Screening for Genetic Variants Associated with Cardiovascular Diseases in Patients with Type 2 Diabetes Mellitus

Diabetes mellitus is associated with a wide range of cardiovascular diseases that comprise the largest cause of both morbidity and mortality for the diabetic patients. Our objective was to study the allelic and genotypic frequencies of genetic variants that have shown a strong association with cardiovascular disease in diabetic patients with and without cardiovascular complications and to assess the additional contribution of genetic variation in determining the risk for such complications. We have used cardiovascular disease StripAssay kit (Vienna Lab) based on polymerase chain reaction and reverse hybridization. The following mutations were studied: FV G1691A (Leiden), FV H1299R (R2), Prothrombin G20210A, Factor XIII V34L, β-Fibrinogen 455 G-A, PAI-1 4G/5G, GPIIIa L33P (HPA-1), MTHFR MTHFR A1298C, ACE I/D, Apo B R3500Q, Apo E2/E3/E4. 36 diabetic patients divided in 2 groups were analyzed: 1) 20 diabetic patients with cardiovascular disease and 2) 16 diabetic patients without cardiovascular disease.


Introduction
Diabetes mellitus (DM) is a very serious health issue that has reached extremely high levels worldwide nowadays. Almost half a billion people are living with the disease. Findings of the current 9 th edition of the International Diabetes Federation (IDF) atlas state that DM is one of the diseases that grows very fast all around the world. It is found that in 2019 463 million people have diabetes and this number is expected to reach 578 million by 2030, and 700 million by 2045. Unfortunately, three out of four patients are of working age and over 4 million people of the age 20-79 years are about to die from diabetes-Page 3/16 related causes in 2019. There is a 15% increase in the people with diabetes in Europe -they are 59 million in 2019 and their number is expected to rise to 66 million in 2030 and to 68 million in 2040, respectively. Type 2 DM accounts for the vast majority -around 90% -of the disease worldwide (1).
It is well known that the long-term complications of diabetes can be present at diagnosis in people with type 2 diabetes. The disease is associated with a wide range of cardiovascular disease (CVD) that comprise the largest cause of both morbidity and mortality for the patients (2). The prevalence of coronary artery disease (CAD) is found to be around 21% and that of any CVD is around 32% in adults with diabetes (3). The morbidity from CVD in diabetic patients is 2 to 4 fold higher in comparison to people without diabetes. Patients with DM without myocardial infarction (MI) have exactly the same risk for CAD as people that have already have MI (4). The most common types of CVD that are found in patients with diabetes are arterial hypertension, coronary heart disease, cerebrovascular disease, peripheral artery disease as well as congestive heart failure. As a whole, CVD contribute for between onethird and one-half of all deaths.
Metabolic syndrome is a speci c set of symptoms, which plays an important role in cardiovascular morbidity and mortality. It is a progressive phenotype which is characterized by insulin resistance, abdominal obesity, hypertension, dyslipidemia or type 2 DM. As it is known, atherosclerosis is a chronic in ammatory and lipid-depository disease that can eventually lead to different cardiovascular events.
Subclinical in ammation is observed in type 2 DM, obesity, insulin resistance. It is characterized by overexpression of cytokines which are produced by adipocytes, activated macrophages and other cells.
In ammatory mediators like plasminogen activator inhibitor -1 (PAI-1), C-reactive protein (CRP), brinogen and others take part in signal pathways, in insulin action and in amplifying the in ammatory response. These cytokines are connected also with chronic in ammatory processes which cause lipid accumulation and development of atherosclerosis and CVD. Atherosclerosis is a complex multifactorial disease and the accelerating of the atherosclerotic process in DM may be explained by hyperglycemia, oxidative stress, accumulation of advanced glycation end products (AGEs), dyslipidemia, hyperinsulinemia, overexpression of in ammatory markers and genetic variabilities (5).
