Calcitonin related polypeptide alpha gene polymorphisms according to plasma total homocysteine levels in ischemic stroke patients of Trakya Region

ABSTRACT The aim of this study was to determine the genotype distributions of calcitonin related polypeptide alpha (CALCA) gene polymorphisms according to the plasma total homocysteine levels in ischemic stroke patients and patient subtypes selected from Trakya Region. The study included 82 patients and 92 healthy controls. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to determine the genotype distributions of CALCA gene polymorphisms. The plasma total homocysteine levels were measured by Immulite 2000XPi homocysteine kits. Significant differences were not found between the group of patients and the control group in terms of CALCA gene polymorphisms genotype distributions (p > 0.05). Significant differences were not found between ischemic stroke patients and healthy controls, in the patient subtypes with ischemic stroke in respect to the CALCA gene polymorphisms genotype distributions according to the plasma total homocysteine levels (p > 0.05). This suggests that the CALCA gene polymorphisms genotype distributions studied according to the plasma total homocysteine levels could not likely be considered a genetic risk factor for ischemic stroke development.


Introduction
Cerebrovascular diseases (CVD) are defined as sudden onset of local or global neurologic symptoms due to ischemia or hemorrhage in cerebral tissues. Ischemia of the cerebral tissue is mainly caused by diseases of the large or small vessels, but also by embolic material arising from the heart (cardioembolism). These three subtypes of cerebral ischemia have different mechanisms of pathogenesis.
The underlying mechanism of pathogenesis in the large cerebral artery is mainly due to the atherothrombosis of the carotid, vertebral and proximal cerebral arteries. On the other hand, the pathology in the small vessel disease is the hypohyalinosis of the vessel wall in distal penetrating branches of arteries, which causes lacunar infarction in the brain. The third subtype cardioembolism emerges from an embolic thrombus arising from a diseased heart valve or endometrium or arrhythmia. All three pathogenic mechanisms ultimately result in a decrease in the cerebral blood flow or a drop below certain values of oxygen and glucose that are required by the brain, leading to cell damage [1][2][3].
Trial of Org 10 172 in Acute Stroke Treatment (TOAST) [2,4,5] additionally described more ischemic stroke subtypes: cryptogenic ischemic stroke and other unclassified stroke. The cryptogenic ischemic stroke subtype is associated with hematologic disorders that cause hypercoagulability conditions. Primary, secondary central nervous system vasculitis, rare small vessel diseases, mitochondrial diseases, trauma and dissection, hypercoagulability conditions and hematologic disorders are found in this subtype. The category of other unclassified ischemic stroke represents 5% of all ischemic strokes and includes patients that have the characteristics of the first three subtypes simultaneously [2,4].
Calcitonin gene-related peptide (CGRP) plays an important role in the dilatation of cerebral arteries in humans and is a key member of the calcitonin peptide family. CGRP and its receptors are effective in central and peripheral nervous system disorders. There are two isoforms of CGRP, calcitonin related polypeptide alpha (CALCA) and beta (CALCB) [6,7]. The human CALCA gene is located on chromosome 11 (11p15.2-p15.1) and it encodes calcitonin and CGRP. The CALCA gene consists of 1 promoter and 6 exons and performs its function via CGRP receptors. Many polymorphisms are observed in the CALCA gene and these polymorphisms may be a genetic risk factor for CVD, such as ischemic stroke, in many reports [6][7][8][9][10].
The most common polymorphism of the CALCA gene is the CALCA T692C gene polymorphism. In addition to the CALCA T692C gene polymorphism, there are also CALCA -1786T > C, -624 (T / C) and -1752C > G polymorphisms. The CALCA T692C gene polymorphism is a single nucleotide polymorphism and it is characterized by a T/C base transition in position 692 of the CALCA gene. There are three genotypes of the CALCA T692C gene polymorphism: 692TT homozygote, 692CT heterozygote and 692CC homozygote. The CALCA -1786T>C gene polymorphism is another single-nucleotide gene polymorphism that belongs to the CALCA gene. This polymorphism is located in the promoter region and arises as a result of a T/C base exchange in position -1786. TT, CT and CC genotypes are observed in the CALCA -1786T > C gene polymorphism. The CALCA 624 (T/C) gene polymorphism occurs as a consequence of a T/C base transition in position -624 of the CALCA gene promoter region. The genotypes observed in the CALCA -624 (T/C) gene polymorphism are TT, CT and CC [6][7][8][9][10]. The CALCA gene polymorphisms are associated with several diseases such as Parkinson disease, ovarian cancer, bone mineral density, migraine, schizophrenia and essential hypertension [6,8]. However, in many studies, there has been reported no relationship between CALCA gene polymorphisms and ischemic stroke [6,8].
Homocysteine is an amino acid containing sulphur and occurs during methionine metabolism. It plays an important role in the metabolic pathways of thiol compounds. The plasma total homocysteine levels are subdivided into four groups. Values of 5-15 mmol/L are considered normal; values of 15-30 mmol/L, moderate; values of 30-100 mmol/L, high and values above 100 mmol/L, severely increased. The plasma total homocysteine levels increase depending on the age and may be higher in men, than in women. The total plasma concentration decreases in women due to estrogen [11].
Hyperhomocysteinemia (values above 15 mmol/L) is an important risk factor for ischemic stroke. Metabolic, chronic diseases, vitamin deficiency and genetic nutritional disorders, personal characteristics and some drugs may cause hyperhomocystenia [12,13]. The aim of this study was to determine the genotype distributions of CALCA gene polymorphisms according to the plasma total homocysteine levels in ischemic stroke patients and patient subtypes in the population of Trakya Region.

