Vascular Genetic Variants and Ischemic Stroke Susceptibility in Albanians from the Republic of Macedonia

BACKGROUND: Acute first-ever ischemic stroke (FIS) is a heterogeneous, polygenic disorder. The contribution of vascular genetic variants as inherited causes of ischemic stroke has remained controversial. AIM: To examine the association of genetic variants in vascular factors with the occurrence of FIS. MATERIAL AND METHODS: The current research was performed in a group of 39 patients with FIS (study group) and 102 healthy volunteers (control group). We analyzed the prevalence of vascular genetic variants in following genes: factor V, prothrombin, methylenetetrahydrofolate reductase (MTHFR), factor XIII, plasminogen activator 1, endothelial protein C receptor (EPCR), apolipoprotein B, apolipoprotein E, β-fibrinogen, human platelet antigen 1, angiotensin-converting enzyme (ACE), endothelial nitric oxide synthase (eNOS) and lymphotoxin alpha. RESULTS: It was found that heterozygous LTA 804C>A and FXIII V34L Leu/Leu were significantly more frequent in patients with FIS than in control group (p = 0.036 and p = 0.017, respectively). The frequency of FXIII V34L Val/Val was significantly lower in patients with FIS than in control group (p = 0.020). Other frequencies of vascular gene variants in patients with FIS and in control group were not significantly different. CONCLUSIONS: This is the first comprehensive study to present data indicating that polymorphism of vascular genes in the prevalence of acute FIS exists in the Albanian population from the Republic of Macedonia. Variations in these genes have been detected in patients with acute FIS, suggesting that their combination might act in a susceptible or protective manner in this Albanian population.


Introduction
Ischemic stroke is a major public health problem with a heritable component to the risk of disease [1][2][3]. The last decade has been marked by initiatives aimed at raising awareness of the inherited predisposition to ischemic stroke. There has been an increase in studies that analysed polymorphic genes responsible for arterial and venous ischemic events. An increased understanding of genetics and molecular biology has fuelled this research and shed light on several genetic risk factors that may help to predict arterial events.
Most of the studies related to arterial thrombosis have actually involved patients with coronary syndromes [4], myocardial infarction [5] and events of the arterial circulatory system [6,7], and they produced conflicting results. To date, most studies on the genetics of ischemic stroke have focused on candidate genes in a control group and unrelated cases. The causes of the majority of ischemic strokes include atherosclerotic, cardiogenic and lacunar (penetrating vessel) mechanisms [8]. Several animal and clinical studies clearly defined genetic predisposition to hereditary thrombophilia or atherosclerosis, in the case of atherosclerosis, and particularly, to the clinical expression of ischemic stroke [9]. Arteriosclerosis of the large vessels is the cause of cerebral infarction in almost half of all ischemic events [10]. Arteriosclerosis involves numerous genes and some pathways thought to be involved in the development and rupture of atherosclerotic lesions [11].
This study is the first comprehensive study of the vascular gene variations associated with first-ever ischemic stroke in the Albanian population from the Republic of Macedonia. The purpose of this research was to examine the association of vascular gene variations in 13 genes and 17 genetic variants in patients with acute FIS and compare the frequencies in the patient and control groups.

