Mol Biol Rep DOI 10.1007/s11033-014-3026-8
ABO blood group polymorphisms and risk for ischemic stroke and peripheral arterial disease Adriano de Paula Sabino • Daniel Dias Ribeiro • Caroline Pereira Domingheti Danyelle Romana Alves Rios • Luci Maria SantAna Dusse • Maria das Grac¸as Carvalho • Ana Paula Fernandes
•
Received: 12 December 2012 / Accepted: 3 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Recent studies have demonstrated association between ABO blood system and thrombosis, indicating that individuals belonging to non-O blood groups (A, B or AB) present an increased risk of venous thrombosis, heart disease, and ischemic stroke (IS) as compared to O blood group carriers. In this study, we investigated the frequency of ABO blood group polymorphisms and its association with IS and peripheral arterial disease. Significant differences were observed for O1 (OR 0.57, 95 % CI 0.35–0.95, p \ 0.05) and O2 (OR 3.47, 95 % CI 1.15–10.28, p \ 0.05) alleles among IS patients while significant differences were observed for B phenotype (26.3 vs 9.5 %, OR 3.42, 95 % CI 1.32–8.76, p = 0.01, patients vs controls, respectively) and alleles A1 (OR 0.31, 95 % CI 0.11–0.84, p \ 0.05), O2 (OR 4.61, 95 % CI 1.59–13.23, p \ 0.01) and B (OR 3.42, 95 % CI 1.62–7.13, p \ 0.001) alleles for PAD patients. O1 allele was an independent variable (OR 0.27, 95 % CI 0.12–0.57, p \ 0.001) for IS patients. These data suggest the relationship of non-O blood groups in pathogenesis of thrombosis events and a possible protective effect of O blood group.
A. de Paula Sabino (&) C. P. Domingheti L. M. S. Dusse M. das Grac¸as Carvalho A. P. Fernandes Faculty of Pharmacy, Federal University of Minas Gerais, Av. Antoˆnio Carlos 6627, Pampulha, Belo Horizonte, MG, Brazil e-mail: [email protected] D. D. Ribeiro University Hospital–Federal University of Minas Gerais, Belo Horizonte, MG, Brazil D. R. A. Rios Health Sciences Center, Federal University of Sa˜o Joa˜o Del Rei, Divino´polis, MG, Brazil
Keywords PAD
Arterial thrombosis ABO genotypes IS
Introduction Antigens of the ABO system consist of an A or a B carbohydrate structure carried on the substrate H antigen. The A or B glycosyltransferase encoded at the ABO locus on chromosome 9 defines which specific carbohydrate is added to the end of the H substance oligosaccharide chains [1]. The ABO gene contains seven exons (range in size from 28 to 688 bp) and six introns spanning about 18–20 kilobase pairs (kbp), in position 9q34.1-9q34.2, being the most of the enzymes encoded by exons 6 and 7, which constitute 77 % of the ABO gene [2, 3]. The major alleles of ABO gene are A1, B and O that provide the four blood groups: A, B, AB and O. Among these alleles there is a large genetic heterogeneity, including the change of amino acid residues in the glycosyltransferase molecule. Recent studies have demonstrated association between ABO blood system and venous thromboembolism (VTE), indicating that individuals belonging to non-O blood groups (A, B or AB) present an increased risk of venous thrombosis, heart disease, and ischemic stroke (IS) as compared to O blood group carriers [4–11]. A meta-analysis showed that the prevalence of non-O blood group was significantly higher in VTE patients compared with controls with a resulting odds ratio of 2.09 (p \ 0,00001) [12]. This association can be explained by higher plasma levels of factor VIII (FVIII) and von Willebrand factor (VWF) observed in ‘‘non O’’ groups [7, 13, 14]. Morelli et al. [7] studied the effect of the genotype of the ABO system and the risk of deep venous thrombosis in 471
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patients who had venous thrombosis and 471 individuals without thrombotic events (controls). In the group of patients, all genotypes ‘‘non O’’ (70.9 %), except A2A2 homozygous or heterozygous A2O1/A2O2 (7.2 %) had high risk of thrombosis compared to those with genotype OO (29.1 %). The risk of venous thrombosis increases with increased levels of VWF and FVIII which are higher in individuals ‘‘non O’’ comparing to group ‘‘O’’. Contrarily, the association between blood group and arterial thrombosis is difficult to prove, since increased FVIII and VWF plasma levels in arterial thrombosis observed may reflect a chronic inflammation, as the result of atherosclerosis [15, 16]. Even with this limitation, several studies have reported an association between ‘‘non O’’ group and risk of arterial thrombosis, such as coronary artery disease and peripheral vascular disease [16–19]. Clark et al. [20] performed a case–control study to examine the frequency of ABH, Lewis and Secretor genotypes and phenotypes in patients with acute cerebral ischemia of arterial origin (CIAO). They observed that non-O and, in particular A, blood groups are more common in those presenting with CIAO, although there was not additional influence of Lewis or Secretor genotypes and phenotypes. Although the risk identified have been modest, the authors emphasize that clarification of the link between blood groups on the occurrence, and perhaps recurrence, of CIAO might ultimately be useful in understanding the link between factors such as VWF, activated protein C (APC) resistance, or lipids and vascular disease. In contrast to these results, an analysis of ABO genotype and IS showed an increased risk in those carrying B allele as compared with all other genotypes [21]. The aim of this study was to investigate the association of the ABO genotype and phenotype with IS and peripheral arterial disease (PAD).
