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Effects of coagulation Factor VII polymorphisms on the coronary artery disease in Japanese
Thrombosis Research 105 (2002) 493 – 498
Regular Article
Effects of coagulation Factor VII polymorphisms on the coronary artery disease in Japanese Factor VII polymorphism and coronary disease Keiko Shimokata a, Takahisa Kondo a,*, Miyoshi Ohno b, Kyosuke Takeshita a, Yasuya Inden a, Shigeo Iino a, Hidehiko Saito c, Makoto Hirai a a
First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 Japan b Division of Cardiology, Nagoya First Red Cross Hospital, Nagoya, Japan c Nagoya National Hospital, Nagoya, Japan Received 26 February 2002; received in revised form 19 April 2002; accepted 21 April 2002 Accepting Editor: S. Iwanagah
Abstract We investigated the relationships among Factor VII coagulant activity (FVIIc), genetic polymorphisms of Factor VII (FVII) and coronary artery disease (CAD) in 380 unrelated Japanese individuals (mean 64 years) who underwent coronary angiography and whose cholesterol levels were within normal range. CAD subjects were defined as those in whom one of the three major coronary arteries showed > 50% narrowing after nitroglycerin administration. FVIIc was measured and the following polymorphisms of FVII were determined: R353Q polymorphism (M1, M2 alleles), 323 0/10 bp polymorphism (0, 10 alleles), hypervariable region 4 of intron 7 (HVR4; H5, H6, H7 alleles). FVIIc was slightly lower in M1M2/M2M2 than M1M1 (89.5 F 8.9%, 93.4 F 17.8%). Those with M2 and/or 10 allele have less chance of developing CAD (M2: OR 0.36, 95% CI 0.18 – 0.69, 10: OR 0.50, 95% CI 0.26 – 0.97). However, both alleles did not associate with myocardial infarction (MI). HVR4 was unrelated with CAD, nor with MI. In conclusion, M2 and/or 10 allele has protective effects on the developing CAD in individuals with a normal cholesterol level. D 2002 Published by Elsevier Science Ltd. Keywords: Polymorphism; Factor VII; Coronary disease; Myocardial infarction
1. Introduction Thrombosis underlies most acute manifestations of coronary atherosclerotic disease, including myocardial infarction (MI) [1]. Plaque disruption, with resulting exposure of tissue factor to blood and binding of tissue factor to circulating coagulation Factor VII (FVII) [2,3], is considered the major cause of thrombosis in MI. During the past two decades, there have been numerous reports Abbreviations: FVII, Factor VII; FVIIc, Factor VII coagulant activity; CAD, coronary artery disease; MI, myocardial infarction; OR, odds ratio; CI, 95% confidence interval; R353Q, substitution of glutamine for arginine at position 353 in the catalytic domain in the FVII gene; 5VF7, a 10-bp insertion in the promoter region in the FVII gene; HVR4, hypervariable region 4 of intron 7 of the FVII gene; BMI, body mass index; HDL, highdensity lipoprotein; Group Zero, no significant coronary artery stenosis group; Group S/D/T, single-, double- or triple-vessel disease group. * Corresponding author. Tel.: +81-52-744-2147; fax: +81-52-744-2157. E-mail address: [email protected] (T. Kondo). 0049-3848/02/$ - see front matter D 2002 Published by Elsevier Science Ltd. PII: S 0 0 4 9 - 3 8 4 8 ( 0 2 ) 0 0 0 6 7 - 1
investigating Factor VII coagulant activity (FVIIc) in coronary artery disease (CAD). Some identified FVIIc as a potent risk indicator for CAD [4 –6], while others did not [7,8]. For example, in the Northwick Park Heart Study, FVIIc was an independent predictor of MI in initially healthy middle-aged men, and particularly of fatal coronary events [4]. However, three large-scale prospective studies, the Prospective Cardiovascular Mu¨nster Study [6], the Edinburg Artery Study [7] and the ECTIM Study [8], failed to identify the FVII level as a predictor of future CAD. Why are their discrepancies in the study of FVIIc and CAD? Three reasons come to mind. First, there is a confounding relation between FVIIc and MI, and no causal relationship has been found to date. The high FVIIc level could be a consequence of atherosclerosis rather than a cause of disease progression. Second, FVIIc is related to age, lipid level and obesity [9 –11]. Therefore, if the lipid levels of subjects in a study group are too high or if these
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subjects are overweight, the causal relation between FVIIc and CAD would be misunderstood. Third, the genetic background in the previous study is not the same [7,8,12,13]. It is a well-known fact that there are genetic differences in the activation of the coagulation cascade [14]. Some polymorphisms reported in Caucasians are not found in the Asian population such as Leiden mutation and thrombin polymorphism [12,13]. Furthermore, Japan is one of the countries where the incidence of CAD is much less common compared with Western countries [15]. In this sense, it is intriguing to investigate the association of FVII polymorphism and CAD in Japan. So far, Kario et al. have reported the association of FVII polymorphism, FVIIc and cerebrovascular disease in Japan. However, the association of FVII polymorphism and CAD in Japanese subjects remains unclear [16 – 19]. Recently, three common polymorphisms in the FVII gene, i.e., the substitution of glutamine for arginine at position 353 in the catalytic domain (R353Q), a 10-bp insertion in the promoter region (5VF7) and a variable number of 37-bp repeats in intron 7 (HVR4), are reported to be responsible for up to one third of the variations in FVIIc among Caucasians and Japanese [20,21] and could be risk factors for MI [22]. If FVIIc really influences the risk for cardiovascular disease, these polymorphisms become good tools because they are determined at the time of birth and do not change throughout life. In this study, we investigated the relation between FVII polymorphisms and the frequency of CAD in Japanese subjects. One advantage of the present study is that Japanese are highly homogenous in genetic background because Japan is an insular country and closed its doors to outside contact for nearly 300 years until about 1876. Secondly, the cholesterol levels of subjects in this study are not high; we could avoid the effect of cholesterol and evaluate the genuine effect of FVIIc on the progression of CAD. The main purposes of this study were (1) to clarify the relation among three common polymorphisms of FVII and MI, and (2) to investigate the effect of these polymorphisms on the progression of coronary artery sclerosis in Japanese whose cholesterol levels are low compared with Caucasian subjects. We also investigated whether there was an association between polymorphisms in the FVII gene and FVIIc in Japanese as is often reported in Caucasians.