Sampling
This study was a case-control study (comparative analytical study) carried out from September 2008 to August 2010. In total, 231 patients with FIS as a study group and 194 healthy volunteers as a control group were enrolled in the initial study. The inclusion criteria were consecutive patients admitted to the Neurology Department with confirmed diagnosis acute FIS, age ranging from 18 to 90 years, an absence of a history of stroke and completion of interview and analysis. All patients gave informed consent prior to participation in this study. The exclusion criteria were patients younger than 18 years, patients, who had experienced an acute hemorrhagic stroke, a known malignancy, acute or chronic renal or failure or acute myocardial infarction and patients who had recent inflammatory or immunologic disease. Acute FIS was defined as a clinical syndrome on the basis of clinical symptoms and (a) recent infarct(s) in the clinically relevant area of the brain, using brain computed tomography (CT) and/or magnetic resonance imaging (MRI).
Healthy controls that were free from cerebrovascular diseases were selected from among local residents, using the same exclusion criterion as for patients. Informed consent was obtained from all participants prior to conducting the study. The protocol of the study was reviewed and approved by the professional college at Clinical Hospital Tetovo (Nr.02-1280/2, 02.07.2008) [28].
Within a study of the genetics of the Macedonian population, a detailed description of the study subjects and study design were performed as previously described [29,30]. Briefly, after signed consent was given to obtain and store a DNA sample, 212 subjects were recruited in total (stratified into 58 healthy controls and 12 patients of Macedonian origin and 103 healthy controls and 39 patients of Albanian origin) all residents of Tetovo and its neighbouring region who met the study inclusion criteria to engage in the proposed national project over a 2-year period (2010-2012), examining gene variations associated with diseases of blood vessels in the Republic of Macedonia, which was originally designed as a prospective, nationwide, multicenter case-control study (ID 13-3589/1, 26.07.2010).
Among others, the objectives were to investigate the distribution of candidate genes in healthy Macedonian and Albanian populations and the association of candidate genes with stroke in Macedonians and Albanians from the Republic of Macedonia. Of these, genetic analyses were completed for 141 (66.51%) participants, all of whom were Albanians, while others are in progress. The present study comprises 39 patients with acute FIS treated at the Clinical Hospital Tetovo, Department of Neurology as a study group and 102 healthy volunteers as a control group. Each individual was interviewed on a one-to-one basis, his/her genealogy was recorded for the past three generations, and their Albanian origin was ascertained by making enquiries as far back as their four grandparents. Admixture, if any, was recorded for each individual. Individuals with only one Albanian parent were excluded from the study.

Blood sampling and processing
A sample of peripheral vein whole blood (10 mL) was collected by venipuncture in standard collection tubes containing anticoagulant ethylenediaminetetraacetic acid (EDTA) in the first 24 h of admission and then dispensed into two tubes (one for complete blood picture and the other for extraction of DNA). The first tube was transferred to the biochemical laboratory for routine analysis and the second tube was separated and referred for genetic analysis. http://www.mjms.mk/ http://www.id-press.eu/mjms/

DNA extraction, molecular genetic screening and storage
At the Institute of Immunobiology and Human Genetics, part of the Faculty of Medicine, Ss Cyril and Methodius University of Skopje, Skopje, Republic of Macedonia [28][29][30], genomic DNA was isolated from peripheral blood leukocytes by the phenol-chloroform extraction method [31] or with BioRobot EZ1 workstation (QIAGEN) [32].
Genetic testing of the candidate genes was based on the polymerase chain reaction and reverse hybridization principle using the CVD SripAssay [ViennaLab (Laboradiagnostica GmbH, Austria)]. The DNA samples were subsequently stored in the anthropology project field of the Macedonian Human DNA Bank (hDNAMKD) [33].

Statistical analysis
The population genetics analysis package, PyPop, developed by the Biostatistics Core for the Workshop [34][35][36] was used for analysis of the genetic data for this report. All analyses were based on available data. The results were collected, tabulated and statistically analysed by personal computer and the commercially available statistical software package SPSS version 17 (SPSS Inc. Chicago, Illinois, USA). The distribution of gene polymorphism frequency was not perfectly normal.
Descriptive statistics were used for the prevalence of genetic polymorphisms. Between-group differences in genotype frequencies were compared using a two-by-two contingency table and analysed by chi-square analysis with Yates' correction test. Pearson P values, crude odds ratios (OR), and Wald's 95% confidence intervals (CI) were calculated to test the associations between mutations/polymorphisms and FIS with free statistical calculators (http://www.quantitativeskills.com/sisa/statistics/two2hl p.htm). The level of statistical significance was set at p = 0.05.

