (odds ratio=4.1; 95% CI, 1.14 to 14.73). Subanalyses stratified by the presence of vascular risk factors and ethnicity did not significantly modify the effect of the PON1 192 polymorphism on AIS risk. No significant differences were found between patients and controls regarding the PON1 55 polymorphism.

Conclusions— These findings suggest that the PON 192RR genotype is independently associated with an increased risk of nonfatal AIS among young adults. Further studies are necessary to understand better the mechanistic implications of these observations in the development of AIS in the young.

Key Words: atherosclerosis • polymorphism • risk factors • stroke, ischemic • young adults

	Introduction

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Arterial ischemic stroke (AIS) in the young is a heterogeneous condition with a wide spectrum of underlying causes; however, its etiology remains unknown in approximately one third of patients, despite extensive clinical and laboratory investigation.¹ The occurrence of AIS among young adults is not uncommon; 2% to 19% of ischemic strokes affect patients aged <45 years worldwide.^2,3 Thus, the identification of novel risk factors and the prevention of cerebral thrombosis in this age group have important public health implications.

Serum paraoxonase (PON1) is a calcium-dependent esterase synthesized by the liver and bound exclusively to HDL in plasma. It has been extensively studied in the field of toxicology owing to its ability to detoxify organophosphate insecticides and nerve gases⁴; its physiological role, however, remains unclear. Recently, PON1 has been implicated in the pathogenesis of atherosclerosis and cardiovascular disease (CVD). It has been shown to preserve HDL function⁵ and to protect LDLs from oxidative modification by hydrolyzing lipid peroxides,⁶ thus exerting antioxidant and antiatherogenic effects. In addition, PON1 protects against the induction of monocyte-endothelial interactions in the artery wall by metabolizing biologically active lipids in oxidized LDL.⁷ Both peroxidation of LDL and the secondary inflammatory responses are key steps in the initiation of atherogenesis. Thus, PON1 is now thought to be responsible, at least in part, for the cardioprotective properties of HDL.⁵ PON1-deficient mice are more susceptible to diet-induced atherosclerosis than their wild-type littermates, and HDL isolated from these animals is unable to prevent LDL oxidation in vitro.⁸ In addition, clinical reports have demonstrated that PON1 activity is reduced in patients with acute myocardial infarction⁹ and conditions associated with accelerated atherogenesis, such as familial hypercholesterolemia and diabetes mellitus.¹⁰

There is a 10- to 40-fold interindividual variability in serum PON1 activity as measured by rates of hydrolysis of the exogenous substrate paraoxon.¹¹ Two common polymorphisms in the coding region of the PON1 gene, which lead to a glutamine (Q) arginine (R) substitution at position 192 and a leucine (L) methionine (M) substitution at position 55, independently influence PON1 activity and have been defined as the molecular basis for this interindividual variability.^11,12 Several case-control studies have investigated the association between the PON1 polymorphisms and coronary artery disease (CAD), yielding conflicting results. While some reports have shown an increased susceptibility to CAD among carriers of the 192R allele, with risk estimates ranging between 1.7 and 8.8, others have reported a lack of association.¹³ Garin and colleagues¹² first demonstrated that homozygosity for the PON1 55L allele also independently increased the risk of CAD, yet results of several later studies failed to confirm this association.^14,15 Far fewer studies have analyzed the role of the PON1 polymorphisms in cerebrovascular disease, but similarly, inconsistent results have been found. The majority of these studies have focused on intima-media thickness and stenosis of the carotid artery,^16–19 while only 3 have been performed among patients with AIS per se.^20–22 In addition, most of the studies have been performed in middle-aged or elderly populations. Therefore, in the present study we sought to investigate a selected population of young adults with AIS of undetermined etiology to assess the possible role of the PON1 polymorphisms in the early development of clinically manifest AIS.

	Subjects and Methods

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Study Subjects
Between January 1996 and December 2000, 278 survivors of a first AIS occurring between the ages of 15 and 45 years were consecutively referred to the Neurology or Hematology clinics of the State University of Campinas Medical School Hospital, in Campinas, Brazil. Arterial IS was defined as an acute, focal, neurological deficit lasting >24 hours or symptoms lasting <24 hours if a brain imaging study showed an ischemic lesion appropriate to the symptoms. The diagnosis of AIS was based on findings of history and physical examination and confirmed by CT and/or MRI of the brain. Patients with cerebral venous thrombosis, transient ischemic attacks, and migrainous events were excluded. After undergoing extensive work-up, those patients for whom an underlying cause for the AIS could be identified were further excluded from the study. These consisted of systemic disease; cancer; cardiac sources of embolism; sickle cell anemia; deficiency of natural anticoagulants, such as protein C, protein S, or antithrombin III; and the presence of antiphospholipid antibodies. The remaining 118 patients were enrolled in a research study for the evaluation of inherited risk factors for AIS of undetermined etiology in the young. This study was approved by the Medical Ethics Committee of the State University of Campinas.