2. Materials and methods 2.1. Study population and laboratory data collection The study subjects were recruited from the First Department of Internal Medicine, Nagoya University School of Medicine, and from the Division of Cardiology at Nagoya First Red Cross Hospital in Aichi Prefecture from January
to May 2000. Blood samples and data were collected from 380 consecutive subjects who underwent heart catheterization because of suspected CAD, arrhythmia, valvular disease or congenital heart disease (133 MI subjects and 247 non-MI subjects). Diagnosis of acute MI was made based on chest symptoms, electrocardiographic changes and serum creatine kinase elevations more than twice the normal upper level. Individual histories were taken concerning diabetes mellitus (DM), hypertension and smoking habits. Body mass index (BMI) was calculated by dividing weight in kilograms by height in meters squared. Levels of red blood cells, white blood cells, platelets, total cholesterol, triglyceride and high-density lipoprotein (HDL) cholesterol were determined with standard procedures (after overnight fast). In MI subjects, we used data sampled over 1 month after the onset of MI. Coronary angiography was performed according to the Judkins method. Coronary artery stenosis was defined as significant when the lumen diameter was >50% narrowed after nitroglycerin administration. Then, relevant CAD was also characterized as single-, double- and triple-vessel disease. We further divided subjects into two groups according to the number of diseased vessels: Group Zero (n = 125) vs. Group S/D/T (n = 255). The Group Zero subjects had no significant coronary artery stenosis, and those in Group S/D/ T had single-, double- or triple-vessel disease. Three cardiologists blinded to the results of genotype analyses made the CAD assessment. Written informed consent was obtained from all subjects studied in the two hospitals. The study was in accord with the guidelines approved by the ethics committee of Nagoya University School of Medicine. 2.2. DNA analysis DNA was isolated according to the manufacturer’s instructions (Gene Trapping by Liquid Extraction, Takara Company, Japan). The R353Q polymorphism was amplified according to a modification of the method of Marchetti et al. [23]. PCR was performed using the described set primers. The amplified fragments were digested with 5 U of MspI (New England Biolaboratories, Beverly, MA, USA) and then subjected to electrophoresis on 3% Nusieve 3:1 agarose gel. Fragments of 205 (the M1 allele) and 272 bp (the M2 allele) were detected. To detect the 5VF7 polymorphism, a 214-bp DNA fragment was amplified using the described primers [24]. Amplified fragments were digested with 10 U of EcoT 14I (Takara Company, Japan) and then subjected to electrophoresis on 1.5% agarose gel. Fragments of 214 (0 allele) and 136 + 88 bp (10 allele) were detected. The HVR4 of the FVII gene was amplified using primers described before [25]. Two alleles were identified: a common allele (the H6 allele) of 443 bp with six monomers, and a less frequent allele (the H7 allele) of 480 bp with seven monomers of 37 bp. We could not find the very rare H5 allele of 406 bp with five monomers.
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2.3. FVIIc level
Table 2 Distribution of three genotypes between Group S/D/T and Group Zero
We measured the FVIIc level of 200 of a total of 380 patients, whose plasma was stored at 80 jC. FVIIc was assayed by a one-stage assay using human placenta thromboplastin.
Polymorphism (a) All subjects R353Q
5VF7
2.4. Statistical analysis
HVR4
Clinical laboratory data were expressed as mean F S.D. and compared between Group S/D/T and Group Zero subjects by Student’s t test or the Mann – Whitney U test when applicable. Differences in means of continuous variables between more than two groups were assessed by Kruskal –Wallis test. Allele frequencies were estimated by the gene counting methods, and the Hardy– Weinberg equilibrium was confirmed by chi-square test. The odds ratio (OR) and 95% confidence interval (CI) were also calculated. Multiple logistic regression analyses were performed and adjusted by age, sex, smoking status, total cholesterol level and presence of hypertension and DM using Stat View 5.0 (SAS Institute, Cary, NC, USA). A P value of < .05 was considered statistically significant.
Genotype
S/D/T
Zero
OR (95% CI)
M1M1 M1M2 and M2M2 0/0 0/10 H6H6 H6H7 H7H7
237 18
103 22
1.00 0.36 (0.18 – 0.69)
234 21 93 113 49
106 19 44 52 29
1.00 0.50 (0.26 – 0.97) 1.00 0.97 (0.60 – 1.58) 1.25 (0.70 – 2.24)
124 5
64 16
1.00 0.16 (0.06 – 0.46)
123 6 47 56 26
67 13 24 33 20
1.00 0.25 (0.09 – 0.69) 1.00 1.15 (0.60 – 2.22) 1.51 (0.70 – 3.23)
(b) Subjects under 65 years old R353Q M1M1 M1M2 and M2M2 5VF7 0/0 0/10 HVR4 H6H6 H6H7 H7H7
Group Zero indicates subjects without significant coronary artery disease; Group S/D/T indicates subjects with single-, double- or triple-vessel disease.
The genotype frequencies of the three polymorphisms examined in this study were all in agreement with those predicted by the Hardy– Weinberg equilibrium.
3. Results 3.1. Baseline characteristics of study population
3.2. Association of CAD and three genotypes at FVII Table 1 listed the clinical characteristics, blood cell counts, lipid level and genotype distribution of FVII in this study group. The number of male subjects was significantly higher and the HDL level was significantly lower in the Group S/D/T than in the Group Zero ( P < .05).
Table 1 Clinical characteristics of Group S/D/T and Group Zero
Male/female Age (year) BMI (kg/m2) BPs (mm Hg) BPd (mm Hg) RBC ( 1012/l) WBC ( 109/l) Plt ( 109/l) T chol (mmol/l) TG (g/l) HDL (mmol/l) DM (%) Smoking (%)
S/D/T (n = 255)
ZERO (n = 125)
P value
214/41 63.4 F 9.38 23.7 F 2.80 137 F 21.1 76.8 F 12.7 4.23 F 0.56 6.13 F 1.64 223 F 73.4 5.05 F 1.03 1.63 F 0.87 1.10 F 0.29 30.2 51.9
85/40 63.6 F 13.1 23.2 F 3.28 134 F 21.1 77.1 F 13.2 4.35 F 0.53 6.10 F 1.60 214 F 56.6 5.24 F 0.97 1.42 F 0.97 1.63 F 0.40 19.9 39.1
< .001 * .926 .250 .291 .886 .052 .899 .195 .074 .046 < .001 *,y .671 .350
Group Zero indicates subjects without significant coronary artery disease; Group S/D/T indicates subjects with single-, double- or triple-vessel disease. BMI, body mass index; BPs, systolic blood pressure; BPd, diastolic blood pressure; RBC, red blood cell; WBC, white blood cell; Plt, platelet; T. chol, total cholesterol; TG, triglyceride; HDL, high-density lipoprotein; FH, family history of coronary artery disease; DM, diabetes mellitus. *m2, statistics; y, Mann-Whitney U test.