auto-immune disorders, as well as those with coagulation disorders were excluded. Baseline information on smoking habits, medication use, and personal history of disease were gathered by trained medical staff during a standardized interview. In addition, all participants underwent an extensive standardized medical examination. Body mass index (BMI) was calculated as weight in kilograms divided by height in square meters. Systolic and diastolic blood pressure was measured on the right arm in a sitting position, according to the recommendations of the American heart association. Persons being aware of having hypertension, taking antihypertensive medication, and/or having blood pressure values 160/90 mmHg at baseline were defined as actual hypertension. Individuals were classified as having diabetes mellitus if plasma glucose was equal or superior to 126 mg/dl in the fasting state or if individuals were receiving oral anti-diabetics or insulin. Hypercholesterolemia were considered if total cholesterol was superior to 239 mg/dl. None of the patients were using lipid lowering therapy. A regular smoker was defined as a subject who currently smoked at least five cigarettes per day. No renal disease was observed or detected by the trained medical staff. Patients had normal levels of urea and creatinine. The control group was composed by 201 subjects with no previous history of arterial or venous thrombosis from the same demographic area, with no familiar relationship with patients. The institutional ethics committee of the federal university of Minas Gerais, approved this study and an informed consent was obtained from all participants. Sample collection
Materials and methods
Venous blood was also collected from all subjects into EDTA (5 ml) tubes using the Vacutainer System (Becton– Dickinson, Franklin Lakes, New Jersey, USA) and was submitted to genomic DNA extraction using the Wizard purification system (Promega Inc., Madison, Wisconsin, USA), following the manufacturer’s instructions.
Patients and control subjects
Genotyping the ABO system
A total of 282 individuals participated of this study. Survival patients to the arterial thrombotic event (n = 81) with diagnosis of IS and PAD were consecutively selected by physicians at the Hematology Unit of the University Hospital (Federal University of Minas Gerais, Belo HorizonteMG) to participate of the present study between July 2007 and December 2010. All of them had the diagnosis of IS and PAD confirmed by magnetic resonance, brain computer tomography and/or arteriography. Patients with major systemic diseases, which are known to predispose to thrombosis such as cancer, infections or hepatic diseases,
ABO polymorphisms were investigated by PCR–RFLP. Briefly, amplification of exons 6 and 7 of the ABO gene were obtained through two separate reactions using oligonucleotides previously described [22]. PCR reactions were performed in a TC-312 thermocycler (Techne Inc.) using 1.5 pMol of each primer for exon 6 and 0.5 pMol of each primer for exon 7 (Invitrogen, Sa˜o Paulo, Brazil), 0.2 mM of dNTPs (GIBCO BRL, Sa˜o Paulo, Brazil), and 0.5 units of Taq polymerase (Phoneutria, Belo Horizonte, Minas Gerais, Brazil). PCR reactions were submitted to 40 cycles consisting of 2 min at 94 °C for initial denaturing, 10 s at
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94 °C for denaturing, 30 s at 62 °C for primer annealing, and 30 s for primer extension. PCR products were submitted to restriction endonuclease digestion for 4 h at 37 °C with enzymes KpnI and MspI (Promega Inc.) in a single reaction. Digested fragments were analyzed in polyacrylamide gel electrophoresis. This method allows the differentiation of alleles A1, A2, B, O1, and O2 [22]. DNAs from previously typed individuals were included in each set of analyzed samples in order to control enzyme activity. Statistical analysis Statistical comparisons were performed using version 6.04 of the Epi Info program [23], Sigma Stat version 2.0. The patient group was tested against the control group for each polymorphism frequency. Odds ratios (OR) were used as a measure of the association between each polymorphism of the ABO system and were interpreted as the risk for VTE. Confidence intervals (95 % CI) were determined for ORs. Significance levels were estimated by applying the Chi square test. Differences were considered significant when p \ 0.05. Multiple regression analysis was used to explore whether the genetic (ABO blood groups) and acquired variables such as smoking, obesity, hypertension, Hypercholesterolemia and diabetes mellitus were independently associated with the development of thrombosis in these patients.
Results Considering the baseline characteristics of patients (Table 1), the majority of them were young adults and 92 % of them developed the first episode of IS and PAD before the age of 45 years. Male was 43 % of the patients and the median age was similar in patients and controls (36 and 32.5 respectively, no significant difference was observed). Smoking (14.8 %), hypertension (7.4 %), were the most frequently acquired risk factors for arterial thrombosis detected among patients. Familiar history of thrombosis was observed among 24.7 % of patients and recurrence in 22.7 %. The results obtained for ABO blood groups phenotypes showed significant differences for B group (9.5 vs 23.5 %, OR 2.94, 95 % CI 1.38–6.24, p \ 0.01) by comparing the control and patients groups, respectively. Instead of the most of ABO groups are non-O, no significant differences were observed among others comparisons. The phenotype of control group was in agreement with the donor’s data from local population of Minas Gerais state [24]. No significant differences were observed for ABO blood groups polymorphisms by comparing controls and patients,
considering the genotypes found: A1A1, A1A2, A1O1, A1O2, A2A2, A2O1, A2O2, A1B, A2B, BB, BO1, BO2, O1O1, O1O2, O2O2 (data not shown). The frequencies of A1, A2, B, O1 and O2 alleles are presented in Table 2. Significant differences were observed for O1 (OR 0.59, 95 % CI 0.40–0.88, p \ 0.01), O2 (OR 4.0, 95 % CI 1.65–9.82, p \ 0.001) and B (OR 2.66, 95 % CI 1.44–4.89, p = 0.001), by comparing patients and control groups. The phenotypes and genotypes were analyzed according to the anatomical site (IS or PAD). No significant differences were observed for ABO blood groups phenotypes and genotypes by comparing IS patients and controls subjects. However, significant differences were observed for O1 (OR 0.57, 95 % CI 0.35–0.95, p \ 0.05) and O2 (OR 3.47, 95 % CI 1.15–10.28, p \ 0.05) alleles (Table 3).