Study cohort
This study included 82 ischemic stroke patients (36 women and 46 men) and 92 healthy controls (57 women and 35 men). The mean age of the patients with ischemic stroke and the healthy controls was 66.70 § 13.08 and 55.37 § 14.75 years, respectively. In the group of patients, the mean age among women and men was respectively 68.50 § 12.44 and 58.98 § 11.91. In the healthy control group, the mean age of women and men was respectively 65.28 § 13.53 and 49.49 § 17.05. The patients were separated into five stroke subtypes as defined in the TOAST classification [2,4,5]. The patients and the control group were selected from Edirne (Turkey) and the surrounding areas. Our group of patients included adult patients, over 18 years old (inclusive of over 65 years), diagnosed with ischemic stroke. The patients younger than 18 years and those who have a history of central nervous system diseases, malignancy or coronary ischemia were excluded from this study.
Informed consent forms were obtained from all patients and control subjects included in this study. This study was carried out following the approval of the Trakya University Medical Faculty Non-Invasive Clinical Researchers Local Ethics Committee.

DNA isolation
Genomic DNA from the peripheral blood of the subjects was isolated in ethylenediaminetetraacetic acid (EDTA). Invitrogen DNA blood kits were used for DNA isolation from patients with ischemic stroke and healthy control subjects. The purity and quality for isolated DNA samples were determined with a Nanodrop spectrophotometer (Allsheng Nano-200, Hangzhou, China). The DNA samples were also checked by 0.8% agarose gel electrophoresis (Minicell Primo EC 320, Cleaver Scientific for electrophoresis tank and EC-105, Cleaver Scientific MP-300 V for power source) [Online supplementary Figure  1S
The PCR conditions for the CALCA T692C gene polymorphism were 5 min for denaturation at 94 C, followed by 38 cycles of denaturation for 50 s at 94 C, annealing for 50 s at 57 C and extension for 1 min at 72 C, followed by 10 min of termination at 72 C. The PCR conditions for the CALCA -1786T > C gene polymorphism were 5 min for denaturation at 94 C, followed by 38 cycles of denaturation for 45 s at 94 C, annealing for 40 s at 62 C and extension for 45 s at 72 C, followed by 10 min of extension at 72 C. For CALCA -624 (T/C) gene polymorphism, the PCR conditions were 10 min for denaturation at 94 C, followed by 35 cycles denaturation for 45 s at 95 C, annealing for 45 s at 60 C, and extension for 45 s at 72 C, followed by 7 min of termination at 72 C. The PCR products were separated in a 2% agarose gel and ethidium bromide-stained fragments were analyzed under ultraviolet (UV) light [Online supplementary Figure 1S

RFLP analysis
For the RFLP analysis, 1x Buffer Tango, PCR reaction products, dH 2 O and 5 U restriction enzyme were used for digestion. The PCR products were digested at 37 C for 3 h with restriction enzymes. PshAI restriction enzyme (Thermo Scientific, Waltham, USA) was used to determine the CALCA T692C gene polymorphism [14,15]. The genotype distributions of CALCA -1786T>C and -624 (T/ C) gene polymorphisms were determined by using BsmAI restriction enzyme (Thermo Scientific, Waltham, USA) [10,16]. The restriction digest products were observed under UV light and the polymorphisms were determined by 2.5% agarose gel electrophoresis [Online supplementary Figure 1S  Determination of plasma total homocysteine levels The patient and control blood samples were centrifuged (Hettich EBA 21, Allegra X-22R) at 4000 g for 10 min and their plasma was separated. The plasma samples of these groups were stored at -86 C (Thermo Forma-86 Cult Freezer) until use. The plasma total homocysteine levels were measured in the Central Laboratory of Biochemistry Department. Immulite 2000XPi homocysteine kits (Siemens, Munich, Germany) are used to determine the plasma total homocysteine level measurements in patients and control subjects. Values of 5-15 mmol/L were accepted as normal. Values above 15 mmol/L were defined as hyperhomocystenemia.