Distributions of R353Q, 5VF7 and HVR4 polymorphisms are shown in Table 2. M1M1, M1M2 and M2M2 genotypes of R353Q were present in 237 (92.9%), 17 (6.7%) and 1 (0.4%) of 255 in Group S/D/T, respectively. M1M1, M1M2 and M2M2 genotypes were present in 103 (82.4%), 20 (16.0%) and 2 (1.6%) of 125 in Group Zero, respectively. Similarly, 0/0, 0/10 genotypes of 5VF7 were present in 234 (91.8%) and 21 (8.2%) of 255 Group S/D/T, and 106 (84.8%) and 19 (15.2%) of 125 Group Zero. We could not find any 10/10 subjects in this study. As for HVR4 polymorphism, H6/H6, H6/H7 and H7/H7 genotypes were present in 93 (36.5%), 113 (44.3%) and 49 (19.2%) of 255 in Group S/D/T and 44 (35.2%), 52 (41.6%) and 29 (23.2%) of 125 in Group Zero, respectively (Table 2). Because there were a few subjects present in M2M2 genotype, we combined M1M2 and M2M2 subjects into one group. Crude and adjusted ORs for CAD with respect to each genotype are shown in Table 2. The frequency of M1M2/M2M2 for Group S/D/T compared with Group Zero was significantly lower (OR = 0.36, 95% CI 0.18– 0.69) and even lower for those under 65 years old (OR = 0.16, 95% CI 0.06 –0.46). A similar trend was obtained as to the 5VF7 polymorphism of FVII. The frequency of 0/10 genotype for Group S/D/T was significantly lower than that of 0/0 genotype (OR = 0.50, 95% CI 0.26 – 0.97) and was even lower among those under the age
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Table 3 Distribution of three genotypes between Group S/D/T and Group Zero among subjects with DM Polymorphism
Genotype
S/D/T
Zero
OR (95% CI)
R353Q
M1M1 M1M2 and M2M2 0/0 0/10 H6H6 H6H7 H7H7
75 3
17 4
1.00 0.17 (0.03 – 0.83)
74 4 26 40 12
17 4 8 8 5
1.00 0.23 (0.05 – 1.01) 1.00 1.54 (0.51 – 4.61) 0.74 (0.20 – 2.74)
5VF7 HVR4
Group Zero indicates subjects without significant coronary artery disease; Group S/D/T indicates subjects with single-, double- or triple-vessel disease.
of 65 years (OR = 0.25, 95% CI 0.09– 0.69). As for the HVR4 polymorphism, there was no relation between genotypes and the presence of CAD. Among CAD subjects, the frequency of the three genotypes was not different between single-vessel disease subjects and double/triple-vessel diseases subjects (data not shown). 3.3. Association of CAD among subjects with DM and FVII polymorphisms Table 3 shows the relation between CAD subjects with DM and FVII polymorphisms. We found a similar trend in DM subjects. The frequency of M1M2/M2M2 for Group S/ D/T was also significantly lower (OR = 0.17, 95% CI 0.03– 0.83) and the frequency of 0/10 for Group S/D/T was also lower (OR = 0.23, 95% CI 0.05 –1.01). There was no relation as to the HVR4 polymorphism. As for subjects without DM, the frequency of M1M2/M2M2 for Group S/D/T was slightly lower (OR = 0.58, 95% CI 0.29 –1.19). However, there was no association between CAD and other two polymorphisms in subjects without DM (data not shown). 3.4. FVIIc level Table 4 shows the FVIIc level of each polymorphism. We measured the FVIIc level in 200 of 380 subjects. There were no clinical differences between the 200 subjects and the remaining 180 (data not shown). The FVIIc was slightly Table 4 FVII procoagulant activity according to genotype Polymorphism
Genotype
FVIIc (%) mean F S.D.
P value
R353Q
M1M1 M1M2 and M2M2 0/0 0/10 H6H6 H6H7 H7H7
93.4 F 17.8 89.5 F 8.99
.33
92.8 F 16.4 85.6 F 11.4 93.8 F 13.3 92.4 F 19.8 87.0 F 39.7
.37
5VF7 HVR4
.79
Table 5 FVII procoagulant activity in each subject FVIIc (%) mean F S.D.
P value
(a) Groups S/D/T and Zero S/D/T Zero
96.7 F 13.0 92.0 F 16.3
< .05 *
(b) MI and non-MI subjects MI Non-MI
95.5 F 13.2 93.6 F 16.7
.387
Group Zero indicates subjects without significant coronary artery disease; Group S/D/T indicates subjects with single-, double- or triple-vessel disease. MI indicates subjects with MI in the past; non-MI indicates subjects with no MI in the past. *, Student t test.
lower in subjects with M1M2/M2M2 than M1M1 (89.5% vs. 93.4%), but not significantly. Similarly, FVIIc was slightly lower in subjects with 0/10 than 0/0 (85.6% vs. 92.3%). There was no difference in FVIIc among HVR4 polymorphisms. We also compared the FVIIc level between Group S/D/T and Group Zero (Table 5a). FVIIc level was significantly lower in Group Zero than in Group S/D/T (92.0% vs. 96.7%, P < .05). There was no difference in the FVIIc level between MI and non-MI subjects (Table 5b).
4. Discussion 4.1. Major findings Our study revealed that the frequency of M1M2/M2M2 was significantly higher compared with the M1M1 genotype among those without significant stenosis of the coronary arteries (Group Zero). This tendency was stronger for subjects under the age of 65, and those with DM. There was no difference between M1M2/M2M2 and M1M1 genotypes in the occurrence of MI (data not shown). This was true of 0/10 genotype. The frequency of 0/10 genotype was also significantly higher than in 0/0 genotype in Group Zero, but we found no difference between the two in terms of MI occurrence (data not shown). Many studies demonstrated that individuals with M1M1 and those with 0/0 have a higher level of FVIIc [24,26 – 28]—findings corroborated by our data. Although FVIIc is affected by many environmental factors [28 – 30], subjects with M1M1 or 0/10 genotype are expected to be exposed to higher levels of FVIIc throughout life. Taking these facts and our data into consideration, FVII genotypes with higher FVIIc could be a risk factor for CAD and not its consequence. When we divide CAD into (I) presence and progression of CAD and (II) onset of MI, effects of FVIIc on these two steps are somewhat different in this study. Subjects with M1M2/M2M2 and/or subjects with 0/10 genotype (lower FVIIc level group) have less probability of contracting CAD. On the other hand, the FVII genotype has no effect on the onset of MI.