Table 1 Baseline characteristics of controls and patients Patients (n = 81)
Control (n = 201)
p value
Age (median)
36
32.5
[0.05
Male (%)
35 (43)
74 (36.8)
[0.05
BMI [30 kg/m2
2
0
–
Diabetes (%)
1(1.2)
1(0.4)
[0.05
Hypertension (%) Regular smoker (%)
6 (7.4) 12 (14.8)
1 (0.4) 9 (4.4)
\0.01 \0.01
BMI body mass index
Table 2 Frequencies of A1, A2, B, O1 and O2 alleles between patients and controls Alleles
Patients 162 (%)
Control 402 (%)
A1
20 (12.0)
74 (18.4)
A2
11 (7.0)
23 (5.7)
O1 O2
88 (55.0) 15 (9.0)
268 (67.0) 10 (2.5)
B
26 (17.0)
27 (6.4)
OR
95 % CI
p value
0.62
0.35–1.09
0.100
1.20
0.53–2.65
0.770
0.59 4.00
0.40–0.88 1.65–9.82
\0.010 \0.001
2.66
1.44–4.89
0.001
Table 3 Frequencies of A1, A2, B, O1 and O2 alleles between IS patients and controls Alleles
Patients 86 (%)
Control 402 (%)
OR
95 % CI
p value
A1
17 (20.0)
74 (18.4)
1.09
0.58–2.03
0.88
A2
5 (6.0)
23 (5.7)
1.02
0.33–2.93
1.00
O1
46 (53.0)
268 (67.0)
0.57
0.35–0.95
\0.05
O2
7 (8.0)
10 (2.5)
3.47
1.15–10.28
\0.05
B
11 (13.0)
27 (6.4)
2.04
0.90–4.51
0.09
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No significant differences were observed for ABO blood groups genotypes by comparing PAD patients and controls subjects, but significant differences were observed for B phenotype (26.3 vs 9.5 %, OR 3.42, 95 % CI 1.32–8.76, p = 0.01, patients vs controls, respectively) and alleles A1 (OR 0.31, 95 % CI 0.11–0.84, p \ 0.05), O2 (OR 4.61, 95 % CI 1.59–13.23, p \ 0.01) and B (OR 3.42, 95 % CI 1.62–7.13, p \ 0.001) alleles (Table 4). After adjustments for lifestyle covariates and alleles, in a multiple regression model, O1 allele was an independent variable (OR 0.27, 95 % CI 0.12–0.57, p \ 0.001) for IS patients. No significance was observed with PAD patient’s analysis (Table 5).
Discussion Recent studies have shown common risk factors for venous and arterial thrombosis including age, obesity, tabagism, diabetes mellitus, hypertension, dyslipidemia and metabolic syndrome. This association and interaction is not completely understood and has been described mainly in patients with idiopathic VTE [25, 26]. Thus considering the multifactorial and multigenic character of the thrombosis, this study investigated the possible association of ABO
Table 4 Frequencies of A1, A2, B, O1 and O2 alleles between PAD patients and controls Alleles
Patients 76 (%)
Control 402 (%)
OR
95 % CI
p vaule
A1
5 (7.0)
74 (18.4)
0.31
0.11–0.84
\0.05
A2
6 (8.0)
23 (5.7)
1.41
0.45–3.74
0.44
O1
42 (55.0)
268 (67.0)
0.62
0.36–1.05
O2
8 (10.0)
10 (2.5)
4.61
1.59–13.23
\0.01
B
15 (20.0)
27 (6.4)
3.42
1.62–7.13
\0.001
0.08
Table 5 Adjusted odds ratio for studied variables and alleles in IS patients Variables
OR
95 % CI
p value
TC
0.95
0.37–2.43
0.91
HDLc LDLcf
1.01 1.06
0.39–2.58 0.41–2.72
0.99 0.91
TG
1.01
0.84–1.22
0.93
A1
0.60
0.08–4.52
0.62
O1
0.27
0.12–0.57
\0.001
TC total cholesterol total, HDLC high density lipoprotein cholesterol, LDLC f low density lipoprotein cholesterol, estimated by Friedewald formula, TG triglycerides
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blood groups polymorphism and arterial thrombosis pathogenesis. We found significant differences for O1 and O2 alleles for IS patients, and A1, O2 and B alleles for PAD patients, respectively (Tables 3 , 4) in a univariate analysis. In a multiple regression analysis O1 was an independent variable for IS patients. Also, the B group was most frequent among PAD patients. The relationship between the ABO blood group and venous thrombosis has been demonstrated by several reports [5–7, 27–30], but its association with arterial thrombosis is not clear. We know that most of effects of ABO blood group in FVIII levels are mediated by VWF, since there are similar oligosaccharides among VWF and ABO antigens, which affect the VWF and VWF/FVIII complex clearance. On the other hand, several factors have been also correlated with increased FVIII plasma levels, such as diabetes mellitus, elevated plasmatic insulin, fibrinogen, triglycerides and oral contraceptive use [31]. Genetic or acquired decrease in FVIII and VWF clearance and alterations in other genes involved in biosynthesis of VWF are also related to elevated FVIII plasma levels [15, 32–34]. Subjects with AB phenotype have increased levels of VWF while AA, AO, BB and BO have intermediate levels [35–37]. Prospective studies evaluating more than 14,000 individuals demonstrated association among increased levels of FVIII and VWF with IS including association with mortality [16, 38]. Although several studies have tried to show the relationship between VWF and FVIII levels and arterial events and the role of blood groups in this process, these are a difficult analysis once the high levels of these proteins can reflect chronic inflammatory process of atherosclerosis and not necessarily blood group influence [16, 39]. O’Donnell et al. [40] found a direct relationship among ABO genotype, transferase A expression and antigen A rate express on circulating VWF. The mechanism that