Polymorphisms -455G/A and -148C/T and Fibrinogen Plasmatic Level as Risk Markers of Coronary Disease and Major Adverse Cardiovascular Events

Some polymorphisms in genes codifying for fibrinogen have been correlated with plasma levels of this protein, and several studies reported their associations with acute cardiovascular events. In the present study, 118 subjects with unstable and stable coronary diseases were enrolled to determinate the associations among fibrinogen gene polymorphisms, plasma fibrinogen levels, and major cardiovascular adverse events in a sample of southwestern Mexico. The groups, including 81 control subjects, were matched for age, sex, body mass index, and sedentarism. Plasma fibrinogen levels and the polymorphisms 455G/A, -148C/T, +1689T/G, and Bcl I of the gene of fibrinogen were compared in all groups. Plasma fibrinogen levels (>465 mg/dl) were significant in patients with coronary disease. Fibrinogen plasma values > 450 mg/dl were associated with cardiovascular mortality during the follow-up analysis of the unstable coronary disease group (p = 0.04). The allelic loads of -455A and -148T were associated with plasma fibrinogen levels > 450 mg/dl (p < 0.003 and p = 0.03, respectively) and with coronary disease (p = 0.016 and p < 0.006, respectively). The follow-up of posterior events after an acute coronary event showed that the genetic load of the -148T allele was associated with major adverse cardiovascular events (RR = 1.8, 95%CI = 1.01‐3.35, p = 0.04). Fibrinogen plasmatic levels > 450 mg/dl and the fibrinogen polymorphisms -455G/A and 148C/T had association with MACE and coronary disease. This study suggests that these gene polymorphisms are associated with cardiovascular risk.


Introduction
The role of fibrinogen gene polymorphisms and protein levels as inflammation markers of cardiovascular risk has been evaluated in several epidemiological studies excluding Latin-American populations [1][2][3][4]. In vitro studies suggest that the synthesis of the β chain of fibrinogen is the limiting factor for the production of fibrinogen. Some polymorphisms of this gene may be associated with elevated levels of plasma fibrinogen. Since studies associating fibrinogen polymorphisms and cardiovascular risk have not included Latin-American populations [5,6], we studied the associations among fibrinogen polymorphisms, plasma levels of the protein, and coronary disease, including unstable coronary disease (UCD) and stable coronary disease (SCD), and their associations with major adverse cardiovascular events (MACE) in a sample of southwestern Mexico.

Study Design.
The study design is prospective and controlled, with a six-month follow-up study. Inclusion criteria are (a) age between 35 and 75 years, (b) UCD and SCD with secondary atherothrombosis, and (c) clinical stability. Exclusion criteria are (a) age greater than 75 years; (b) UCD secondary effort stress, uncontrolled hypertension, aortic stenosis, acute or chronic pulmonary arterial hypertension, thyrotoxicosis, anemia, heart failure, excessive vasodilatation, etc.; (c) Killip class III or IV; (d) acute coronary event in the last three months; (e) ejection fraction < 35%; (f) SCD without atherothrombosis; (g) hematological, hepatic, or neoplastic disease; (h) acute or chronic inflammatory activity; and (i) excessive alcohol consumption. The control group is composed of apparently healthy subjects. All groups were matched for age, sex, body mass index, and sedentarism, and all samples were obtained under similar conditions. This study was conducted in accordance with the Declaration of Helsinki principles and was approved by the Ethics Committee of the Hospital Regional de Alta Especialidad "Ciudad Salud" in Tapachula, Mexico. All subjects signed an informed consent before entering the study.

