Screening for Genetic Variants Suggests β-brinogen -455 G/A Genotype as a Contributor to Cardiovascular Complications in Type 2 Diabetes Mellitus


 Background

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.
Methods

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 C677T, 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.
Results

We found higher than population frequency for the following alleles/genotypes – 5.5% for FV Leiden allele, 9.7% for FVR2 allele, 38.9% for β-Fibrinogen genotype − 455G/A, 58.9% for PAI-1 4G allele, 36.1% for ACE D/D genotype. Statistically higher frequency was established for β-Fibrinogen − 455 G-A in the patients with cardiovascular disease compared to non- cardiovascular disease (55% vs. 18.7%).
Conclusions

We detected high frequency of β-Fibrinogen − 455 G/A genotype in diabetic patients, especially in these with cardiovascular disease. Based on its pro-inflammatory role and its connection to possible thrombotic events, patients would benefit from anti-inflammatory treatment.


Abstract
Background 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.

Conclusions
We detected high frequency of β-Fibrinogen − 455 G/A genotype in diabetic patients, especially in these with cardiovascular disease. Based on its pro-in ammatory role and its connection to possible thrombotic events, patients would bene t from anti-in ammatory treatment.

Background
Diabetes mellitus (DM) is a very serious health issue that has reached extremely high incidence worldwide nowadays (1). Almost half a billion people are living with the disease. Findings of the current 9th 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 millions people have diabetes and this number is expected to reach 578 millions by 2030, and 700 millions by 2045. It is well known that the long-term complications of diabetes can be present at the time of diagnosis in people with type 2 diabetes. DM is associated with a wide range of cardiovascular disease (CVD) comprising 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% in adults with diabetes, the percentage increasing to 32% when consider any CVD (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 the people already affected by MI (4). The most common types of CVD 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 to mortality of one-third to one-half of DM patients.
As it is known, atherosclerosis is a lipid-depository condition leading to different cardiovascular diseases and is associated with chronic in ammation. Subclinical in ammation is observed in type 2 DM, obesity, and metabolic syndrome with insulin resistance as well. It is characterized by overexpression of cytokines 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 signaling pathways, connected to insulin action and in ammatory response (5). A common mutation − 455 G/A in the promoter region of the beta-brinogen gene has been associated with elevated brinogen Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10methylenetetrahydrofolate in 5-methyltetrahydrofolate. It plays role in the metabolism of folate and in the regulation of homocysteine levels. Frequent C677T polymorphism in MTHFR is associated with high risk of CVD development. The mutation leads to hyperhomocysteinaemia -a risk factor for atherosclerosis (8). On the other hand, it was found that common polymorphisms and mutations in the genes encoding Factor V Leiden (FVL) and MTHFR can contribute to deep vein thrombosis -a condition caused by hypercoagulability, which can be genetic or acquired. A study determined the incidence of FVL, MTHFR C677T and MTHFR A1298C gene polymorphisms in patients with the abovementioned disease. The results detected MTHFR A1298C polymorphism in 77% of cases, followed by polymorphisms MTHFR C677T (67%) and FVL (17%) (9).
Renin-angiotensin-aldosterone system (RAAS) regulates the blood volume and pressure and take part in the development of arterial hypertension. It also has a role in the pathogenesis of atherosclerosis, vascular and systemic in ammation, as well as in insulin resistance, DM and obesity. Genetic polymorphism of the gene for angiotensin converting enzyme (ACE), belonging to RAAS, has a role in atherosclerosis pathogenesis (10). The DD genotype of ACE is known to be connected to higher serum activity of ACE as well as to high risk of left ventricular hypertrophy, arterial hypertension and CAD (11,12). Increased levels of apolipoprotein B (apoB) -containing lipoproteins like LDL and chylomicron remnants cause atherosclerosis as well (13). 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 apolipoprotein E (apoE) accumulate lipoprotein remnants, whereas these with apoE stimulate accumulation of cholesterol esters in macrophages (14).
The aim of our study was to investigate the allelic and genotypic frequencies of variants in the genes 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.