explains how ABH determinants influence VWF plasma levels is still unclear and some hypotheses have been proposed to justify this phenomenon [41]. The first one admits that ABO antigens influence the rate of synthesis or secretion of VWF by endothelial cells [42]. The second suggest that the determinants of blood group O are associated to increased VWF hepatic clearance [43]. Finally, recent studies have suggested that the determinants of the ABO blood group may influence the susceptibility of plasma VWF to proteolysis by ADAMTS13 [41, 44, 45] that promotes the cleavage of this factor [46–48]. Thus, FVIII plasma levels can be modulated by VWF gene polymorphisms and ADAMTS 13, a metalloproteinase that modulates the VWF multimers clearance, preventing platelet aggregation and thrombus formation [49, 50]. Meade et al. [18] reported association between FVIII levels
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and ischemic coronary disease after adjustment for blood group and Jager et al. [51] demonstrated association between increased levels of VWF and mortality by cardiovascular disease. Taken together these data show that high levels of these proteins are a risk factor for arterial thrombosis, but the ABO blood group influence in this pathogenesis is not clear. Thus genetic evaluation of ABO polymorphism can be useful for elucidate the role of blood group in this process, since the genetic profile is not modified by factors such as FVIII plasma levels. In this study the O genotype and allele was the most frequent in control subjects. The O blood group and O1O1 genotype are the most common in general populations. Batissoco et al. [3] reported the frequencies of O1 and O2 alleles in blood donors of Sa˜o Paulo city, showing 90.24 % of O1O1, 8.53 % O1O2 and 1.22 % O2O2. Novaretti et al. [52], showed the prevalence of 49.23 % of O group, 33.71 % A, 13.93 % B and 3.13 % AB in Sa˜o Paulo, which were similar to control group of this study and data from Hemominas foundation in Minas Gerais State, showing the high frequency of O group in our population when comparing with European population, where the A group is more frequent [5, 7, 30, 53]. High frequency of non-O groups was observed among patients with arterial thrombosis, mainly B group (OR 2.94, 95 % CI 1.38–6.24, p \ 0.01). Significant differences were observed for O1 (OR 0.59, 95 % CI 0.40–0.88, p \ 0.01), O2 (OR 4.0, 95 % CI 1.65–9.82, p \ 0.001) and B (OR 2.66, 95 % CI 1.44–4.89, p = 0,001) alleles, by comparing patients and control groups. It is important to note that there was significant difference for O2 allele frequency, however considering the factors that the standard of O genes heterogeneity and that this group is originated from glicosyltransferases deficiency, that convert the H substance into A and B antigens and the similar oligosaccharides on VWF, we cannot confirm that O2 is associated with VWF levels and occurrence of thrombosis. This observation of increased O2 allele frequency can be justified by increased frequency of BO2 genotype. O2 allele analysis is a somatory from the genotypes BO2, O1O2 and O2O2 among patients. Thus, the increased OR for O2 allele comes from mainly BO2 heterozygous, that do not justify the association between O2 allele and risk for arterial thrombosis. Ohira et al. [8] investigated ABO blood groups in 1,500 subjects (patients and controls) and showed increased frequencies of non-O blood groups and high levels of FVIII and VWF among patients and its relationship with VTE mainly in presence of factor V Leiden. Schleef et al. [5] associated O1O1, O1O2 and A2O1 genotypes with low risk for thrombosis development while A1A1, A1B, A1O1 and BO1 were associated with high risk and there was a correlation with FVIII and VWF plasma levels with non-O groups, mainly A1 and B alleles carriers. Similar results
were reported by Morelli et al. [7] with exception of A2A2, A2O1 and A2O2 genotypes that were associated with high risk for VTE. In our study, after adjustment in multiple regression model, the O1 allele was a independent variable with adjusted OR 0.27, 95 % CI 0.12–0.57, p \ 0.001, for IS patients. Limitation of this study should be considered such as small sample size, population studied, no determination of FVIII and VWF levels (limitation discussed above in arterial events). The data obtained in this study is in agreement with previous reports that suggest the relationship of non-O blood groups in pathogenesis of thrombosis events and a possible protective effect of O blood group, mainly in young-adults patients with diagnosis of IS and PAD. Studies with a large number of patients are necessary to better understand the intrinsic mechanisms and etiology associated with ABO blood groups and arterial and venous thrombosis in our population. Acknowledgments Authors are grateful to Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG) and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) for sponsoring this investigation.