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4.2. Possible mechanism by which FVIIC affects CAD occurrence Surprisingly, FVII genotype did not affect the occurrence of MI, most probably because it is widely acknowledged that most MI result from thrombotic occlusion after plaque disruption and exposure of tissue factor to blood [1– 3]. Thus, it is biologically plausible that among subjects with lower levels of FVII, because of this polymorphism, thrombus formation could become less likely, and even if formed, it would be only temporary and liable to dissolve. However, our results did not agree with this idea. On the other hand, FVIIc affects atherosclerosis for more complicated reasons. In our study, those with M2 allele and those with 10 allele had a lower frequency of contracting CAD. It is theoretically possible that through the generation of thrombin and fibrin formation, FVII may contribute to atherosclerosis. Development of coagulation in the vessel wall may result in production of thrombin and activation of platelet, leading to the release of various cytokines and the proliferation of smooth muscle cells in the vessel wall. Thus, someone with a lower FVIIc level may have less chance of developing CAD. 4.3. Comparison with previous studies The many reports thus far investigating the relation between FVII polymorphism and CAD have been rather contradictory. Girelli et al. [31] reported that M2 allele of FVII polymorphisms had a protective effect on MI but not on the severity of coronary atherosclerosis. The Northwick Park Heart Study showed that FVIIc is a strong indicator of MI susceptibility [4]. Yet other studies have failed to show any relation between FVII polymorphisms and CAD [7,32 – 34]. The genetic heterogeneity in the previously studied populations may be one reason for the great discrepancy. The subjects who were recruited for our study were from the same district and were believed to share the same ancestry and a homogeneous genetic background. Also, subjects in previous reports had a higher serum lipid profile [31 – 35], the strongest risk factor for CAD. Therefore, in the context of subjects with high serum lipid profile, the effect of the coagulation system on CAD progression might be relatively low. In this sense, the studies of Kario et al. might be helpful in understanding our results, since they examined the association among FVII R353Q polymorphism, FVIIc and the incidence of cerebrovascular disease in Japan [16 – 18]. Their subject profiles might be similar to ours. However, they did not examine the association between FVII polymorphisms and CAD. Many other reports focused on subjects with high serum cholesterol levels, and thus, the results were so contradictory. In contrast, Japanese subjects, especially those in our study, showed normal cholesterol levels. Both Group S/D/T and Group Zero subjects showed cholesterol levels under 5.2 mmol/l (200 mg/dl). Thus, our study is an ideal setting in which to investigate whether a
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coagulation factor actually tends to decrease CAD or the susceptibility of MI with low to normal lipid profile. Consistent with our results, Tamaki et al. [19] reported that FVII polymorphisms were not associated with MI in Japanese. However, they did not elucidate the relation between FVII polymorphisms and the presence of CAD. Hence, to the best of our knowledge, ours is the first study to demonstrate that M1M2, M2M2 and/or 0/10 genotype of FVII polymorphisms had a protective effect against CAD in those with a normal cholesterol level. 4.4. Study limitations This study is not a randomized case –control one, and some selection bias can influence the results of association studies. However, our study population in Nagoya City in central Japan has a homogeneous genetic background. Moreover, the distribution of FVII genotypes in the population group under study was in Hardy– Weinberg equilibrium, which strongly suggests that we avoided selection bias. A second limitation is the small sample, which may have produced some bias because of the low statistical power. A third limitation is that we could only assess survivors of MI. To overcome these limitations, a large prospective study will be needed to examine the clinical value of FVII polymorphisms in CAD.
5. Conclusions Based on the results of this study, we consider that Japanese subjects, especially those with low to normal serum cholesterol levels, have an inherent predisposition by which FVII polymorphisms (R353Q and 5VF7) affect CAD. In other words, M1M2, M2M2 and/or 0/10 genotypes of FVII protect against CAD.
Acknowledgements We thank Hiroaki Sano, Mitutaka Makino, Noriko Niwa and Noriyuki Suzuki for their generous assistance in the course of this study. We also thank Keiko Kinoshita and Yuka Okajima for their work on the manuscript.
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[20] Bernardi F, Marchetti G, Pinotti M, Arcieri P, Baroncini C, Papacchini M, Zepponi E, Ursicino N, Chiarotti F, Mariani G. Factor VII gene polymorphisms contribute about one third of the factor VII level variation in plasma. Arterioscler, Thromb, Vasc Biol 1996;16:72 – 6. [21] Takamiya O. Genetic polymorphism (Arg353 – Gln) in coagulation factor VII gene and factor VII levels (coagulant activity, antigen and binding ability to tissue factor) in 101 healthy Japanese. Scand J Clin Lab Invest 1995;55:211 – 5. [22] Iacoviello L, Di Castelnuovo A, De Knijff P, D’Orazio A, Amore C, Arboretti R, Kluft C, Donati MB. Polymorphisms in the coagulation factor VII gene and the risk of myocardial infarction. N Engl J Med 1998;338:79 – 85. [23] Marchetti G, Patracchini P, Gemmati D, DeRosa V, Pinotti M, Rodorigo G, Casonato A, Girolami A, Bernardi F. Detection of two missense mutations and characterization of a repeat polymorphism in factor VII gene (F7). Hum Genet 1992;89:497 – 502. [24] Di Castelnuovo A, D’Orazio A, Amore C, Falanga A, Kluft C, Donati MB, Iacoviello L. Genetic modulation of coagulation factor VII plasma levels: contribution of different polymorphisms and gender-related effects. Thromb Haemostasis 1998;80:592 – 7. [25] De Knijff P, Green F, Johansen LG, Grootendorst D, Temple A, Cruickshank JK, Humphries SE, Jespersen J, Kluft C. New alleles in F7 VNTR. Hum Mol Genet 1994;3:384. [26] Green F, Kelleher C, Wilkes H, Temple A, Meade T, Humphries S. A common genetic polymorphism associated with lower coagulation factor VII levels in healthy individuals. Arterioscler Thromb 1991;11: 540 – 6. [27] Ghaddar HM, Folsom AR, Aleksic N, Hearne LB, Chambless LE, Morrissey JH, Wu KK. Correlation of factor VIIa values with factor VII gene polymorphism, fasting and postprandial triglyceride levels, and subclinical