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10methylenetetrahydrofolate in 5-methyltetrahydrofolate. MTHFR plays role in the metabolism of folate and in the homeostasis of homocysteine. Frequent C677T polymorphism in MTHFR is connected with high risk of development of CVD, DM. The mutation leads to hyperhomocysteinemia -a risk factor for atherosclerosis (6). On the other hand, it is found that a number of common polymorphisms and mutations in the genes coding for Factor V Leiden (FVL) and MTHFR can contribute to deep vein thrombosis -a condition that can be associated with hypercoagulability, which can be genetic or acquired. A study determines the prevalence of FVL, MTHFR C677T and MTHFR A1298C gene polymorphisms in patients with the disease. The results show that MTHFR A1298C gene was found in 77% among cases, followed by MTHFR C677T (67%) and FVL (17%) (7).
Renin-angiotensin-aldosterone system (RAAS) regulates the blood volume and pressure. It also has a role in the pathogenesis of atherosclerosis and can take part in the development of arterial hypertension, insulin resistance, DM, obesity, vascular and systemic in ammation. Angiotensin II activates intracellular signal pathways which cause atherothrombosis through in ammation, endothelial dysfunction, impaired brinolysis. Genetic polymorphism of RAAS genes including of angiotensin converting enzyme (ACE), angiotensin II type I receptor, angiotensinogen take part in atherosclerosis pathogenesis (8). The DD genotype of ACE is known to be connected to higher serum activity of ACE as well as the risk of left ventricular hypertrophy, arterial hypertonia and CAD occurrence (9,10).
Plasma lipoproteins are made of hydrophobic core that consists of triglycerides and cholesterol esters, and of super cial monolayer of phospholipids, unesteri ed cholesterol and apolipoprotens. Increased levels of apolipoprotein B (apoB) -containing lipoproteins like LDL and chylomicron remnants cause atherosclerosis. Chylomicrons which contain apoB 48 are secreted in guts after meal while VLDL which have apoB 100 come from the liver. The metabolism of chylomicron remnants and VLDL in liver is facilitated by apolipoprotein E (apoE). ApoB 100 is responsible for LDL uptake in liver (11). Genetic defect of apoB 100 causes increased level of LDL which accumulates in plasma and leads to hypercholesterolemia and premature atherosclerosis. On the other hand, patients which lack apoE accumulate lipoprotein remnants. Lipoprotein remnants with apoE stimulate accumulation of cholesterol esters in macrophages. In lesions most of apoE molecules are synthesized locally by macrophages (12).
Different studies evaluate hemostatic gene variants and atherothrombotic and cardiovascular complications. Diabetic patients are affected by abnormalities of the coagulation cascade and are predisposed to thrombotic events because of metabolic changes and acquired or inherited coagulation defects (13). Factor V (FV) Leiden is a procoagulant mutation that is associated with venous and arterial thrombosis as well as pregnancy complications. The relationship between the factor V Leiden mutation and atherosclerosis is a matter of debate due to con icting data. A study found a relevant increase in the prevalence of diabetes between patients with venous thromboembolism carriers of FVL compared to noncarriers of FVL although this was not statistically signi cant (14). Persistent hyperglycaemia in diabetes mellitus causes coagulopathies due to glycation of haemoglobin, prothrombin, brinogen and other proteins that are involved in the clotting pathway. Shortened activated partial thromboplastin time (aPTT) and prothrombin time (PT) re ect hypercoagulable state, which is associated with an increased thrombotic risk and different CVD (15). Plasminogen activator inhibitor-1 (PAI-1) also known as endothelial plasminogen activator inhibitor or serpin E1 is a serine protease inhibitor (serpin) that functions as the main inhibitor of tissue plasminogen activator (tPA) and urokinase (uPA), the activators of plasminogen and the process of brinolysis. Elevated PAI-1 is an important risk factor for thrombosis and atherosclerosis (16). Circulating PAI-1 levels are found to be elevated in patients with CAD. There are studies that show that insulin resistance may be a regulator of PAI-1 expression. The production of PAI-1 by adipose could be an important contributor to the elevated plasma PAI-1 levels that are seen in patients with insulin resistance (17). Patients with metabolic syndrome typically present with signi cantly higher levels of PAI-1 (18). Prospective studies of patients with