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ABO blood group polymorphisms and risk for ischemic stroke and peripheral arterial disease Adriano de Paula Sabino • Daniel Dias Ribeiro • Caroline Pereira Domingheti Danyelle Romana Alves Rios • Luci Maria SantAna Dusse • Maria das Grac¸as Carvalho • Ana Paula Fernandes
•
Received: 12 December 2012 / Accepted: 3 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Recent studies have demonstrated association between ABO blood system and thrombosis, indicating that individuals belonging to non-O blood groups (A, B or AB) present an increased risk of venous thrombosis, heart disease, and ischemic stroke (IS) as compared to O blood group carriers. In this study, we investigated the frequency of ABO blood group polymorphisms and its association with IS and peripheral arterial disease. Significant differences were observed for O1 (OR 0.57, 95 % CI 0.35–0.95, p \ 0.05) and O2 (OR 3.47, 95 % CI 1.15–10.28, p \ 0.05) alleles among IS patients while significant differences were observed for B phenotype (26.3 vs 9.5 %, OR 3.42, 95 % CI 1.32–8.76, p = 0.01, patients vs controls, respectively) and alleles A1 (OR 0.31, 95 % CI 0.11–0.84, p \ 0.05), O2 (OR 4.61, 95 % CI 1.59–13.23, p \ 0.01) and B (OR 3.42, 95 % CI 1.62–7.13, p \ 0.001) alleles for PAD patients. O1 allele was an independent variable (OR 0.27, 95 % CI 0.12–0.57, p \ 0.001) for IS patients. These data suggest the relationship of non-O blood groups in pathogenesis of thrombosis events and a possible protective effect of O blood group.
A. de Paula Sabino (&) C. P. Domingheti L. M. S. Dusse M. das Grac¸as Carvalho A. P. Fernandes Faculty of Pharmacy, Federal University of Minas Gerais, Av. Antoˆnio Carlos 6627, Pampulha, Belo Horizonte, MG, Brazil e-mail: [email protected] D. D. Ribeiro University Hospital–Federal University of Minas Gerais, Belo Horizonte, MG, Brazil D. R. A. Rios Health Sciences Center, Federal University of Sa˜o Joa˜o Del Rei, Divino´polis, MG, Brazil
Keywords PAD
Arterial thrombosis ABO genotypes IS
Introduction Antigens of the ABO system consist of an A or a B carbohydrate structure carried on the substrate H antigen. The A or B glycosyltransferase encoded at the ABO locus on chromosome 9 defines which specific carbohydrate is added to the end of the H substance oligosaccharide chains [1]. The ABO gene contains seven exons (range in size from 28 to 688 bp) and six introns spanning about 18–20 kilobase pairs (kbp), in position 9q34.1-9q34.2, being the most of the enzymes encoded by exons 6 and 7, which constitute 77 % of the ABO gene [2, 3]. The major alleles of ABO gene are A1, B and O that provide the four blood groups: A, B, AB and O. Among these alleles there is a large genetic heterogeneity, including the change of amino acid residues in the glycosyltransferase molecule. Recent studies have demonstrated association between ABO blood system and venous thromboembolism (VTE), indicating that individuals belonging to non-O blood groups (A, B or AB) present an increased risk of venous thrombosis, heart disease, and ischemic stroke (IS) as compared to O blood group carriers [4–11]. A meta-analysis showed that the prevalence of non-O blood group was significantly higher in VTE patients compared with controls with a resulting odds ratio of 2.09 (p \ 0,00001) [12]. This association can be explained by higher plasma levels of factor VIII (FVIII) and von Willebrand factor (VWF) observed in ‘‘non O’’ groups [7, 13, 14]. Morelli et al. [7] studied the effect of the genotype of the ABO system and the risk of deep venous thrombosis in 471
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patients who had venous thrombosis and 471 individuals without thrombotic events (controls). In the group of patients, all genotypes ‘‘non O’’ (70.9 %), except A2A2 homozygous or heterozygous A2O1/A2O2 (7.2 %) had high risk of thrombosis compared to those with genotype OO (29.1 %). The risk of venous thrombosis increases with increased levels of VWF and FVIII which are higher in individuals ‘‘non O’’ comparing to group ‘‘O’’. Contrarily, the association between blood group and arterial thrombosis is difficult to prove, since increased FVIII and VWF plasma levels in arterial thrombosis observed may reflect a chronic inflammation, as the result of atherosclerosis [15, 16]. Even with this limitation, several studies have reported an association between ‘‘non O’’ group and risk of arterial thrombosis, such as coronary artery disease and peripheral vascular disease [16–19]. Clark et al. [20] performed a case–control study to examine the frequency of ABH, Lewis and Secretor genotypes and phenotypes in patients with acute cerebral ischemia of arterial origin (CIAO). They observed that non-O and, in particular A, blood groups are more common in those presenting with CIAO, although there was not additional influence of Lewis or Secretor genotypes and phenotypes. Although the risk identified have been modest, the authors emphasize that clarification of the link between blood groups on the occurrence, and perhaps recurrence, of CIAO might ultimately be useful in understanding the link between factors such as VWF, activated protein C (APC) resistance, or lipids and vascular disease. In contrast to these results, an analysis of ABO genotype and IS showed an increased risk in those carrying B allele as compared with all other genotypes [21]. The aim of this study was to investigate the association of the ABO genotype and phenotype with IS and peripheral arterial disease (PAD).