Unrelated blood donors and volunteers from the same geographical area without a clinical history of coronary, peripheral, or cerebrovascular disease were invited to participate in the study as control subjects and were matched 1:1 to patients with respect to age (±2 years) and sex. All controls were interviewed by trained personnel to obtain information on past medical history, conventional vascular risk factors (hypertension, diabetes, current or former smoking, hyperlipidemia, and use of oral contraceptives), and the use of medications and illicit drugs. Blood pressure was measured, and a blood sample for laboratory testing and genotyping was obtained.

Brazil has an ethnically heterogeneous population. In the Campinas area, most of the population descends from Caucasian immigrants from southern and central Europe. In addition, part of the population is of African origin, and a minor portion is of Asian background. We defined ethnic background according to the subjects’ self-determination.

Genotype Determinations
Peripheral blood samples were collected after study subjects’ consent. Genomic DNA was obtained from leukocytes by standard phenol-chloroform extraction. Determination of the PON1 polymorphisms was achieved by polymerase chain reaction (PCR) followed by restriction digestion with 5 U of AlwI for the 192 polymorphism and 2.5 U of NlaIII in the presence of 0.1 mg/mL BSA for the 55 polymorphism, as described by Ombres et al²³ and Humbert et al,¹¹ respectively. Factor V Leiden, the G20210A mutation in the prothrombin gene, and the 677 CT substitution in the methylenetetrahydrofolate reductase (MTHFR) gene were determined by PCR and restriction digestion, according to the methods of Bertina and colleagues,²⁴ Poort and colleagues,²⁵ and Frosst and coworkers,²⁶ respectively. Digested PCR products were separated by electrophoresis on 2.5% ethidium bromide–stained agarose gel.

Statistical Analysis
Differences in demographic characteristics and vascular risk factors between patients and controls were initially compared by univariate analysis with the use of Student’s t test for age and the ² test for all categorical variables. Odds ratios (ORs) and their corresponding 95% CIs were calculated to determine crude associations among categorical predictors. Allele frequencies were determined by the gene counting method, and Hardy-Weinberg equilibrium was tested by the ² test.

A conditional logistic regression model, stratified by a variable that accounted for matching on age (±2 years) and sex, was used to evaluate the association of the PON1 polymorphisms with stroke risk. The 192RR and 55LL genotypes were analyzed with the use of the 192QR+QQ genotypes and the 55LM+MM genotypes, respectively, as reference groups. Adjustments were made for hypertension, diabetes, smoking, hyperlipidemia, and ethnicity. The extent to which associations with the PON1 polymorphisms were modified by other risk factors was assessed through analyses stratified by these risk factors. Heterogeneity between the strata was evaluated by the Breslow-Day test. Interaction terms were created as the product of 2 predictor variables and were included in the regression models. All statistical analyses were performed with the use of Statistical Analysis Systems (SAS version 8.2).

	Results

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The demographic characteristics and the prevalence of vascular and inherited prothrombotic risk factors among patients and controls are shown in Table 1. The groups were well matched for age, sex, and ethnic background. Caucasians were the most prevalent ethnic group among both patients and controls. As expected, all conventional vascular risk factors were more prevalent among patients than controls; however, only hypertension and smoking reached statistically significant differences by univariate analysis (P<0.0001 and P=0.0026, respectively). Regarding the inherited prothrombotic risk factors, factor V Leiden was equally distributed in both groups, being present in 3.4% of patients and controls. Heterozygosity for the prothrombin G20210A mutation, as well as homozygosity for the C677T substitution in the MTHFR gene, was more prevalent among patients than controls; however, neither difference was statistically significant (P=0.47 and P=0.23, respectively). None of the study subjects was found to be homozygous for factor V Leiden or the prothrombin G20210A mutation.

View this table: [in this window] [in a new window]   Table 1. Demographic and Clinical Characteristics of Patients With AIS and Controls

Table 2 shows the genotype distribution and allele frequencies of the PON1 polymorphisms in AIS patients and controls. The distributions of the PON1 192 and 55 genotypes were in Hardy-Weinberg equilibrium in both groups. Regarding the 192 polymorphism, the genotypic distribution differed significantly between patients and control subjects (P=0.011). The RR genotype was overrepresented among patients (16.1%) as compared to controls (5.1%; P=0.006), yielding an OR of 3.6 (95% CI, 1.4 to 9.3) by univariate analysis and a significantly higher frequency of the 192R allele among patients than control subjects (0.43 versus 0.31; P=0.01; OR=1.64; 95% CI, 1.1 to 2.4). The distribution of the PON1 55 genotypes and alleles did not differ significantly between the 2 groups.

