الفهرس 
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Abstract
Cardiovascular disease (CVD) is the leading cause of death globally, accounting for almost one-third of all deaths. In Egypt, CVDs account for 40% of all deaths. CVD is a multifactorial disorder influenced by genetics, environmental factors and risk factors. Inflammation plays a key role in the production of atherosclerosis, which increases the risk of cardiovascular disease. Hypertension, dyslipidemia, diabetes, and lifestyle factors such as nutrition (diet), exercise, smoking, and psychosocial stress are the most significant CVD risk factors.
VitD is essential for a variety of functions besides bone health, such as cardiovascular health. The VDR polymorphism has been proposed as a potential new genetic marker for cardiovascular disease, as the disease has been linked to low VitD levels. Although, several studies in recent years have attempted to elucidate the possible relationship between VitD levels, VDR gene polymorphisms, and CVD, but the findings are still controversial, moreover existence significant differences have been observed between SNPs and the ethnic groups studied, highlighting the need for further research into the proposed triangular relationships between SNPs, VitD, and CVD in different ethnic populations.
The objective of this study was to assess and correlate vitamin D level and frequency of the VDR gene polymorphisms (rs2228570 FokI and rs1544410 BsmI) among cardiovascular Egyptian patients with and without diabetes mellitus, to the clinical characteristics and laboratory finding at diagnosis of CVD. Beside, the aim was also to study the association of interleukin 6 and interleukin 10 with CVD in Egyptian Patients.
In this study, blood samples were obtained under fasting conditions from 69 adult healthy volunteers without any cardiac complains or dyspnea on exertion served as controls (36 men and 33 women; mean age 53.5 ± 6.4 years [mean ±SD], range 41-65years), and 69 previously diagnosed T2D patients without cardiovascular disease, this group do not suffer from any cardiac complain and had normal ECG and echocardiography (27men and 42 women; mean age (50.6 ± 7.2) years[mean ±SD], range 40 – 65 years), 124 CVD patients (94 men and 30 women; mean age (56.8 ± 6.5) years [mean ±SD], range 40 – 65 years) and 120 CVD patients with T2D; 52 men and 68 women, mean age (55.9± 6.9) years[mean ±SD], and range 40 – 65 years). CVD patients were verified by either a history of myocardial infarction or coronary angiography and met World Health Organization (WHO) criteria.
Patients were consecutively enrolled from Sayed Galal Hospital, Cairo, Egypt from January to May 2017. All subjects undergone the following: complete clinical examination (including measurement of weight, height, diastolic blood pressure (DBP), systolic blood pressure (SBP), 12 leads resting electrocardiogram (ECG), echocardiography, and coronary angiography and answered a short form questionnaire including family history, the presence of hypertension, diabetes, and whether they were smokers or not.
The exclusion criteria were: Patients suffer from rheumatic and congenital heart diseases, endocrinal and genetic obesity, hepatic diseases, renal diseases, chronic lung diseases, mal-absorptive disorders, cancer or had history of any psychiatric disorder. Patients have taken medications as anticonvulsants, systemic glucocorticoids, antidepressants, antipsychotics drugs or ingestion of vitamin D or multivitamin supplements were also excluded.
The investigated parameters were:
• Fasting blood glucose, fasting blood insulin, serum (TG, Chol, HDL, and LDL), serum vitamin D and serum interlukin 6 and 10.
• Genomic DNA was extracted from whole blood samples and (rs2228570 C >T FokI) and (rs1544410 A >G BsmI) gene polymorphisms were identified by (PCR-RFLP) assay.
The results revealed that:
• VitD was significantly decreased in CVD without T2D group when compared to healthy controls (P < 0.001), and was also significantly decreased in CVD with T2D group when compared to diabetic group (P < 0.001). Interestingly, CVD patients with T2D had the lowest levels of serum VitD in comparison with those without T2D with a statically difference between them (P < 0.001).
• VitD was negatively significantly correlated with BMI and IL- 6 in diabetic, CVD without T2D and with CVD with T2D groups (p ˂0.001) and (p˂0.01) respectively, with FBG in diabetic and CVD with T2D groups (p˂0.001), with insulin in CVD with and without T2D groups (p˂ 0.001), with HOMA-IR in diabetic and CVD with and without T2D groups (p˂ 0.001). With TC, TG, and LDL-C in all studied groups whereas VitD level was positively correlated with HDL-C in all studied groups and with IL-10 in diabetic, CVD without and with T2D groups (p˂0.01).