auto-immune disorders, as well as those with coagulation disorders were excluded. Baseline information on smoking habits, medication use, and personal history of disease were gathered by trained medical staff during a standardized interview. In addition, all participants underwent an extensive standardized medical examination. Body mass index (BMI) was calculated as weight in kilograms divided by height in square meters. Systolic and diastolic blood pressure was measured on the right arm in a sitting position, according to the recommendations of the American heart association. Persons being aware of having hypertension, taking antihypertensive medication, and/or having blood pressure values 160/90 mmHg at baseline were defined as actual hypertension. Individuals were classified as having diabetes mellitus if plasma glucose was equal or superior to 126 mg/dl in the fasting state or if individuals were receiving oral anti-diabetics or insulin. Hypercholesterolemia were considered if total cholesterol was superior to 239 mg/dl. None of the patients were using lipid lowering therapy. A regular smoker was defined as a subject who currently smoked at least five cigarettes per day. No renal disease was observed or detected by the trained medical staff. Patients had normal levels of urea and creatinine. The control group was composed by 201 subjects with no previous history of arterial or venous thrombosis from the same demographic area, with no familiar relationship with patients. The institutional ethics committee of the federal university of Minas Gerais, approved this study and an informed consent was obtained from all participants. Sample collection
Materials and methods
Venous blood was also collected from all subjects into EDTA (5 ml) tubes using the Vacutainer System (Becton– Dickinson, Franklin Lakes, New Jersey, USA) and was submitted to genomic DNA extraction using the Wizard purification system (Promega Inc., Madison, Wisconsin, USA), following the manufacturer’s instructions.
Patients and control subjects
Genotyping the ABO system
A total of 282 individuals participated of this study. Survival patients to the arterial thrombotic event (n = 81) with diagnosis of IS and PAD were consecutively selected by physicians at the Hematology Unit of the University Hospital (Federal University of Minas Gerais, Belo HorizonteMG) to participate of the present study between July 2007 and December 2010. All of them had the diagnosis of IS and PAD confirmed by magnetic resonance, brain computer tomography and/or arteriography. Patients with major systemic diseases, which are known to predispose to thrombosis such as cancer, infections or hepatic diseases,
ABO polymorphisms were investigated by PCR–RFLP. Briefly, amplification of exons 6 and 7 of the ABO gene were obtained through two separate reactions using oligonucleotides previously described [22]. PCR reactions were performed in a TC-312 thermocycler (Techne Inc.) using 1.5 pMol of each primer for exon 6 and 0.5 pMol of each primer for exon 7 (Invitrogen, Sa˜o Paulo, Brazil), 0.2 mM of dNTPs (GIBCO BRL, Sa˜o Paulo, Brazil), and 0.5 units of Taq polymerase (Phoneutria, Belo Horizonte, Minas Gerais, Brazil). PCR reactions were submitted to 40 cycles consisting of 2 min at 94 °C for initial denaturing, 10 s at
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94 °C for denaturing, 30 s at 62 °C for primer annealing, and 30 s for primer extension. PCR products were submitted to restriction endonuclease digestion for 4 h at 37 °C with enzymes KpnI and MspI (Promega Inc.) in a single reaction. Digested fragments were analyzed in polyacrylamide gel electrophoresis. This method allows the differentiation of alleles A1, A2, B, O1, and O2 [22]. DNAs from previously typed individuals were included in each set of analyzed samples in order to control enzyme activity. Statistical analysis Statistical comparisons were performed using version 6.04 of the Epi Info program [23], Sigma Stat version 2.0. The patient group was tested against the control group for each polymorphism frequency. Odds ratios (OR) were used as a measure of the association between each polymorphism of the ABO system and were interpreted as the risk for VTE. Confidence intervals (95 % CI) were determined for ORs. Significance levels were estimated by applying the Chi square test. Differences were considered significant when p \ 0.05. Multiple regression analysis was used to explore whether the genetic (ABO blood groups) and acquired variables such as smoking, obesity, hypertension, Hypercholesterolemia and diabetes mellitus were independently associated with the development of thrombosis in these patients.
Results Considering the baseline characteristics of patients (Table 1), the majority of them were young adults and 92 % of them developed the first episode of IS and PAD before the age of 45 years. Male was 43 % of the patients and the median age was similar in patients and controls (36 and 32.5 respectively, no significant difference was observed). Smoking (14.8 %), hypertension (7.4 %), were the most frequently acquired risk factors for arterial thrombosis detected among patients. Familiar history of thrombosis was observed among 24.7 % of patients and recurrence in 22.7 %. The results obtained for ABO blood groups phenotypes showed significant differences for B group (9.5 vs 23.5 %, OR 2.94, 95 % CI 1.38–6.24, p \ 0.01) by comparing the control and patients groups, respectively. Instead of the most of ABO groups are non-O, no significant differences were observed among others comparisons. The phenotype of control group was in agreement with the donor’s data from local population of Minas Gerais state [24]. No significant differences were observed for ABO blood groups polymorphisms by comparing controls and patients,
considering the genotypes found: A1A1, A1A2, A1O1, A1O2, A2A2, A2O1, A2O2, A1B, A2B, BB, BO1, BO2, O1O1, O1O2, O2O2 (data not shown). The frequencies of A1, A2, B, O1 and O2 alleles are presented in Table 2. Significant differences were observed for O1 (OR 0.59, 95 % CI 0.40–0.88, p \ 0.01), O2 (OR 4.0, 95 % CI 1.65–9.82, p \ 0.001) and B (OR 2.66, 95 % CI 1.44–4.89, p = 0.001), by comparing patients and control groups. The phenotypes and genotypes were analyzed according to the anatomical site (IS or PAD). No significant differences were observed for ABO blood groups phenotypes and genotypes by comparing IS patients and controls subjects. However, significant differences were observed for O1 (OR 0.57, 95 % CI 0.35–0.95, p \ 0.05) and O2 (OR 3.47, 95 % CI 1.15–10.28, p \ 0.05) alleles (Table 3).