MI or CAD have showed the association between increased plasma PAI-1 levels and the risk of coronary events (17). A recent metaanalysis has also proved that PAI-1 polymorphism (4G/5G) is associated with MI (19). PAI -1 is linked to RAAS too, which is an important contributor to vascular disease initiation and progression (20). Small drug molecules have been developed for PAI-inhibition. Tiplaxtinin, (PAI-039), and piperazine-chemotype molecules have been studied (21). Small molecules anti-PAI-1 that are orally bioavailable as TM5001, TM5007, TM5275, were tested in animal models, with some in vitro good results, but unfortunately they did not achieve enough data to be used (22). Factor XIII or brin stabilizing factor that is activated by thrombin to factor XIIIa. XIIIa is an enzyme of the blood coagulation system that crosslinks brin. De ciency of XIII increases bleeding tendency (23). Human platelet antigens (HPA) are polymorphisms in platelet antigens. Platelets play a very important role in acute arterial occlusion and platelet hyperactivity may contribute to an increased risk for CVD. Platelets attach to subendothelial structures by speci c receptors such as the collagen receptor glycoprotein (GP) Ia/IIa, or the primary von Willebrand factor (vWF) receptor GPIb/IX. After that they become activated and aggregate by cross-linking via the brinogen receptor GPIIb/IIIa (24). There is data that GPIIIa (HPA-1) may play a protective role in CVD. Iniesta et al. found that the platelet GP IIIa Polymorphism HPA-1 protects against subarachnoid hemorrhage and that the suggested platelet hyper-reactivity that is determined by this allele could reduce the risk to suffer that disease (25).
Fibrinogen (factor I) is a glycoprotein complex that is produced by the liver. In case of tissue and vascular injury it is converted by thrombin to brin and then to a brin-based blood clot which acts to occlude blood vessels and stop bleeding. Fibrinogen is a "positive" acute-phase protein and its blood levels rise in response to certain conditions like systemic in ammation or tissue injury (26). Studies have shown that high levels of brinogen are associated with CAD and may contribute to vascular disease by increasing blood viscosity thus stimulating brin formation, or increasing platelet-platelet interaction (27).
Fibrinogen is considered as being involved in thrombotic occlusion and in the nal stage of atherothrombosis. There are studies suggesting that brinogen may play a more active role in the development and progression of atherosclerotic plaque (28). On the other hand, brinogen production and plasma concentration are increased in type 2 DM. It is not known whether altered response to insulin contributes to hyper brinogenemia in diabetic patients. Fibrinogen production is acutely increased by insulin when euglycemia and euaminoacidemia are maintained in type 2 diabetic individuals but not in people without the disease. Enhanced brinogen production by insulin is supposed to be a main alteration leading to hyper brinogenemia and to cardiovascular risk in type 2 DM (29). Fibrinogen expression and deposition is also increased in obese people. The increase in brinogen expression and brin deposition leads to increased adipocyte in ammation and macrophage in ltration which suppresses glucose uptake and may lead to adipose tissue brosis. However, relationship between brinogen and insulin resistance is controversial. Free fatty acids may explain the relationship between brinogen and insulin resistance because a simultaneous increase in free fatty acids and brinogen is seen in variety of clinical and experimental condition. This relationship might also result from an in ammatory reaction that accompanies atherosclerosis (30). A common mutation -455 G/A in the promoter region of the beta-brinogen gene has been associated with elevated brinogen in plasma. showed that brinogen levels were signi cantly higher in the patients with CAD than those without. The data suggested a relationship between the -455 G/A beta-brinogen gene polymorphism and the development of CAD in DM (31). Lam et al. investigated the relation between the G/A-b-brinogen gene polymorphism and plasma brinogen concentration and its role in CAD in patients with type 2 DM and in non-diabetic control subjects. They concluded that the G/A455 polymorphism of the b-brinogen gene is a genetic determinant of plasma brinogen concentrations and CAD in their cohort (32).