carotid atherosclerosis. Circulation 1998;98:2815 – 21. [28] Folsom AR, Wu KK, Rasmussen M, Chambless LE, Aleksic N, Nieto FJ. Determinants of population changes in fibrinogen and factor VII over 6 years, the Atherosclerosis Risk in Communities (ARIC) Study. Arterioscler, Thromb, Vasc Biol 2000;20:601 – 6. [29] Kario K, Miyata T, Sakata T, Matsuo T, Kato H. Fluorogenic assay of activated factor VII, plasma factor VIIa levels in relation to arterial cardiovascular diseases in Japanese. Arterioscler Thromb 1994;14: 265 – 74. [30] Saha N, Liu Y, Heng CK, Hong S, Low PS, Tay JSH. Association of factor VII genotype with plasma factor VII activity and antigen levels in healthy Indian adults and interaction with triglycerides. Arterioscler Thromb 1994;14:1923 – 7. [31] Girelli D, Russo C, Ferraresi P, Olivieri O, Pinotti M, Friso S, Manzato F, Mazzucco A, Bernardi F, Corrocher R. Polymorphisms in the factor VII gene and the risk of myocardial infarction in patients with coronary artery disease. N Engl J Med 2000;343:774 – 80. [32] Wang XL, Wang J, McCredie RM, Wilcken DEL. Polymorphisms of factor V, factor VII, and fibrinogen genes: relevance to severity of coronary artery disease. Arterioscler, Thromb, Vasc Biol 1997;17: 246 – 51. [33] Doggen CJM, Cats VM, Bertina RM, Reitsma PH, Vandenbroucke JP, Rosendaal FR. A genetic propensity to high factor VII is not associated with the risk of myocardial infarction in men. Thromb Haemostasis 1998;80:281 – 5. [34] Lievers KJA, Mennen LI, Rattink AP, Zwinderman AH, Jukema JW, Schouten EG, De Maat MPM. The 323Ins10 polymorphism for factor VII is not associated with coronary atherosclerosis in symptomatic men. Thromb Res 2000;97:275 – 80. [35] Feng D, Tofler GH, Larson MG, O’Donnell CJ, Lipinska I, Schmitz C, Sutherland PA, Johnstone MT, Muller JE, D’Agostino RB, Levy D, Lindpaintner K. Factor VII gene polymorphism, factor VII levels, and prevalent cardiovascular disease: the Framingham Heart Study. Arterioscler, Thromb, Vasc Biol 2000;20:593 – 600.
Regular Article
Effects of coagulation Factor VII polymorphisms on the coronary artery disease in Japanese Factor VII polymorphism and coronary disease Keiko Shimokata a, Takahisa Kondo a,*, Miyoshi Ohno b, Kyosuke Takeshita a, Yasuya Inden a, Shigeo Iino a, Hidehiko Saito c, Makoto Hirai a a
First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 Japan b Division of Cardiology, Nagoya First Red Cross Hospital, Nagoya, Japan c Nagoya National Hospital, Nagoya, Japan Received 26 February 2002; received in revised form 19 April 2002; accepted 21 April 2002 Accepting Editor: S. Iwanagah
Abstract We investigated the relationships among Factor VII coagulant activity (FVIIc), genetic polymorphisms of Factor VII (FVII) and coronary artery disease (CAD) in 380 unrelated Japanese individuals (mean 64 years) who underwent coronary angiography and whose cholesterol levels were within normal range. CAD subjects were defined as those in whom one of the three major coronary arteries showed > 50% narrowing after nitroglycerin administration. FVIIc was measured and the following polymorphisms of FVII were determined: R353Q polymorphism (M1, M2 alleles), 323 0/10 bp polymorphism (0, 10 alleles), hypervariable region 4 of intron 7 (HVR4; H5, H6, H7 alleles). FVIIc was slightly lower in M1M2/M2M2 than M1M1 (89.5 F 8.9%, 93.4 F 17.8%). Those with M2 and/or 10 allele have less chance of developing CAD (M2: OR 0.36, 95% CI 0.18 – 0.69, 10: OR 0.50, 95% CI 0.26 – 0.97). However, both alleles did not associate with myocardial infarction (MI). HVR4 was unrelated with CAD, nor with MI. In conclusion, M2 and/or 10 allele has protective effects on the developing CAD in individuals with a normal cholesterol level. D 2002 Published by Elsevier Science Ltd. Keywords: Polymorphism; Factor VII; Coronary disease; Myocardial infarction
1. Introduction Thrombosis underlies most acute manifestations of coronary atherosclerotic disease, including myocardial infarction (MI) [1]. Plaque disruption, with resulting exposure of tissue factor to blood and binding of tissue factor to circulating coagulation Factor VII (FVII) [2,3], is considered the major cause of thrombosis in MI. During the past two decades, there have been numerous reports Abbreviations: FVII, Factor VII; FVIIc, Factor VII coagulant activity; CAD, coronary artery disease; MI, myocardial infarction; OR, odds ratio; CI, 95% confidence interval; R353Q, substitution of glutamine for arginine at position 353 in the catalytic domain in the FVII gene; 5VF7, a 10-bp insertion in the promoter region in the FVII gene; HVR4, hypervariable region 4 of intron 7 of the FVII gene; BMI, body mass index; HDL, highdensity lipoprotein; Group Zero, no significant coronary artery stenosis group; Group S/D/T, single-, double- or triple-vessel disease group. * Corresponding author. Tel.: +81-52-744-2147; fax: +81-52-744-2157. E-mail address: [email protected] (T. Kondo). 0049-3848/02/$ - see front matter D 2002 Published by Elsevier Science Ltd. PII: S 0 0 4 9 - 3 8 4 8 ( 0 2 ) 0 0 0 6 7 - 1
investigating Factor VII coagulant activity (FVIIc) in coronary artery disease (CAD). Some identified FVIIc as a potent risk indicator for CAD [4 –6], while others did not [7,8]. For example, in the Northwick Park Heart Study, FVIIc was an independent predictor of MI in initially healthy middle-aged men, and particularly of fatal coronary events [4]. However, three large-scale prospective studies, the Prospective Cardiovascular Mu¨nster Study [6], the Edinburg Artery Study [7] and the ECTIM Study [8], failed to identify the FVII level as a predictor of future CAD. Why are their discrepancies in the study of FVIIc and CAD? Three reasons come to mind. First, there is a confounding relation between FVIIc and MI, and no causal relationship has been found to date. The high FVIIc level could be a consequence of atherosclerosis rather than a cause of disease progression. Second, FVIIc is related to age, lipid level and obesity [9 –11]. Therefore, if the lipid levels of subjects in a study group are too high or if these