Statistical analysis
Descriptive statistics was used to describe the cases' characteristics; the continuous variables were expressed as means with standard deviation ( §SD); categorical data were expressed as counts and proportions. The continuous variables were compared by independent Student's t-test or Mann-Whitney test or one-way analysis of variance (ANOVA) or Kruskal-Wallis test dependent on normal distribution and groups number. The categorical data were compared using Fisher's exact test/chisquared test, whichever appropriate. All data analyses were performed with using SPSS (Statistics Package of Social Science) v.20 (IBM) and were reported with 95% confidence intervals; p < 0.05 was considered significant.

Results and discussion
The risk factors such as hypertension, diabetes mellitus, prior cardiovascular disease, smoking, alcohol consumption, fasting blood glucose, cholesterol, trygliceride, high density lipoprotein and low density lipoprotein levels are presented in Table 1. The statistical analysis showed The CALCA T692C, -1786T>C, -624 (T/C) gene polymorphisms genotype distributions within the group of patients with ischemic stroke and the healthy control group are shown in Tables 2-4. CALCA T692C, -1786T>C and -624 (T/C) gene polymorphisms were not identified as a genetic risk factor in the ischemic stroke patients as compared to the healthy control subjects (p > 0.05).
The CALCA T692C, -1786T>C, -624 (T/C) gene polymorphisms genotype distributions according to the plasma total homocysteine levels in the group of ischemic stroke patients and the healthy control group are presented in Tables 5-7. Significant difference was not found between the patients and the control subjects for each genotype of CALCA T692C, -1786T>C, -624 (T/ C) gene polymorphisms according to the plasma total homocysteine levels (p > 0.05).
The CALCA T692C, -1786T>C, -624 (T/C) gene polymorphisms genotype distributions according to the plasma total homocysteine levels in ischemic stroke patient subtypes are presented in Tables 8-10. There was no significant difference in the genotype distributions of CALCA T692C, -1786T>C, -624 (T/C) gene polymorphisms according to the plasma total homocysteine levels for each subtype of patients (p > 0.05).       Ischemic stroke may cause death and long-term disability. It arises as a result of obstruction of the cerebral blood flow of any region in the brain and may be focal or global. The genetics of ischemic stroke is dependent on several genes and peripheral factors [13].
CGRP is the strongest vasodilatator neuropeptide. It has two isoforms, CALCA and CALCB. CGRP plays an important role in the pathophysiology of hypertension [6,7]. Differences in the CGRP plasma concentration between healthy subjects and hypertensive patients are not known until now. In some studies, the plasma CGRP concentrations in normotensive controls, compared with hypertensive patients and preeclamptic pregnant women were found to be significantly low [14]. In other studies, there has been observed a significant positive correlation between elevated plasma CGRP levels and systolic and diastolic blood pressure in patients with hypertension [9]. CGRP has been associated with essential hypertension in Japanese and Chinese populations [14].
CALCA is a neuropeptide and includes 37 amino acids. The polymorphisms of the CALCA gene have been found to be associated with many diseases, such as ischemic stroke, Parkinson disease, ovarian cancer and essential hypertension [14]. However, there are not many studies investigating the relationship between the CALCA gene polymorphisms and ischemic stroke [9,15].
In a study performed by Guldiken et al. [15] in Medical Faculty of Trakya University, the relationship between the CALCA T692C gene polymorphism and migraine was studied. The study included 134 women with migraine and 96 healthy subjects. As a result, the CALCA T692C gene polymorphism was not identified as a genetic risk factor in the development of migraine [15]. The study carried out by Wedemeyer et al. [10] in Germany, showed no significant relationship between the CALCA-1786T> C gene polymorphism and aseptic loose total hip arthroplasty. Sutherland et al. [6] found no association between the genotype distributions of the CALCA -624 (T/C) gene polymorphism and migraine. On the other hand, a study of ovarian cancer found that women with any CALCA C allele were at a significantly higher risk of ovarian cancer than women with the TT genotype [16]. A clear positive correlation was found between the CT genotype of the CALCA -624 (T/C) gene polymorphism and ovarian cancer among Japanese subjects [16].
Homocysteine is an amino acid containing sulphur. It is known that hyperhomocystenemia is a risk factor for ischemic stroke [17][18][19][20]. Relationship was found between various levels of serum hyperhomocysteinemia (100 mmol/L) and cerebral thromboembolism. Moderate hyperhomocystenemia (20-100 mmol/L) may be a vascular risk factor, as it may cause obstructive vascular diseases, cancer and cerebral diseases [11]. For example, hyperhomocysteinemia has been reported in 42% of cerebrovascular diseases [11]. A significant relationship has been found between elevated homocysteine levels and cerebral infarction in the same study [11]. In addition, the plasma total homocysteine levels were found to be associated with vascular diseases [17]. Elevated homocysteine levels create conditions that stimulate endothelium dysfunction [18]. Thus, elevated homocysteine levels predispose to ischemic and hemorrhagic strokes [18]. Hyperhomocysteinemia is especially known as a risk factor for cerebrovascular diseases and disorders such as peripheral vascular and coronarythrombosis [17][18][19]. Table 9. CALCA -1786T>C gene polymorphisms genotype distributions according to plasma total homocysteine levels (mmol/L) in the subtypes of patients  In a meta-analysis, an association was found between elevated homocysteine levels and the risk of atherosclerotic vascular disease [20]. In coronary artery patients, when the plasma total homocysteine levels were higher than 15mmol/L, the coronary artery risk was found to be 2.1-2.4-fold higher [20]. Premature infarction and coronary artery disease have been revealed to cause an increase in homocysteine levels in a cohort of Turkish subjects [20]. Elevated homocysteine levels have also been identified as a risk factor for atherosclerotic vascular diseases, arterial and venous thromboembolism and cerebral and peripheral arterial diseases [20]. In the same study, elevated plasma total homocysteine levels have been found in hypertensive patients or patients with diabetes mellitus stroke as well as high plasma total homocysteine levels in stroke patients without hypertension or diabetes mellitus [20]. Thus, the plasma total homocysteine level has been suggested to be a risk factor independent of hypertension or diabetes mellitus [20]. On the other hand, in our study, the CALCA gene polymorphisms genotype distributions according to plasma total homocysteine levels were not identified as a risk factor independent of hypertension or diabetes mellitus.
There are different results in relation to high plasma total homocysteine levels and stroke subtypes [21]. The plasma total homocysteine levels have been reported to be significantly higher in large vessel disease and small vessel disease than in other subtypes among ischemic stroke patients [21]. In the study performed by Ashjazadeh et al. [22] the in Iranian province of Fars, elevated plasma total homocysteine levels were determined to be an independent risk factor for ischemic stroke and a significant relationship was observed between increased plasma total homocysteine levels and cardioembolic stroke risk. In a meta-analyses study [23] and a study in China [24], elevated plasma total homocysteine levels were found to be associated with ischemic stroke patients and patient subtypes. Lindgren et al. [25] did not find a significant correlation between the plasma total homocysteine levels and ischemic stroke subtypes. Hyperhomocystenemia has been reported as an independent risk factor for ischemic stroke in young Asian adults. The association of increased homocysteine levels with large vessel strokes suggests that hyperhomosystenemia may increase stroke risk [26]. No significant difference was found between major vessel infarction and small vessel infarction subtypes in the Malaysian [27] and Asian population [27].
The plasma total homocysteine levels were found to be significantly high in the cases of large and small vessels disease compared to cardioembolism and hemorrhagic subtypes [21]. Many studies have indicated a correlation between elevated plasma total homocysteine and atherosclerotic vascular disease [21]. The homocysteine levels were not found to be significantly correlated with stroke [21]. The elevated plasma total homocysteine levels have been reported as an independent risk factor for vascular diseases including ischemic cerebral stroke [21]. On the other hand, a strong relationship was found between elevated plasma total homocysteine levels and ischemic stroke in the study carried out by Tan et al. [26]. In another study, different results were found in Turkish and Malay populations with ischemic stroke [27].
Biswas et al. [28] found a significant association between elevated total plasma homocysteine levels and ischemic stroke in 120 Indian patients. Omrani et al. [29] suggested increased plasma total homocysteine levels as a risk factor for acute ischemic stroke in 93 Iranian patients. The authors did not observe a relationship between increased plasma total homocysteine levels and ischemic stroke subtypes, but there was a significant relationship between elevated plasma total homocysteine levels and smoking in their cohort of patients.
Although there are limited studies related to CALCA gene polymorphisms or plasma total homocysteine levels in patients with ischemic stroke, so far there is no study in terms of correlation between CALCA gene polymorphisms and plasma total homocysteine levels in patients and patient subtypes with ischemic stroke. The results obtained in the present study suggest that the CALCA gene polymorphisms genotype distributions studied here according to the plasma total homocysteine levels are not likely to be considered a genetic risk factor for development of ischemic stroke.

Conclusions
In this study, we did not observe significant differences between the patients with ischemic stroke and the healthy control group in terms of the CALCA gene polymorphisms genotype distributions according to the plasma total homocysteine levels. Significant differences were not found in the subtypes of patients with ischemic stroke either. The results from this study suggest that the CALCA T692C, -1786T>C and -624 (T/C) gene polymorphisms according to the plasma total homocysteine levels could not likely be identified as a genetic risk factor for ischemic stroke patients and their subtypes selected from Trakya Region.

Conflict of interest
We declare that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.