Aim of the study
To investigate the allelic and genotypic frequencies of genetic variants that have shown strong association with CVD in patients with type 2 DM and the presence or absence of cardiovascular complications in order to estimate the additional contribution of the genetic variations in determining the risk of such complications.

Materials And Methods
We collected probes from peripheral venous blood of patients with type 2 diabetes mellitus. They were divided in two groups according to their cardiovascular status: i) with type 2 DM and CVD, middle age 56,3±10,8, and ii) with type 2 DM without CVD, middle age 42,5±10, 8 We have used CVD StripAssay kit (Vienna Lab) based on polymerase chain reaction (PCR) and reverse hybridization. The procedure included three steps: 1. DNA isolation; 2. PCA ampli cation with biotinized primers; 3. Hybridization of ampli cated products on test strip containing speci c for the allele oligonucleotide probe immobilized on a massive of parallel bands ( gure 1). The bound biotinized sequence are found with the help of streptavidin -alkaline phosphatase and colour substrates.

Principle of the test:
In vitro ampli cation (PCR; 2 separate reactions of a probe) The ampli cated products are stored in ice or at 2-8 o C for later use.

Test with electrophoresis
Analysis of the products of ampli cation with gel electrophoresis (2,8% agar gel).
Hybridization (45 o C; water bath system)

Result interpretation
The genotype of the probe is determined with the help of Collector TM sheet.
The processed Test strip is put in one of the elds, it is leveled up to the schematic draught with the help of the red marker line (top) and the green one (bottom) and is xed with glue.
A positive reaction of the top control line shows the right function of Conjugate Solution and Color Developer. That line must always be positively coloured.
For each polymorphic position one of the following colour models must be present and the intensity of the positive lines may vary ( gure 2).
The allelic frequencies of each of the investigated genetic variants were determined and were compared to the population frequencies from genomic databases -The Genome Aggregation Database (gnomAD), 1000 Genomes Project phase 3 database, Ensembl Genome Browser.
In our cohort the number of patients studied is 36, that are 72 alleles -these are 20 patients from the rst group (40 alleles) and 16 from the second one (32 alleles). For some genetic variants the number is less due to unsuccessful analysis. Figure 4 and 5 show the results from the genotyping of 12 genetic variants in risk genes in diabetic patients with and without CVD.

Results from the genotyping of Factor V Leiden and HR2
Altogether for all patients a frequency of 5,5% is found -more than two-fold increase than the world population frequency of 1,9% and 2,9% in Europe. According to 1000 Genomes database the frequency of the heterozygotes is 2% and in our cohort we found it 11%. No connection between the mutation of FV Leiden and cardiovascular complications has been established, even in the group of the patients without CVD a higher frequency of the mutation is found -9,4%. We found also a higher than population frequency for FV H1299R (R2) -9,7% in comparison to world frequency 5,7% and 6% in Europe.
Allele/Genotype DM with CVD DM without CVD All In order to conclude about the factors contributing to congenital thrombophilia we found higher frequencies for most of them than in the world population frequency but not reaching statistical signi cance. The highest frequency is that of the PAI-1 variant in patients with DM. The frequency of Factor XIII polymorphism is lower than that in world population frequency which is in accordance to the suggested protective role of the polymorphism. When comparing the frequencies in the groups with and without CVD only the variants of PAI-1 and Fibrinogen show higher frequency in the group with CVDgure 13.

Results from the genotyping of MTHFR
The allelic frequency of MTHFR 677T we found is 25% and is a little lower than that of world population -31%, and in Europe -32%. The allelic frequency of MTHFR 1298C in our study is 38,9% and is higher than that in world -29%, and in Europe -32%. The mutation was not found in any of the patients and its world population frequency is 1:5000.

Discussion
The investigation of the allelic frequency of 12 genetic variants connected to cardiovascular risk in Bulgarian patients with type 2 DM showed more than two-fold increase in comparison to population frequency for the following alleles: -5,5% for FV (Leiden) mutation compared to 1,9% in world population and 2,9% in Europe population (no connection between FV (Leiden) mutation and cardiovascular complications has been established -in the group of patients without CVD the frequency of the mutation is higher -9,4% compared to 2,5% in the group of patients with CVD) -58,6% for PAI-1 4G in comparison to 26,9% in world population Increased frequency in comparison to world population for the following genotypes has been found: -38,9% for β-brinogen 455 G/A compared to 22% in world population -36,1% for ACE D/D compared to 25% in world population Lower frequency for Factor XIII 34L and MTHFR C677T in the investigated groups in comparison to world population has been found. It is suggested that Factor XIII 34L may have protective role in CVD development.
No statistically signi cant difference between the investigated groups with and without CVD in the allelic and genotypic frequency in 11 out of 12 studied genetic variants has been found.
A statistically signi cant higher frequency in heterozygotes for β-brinogen 455 G/A in the group of patients with DM and CVD has been found -55% in comparison to 18,7% in the group without CVD. It is supposed that the role of β-brinogen as pro-in ammatory protein along with its thrombotic effects may increase the risk for CVD in patients with DM.

Conclusion
In our study we aimed at investigating the allelic and genotypic frequencies of genetic variants that have are supposed to have strong association with CVD in patients with type 2 DM with and without cardiovascular complications in order to try to estimate the additional contribution of the genetic variations in determining the risk of such complications. We found a statistically signi cant higher frequency in heterozygotes for β-brinogen 455 G/A in the group of patients with DM and CVD which is also seen in other studies. This comes to show that brinogen is really an important contributor to the pathogenesis of CVD, especially in patients with type 2 DM.

Declarations
Ethics approval and consent to participate: The collection of patients' samples was approved by the institutional ethical committee (Medical University So a) with the approval No1209/2018. Each patient signed a written Informed consent.