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Paraoxonase Polymorphism (Gln¹⁹²-Arg) Is Associated with Coronary Heart Disease in Japanese Noninsulin-Dependent Diabetes Mellitus

Institute of Clinical Medicine (M.O., Y.T., K.Y.), University of Tsukuba, Ibaraki, Japan; The Wellcome Trust Centre for Human Genetics (M.O.), University of Oxford, Oxford, United Kingdom

Address correspondence and requests for reprints to: Masato Odawara, The Wellcome Trust Centre for Human Genetics, University of Oxford, Windmill Road, Oxford, OX3 7BN, United Kingdom.

	Abstract

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Serum paraoxonase/arylesterase (PONA) is associated with high-density lipoprotein and may prevent oxidation of low-density lipoprotein by hydrolyzing lipid peroxides. A recent report suggested an association of glutamine (A type)/arginine (B type) polymorphism at position 192 of PONA gene with coronary heart disease (CHD) among Caucasian patients with noninsulin-dependent diabetes mellitus (NIDDM). However, conflicting results have also been reported. To investigate the significance of this polymorphism in the pathogenesis of CHD, we performed an association study of this polymorphism with CHD in Japanese NIDDM patients. We genotyped 164 patients with NIDDM, 42 with CHD, and 122 without CHD. Other known risk factors for CHD were matched between the 2 groups. AB+BB isoforms were detected in 41 of 42 diabetic patients with CHD. The proportion of B allele carriers (AB+BB) was significantly higher than that of AA carriers among diabetic patients with CHD compared with those without CHD (² = 7.68, P = 0.003). Multivariate logistic regression analyses showed a markedly increased odds ratio (OR: 8.823, CI, 1.13–68.7) in B allele carriers, while ORs of other risk factors remained between 1.01 and 1.92. Carriers of the B allele of the Gln192Arg polymorphism in the PONA gene proved to be at increased risk for developing CHD in Japanese NIDDM patients. This association was independent of other known risk factors for CHD, suggesting an important role of the paraoxonase B isoform in the pathogenesis of CHD.

	Introduction

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NONINSULIN-dependent diabetes mellitus (NIDDM) is a major risk factor for the development of coronary heart disease (CHD), which is one of the leading causes of death in Japan as well as in western countries (1). Previous studies suggested that there may be a genetic predisposition (2, 3). Many genes are likely to be involved in the pathogenesis of CHD, including those involved in lipoprotein metabolism (4). Human paraoxonase/arylesterase (PONA) is a Ca^{2-dependent glycoprotein that binds to high-density lipoproteins (HDL) and has been shown to prevent LDL oxidation in vitro (5, 6, 7, 8, 9). Decreased PONA activity has recently been attributed to the presence of an A/G polymorphism in the coding region of the gene encoding this enzyme. The A/G polymorphism corresponds to glutamine (Gln)/arginine (Arg) polymorphism at amino acid position 192 (10, 11, 12). Recently, the presence of the Gln192}-Arg polymorphism in the PONA gene was reported to be an independent risk factor for CHD in a French Caucasian population with NIDDM (13). However, conflicting results have also been reported (14). To investigate whether this polymorphism is of pathological significance in Japanese, we examined an association of Gln¹⁹²-Arg polymorphism of paraoxonase gene with CHD in Japanese patients with NIDDM.

	Subjects and Methods

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Experimental subjects

We recruited 164 unrelated Japanese patients with NIDDM (89 males/75 females) who fulfilled the World Health Organization criteria for diabetes mellitus (15) from out-patient clinics of the University of Tsukuba Hospital and its affiliated hospitals. Informed consent was obtained from all of the patients before the study. Of these patients, 42 (22 males/20 females, P = 0.74, chi squared test: 63.7 \ 8.8 yr: range 42–75 yr) were diagnosed as having coronary heart disease (CHD) (CHD group), because they had a past history of acute myocardial infarction or had at least 75% stenosis in 1 or more major coronary branches on coronary angiography. The CHD- group included 122 patients (67 males/55 females: 61.8 \ 7.7 yr: range 48–82 yr) who appeared to be free from CHD (CHD- group), judging from normal electrocardiograms (ECG) at rest and lack of any history or family history of coronary heart disease in the first and second degree relatives.

Characteristics of subjects

Age, sex, body mass index (t test), the duration of diabetes (t test), proportion of smokers (chi-squared test), systolic and diastolic blood pressure (t test), serum total cholesterol (t test), triglycerides (Mann-Whitney’s U-test), Apo A1, Apo B, HDL cholesterol, and HbA_1c were not significantly different between the groups (t test) (Table 1).

DNA was extracted from peripheral leukocytes using standard procedures (16). Two primers (PON-192-S; 5'-TATTGTTGCTGTGGGACCTGAG-3' and PON-192-AS; 5'-CACGCTAAACCCAAATACATCTC-3') were synthesized for polymerase chain reaction (PCR) amplification of a 99 bp DNA fragment covering the region containing the Gln¹⁹² or Arg¹⁹². Aliquots of 200 ng of genomic DNA were used for PCR amplification as described previously (13), except that secondary amplification of 16 cycles was not performed. The amplified DNA fragments were digested with Alwl for 2–3 hr at 37 C, separated on nondenaturing 8% acrylamide gels, and stained with ethidium bromide. Allele A (glutamine: Gln) corresponded to a 99 bp fragment and allele B (arginine: Arg) to 65 and 34 bp fragments (Fig. 1).

View larger version (78K): [in this window] [in a new window]   Figure 1. Clinical characteristics of the Japanese patients with noninsulin-dependent diabetes mellitus (NIDDM) with or without coronary heart disease (CHD). Age, sex, body mass index, the duration of diabetes, proportion of smokers, systolic and diastolic blood pressure, serum total cholesterol, triglycerides, Apo A1, Apo B, HDL cholesterol, and HbA1c were not significantly different between the groups.