Blood Sample and Laboratory Analysis.
Because of the exquisite sensitivity of the plasma markers in in vitro manipulations, the samples were obtained with extreme care. Once the patient signed the informed consent, two people with special training did a venous puncture with a vacuum test tube. 3 milliliters of venous blood was collected in a tube with sodium citrate (ACD) and another 10 milliliters in a tube with EDTA. The puncture was done with a 20-gauge needle in a region of the arm with no cracks, folds, or skin breaks. The sample was always obtained before starting any fibrinolytic, antithrombotic, and antiplatelet treatments or invasive procedures, including placement of intravenous lines. The anticoagulated samples with ACD were centrifuged immediately at a temperature of 4°C, and the plasma obtained was used to determine the levels of plasma fibrinogen by the von Clauss method [7]. The levels of plasma fibrinogen were divided into three groups: low (<350 mg/dl), intermediate (350 to 450 mg/dl), and high (>450 mg/dl). The anticoagulated material with EDTA was used for the isolation of DNA. Laboratory personnel were blinded to the clinical diagnosis of the subjects. PCR was carried out in volumes of 25 μl containing 100 ng of DNA, 1x reaction buffer, 1.5 μM of MgCl 2 , 0.5 μM of dNTPs, 0.5 μM of Taq polymerase, and 0.5 μM of each initiator. The alignment temperatures varied from 55 to 61°C. The amplified and digested products were analyzed in 2 and 4% agarose gels, respectively.
In order to assure the genotyping quality, the results were validated in a posterior screening in 10% of randomly selected samples and all those with a homozygotic pattern for the mutant alleles, as proposed by Bladbjerg et al. [9]. Two independent observers analyzed the genotypes, and an additional observer solved any discrepancy.    using a two-tailed Student t-test, evaluating the result by the Wilcoxon test. The discreet variables were analyzed by Chisquare using the Yates correction. A two-way analysis of variance (ANOVA) for repeated measures was performed to analyze differences in levels of fibrinogen between UCD, SCD, and healthy subjects. Logistic and multiple regression analysis models were used to assess the effect of fibrinogen plasma levels and its polymorphisms on MACE in the acute phase and in the follow-up. Survival Kaplan-Meier curves were considered, and odds ratio (OR) and relative risk (RR) with confidence intervals (CI) of 95% were also considered. Statistical significance was established for values of p < 0 05. The data were reported in percentages, averages, standard deviations, CI, and OR. All the analyses were done using a commercial statistical package (GB-STAT version 10.0 of Dynamic Microsystems Inc., Copyright 2004).

The Characteristics of Patients and Controls.
From January 2015 to December 2017, 199 subjects were included in the study. Fifty patients with UCD and 68 with SCD, respectively, were admitted to the Hospital Regional de Alta Especialidad "Ciudad Salud," for a total of 118 patients and 81 subjects in the control group. Demographic characteristics of the groups are shown in Table 1. No intergroup differences in age and gender were observed. The UCD group had more history of a previous acute coronary event, diabetes, hypertension, smoking, and lipid abnormalities. Over 50% in all groups did not have physical activity, and obesity was observed in all of them. Fibrinogen levels were significantly higher in patients with coronary disease in general with respect to controls (p = 0 001). In the six-month follow-up of UCD patients, high plasmatic fibrinogen levels correlated with cardiovascular mortality (p = 0 04) (Figure 1).

Genotype and Allele
Frequency Distribution. Table 2 shows the distribution of genotypes and alleles in the three analyzed groups. All analyzed fibrinogen gene polymorphisms were in Hardy-Weinberg equilibrium (p > 0 05). Of the four studied polymorphisms, only those located in the fibrinogen B gene promoter (455G/A and -148C/T) were different between UCD, SCD, and controls. The frequency of the genotypes -455G/A and -148C/T was significantly more represented in the patients with coronary disease (p = 0 016 and p < 0 006, respectively). Nevertheless, when the analysis focused on allele frequencies, the variants -455 and -148T were significantly more frequent in patients with UCD and SCD when compared with controls (p = 0 01 and p = 0 0005, respectively). The rest of the alleles did not show significant values for a particular clinical condition. The determinations of genetic loads of an individual allele among the possible studied genotypes demonstrated an increased OR for coronary disease. The load of the -455A allele was associated with SCD (OR = 3 35, 95%CI = 1 6-7 1, p = 0 002) and UCD (OR = 3 19, 95%CI = 1 4-7 2, p = 0 005), and an association was also observed for the load of the -148T allele with SCD (OR = 2 21, 95%CI = 1 1-4 3, p = 0 02) and UCD (OR = 3 10, 95%CI = 1 5-6 5, p = 0 003) ( Table 3). Table 4. Levels > 450 mg/dl of fibrinogen were associated with the polymorphisms -455G/A, -148C/T, +1689T/G, and Bcl-1. The genotypes -455G/A and +1689 were associated with high plasma levels of the protein (p < 0 003 and p = 0 02).

Multiple Regression Models.
A multiple regression analysis determined that the genetic load of the -455A and -148T alleles is an independent predictor of elevated levels of fibrinogen (r = 0 3, p = 0 008), after considering other risk factors such as age, sex, BMI, dyslipidemia, diabetes, smoking, and sedentarism.