View this table: [in this window] [in a new window]   Table 2. Genotype Distribution and Allele Frequencies of PON1 192 and 55 Polymorphisms in Patient and Control Groups

Table 3 shows the distribution of the PON1 192 and 55 genotype combinations in the patient and control groups. Methionine at position 55 (M allele) was rarely associated with arginine (R allele) at position 192. None of the subjects was found to carry both the 192RR and 55MM genotypes. When we calculated the number of 192R and 55L risk alleles each subject carried, a trend to an increased susceptibility to AIS was found among carriers of 2 or more risk alleles (P=0.06; OR=1.7; 95% CI, 0.94 to 3.2).

View this table: [in this window] [in a new window]   Table 3. Distribution of PON1 192 and 55 Genotype Combinations in Patient and Control Groups

The findings from the univariate analyses were further investigated in a conditional multiple logistic regression model incorporating the PON1 192 and 55 polymorphisms, ethnicity, and the vascular and inherited prothrombotic risk factors. The results of this analysis are shown in Table 4. The 192RR genotype independently increased the susceptibility to AIS 4-fold (OR=4.1; 95% CI, 1.14 to 14.73). Hypertension showed the strongest association with AIS, increasing the susceptibility more than 10-fold (OR=10.85; 95% CI, 3.55 to 33.18), while the risk estimate associated with smoking was 2.65 (95% CI, 1.25 to 5.62).

View this table: [in this window] [in a new window]   Table 4. Conditional Multiple Logistic Regression Model Incorporating PON1 192 and 55 Polymorphisms and Vascular and Inherited Prothrombotic Risk Factors

To assess whether smoking modified the risk of AIS associated with the 192 polymorphism, we analyzed the distribution of the 192RR genotype after stratification for smoking status (Table 5). Crude analyses accounting for age and sex revealed that there was a nonsignificant 2.5-fold increase in the risk of AIS among nonsmokers who had the RR genotype. Smoking alone doubled the risk of AIS as compared to the reference group (P=0.0089). Among smokers, the presence of the RR genotype further increased the risk of AIS >8-fold; however, the sample size for this analysis was small, yielding very wide CIs. Thus, these results must be interpreted with caution. When we tested for interaction in a conditional logistic regression model adjusting for all other vascular risk factors, we found no significance for the interaction variable (P=0.27). All other risk factors were analyzed for interactions, yielding no significant results (data not shown).

View this table: [in this window] [in a new window]   Table 5. Effect of PON1 192RR Genotype on Risk of AIS Among Smokers and Nonsmokers

Although the patient and control groups were well matched regarding ethnicity (P=0.43), we performed separate subanalyses among Caucasians and subjects of African descent, owing to the well-described interethnic variability of the PON1 allele frequencies. The genotype distributions remained in Hardy-Weinberg equilibrium in all groups. Among Caucasians, the risk estimates for AIS associated with the 192RR genotype (14.8% in patients versus 4.2% in controls; P=0.013; OR=4.0; 95% CI, 1.1 to 15.2) and the frequency of the R allele (0.42 in patients versus 0.28 in controls; P=0.0036; OR=1.9; 95% CI, 1.2 to 3.0) were slightly increased compared with those found in the overall analysis. Among the small number of subjects of African origin, the prevalence of the 192RR genotype was twice as high in patients (28.6%) as in controls (14.3%), yet this difference was not significant (P=0.13). Regarding the distribution of the PON1 55 genotypes and alleles, no significant differences were found between patients and controls in both ethnic groups.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The currently established risk factors for atherothrombosis are largely environmental in nature and are not alone sufficient to explain completely the variations in incidence and risk of developing CAD or AIS.²⁷ Several reports indicate that there is a significant genetic component underlying the occurrence of AIS, yet the specific genes contributing to disease risk remain largely undefined.²⁸ Kittner²⁹ suggested that stroke in the young can be used as a model system for studying novel risk factors for stroke, including the genetic basis for stroke risk. Because conventional vascular risk factors are less common in this young age group, it is likely that a genetic influence on the development of atherothrombosis is most clearly evident. In addition, understanding the early onset of AIS offers promising clues to mechanisms relevant to disease across the entire age spectrum.²⁹