Results
We analysed genetic tests for a total of 141 participants, divided into two different groups: data for 39 patients with FIS (mean age: 62.56 years; range: 44-82 years), and the results of 102 healthy subjects (mean age: 48.86 years; range: 27-71 years).
The baseline and demographic characteristics of the study participants are presented in Table 1.
The mean age at first onset FIS was significantly higher in the patient groups older than 60 years than in the age-matched control group, and the difference was statistically significant (t = 7.836; p = 0.000). Genotype frequencies were compared separately for all vascular gene variations between cohorts of patients and controls. The genotype distributions in the study and control groups are provided in Table 2.
When genotype frequencies were stratified into distribution frequencies, we observed that in most cases, higher frequencies of gene mutations were detected in the control group than in the patient group. The APOE E3/E3 polymorphism was present in only one patient, and the fibrinogen beta (-455G>A) mutation was not present in any patient, whereas the frequencies of heterozygote carriers of factor V (Leiden-G1691A; R2-H1299R), PTH, MTHFR-677, MTHFR-1298, FXIII, LTA, HPA 1, ACE, PAI-1, eNOS-T786C and eNOS-G894T were lower in patients than in control subjects.
Heterozygotes for the factor V Leiden mutation were more frequent in control healthy subjects (8.82%) than in the group of patients with FIS (3 or 7.69%).
The prevalence of heterozygote variants in PTH 20210G>A was higher among controls than among patients with FIS (6.86% vs. 2.56%, respectively).
Analysis of the study group for the MTHFR C677T polymorphism indicated that 15 (38.46%) patients were homozygous for the wild-type (CC), 21 (53.84%) were heterozygous (CT), and the remaining 3 (7.69%) were homozygous for the mutant allele (TT). In the control group of 102 analyzed subjects, the following results were obtained: 27 persons (26.47%) were homozygous for the wild-type (CC), 55 individuals (53.92%) were heterozygous for the polymorphism (CT), and 20 individuals (19.61%) were homozygous for the variant type (TT).
The prevalence of wild-type homozygotes and heterozygotes for the FXIII V34L Leu34 polymorphism differed significantly between patients with acute FIS and their respective controls.
In the EPCR gene, we observed a significant difference in the distribution of the CC and CG/GG genotype groups between the control and patient groups. The carriers of the EPCR 4600AG genotype were more frequent in patients with FIS (10 or 25.64%) than in control healthy subjects (20 or 19.61%). In contrast, carriers of the EPCR 4678CG genotype were more frequent in control healthy subjects (50 or 49.02%) than in patients with FIS (18 or 46.15%), except for carriers of the EPCR 4678GG genotype, which were more frequent in patients with FIS (16 or 41.03%) than in healthy subjects (24 or 23.53%). The eNOS mutation frequencies were higher in the patient group. There were no significant differences in genotype distribution between patient and control groups.

Heterozygotes
for the LTA C804A polymorphism were more frequent in patients with FIS (13 or 33.33%) than in control healthy subjects (20 or 19.61%).
HPA-1 1a/1b and ACE I/D mutation frequencies were higher in the control than in the study group, but the polymorphism frequency was not significantly different between the study and control groups.
No homozygous or heterozygous mutants for APO B were detected in any of the groups (in all patients and controls, the APO-B genotype distribution was normal; these data are not shown in Table 2).

PAI-1, 4G/5G
and 5G/5G mutation frequencies were higher in the control than in the study group, but the polymorphism frequency was not significantly different between the study and control groups.
The prevalence of all investigated gene variations did not differ significantly between the studied groups (Pearson's P value > 0.05), except for the prevalence of the homozygous wild-type and heterozygous genotypes, FXIII V34L Leu34 carriers and homozygotes (GG) for the factor EPCR G4678C polymorphism. There were also no significant differences with regard to ethnicity (all participants were Albanians from the Republic of Macedonia). The frequency of ACE I/D carriers was 67.65%, that of eNOS-G894T heterozygotes was 61.54% and the MTHFR C677T CT genotype was detected in 53.84% of patients.
The most frequent ACE genotype in patients with FIS was I/D, with an observed frequency of 67.65%; a lower frequency was found for the D/D genotype (29.41%) and the lowest frequency was found for the I/I genotype (2.94%).