• Low circulating levels of VitD have been linked to an increased risk of CVD.
These could be explained by that VitD suppresses rennin gene expression, regulates the growth and proliferation of vascular smooth muscle cells and cardiomyocytes. Therefore, the absence of VitD receptor activation results in up-regulation of the renin angiotensin system, hypertension, endothelial dysfunction, and left ventricular hypertrophy.
• With regard to Egyptian population, the homozygous (mutant) TT and the heterozygous CT genotypes of VDR (rs2228570C > T) were significantly more frequent in CVD patients with and without diabetes compared to diabetic and control groups whereas, the homozygous (wild) CC was significantly less frequent in CVD patients with and without T2D compared to diabetic and control groups, (p˂0.01). while there was no significance difference in genotype distribution between CVD with and without diabetes groups (p=0.65), and the mutant T allele was significantly more frequently in CVD patients with and without T2D and associated with 2.5 times increased risk for CVD more than diabetic and control groups (OR =2.397 and 2.289 respectively).
While on the other hand the genotype AG within VDR( rs 1544410A > G) occurred less frequently in CVD patients with and without T2D vs controls, while GG genotype found more frequently in CVD with and without T2D patients and the A allele was less frequently in CVD patients with and without T2D while the mutant G allele was significantly more frequently in CVD patients with and without T2D and associated with 6 times in CVD more than healthy group (OR =5.745) and with 2 times in CVD with T2D more than CVD without T2D (OR= 1.679), presumably that the A allele might have a protective effect regarding CV risk while the G allele may represent increased risk. These polymorphisms might represent a new cardiac risk factor with our finding.
• Furthermore, when comparing VitD levels to VDR polymorphisms, the heterozygous CT/AG or homozygous TT/GG mutants of FokI and BsmI polymorphisms, respectively, had lower VitD levels and were significantly more prevalent in CVD patients with T2D compared to those without T2D, indicating that VitD and VDR variants significantly altered VitD signaling process leading to heart-related deformities beside that hypo-vitaminosis D may lead to beta-cell dysfunction and insulin resistance.
• Relating to clinical parameter and VDR polymorphism, higher BMI, FBG, HOMA-IR, TC, TG, LDL-C, IL-6 ,IL-10 and lower HDL-C were seen in patients with TT /GG and CT/ AG genotype than those with CC and AA genotype.
• Serum IL-6 level (proinflamatory marker) was highly significant in CVD patient with and without T2D in comparison of non-CVD subjects (P < 0.001), which confirmed by positive correlation of IL-6 with cardiovascular risk factors as HOMA-IR, TC, TG, LDL and BMI in cardiovascular with and without diabetes groups. Moreover there was a significant statistical difference in IL-6 level between CVD patients with and without T2D groups (P < 0.001), this could be explained on the basis of increased IL-6 level is associated with increased blood viscosity leading to the deposition of fibrinogen, decreases the activity of lipoprotein lipase and stimulates the hypothalamic-pituitary-adrenal axis which associated with obesity, hypertension, and insulin resistance, all of these action are strong risk factors for CVD.
• Serum IL-10 (anti- inflammatory marker) was significantly decreased in CVD patient with and without T2D, when compared to non-CVD patients, also, IL-10 was negatively correlated with cardiovascular risk factor (BMI, HOMA-IR, TC and LDL-C and by positively correlated with the cardio-protective parameter (HDL-C).
• Relating to the area under the Receiver characteristic curve of IL-6 was 0.833 indicating a good ability of IL-6 values for distinguishing cardiovascular patient from non cardiovascular patient, whereas, the area under the Receiver characteristic curve of IL-10 was 0.657 indicating a poor ability of IL-10 values for distinguishing CVD patient from non-CVD patient. Thus IL-6 may be considered as biochemical marker that can be used to predict the CV risk.
The study concludes that the level of VitD decreased in CVD patients and was extremely decreased in CVD with T2D. VitD level and the distribution VDR polymorphisms were associated with risk of CVD in Egyptian patients with or without diabetes considering that the mutant T allele of VDR (rs2228570C > T) and the mutant G allele of VDR (rs 1544410A > G) may represent increased risk of CVD. These results suggest VDR polymorphisms as potential diagnostic biomarkers for CVD susceptibility. Furthermore, both IL-6 and IL-10 were associated with CVD risk and IL-6 could be used as a prognostic marker of CVD in Egyptian patients.