Chi-squared tests were performed to examine the differences in sex and the prevalence of smokers; Mann-Whitney U test was performed to examine triglyceride levels between two groups; Kruskal-Wallis test was used for a comparison of triglycerides among three groups; and Student’s t-test was used for analysis of other parameters. Chi-squared test and Fisher’s exact test were performed to compare the distribution of PONA genotypes and allele frequencies, respectively. Student’s t-test was used to analyze the parameters of AA or ABBB carriers except for triglycerides, which were analyzed by Mann-Whitney U test. Multivariate logistic regression analyses were performed on PONA genotypes, sex, age, systolic and diastolic blood pressure, proportion of smokers, and serum total cholesterol.

	Results

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Association of the PONA genotypes and CHD

Genetic analysis revealed that, among the CHD patients, only one had AA (Gln/Gln) isoform and 41 had AB (Gln/Arg, 24 patients) or BB (Arg/Arg, 17 patients) isoforms (Table 2). Twenty-five of 122 had AA isoform, and 97 had AB (53 patients) or BB (44 patients) isoforms. The ABBB isoforms were detected with significantly higher prevalence in patients with than in those without CHD (² = 7.68, P = 0.003) (Table 2). There was a trend toward an increased prevalence of the B allele in the CHD group (A allele: 31.0%; B allele: 69.0%) compared with the CHD- group (A allele: 42.2%; B allele: 57.8%; P = 0.07), although this was not statistically significant. The genotype frequencies in the CHD- group were in Hardy-Weinberg equilibrium. Multivariate logistic regression analyses revealed that the PONA genotype contributed to the pathogenesis of CHD independently of other known risk factors, such as age, sex, blood pressure, cigarette smoking, and serum total cholesterol (Table 3).

A previous investigation indicates that the genotypes of this polymorphism were significantly associated with variation of plasma concentrations of total HDL, non-HDL, and LDL cholesterol, total triglycerides, and apolipoprotein B (17). We investigated risk factors related to CHD between the two groups (AA vs. ABBB). There were no significant differences between the 2 groups (Table 4). Next, we classified all of the 164 patients with NIDDM with or without CHD, into three genotype groups (AA, AB, or BB groups). Clinical data were investigated in these patients. However, we could not observe significant differences among the three genotype groups, such as in total cholesterol (P = 0.5), HDL cholesterol (P = 0.9), triglycerides (P = 0.38), Apo A1 (P = 0.54), and Apo B (P = 0.6) (Table 5). Total cholesterol/HDL cholesterol, and Apo B/Apo A1 ratios were not different among the 3 groups.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We performed an association study of the Gln¹⁹²-Arg polymorphism of paraoxonase/arylesterase gene with CHD in 164 unrelated Japanese patients with NIDDM. Forty-two patients had a past history of acute myocardial infarction or had angiographically documented CHD, and 122 patients were free from CHD as determined from normal electrocardiograms at rest and lack of a history of CHD. Although the number of patients analyzed was limited, a highly significant increase in ABBB genotypes was observed in diabetic patients with CHD compared with those without CHD. There was also a trend toward an increased frequency of the B allele in patients with CHD, although this did not reach statistical significance, presumably because of the relatively limited number of patients analyzed. Multivariate logistic regression analyses revealed that the association of ABBB genotypes with CHD group was independent of age, sex, systolic and diastolic blood pressure, smoking, and serum total cholesterol level. To exclude the possibility that B allele carriers (ABBB) have other risk factors related to CHD, we investigated these factors between the 2 groups (AA vs. ABBB). There were no significant differences between carriers of the B allele and those homozygous for AA with respect to plasma cholesterol, triglycerides, or Apo B concentrations, nor to HDL-cholesterol or Apo A-1, in contrast to previous reports (13, 17) (Table 4). These differences from the observations of previous reports, suggest that the association of paraoxonase polymorphism and lipid profile may be variable depending on patients examined. By an analysis of all 164 patients, no significant differences were observed among the carriers of the 3 genotypes in clinical features, including plasma lipid concentrations (Table 5) and associated diseases. These differences from previous reports suggest that the association of paraoxonase polymorphism and lipid profile may be variable depending on patients examined. Whether BB carriers have earlier onset of CHD or more severe clinical phenotypes was not clear, partly due to an insidious onset of coronary stenosis or acute myocardial infarction among diabetic patients. Previous reports indicated that the Gln¹⁹²-Arg polymorphism is associated with CHD among French Caucasian NIDDM (13), U.S. Caucasian populations without any evidence of an association of NIDDM (18), or with Caucasian ancestry (19). However, conflicting results have also been reported by another group (14). Although the number of patients analyzed was not large in our study, the highly significant results suggest an important role of the PONA gene Gln¹⁹²-Arg polymorphism in the pathogenesis of CHD. The prevalence of CHD has been reported to be much lower in Japanese NIDDM than in Caucasian patients. However, these observations suggest that Gln¹⁹²-Arg variant is a common risk factor that contributes to the pathogenesis of CHD in both Japanese and Caucasian NIDDM patients. We conclude that the Gln¹⁹²-Arg polymorphism of the paraoxonase gene (B allele) is associated with coronary heart disease in Japanese patients with NIDDM. The precise mechanism by which the Gln¹⁹²-Arg polymorphism influences susceptibility to CHD remains unknown. However, our observations suggest that there is an association between this polymorphism and diabetic patients with CHD from at least 2 different ethnic groups, and that the paraoxonase B isoform plays an important role in the pathogenesis of coronary heart disease.

Received September 5, 1996.

Revised February 27, 1997.

Accepted April 10, 1997.

	References

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