Table 1 Baseline characteristics of controls and patients Patients (n = 81)
Control (n = 201)
p value
Age (median)
36
32.5
[0.05
Male (%)
35 (43)
74 (36.8)
[0.05
BMI [30 kg/m2
2
0
–
Diabetes (%)
1(1.2)
1(0.4)
[0.05
Hypertension (%) Regular smoker (%)
6 (7.4) 12 (14.8)
1 (0.4) 9 (4.4)
\0.01 \0.01
BMI body mass index
Table 2 Frequencies of A1, A2, B, O1 and O2 alleles between patients and controls Alleles
Patients 162 (%)
Control 402 (%)
A1
20 (12.0)
74 (18.4)
A2
11 (7.0)
23 (5.7)
O1 O2
88 (55.0) 15 (9.0)
268 (67.0) 10 (2.5)
B
26 (17.0)
27 (6.4)
OR
95 % CI
p value
0.62
0.35–1.09
0.100
1.20
0.53–2.65
0.770
0.59 4.00
0.40–0.88 1.65–9.82
\0.010 \0.001
2.66
1.44–4.89
0.001
Table 3 Frequencies of A1, A2, B, O1 and O2 alleles between IS patients and controls Alleles
Patients 86 (%)
Control 402 (%)
OR
95 % CI
p value
A1
17 (20.0)
74 (18.4)
1.09
0.58–2.03
0.88
A2
5 (6.0)
23 (5.7)
1.02
0.33–2.93
1.00
O1
46 (53.0)
268 (67.0)
0.57
0.35–0.95
\0.05
O2
7 (8.0)
10 (2.5)
3.47
1.15–10.28
\0.05
B
11 (13.0)
27 (6.4)
2.04
0.90–4.51
0.09
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No significant differences were observed for ABO blood groups genotypes by comparing PAD patients and controls subjects, but significant differences were observed for B phenotype (26.3 vs 9.5 %, OR 3.42, 95 % CI 1.32–8.76, p = 0.01, patients vs controls, respectively) and alleles A1 (OR 0.31, 95 % CI 0.11–0.84, p \ 0.05), O2 (OR 4.61, 95 % CI 1.59–13.23, p \ 0.01) and B (OR 3.42, 95 % CI 1.62–7.13, p \ 0.001) alleles (Table 4). After adjustments for lifestyle covariates and alleles, in a multiple regression model, O1 allele was an independent variable (OR 0.27, 95 % CI 0.12–0.57, p \ 0.001) for IS patients. No significance was observed with PAD patient’s analysis (Table 5).
Discussion Recent studies have shown common risk factors for venous and arterial thrombosis including age, obesity, tabagism, diabetes mellitus, hypertension, dyslipidemia and metabolic syndrome. This association and interaction is not completely understood and has been described mainly in patients with idiopathic VTE [25, 26]. Thus considering the multifactorial and multigenic character of the thrombosis, this study investigated the possible association of ABO
Table 4 Frequencies of A1, A2, B, O1 and O2 alleles between PAD patients and controls Alleles
Patients 76 (%)
Control 402 (%)
OR
95 % CI
p vaule
A1
5 (7.0)
74 (18.4)
0.31
0.11–0.84
\0.05
A2
6 (8.0)
23 (5.7)
1.41
0.45–3.74
0.44
O1
42 (55.0)
268 (67.0)
0.62
0.36–1.05
O2
8 (10.0)
10 (2.5)
4.61
1.59–13.23
\0.01
B
15 (20.0)
27 (6.4)
3.42
1.62–7.13
\0.001
0.08
Table 5 Adjusted odds ratio for studied variables and alleles in IS patients Variables
OR
95 % CI
p value
TC
0.95
0.37–2.43
0.91
HDLc LDLcf
1.01 1.06
0.39–2.58 0.41–2.72
0.99 0.91
TG
1.01
0.84–1.22
0.93
A1
0.60
0.08–4.52
0.62
O1
0.27
0.12–0.57
\0.001
TC total cholesterol total, HDLC high density lipoprotein cholesterol, LDLC f low density lipoprotein cholesterol, estimated by Friedewald formula, TG triglycerides
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blood groups polymorphism and arterial thrombosis pathogenesis. We found significant differences for O1 and O2 alleles for IS patients, and A1, O2 and B alleles for PAD patients, respectively (Tables 3 , 4) in a univariate analysis. In a multiple regression analysis O1 was an independent variable for IS patients. Also, the B group was most frequent among PAD patients. The relationship between the ABO blood group and venous thrombosis has been demonstrated by several reports [5–7, 27–30], but its association with arterial thrombosis is not clear. We know that most of effects of ABO blood group in FVIII levels are mediated by VWF, since there are similar oligosaccharides among VWF and ABO antigens, which affect the VWF and VWF/FVIII complex clearance. On the other hand, several factors have been also correlated with increased FVIII plasma levels, such as diabetes mellitus, elevated plasmatic insulin, fibrinogen, triglycerides and oral contraceptive use [31]. Genetic or acquired decrease in FVIII and VWF clearance and alterations in other genes involved in biosynthesis of VWF are also related to elevated FVIII plasma levels [15, 32–34]. Subjects with AB phenotype have increased levels of VWF while AA, AO, BB and BO have intermediate levels [35–37]. Prospective studies evaluating more than 14,000 individuals demonstrated association among increased levels of FVIII and VWF with IS including association with mortality [16, 38]. Although several studies have tried to show the relationship between VWF and FVIII levels and arterial events and the role of blood groups in this process, these are a difficult analysis once the high levels of these proteins can reflect chronic inflammatory process of atherosclerosis and not necessarily blood group influence [16, 39]. O’Donnell et al. [40] found a direct relationship among ABO genotype, transferase A expression and antigen A rate express on circulating VWF. The mechanism that explains how ABH determinants