Paraoxonase has emerged as a novel risk factor for CVD in the past decade, yet the risk associated with the PON1 192 and 55 polymorphisms remains unclear. We found that the 192RR genotype was independently associated with a 4-fold increase in risk of nonfatal AIS. This result is in accordance with several case-control studies that have demonstrated an increased susceptibility to CAD among carriers of the 192R allele. Although other studies reported a lack of association, a recent meta-analysis performed by Mackness and colleagues¹³ determined that, overall, the 192R allele is associated with an increased risk of CVD. These data are consistent with laboratory studies showing that HDL containing the 192R isoform is less effective in protecting LDL from oxidative modification.³⁰ Furthermore, clinical studies have shown that homozygotes for the 192Q allele have a better plasma lipoprotein profile than carriers of the R allele.²³

Regarding the PON1 55 polymorphism, we found no significant difference in genotype or allele distributions between patients and controls, which is in agreement with the majority of studies performed in CAD.^14,15 In the largest study that investigated the role of the 55 polymorphism in cerebrovascular disease, Imai and colleagues²¹ found no association with AIS among elderly Japanese individuals. In the Austrian Stroke Prevention Study, the 55LL genotype was associated with the progression of cerebral white matter lesions, a marker of small-vessel disease,³¹ and the frequency and severity of carotid stenosis¹⁶; however, these patients had no clinically manifest ischemic event.

A cumulative effect of the PON1 192 and 55 polymorphisms on the protective effect of HDL³⁰ and the risk of CVD¹⁷ has been proposed. When we calculated the number of 192R and 55L risk alleles each subject carried, we found a trend to an almost doubled susceptibility to AIS among carriers of 2 or more risk alleles (OR=1.7; 95% CI, 0.94 to 3.2). Interestingly, the 55M allele was rarely associated with the 192R allele. This nonrandom association is due to a linkage disequilibrium, first described by Garin and colleagues,¹² that links the rare R allele at position 192 to the frequent L allele at position 55, thus giving rise to a common haplotype carrying both risk alleles.

Genetic and environmental risk factors may interact in various ways to either decrease or enhance the absolute susceptibility to CVD. Because PON1 exerts its antiatherogenic effects primarily by protecting LDL against oxidative modification, it has been suggested that the presence of the 192R and 55L risk alleles in individuals simultaneously exposed to other factors that enhance oxidative stress, such as diabetes or smoking, may potentiate the risk of CVD. Both diabetes and smoking increase the susceptibility of LDL to in vitro peroxidation and reduce serum PON1 activity in vivo.^10,32 Among smokers, even the low-risk 192Q and 55M alleles have been demonstrated to increase the susceptibility to CAD, conceivably because these individuals are subject to increased oxidative stress^33,34; however, other groups reported an association of the PON1 polymorphisms with cardiovascular disease only in nonsmokers,^35,19 while some investigators found no effect after stratification for smoking status.^18,23 Our results indicate that in both nonsmokers and smokers, there is an increase in risk associated with the presence of the PON1 192R allele; however, we could not establish evidence for an interaction by conditional logistic regression analysis, possibly because of the weak statistical power of these subgroup analyses.

Paraoxonase activity and allele frequencies vary greatly across human populations. This genotypic variability may account for some of the inconsistent results found in previous association studies between the PON1 polymorphisms and CVD. In our study, restricting the analysis to Caucasians did not appreciably alter the risk estimates for AIS. Among subjects of African origin, the prevalence of the 192RR genotype was twice as high in patients as in controls. This difference was consistent with our overall findings yet did not reach statistical significance, most likely owing to the small sample size. No differences were found in the prevalence of the 55 polymorphism.

Some limitations of our study should be noted. First, this study includes only survivors of AIS, and the possibility of survival bias must be considered. Second, we analyzed the effect of the PON1 polymorphisms on AIS without taking into consideration specific pathogenic subtypes of AIS. Finally, this is an allelic association study and cannot provide evidence for the mechanism by which the PON1 polymorphisms predispose to AIS.

In conclusion, ours is the first study to investigate the association of the PON1 polymorphisms with the development of nonfatal AIS in adults younger than 45 years of age. We found that the PON1 192RR genotype independently increased the risk of AIS by 4-fold in this population. The presence of conventional vascular risk factors did not significantly modify the effect of this genetic marker on AIS risk. Confirmation of these results requires further studies involving larger numbers of subjects and should attempt to assess the mechanistic implications of these observations, especially under conditions of increased oxidant stress.

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL55993, HL58976, and HL61795 to Dr Loscalzo. Dr Voetsch was supported in part by funding from Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior BEX 1032/98-4, Brasília, Brazil.

Received December 14, 2001; revision received February 27, 2002; accepted March 5, 2002.

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