Follow-Up and Major Cardiovascular Adverse Events.
The load of the T allele of the -148 variant was a predictor of MACE in the acute phase and recurrent ischemia in the one-year follow-up in the UCD group (Table 5). A logistic multiple regression analysis determined that the -148T allele is an independent predictor of in-hospital recurrent ischemia (RR = 1 8, 95%CI = 1 01-3 35, p = 0 04) and MACE at the one-year follow-up (RR = 1 9, 95%CI = 1 07-3 52, p = 0 02). Several modeling of the -455G/A, Bcl-1, Taq I, and +1689G/T polymorphisms showed poor or null correlations with plasma fibrinogen levels. The Kaplan-Meier survival analysis suggests a reduced survival rate in patients with UCD and highest plasma fibrinogen levels compared to those with the lowest levels ( Figure 1).

Discussion
These results provide reliable information about baseline fibrinogen levels and gene polymorphisms and cardiovascular events in acute phases and in the 1-year follow-up in Mexican patients. The elevated fibrinogen levels correlated with coronary disease, as has been reported [2][3][4]. This study provides important insights about genetic and inflammatory risk profiles in UCD and SCD in Mexico and probably represents the first study in Latin-America [5].
Notably, the average plasma levels of this protein in subjects with coronary disease (465 mg/dl) and in controls (416 mg/dl) are greater than those reported in other populations using the von Clauss method to measure fibrinogen [10][11][12]. Although they are within reference values [13,14], nevertheless, other evidence reports establish similar values [15,16]. In any case, the differences observed among controls, UCD, and SCD suggest phenotypic stratifications that may be associated with an increased cardiovascular risk.
Another observation was that fibrinogen levels were predictors of cardiovascular mortality during the six-month follow-up in the UCD group. The values observed in UCD subjects who died during the study (468 3 ± 185 8 mg/dl) are similar to those reported in Israeli [1], Japanese-American [3], American [17], and Spanish populations [18].
Analysis of the allelic frequencies of the studied polymorphisms shows that only the -455A and -148T alleles are associated with coronary disease in general (UCD and SCD together), whereas the study of genotypes indicates that the genotypes -455G/A and -148 C/T and T/T are associated with coronary disease. The association between coronary disease and the -455 polymorphism has been reported in the majority of the ethnic groups around the world [5,[19][20][21][22][23]; however, other studies did not find this association [4,24]. Although the evidence on the -148 variant as a cardiovascular risk marker is scarce, it has been associated with myocardial infarction in an Asian population [22] and has been considered as a predictor of carotid atherosclerosis [25]. The alleles +1689G and Bcl-1 B2 did not show associations with cardiovascular risk, as has been reported [4].
The genetic loads of the -455A and -148T alleles conferred relevant ORs for SCD and UCD, corroborating their clinical effect. These polymorphisms were associated with fibrinogen levels > 450 mg/dl, and the genotype -455G/A was the most significant (p < 0 003) followed by +1689G/T, -148C/T, and Bcl-1 (p = 0 02, p = 0 03, and p = 0 03, respectively). It is important to emphasize that these results derive  from a comparison between polymorphisms and fibrinogen levels without discriminating particular clinical conditions. The most relevant finding of this work is the almost twice greater risk of recurrent ischemia and MACE in the 1-year follow-up in UCD patients carrying the -148T allele. Our study shows that the -148T allele is highly associated with increased levels of plasmatic fibrinogen, coronary disease, and adverse events after a UCD event. In addition, the -455A allele is associated with high values of plasmatic fibrinogen and coronary disease. The simultaneous association of the alleles -455A and -148T agrees with the hypothesis of a genetic linkage, as reported in Caucasian populations [26,27].
The two gene polymorphisms relevant for this study are located in the promoter region of the fibrinogen B gene, very close to the elements of response to IL-6 and C/EBP and of the HNF1 and HNF3 [4,5,[28][29][30][31]. They may modify the interaction of the promoter with IL-6, a cytosine responsible for the inflammatory response in the acute phase of a coronary event, and this explains the associations observed in this study.

Conclusions
Plasmatic fibrinogen levels > 450 mg/dl and the polymorphisms -455G/A and 148C/T of the fibrinogen gene are association with MACE and coronary disease in a sample of a Mexican population.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Disclosure
A basic study of this manuscript was presented in the School of Medicine of Universidad Autonoma de Nuevo Leon.

Conflicts of Interest
The authors declare that there is no conflict of interests regarding the publication of this paper.