Discussion
Traditionally, the greatest clinical challenge in the prevention of ischemic stroke is focused on the modification of the main risk factors for atherosclerosis, including symptomatic treatment of hypertension, diabetes, hypercholesterolemia and smoking [10][11][12].
For the first time in the Republic of http://www.mjms.mk/ http://www.id-press.eu/mjms/ Macedonia, the determination of a heritable component to the risk of acute FIS was carried out in unrelated cohorts from an Albanian population living in the Republic of Macedonia. We performed a genetic analysis of patients presenting with ischemic stroke that clinically was their first stroke episode and community controls who were subsequently included in a national project [28][29][30]. The purpose of this study was to investigate the role of specific genes and to obtain data that would support an association of gene polymorphisms regarding the aetiology of ischemic stroke.
Excluding FXIII Leu34 carriers and homozygotes (GG) for the factor EPCR G4678C polymorphism, these preliminary results of genetic analysis in the present study did not find any significant positive (susceptible) association between the investigated gene polymorphisms and FIS (Pearson's P value greater than 0.05) in this cohort of Albanians from the Republic of Macedonia. These results are of interest because they differ from those of other studies.
Many of the traditional risk factors that increase the risk of ischemic strokes, such as hypertension, atrial fibrillation, diabetes mellitus and cigarette smoking, are well known and are modifiable or avoidable [37]. Genetic risk factors are often considered not to be modifiable; however, knowledge of genetic risk factors can provide insights into pathophysiological pathways and targets for drug therapy [10][11][12]. In the general population and in patient cohorts, risk factor profiles change with increasing age [38,39]. Ischemic stroke is the most common cerebrovascular disease, and it is often attributable to the inherited predisposition, especially when apparent at a relatively early age. Approximately 10% of ischemic strokes occur at ages under 45 years [40]. Ischemic stroke is more frequent in women aged 20-30 years and in men older than 35 years [41].
Although many of our patients were middleaged or elderly (see Table 1), it is clear that in our patient group, the number of elderly participants exceeded the number of middle-aged and younger patients. However, further research would provide clear evidence of the impact of gene polymorphisms and their role in the aetiology of ischemic stroke as the combined effects of multiple susceptibility genes. Heritability seems to have a major role in younger stroke patients and patients with a family history of stroke [42][43][44][45][46][47][48].
The present study shows that the PTH G20210A polymorphism is not important in the case of ischemic stroke, similarly to Erten et al. [54].
Two meta-analyses, one from individuals of European descent [55] and another from those of non-European descent [48] report an association with a modest effect for common variants in the genes for coagulation factor V, MTHFR, PTH and ACE.
The MTHFR C677T mutation has been linked to an increased risk for ischemic medical events in a recent population study [56]. Studies of some populations with certain ethnic backgrounds from Asian countries, North America and Europe [57][58][59][60] and from the Eastern Mediterranean region found that the prevalence of the MTHFR C677T polymorphism in its homozygous state is variable [61][62][63].
An elevated total homocysteine (tHcy) level in plasma (hyperhomocysteinemia) is the consequence of decreased activity of MTHFR and is associated with a slight increase in the risk of thrombosis, whereas the common C677T polymorphism in MTHFR has been associated with an increased risk of the development of different cardiovascular diseases [64], including ischemic stroke.
The population frequencies of MTHFR C677T homozygotes vary in the United States from 44.6% in Caucasians to 42% of Hispanics and 25.6% African-Americans [65]. The frequency of the MTHFR C677T genotype is often reported to be high in European, Asian and Central and South American (10%-32%) populations and low in different African populations (0-3%) and also to show geographical gradients among Chinese Han populations [66]. The frequency of the MTHFR C677T genotype is variable in different geographic and ethnic groups: 28%-32.8% in Indian [67][68][69] and 12.3% in Chinese populations [70].
The prevalence of a homozygous gene mutation in MTHFR C677T was high in Turkish stroke patients [71]. In a study in Tamilians [72] the CT genotype was seen in 18.1% and the TT genotype in 1.38%, whereas the MTHFR A1298C polymorphism was more prevalent (47.2%) in a Hungarian study [73].
Previously published studies failed to find an association between MTHFR (MTHFR-677 and MTHFR-1298) genotypes and haplotypes and plasma homocysteine levels in patients with occlusive artery disease and deep venous thrombosis [74] or between MTHFR (MTHFR-677 and MTHFR-1298) [75] and PAI-1 polymorphisms [76] and occlusive artery disease or deep venous thrombosis in Macedonians.