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subjects are overweight, the causal relation between FVIIc and CAD would be misunderstood. Third, the genetic background in the previous study is not the same [7,8,12,13]. It is a well-known fact that there are genetic differences in the activation of the coagulation cascade [14]. Some polymorphisms reported in Caucasians are not found in the Asian population such as Leiden mutation and thrombin polymorphism [12,13]. Furthermore, Japan is one of the countries where the incidence of CAD is much less common compared with Western countries [15]. In this sense, it is intriguing to investigate the association of FVII polymorphism and CAD in Japan. So far, Kario et al. have reported the association of FVII polymorphism, FVIIc and cerebrovascular disease in Japan. However, the association of FVII polymorphism and CAD in Japanese subjects remains unclear [16 – 19]. Recently, three common polymorphisms in the FVII gene, i.e., the substitution of glutamine for arginine at position 353 in the catalytic domain (R353Q), a 10-bp insertion in the promoter region (5VF7) and a variable number of 37-bp repeats in intron 7 (HVR4), are reported to be responsible for up to one third of the variations in FVIIc among Caucasians and Japanese [20,21] and could be risk factors for MI [22]. If FVIIc really influences the risk for cardiovascular disease, these polymorphisms become good tools because they are determined at the time of birth and do not change throughout life. In this study, we investigated the relation between FVII polymorphisms and the frequency of CAD in Japanese subjects. One advantage of the present study is that Japanese are highly homogenous in genetic background because Japan is an insular country and closed its doors to outside contact for nearly 300 years until about 1876. Secondly, the cholesterol levels of subjects in this study are not high; we could avoid the effect of cholesterol and evaluate the genuine effect of FVIIc on the progression of CAD. The main purposes of this study were (1) to clarify the relation among three common polymorphisms of FVII and MI, and (2) to investigate the effect of these polymorphisms on the progression of coronary artery sclerosis in Japanese whose cholesterol levels are low compared with Caucasian subjects. We also investigated whether there was an association between polymorphisms in the FVII gene and FVIIc in Japanese as is often reported in Caucasians.
2. Materials and methods 2.1. Study population and laboratory data collection The study subjects were recruited from the First Department of Internal Medicine, Nagoya University School of Medicine, and from the Division of Cardiology at Nagoya First Red Cross Hospital in Aichi Prefecture from January
to May 2000. Blood samples and data were collected from 380 consecutive subjects who underwent heart catheterization because of suspected CAD, arrhythmia, valvular disease or congenital heart disease (133 MI subjects and 247 non-MI subjects). Diagnosis of acute MI was made based on chest symptoms, electrocardiographic changes and serum creatine kinase elevations more than twice the normal upper level. Individual histories were taken concerning diabetes mellitus (DM), hypertension and smoking habits. Body mass index (BMI) was calculated by dividing weight in kilograms by height in meters squared. Levels of red blood cells, white blood cells, platelets, total cholesterol, triglyceride and high-density lipoprotein (HDL) cholesterol were determined with standard procedures (after overnight fast). In MI subjects, we used data sampled over 1 month after the onset of MI. Coronary angiography was performed according to the Judkins method. Coronary artery stenosis was defined as significant when the lumen diameter was >50% narrowed after nitroglycerin administration. Then, relevant CAD was also characterized as single-, double- and triple-vessel disease. We further divided subjects into two groups according to the number of diseased vessels: Group Zero (n = 125) vs. Group S/D/T (n = 255). The Group Zero subjects had no significant coronary artery stenosis, and those in Group S/D/ T had single-, double- or triple-vessel disease. Three cardiologists blinded to the results of genotype analyses made the CAD assessment. Written informed consent was obtained from all subjects studied in the two hospitals. The study was in accord with the guidelines approved by the ethics committee of Nagoya University School of Medicine. 2.2. DNA analysis DNA was isolated according to the manufacturer’s instructions (Gene Trapping by Liquid Extraction, Takara Company, Japan). The R353Q polymorphism was amplified according to a modification of the method of Marchetti et al. [23]. PCR was performed using the described set primers. The amplified fragments were digested with 5 U of MspI (New England Biolaboratories, Beverly, MA, USA) and then subjected to electrophoresis on 3% Nusieve 3:1 agarose gel. Fragments of 205 (the M1 allele) and 272 bp (the M2 allele) were detected. To detect the 5VF7 polymorphism, a 214-bp DNA fragment was amplified using the described primers [24]. Amplified fragments were digested with 10 U of EcoT 14I (Takara Company, Japan) and then subjected to electrophoresis on 1.5% agarose gel. Fragments of 214 (0 allele) and 136 + 88 bp (10 allele) were detected. The HVR4 of the FVII gene was amplified using primers described before [25]. Two alleles were identified: a common allele (the H6 allele) of 443 bp with six monomers, and a less frequent allele (the H7 allele) of 480 bp with seven monomers of 37 bp. We could not find the very rare H5 allele of 406 bp with five monomers.
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2.3. FVIIc level
Table 2 Distribution of three genotypes between Group S/D/T and Group Zero
We measured the FVIIc level of 200 of a total of 380 patients, whose plasma was stored at 80 jC. FVIIc was assayed by a one-stage assay using human placenta thromboplastin.
Polymorphism (a) All subjects R353Q
5VF7
2.4. Statistical analysis
HVR4
Clinical laboratory data were expressed as mean F S.D. and compared between Group S/D/T and Group Zero subjects by Student’s t test or the Mann – Whitney U test when applicable. Differences in means of continuous variables between more than two groups were assessed by Kruskal –Wallis test. Allele frequencies were estimated by the gene counting methods, and the Hardy– Weinberg equilibrium was confirmed by chi-square test. The odds ratio (OR) and 95% confidence interval (CI) were also calculated. Multiple logistic regression analyses were performed and adjusted by age, sex, smoking status, total cholesterol level and presence of hypertension and DM using Stat View 5.0 (SAS Institute, Cary, NC, USA). A P value of < .05 was considered statistically significant.
Genotype
S/D/T
Zero
OR (95% CI)
M1M1 M1M2 and M2M2 0/0 0/10 H6H6 H6H7 H7H7
237 18
103 22
1.00 0.36 (0.18 – 0.69)
234 21 93 113 49
106 19 44 52 29
1.00 0.50 (0.26 – 0.97) 1.00 0.97 (0.60 – 1.58) 1.25 (0.70 – 2.24)
124 5
64 16
1.00 0.16 (0.06 – 0.46)
123 6 47 56 26
67 13 24 33 20
1.00 0.25 (0.09 – 0.69) 1.00 1.15 (0.60 – 2.22) 1.51 (0.70 – 3.23)
(b) Subjects under 65 years old R353Q M1M1 M1M2 and M2M2 5VF7 0/0 0/10 HVR4 H6H6 H6H7 H7H7
Group Zero indicates subjects without significant coronary artery disease; Group S/D/T indicates subjects with single-, double- or triple-vessel disease.