Patients' selection
We conducted a cross-sectional study with patients from the Department of Endocrinology, Clinics of Diabetology in Medical University of So a. The patients were given an informed consent which they signed and were acquainted with the aims, conditions and risks from their participation in the study  The statistical analysis of the data was performed through SPSS v.20.0 (SPSS, Chicago, USA). The data are expressed as mean value ± standard deviation (SD). The results were tested for normality of distribution and the parametric T-test has been applied. Pearson's chi-squared test is used for proportional comparisons. P-value less than 0,05 is statistically signi cant.
Our data do not show statistically signi cant difference in sex, middle age, BMI, HbA1c, the presence of metabolic syndrome so these variables cannot in uence the results from the DNA analysis.

Molecular-genetic analysis
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 strip of parallel bands (Fig. 1). The bound biotinized sequence are detected by streptavidin -alkaline phosphatase and colour substrates.

Results
In our cohort the number of patients studied is 36, corresponding to 72 alleles for each gene -these are distributed in 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 1 and 2 show the results from the genotyping of 12 genetic variants at risk genes in diabetic patients with and without CVD, respectively.
Page 6/14 1.1 Results from the genotyping of factors for congenital thrombophilia - Table 2 Results from the genotyping of Factor V Leiden and R2 Altogether for all patients a frequency of 5,5% was found for Factor V Leiden mutation (Table 2) -it is two-fold increase than the population frequency in the world (1,9%) and in Europe (2,9%). According to 1000 Genomes database the frequency of the heterozygotes is 2% and in our cohort it was 11%. We found also a higher than population frequency for FV H1299R (R2) -9,7% in comparison to frequency of 5,7% in the world and 6% in Europe. No connection between both FV mutations and cardiovascular complications has been established. We found a frequency of 1,4% and it is comparable to the world population frequency of 0,8% and to that in Europe -1,1% ( Table 2). The mutation was found only in DM patients with CVD not reaching statistical signi cance.
Results from the genotyping of PAI -14G/5G We found higher frequency of the pathogenic allele 4G -58,6% ( Results from the genotyping of Factor XIII V34L In our cohort we found lower frequency of 11,1% for the minor allele (Table 2) compared to 21,9% world population frequency and 25,2% in Europe. It is important to note that in the group with CVD the frequency is even lower -7,5%, which suggests a protective role of this genetic variant.
Results from the genotyping of β-Fibrinogen-455 G/A The overall allelic frequency of the pathological allele in our group was 22,2% which is higher than the world population frequency -16,9%, and close to that in Europe -20,3%. According to 1000 Genomes database the population frequency of the heterozygotes is 22% and we found it 38,9% (Table 2). It increases signi cantly in the group with CVD compared to the one without CVD -55% versus 18,7%gure 3 Results from the genotyping of HPA1(GPIIIa L33P) We found allelic frequency of 12,5% (Table 2) which is comparable to the world population frequency -12,1%; and that in Europe -15,2%.
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 ( Figure 4A). The highest frequency was found for PAI-1 variant in patients with DM. The frequency of Factor XIII polymorphism was lower than that in the world 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 4B.

Results from the genotyping of MTHFRC677T and A1298C
The allelic frequency of MTHFR 677T we found was 25% (Table 3) and is a little lower than that of world population -31%, and that in Europe -32%. The allelic frequency of MTHFR 1298C in our study was 38,9% and it is higher than that in world -29%, and in Europe -32%. We found a frequency of the homozygotes for the pathologic allele of 36,1% (Table 4) which is higher than population frequency in Europe -25%.