Previous studies in Albanians with ischemic stroke also found that elevated plasma homocysteine levels (hyperhomocysteinaemia) were associated with the presence of MTHFR (MTHFR-677 and MTHFR-1298) gene polymorphisms [28,30]. In addition, meat with a high fat content and hyperhomocysteinemia are not the only causes of acute FIS in the Republic of Macedonia [77]. Moreover, MTHFR-677 and MTHFR-1298 gene polymorphisms were not associated with occurrence rates of FIS [28,30], but there is no data on the Albanian population regarding the PAI-1 gene and other susceptibility genes that are involved in the development and progression of ischemic stroke and their possible association with ischemic stroke. Pezzini et al. [78] showed that the risk of ischemic stroke increased 3-fold with the presence of two or more mutations. Co-occurrence of homozygous MTHFR TT and ACE D/D genotypes yielded a highly significant moderate risk of leukoaraiosis (ischemic white matter demyelination). A synergistic effect of the MTHFR TT and ACE D/D genotypes and drinking or smoking has been found [79].
Our results demonstrate that the Factor XIII Val34Leu polymorphism influences the occurrence of acute FIS, in agreement with one study [12] and in contrast to another [13]. In the recent study was concluded that significant association of Factor V Leiden (G1691A), Factor R2 (A4070G), and Prothrombin (G20210A) genetic polymorphism with occlusive artery disease or deep venous thrombosis in Macedonians was not found [80].
We observed a difference in the frequency of the ACE I/I, I/D and D/D polymorphisms between stroke patients and controls, confirming that homozygosity for the ACE D/D polymorphism does not confer a higher risk of ischemic stroke [54] than the I/D and I/I genotypes. Conversely, other studies reported that homozygosity for the ACE D/D polymorphism is a strong risk factor for ischemic stroke [24,25]. Several studies have reported that the HPA-1b polymorphism is associated with ischemic stroke [22,23]; however, GP 1a (HPA5) is correlated weakly with ischemic stroke [43]. In the present study, the frequencies of the 1a/1a, 1b/b and 1a/1b genotypes differed between stroke patients and controls, indicating that the 1b/b genotype is not a risk factor for ischemic stroke.
Results of the present study demonstrated that the frequency of the APOE, ɛ2/ɛ3/ɛ43 polymorphisms were not significantly different between stroke patients and controls and support the notion that APOE, E3/E3 polymorphisms are not associated with ischemic stroke [20,54]. Excluding FXIII Leu34 carriers and homozygotes (GG) for the factor EPCR G4678C polymorphism, the preliminary results of genetic analysis in the present study did not find any significant positive (susceptible) association between the investigated gene polymorphisms and FIS (Pearson's P value > 0.05) in this cohort of Albanians from Macedonia.
The small number of participants in each group limited our power of analysis. Consequently, the genotype distribution of the investigated singlesusceptibility polymorphisms was under-represented or not present, particularly in the patient group. On the other hand, the very weak statistical findings may suggest that if we had a large number in each group, we could find much stronger associations and differences that we missed in this small study population. However, in the control group, the single susceptibility polymorphisms were present, which means that differences between the tested groups are probably due to ethnic differences rather than recruitment bias. There are possibilities that some negative findings might be a consequence of low statistical power. Such results have previously been reported in studies with certain patient groups that were also without genotype distribution of the investigated single-susceptibility polymorphisms. Larger samples may be needed in order to resolve these limitations, validate our results and confirm our conclusions, which are our next goals. In this casecontrol cohort with FIS, in which many of our patients were middle-aged or elderly, the single susceptibility polymorphisms with FIS had the lowest prevalence. These data emphasise the need for primary and secondary prevention measures in middle-aged and elderly populations targeting modifiable lifestyle vascular risk factors.
Although previous studies have identified susceptibility genes that are involved in the development and progression of ischemic stroke [6,41,48,55,78], the present study showed that the examination of individual variants of FXIII Leu34 and homozygotes (GG) for the factor EPCR G4678C polymorphism could affect the occurrence of ischemic stroke. Other analysed single gene polymorphisms in this study do not represent risk factors in the aetiology of ischemic stroke in patients. However, some results indicate that the ACE I/D, eNOS-G894T heterozygous and MTHFR C677T CT genotype combination may result in a significantly higher risk of FIS in this Albanian population from Macedonia.
This was the first comprehensive study in an Albanian population from the Republic of Macedonia to indicate polymorphic variation in the prevalence of acute FIS. Mutations in these genes in the percentage of patients who have acute FIS have been observed, suggesting that their combination might act in a synergistic and cumulative manner in this Albanian population, the study of which is our next goal and should be tested in groups of different ethnic origin.
In summary, the results of mutant genotype frequency analysis in a sample of Albanians from the Tetovo region can be used for characterization of the current genetic profile of Albanians, anthropological comparisons, and association studies with different diseases.