The genotype frequencies of the three polymorphisms examined in this study were all in agreement with those predicted by the Hardy– Weinberg equilibrium.
3. Results 3.1. Baseline characteristics of study population
3.2. Association of CAD and three genotypes at FVII Table 1 listed the clinical characteristics, blood cell counts, lipid level and genotype distribution of FVII in this study group. The number of male subjects was significantly higher and the HDL level was significantly lower in the Group S/D/T than in the Group Zero ( P < .05).
Table 1 Clinical characteristics of Group S/D/T and Group Zero
Male/female Age (year) BMI (kg/m2) BPs (mm Hg) BPd (mm Hg) RBC ( 1012/l) WBC ( 109/l) Plt ( 109/l) T chol (mmol/l) TG (g/l) HDL (mmol/l) DM (%) Smoking (%)
S/D/T (n = 255)
ZERO (n = 125)
P value
214/41 63.4 F 9.38 23.7 F 2.80 137 F 21.1 76.8 F 12.7 4.23 F 0.56 6.13 F 1.64 223 F 73.4 5.05 F 1.03 1.63 F 0.87 1.10 F 0.29 30.2 51.9
85/40 63.6 F 13.1 23.2 F 3.28 134 F 21.1 77.1 F 13.2 4.35 F 0.53 6.10 F 1.60 214 F 56.6 5.24 F 0.97 1.42 F 0.97 1.63 F 0.40 19.9 39.1
< .001 * .926 .250 .291 .886 .052 .899 .195 .074 .046 < .001 *,y .671 .350
Group Zero indicates subjects without significant coronary artery disease; Group S/D/T indicates subjects with single-, double- or triple-vessel disease. BMI, body mass index; BPs, systolic blood pressure; BPd, diastolic blood pressure; RBC, red blood cell; WBC, white blood cell; Plt, platelet; T. chol, total cholesterol; TG, triglyceride; HDL, high-density lipoprotein; FH, family history of coronary artery disease; DM, diabetes mellitus. *m2, statistics; y, Mann-Whitney U test.
Distributions of R353Q, 5VF7 and HVR4 polymorphisms are shown in Table 2. M1M1, M1M2 and M2M2 genotypes of R353Q were present in 237 (92.9%), 17 (6.7%) and 1 (0.4%) of 255 in Group S/D/T, respectively. M1M1, M1M2 and M2M2 genotypes were present in 103 (82.4%), 20 (16.0%) and 2 (1.6%) of 125 in Group Zero, respectively. Similarly, 0/0, 0/10 genotypes of 5VF7 were present in 234 (91.8%) and 21 (8.2%) of 255 Group S/D/T, and 106 (84.8%) and 19 (15.2%) of 125 Group Zero. We could not find any 10/10 subjects in this study. As for HVR4 polymorphism, H6/H6, H6/H7 and H7/H7 genotypes were present in 93 (36.5%), 113 (44.3%) and 49 (19.2%) of 255 in Group S/D/T and 44 (35.2%), 52 (41.6%) and 29 (23.2%) of 125 in Group Zero, respectively (Table 2). Because there were a few subjects present in M2M2 genotype, we combined M1M2 and M2M2 subjects into one group. Crude and adjusted ORs for CAD with respect to each genotype are shown in Table 2. The frequency of M1M2/M2M2 for Group S/D/T compared with Group Zero was significantly lower (OR = 0.36, 95% CI 0.18– 0.69) and even lower for those under 65 years old (OR = 0.16, 95% CI 0.06 –0.46). A similar trend was obtained as to the 5VF7 polymorphism of FVII. The frequency of 0/10 genotype for Group S/D/T was significantly lower than that of 0/0 genotype (OR = 0.50, 95% CI 0.26 – 0.97) and was even lower among those under the age
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Table 3 Distribution of three genotypes between Group S/D/T and Group Zero among subjects with DM Polymorphism
Genotype
S/D/T
Zero
OR (95% CI)
R353Q
M1M1 M1M2 and M2M2 0/0 0/10 H6H6 H6H7 H7H7
75 3
17 4
1.00 0.17 (0.03 – 0.83)
74 4 26 40 12
17 4 8 8 5
1.00 0.23 (0.05 – 1.01) 1.00 1.54 (0.51 – 4.61) 0.74 (0.20 – 2.74)
5VF7 HVR4
Group Zero indicates subjects without significant coronary artery disease; Group S/D/T indicates subjects with single-, double- or triple-vessel disease.
of 65 years (OR = 0.25, 95% CI 0.09– 0.69). As for the HVR4 polymorphism, there was no relation between genotypes and the presence of CAD. Among CAD subjects, the frequency of the three genotypes was not different between single-vessel disease subjects and double/triple-vessel diseases subjects (data not shown). 3.3. Association of CAD among subjects with DM and FVII polymorphisms Table 3 shows the relation between CAD subjects with DM and FVII polymorphisms. We found a similar trend in DM subjects. The frequency of M1M2/M2M2 for Group S/ D/T was also significantly lower (OR = 0.17, 95% CI 0.03– 0.83) and the frequency of 0/10 for Group S/D/T was also lower (OR = 0.23, 95% CI 0.05 –1.01). There was no relation as to the HVR4 polymorphism. As for subjects without DM, the frequency of M1M2/M2M2 for Group S/D/T was slightly lower (OR = 0.58, 95% CI 0.29 –1.19). However, there was no association between CAD and other two polymorphisms in subjects without DM (data not shown). 3.4. FVIIc level Table 4 shows the FVIIc level of each polymorphism. We measured the FVIIc level in 200 of 380 subjects. There were no clinical differences between the 200 subjects and the remaining 180 (data not shown). The FVIIc was slightly Table 4 FVII procoagulant activity according to genotype Polymorphism
Genotype
FVIIc (%) mean F S.D.
P value
R353Q
M1M1 M1M2 and M2M2 0/0 0/10 H6H6 H6H7 H7H7
93.4 F 17.8 89.5 F 8.99
.33
92.8 F 16.4 85.6 F 11.4 93.8 F 13.3 92.4 F 19.8 87.0 F 39.7
.37
5VF7 HVR4
.79
Table 5 FVII procoagulant activity in each subject FVIIc (%) mean F S.D.