Results from the genotyping of ApoBR3500Q and ApoE
The ApoB mutation was not found in any of the patients and its world population frequency is 1:5000.
The frequency of the risk ApoE allele E4 we found was 13,9% (Table 5) and is comparable to the world population frequency -13,8% and that in Europe -16,1%

Discussion
Different studies evaluate thrombophilia's 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 (16). FVL is a procoagulant mutation associated with venous and arterial thrombosis as well as with pregnancy complications. Persistent hyperglycaemia in diabetes mellitus causes coagulopathies due to haemoglobin glycation, prothrombin, brinogen and other proteins involved in the coagulation 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 (17). The relationship between the factor V Leiden mutation and atherosclerosis is a matter of debate due to con icting data. We found higher frequency of 5.5% for FVL in all studied diabetic patients, without correlation to cardiovascular complications. A study found a relevant increase in the prevalence of diabetes among patients with venous thromboembolism carriers of FVL compared to non-carriers of FVL although this was not statistically signi cant (18).
Our study revealed high frequency of 58.6% for the pathologic allele in the gene for Plasminogen activator inhibitor-1 (PAI-1) also known as endothelial plasminogen activator inhibitor or serpin E1 -a serine protease inhibitor (serpin) that functions as the main inhibitor of tissue plasminogen activator (tPA) and urokinase, the activators of plasminogen-related process of brinolysis. Elevated PAI-1 is an important risk factor for thrombosis and atherosclerosis (19). Circulating PAI-1 levels are found to be elevated in patients with CAD. Couple of studies showed that insulin resistance may be a regulator of PAI-1 expression. The production of PAI-1 by adipose tissue could be an important contributor to the elevated plasma PAI-1 levels that are seen in patients with insulin resistance (20). Patients with metabolic syndrome typically present with signi cantly higher levels of PAI-1 (21). Prospective studies of patients with MI or CAD have showed the association between increased plasma PAI-1 levels and the risk of coronary disease (20). A recent meta-analysis has also proved that PAI-1 polymorphism (4G/5G) is associated with MI (22). PAI − 1 is linked to RAAS too, which is an important contributor to vascular disease initiation and progression (23). Taken this data together, we assumed that diabetic carriers of PAI-1 4G polymorphism are highly predisposed to its adverse effects and targeted treatment is worthy to be investigated. Small drug molecules have been developed for PAI-inhibition -Tiplaxtinin, (PAI-039), and piperazine-chemotype molecules have been studied (24). Small anti-PAI-1 molecules have been tested in animal models, with some good results in vitro, but unfortunately they did not achieve enough data to be used (25).
One important nding from our study is the signi cantly higher frequency of β-Fibrinogen − 455 G/A heterozygotes in diabetic patients with CVD compared to non-CVD patients. Fibrinogen (factor I) is a glycoprotein 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 by 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 even in individuals with controlled type 2 diabetes but not in people without the disease. Increased brinogen production by insulin is supposed to be a main cause for hyper brinogenemia and associated cardiovascular risk in type 2 DM (29). Fibrinogen production and deposition is also increased in obese people. The increased brinogen production and brin deposition lead to increased adipocyte in ammation and macrophage in ltration which suppresses glucose uptake. However, relationship between brinogen and insulin resistance is controversial. The possible explanation is the increase in free fatty acids which has been seen in variety of clinical and experimental condition of insulin resistance. This relationship might also result from an in ammatory reaction that accompanies atherosclerosis (30). We 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.

Conclusions
In our study we aimed at investigating the allelic and genotypic frequencies of genetic variants that are supposed to have strong association with CVD, in patients with type 2 DM with and without cardiovascular complications in order to estimate the additional contribution of the genetic variations in determining the risk of such complications. We found a signi cantly higher frequency in heterozygotes for β-brinogen − 455 G/A polymorphism in the group of patients with T2DM and CVD. 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.

Consent for publication:
It is included in the text of the Informed consent signed by the patient. All participants in the study signed the Informed consent.
Availability of data and materials: All data and material are available in the Molecular Medicine Centre, Medical University So a