influence VWF plasma levels is still unclear and some hypotheses have been proposed to justify this phenomenon [41]. The first one admits that ABO antigens influence the rate of synthesis or secretion of VWF by endothelial cells [42]. The second suggest that the determinants of blood group O are associated to increased VWF hepatic clearance [43]. Finally, recent studies have suggested that the determinants of the ABO blood group may influence the susceptibility of plasma VWF to proteolysis by ADAMTS13 [41, 44, 45] that promotes the cleavage of this factor [46–48]. Thus, FVIII plasma levels can be modulated by VWF gene polymorphisms and ADAMTS 13, a metalloproteinase that modulates the VWF multimers clearance, preventing platelet aggregation and thrombus formation [49, 50]. Meade et al. [18] reported association between FVIII levels
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and ischemic coronary disease after adjustment for blood group and Jager et al. [51] demonstrated association between increased levels of VWF and mortality by cardiovascular disease. Taken together these data show that high levels of these proteins are a risk factor for arterial thrombosis, but the ABO blood group influence in this pathogenesis is not clear. Thus genetic evaluation of ABO polymorphism can be useful for elucidate the role of blood group in this process, since the genetic profile is not modified by factors such as FVIII plasma levels. In this study the O genotype and allele was the most frequent in control subjects. The O blood group and O1O1 genotype are the most common in general populations. Batissoco et al. [3] reported the frequencies of O1 and O2 alleles in blood donors of Sa˜o Paulo city, showing 90.24 % of O1O1, 8.53 % O1O2 and 1.22 % O2O2. Novaretti et al. [52], showed the prevalence of 49.23 % of O group, 33.71 % A, 13.93 % B and 3.13 % AB in Sa˜o Paulo, which were similar to control group of this study and data from Hemominas foundation in Minas Gerais State, showing the high frequency of O group in our population when comparing with European population, where the A group is more frequent [5, 7, 30, 53]. High frequency of non-O groups was observed among patients with arterial thrombosis, mainly B group (OR 2.94, 95 % CI 1.38–6.24, p \ 0.01). Significant differences were observed for O1 (OR 0.59, 95 % CI 0.40–0.88, p \ 0.01), O2 (OR 4.0, 95 % CI 1.65–9.82, p \ 0.001) and B (OR 2.66, 95 % CI 1.44–4.89, p = 0,001) alleles, by comparing patients and control groups. It is important to note that there was significant difference for O2 allele frequency, however considering the factors that the standard of O genes heterogeneity and that this group is originated from glicosyltransferases deficiency, that convert the H substance into A and B antigens and the similar oligosaccharides on VWF, we cannot confirm that O2 is associated with VWF levels and occurrence of thrombosis. This observation of increased O2 allele frequency can be justified by increased frequency of BO2 genotype. O2 allele analysis is a somatory from the genotypes BO2, O1O2 and O2O2 among patients. Thus, the increased OR for O2 allele comes from mainly BO2 heterozygous, that do not justify the association between O2 allele and risk for arterial thrombosis. Ohira et al. [8] investigated ABO blood groups in 1,500 subjects (patients and controls) and showed increased frequencies of non-O blood groups and high levels of FVIII and VWF among patients and its relationship with VTE mainly in presence of factor V Leiden. Schleef et al. [5] associated O1O1, O1O2 and A2O1 genotypes with low risk for thrombosis development while A1A1, A1B, A1O1 and BO1 were associated with high risk and there was a correlation with FVIII and VWF plasma levels with non-O groups, mainly A1 and B alleles carriers. Similar results
were reported by Morelli et al. [7] with exception of A2A2, A2O1 and A2O2 genotypes that were associated with high risk for VTE. In our study, after adjustment in multiple regression model, the O1 allele was a independent variable with adjusted OR 0.27, 95 % CI 0.12–0.57, p \ 0.001, for IS patients. Limitation of this study should be considered such as small sample size, population studied, no determination of FVIII and VWF levels (limitation discussed above in arterial events). The data obtained in this study is in agreement with previous reports that suggest the relationship of non-O blood groups in pathogenesis of thrombosis events and a possible protective effect of O blood group, mainly in young-adults patients with diagnosis of IS and PAD. Studies with a large number of patients are necessary to better understand the intrinsic mechanisms and etiology associated with ABO blood groups and arterial and venous thrombosis in our population. Acknowledgments Authors are grateful to Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG) and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) for sponsoring this investigation.
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