P value
(a) Groups S/D/T and Zero S/D/T Zero
96.7 F 13.0 92.0 F 16.3
< .05 *
(b) MI and non-MI subjects MI Non-MI
95.5 F 13.2 93.6 F 16.7
.387
Group Zero indicates subjects without significant coronary artery disease; Group S/D/T indicates subjects with single-, double- or triple-vessel disease. MI indicates subjects with MI in the past; non-MI indicates subjects with no MI in the past. *, Student t test.
lower in subjects with M1M2/M2M2 than M1M1 (89.5% vs. 93.4%), but not significantly. Similarly, FVIIc was slightly lower in subjects with 0/10 than 0/0 (85.6% vs. 92.3%). There was no difference in FVIIc among HVR4 polymorphisms. We also compared the FVIIc level between Group S/D/T and Group Zero (Table 5a). FVIIc level was significantly lower in Group Zero than in Group S/D/T (92.0% vs. 96.7%, P < .05). There was no difference in the FVIIc level between MI and non-MI subjects (Table 5b).
4. Discussion 4.1. Major findings Our study revealed that the frequency of M1M2/M2M2 was significantly higher compared with the M1M1 genotype among those without significant stenosis of the coronary arteries (Group Zero). This tendency was stronger for subjects under the age of 65, and those with DM. There was no difference between M1M2/M2M2 and M1M1 genotypes in the occurrence of MI (data not shown). This was true of 0/10 genotype. The frequency of 0/10 genotype was also significantly higher than in 0/0 genotype in Group Zero, but we found no difference between the two in terms of MI occurrence (data not shown). Many studies demonstrated that individuals with M1M1 and those with 0/0 have a higher level of FVIIc [24,26 – 28]—findings corroborated by our data. Although FVIIc is affected by many environmental factors [28 – 30], subjects with M1M1 or 0/10 genotype are expected to be exposed to higher levels of FVIIc throughout life. Taking these facts and our data into consideration, FVII genotypes with higher FVIIc could be a risk factor for CAD and not its consequence. When we divide CAD into (I) presence and progression of CAD and (II) onset of MI, effects of FVIIc on these two steps are somewhat different in this study. Subjects with M1M2/M2M2 and/or subjects with 0/10 genotype (lower FVIIc level group) have less probability of contracting CAD. On the other hand, the FVII genotype has no effect on the onset of MI.
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4.2. Possible mechanism by which FVIIC affects CAD occurrence Surprisingly, FVII genotype did not affect the occurrence of MI, most probably because it is widely acknowledged that most MI result from thrombotic occlusion after plaque disruption and exposure of tissue factor to blood [1– 3]. Thus, it is biologically plausible that among subjects with lower levels of FVII, because of this polymorphism, thrombus formation could become less likely, and even if formed, it would be only temporary and liable to dissolve. However, our results did not agree with this idea. On the other hand, FVIIc affects atherosclerosis for more complicated reasons. In our study, those with M2 allele and those with 10 allele had a lower frequency of contracting CAD. It is theoretically possible that through the generation of thrombin and fibrin formation, FVII may contribute to atherosclerosis. Development of coagulation in the vessel wall may result in production of thrombin and activation of platelet, leading to the release of various cytokines and the proliferation of smooth muscle cells in the vessel wall. Thus, someone with a lower FVIIc level may have less chance of developing CAD. 4.3. Comparison with previous studies The many reports thus far investigating the relation between FVII polymorphism and CAD have been rather contradictory. Girelli et al. [31] reported that M2 allele of FVII polymorphisms had a protective effect on MI but not on the severity of coronary atherosclerosis. The Northwick Park Heart Study showed that FVIIc is a strong indicator of MI susceptibility [4]. Yet other studies have failed to show any relation between FVII polymorphisms and CAD [7,32 – 34]. The genetic heterogeneity in the previously studied populations may be one reason for the great discrepancy. The subjects who were recruited for our study were from the same district and were believed to share the same ancestry and a homogeneous genetic background. Also, subjects in previous reports had a higher serum lipid profile [31 – 35], the strongest risk factor for CAD. Therefore, in the context of subjects with high serum lipid profile, the effect of the coagulation system on CAD progression might be relatively low. In this sense, the studies of Kario et al. might be helpful in understanding our results, since they examined the association among FVII R353Q polymorphism, FVIIc and the incidence of cerebrovascular disease in Japan [16 – 18]. Their subject profiles might be similar to ours. However, they did not examine the association between FVII polymorphisms and CAD. Many other reports focused on subjects with high serum cholesterol levels, and thus, the results were so contradictory. In contrast, Japanese subjects, especially those in our study, showed normal cholesterol levels. Both Group S/D/T and Group Zero subjects showed cholesterol levels under 5.2 mmol/l (200 mg/dl). Thus, our study is an ideal setting in which to investigate whether a
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coagulation factor actually tends to decrease CAD or the susceptibility of MI with low to normal lipid profile. Consistent with our results, Tamaki et al. [19] reported that FVII polymorphisms were not associated with MI in Japanese. However, they did not elucidate the relation between FVII polymorphisms and the presence of CAD. Hence, to the best of our knowledge, ours is the first study to demonstrate that M1M2, M2M2 and/or 0/10 genotype of FVII polymorphisms had a protective effect against CAD in those with a normal cholesterol level. 4.4. Study limitations This study is not a randomized case –control one, and some selection bias can influence the results of association studies. However, our study population in Nagoya City in central Japan has a homogeneous genetic background. Moreover, the distribution of FVII genotypes in the population group under study was in Hardy– Weinberg equilibrium, which strongly suggests that we avoided selection bias. A second limitation is the small sample, which may have produced some bias because of the low statistical power. A third limitation is that we could only assess survivors of MI. To overcome these limitations, a large prospective study will be needed to examine the clinical value of FVII polymorphisms in CAD.
5. Conclusions Based on the results of this study, we consider that Japanese subjects, especially those with low to normal serum cholesterol levels, have an inherent predisposition by which FVII polymorphisms (R353Q and 5VF7) affect CAD. In other words, M1M2, M2M2 and/or 0/10 genotypes of FVII protect against CAD.
Acknowledgements We thank Hiroaki Sano, Mitutaka Makino, Noriko Niwa and Noriyuki Suzuki for their generous assistance in the course of this study. We also thank Keiko Kinoshita and Yuka Okajima for their work on the manuscript.
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