Polymorphism -238 G/A in Promotor Region of Tumor Necrosis Factor alpha Gene Is a Risk Factor for Ischemic Cerebrovascular Disease in Spanish Population.
Authors: De Velasco Jaime Fernandez , Llamas Pilar , De Ona Raquel , Santos Ana Belen , Meseguer Elena , Garcia-Raso Arancha , Mata Raquel , Tomas Jose Francisco 
Published: April 2020

Abstract
Introduction: Cerebrovascular disease (CVD) is a multifactorial disease caused by the interaction of genetic and environmental factors. The atherosclerotic plaque, the pathological hallmark of CVD, is an inflammatory process, where pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα). TNFα secretion shows a high degree of interindividual variability, which is at least partly genetically determined. We have analysed the prevalence of −238 G/A and −308 G/A polymorphisms in the regulatory region of the TNFα gene promoter in CVD.
Patients and methods: Genotypic analyses were performed on 308 consecutive unrelated patients diagnosed with ischemic CVD, 147 women and 161 men, mean age 70±0.8 years, who were diagnosed according to the Trial of Org 10172 in Acute Stroke Treatment. All included cases were age and sex matched to a control from the same geographic area who had no history of vascular disease. Patients and controls completed a questionnaires including blood pressure, diabetes status, total serum cholesterol level and smoking history. The TNFα variants were detected by PCR using primers containing a single base-pair mismatch to introduce a restriction site into the wild-type nucleotide sequences after amplification. PCR products were digested with NcoI and MspI to detect −308 and −238 variants, respectively. The strength of the association of the polymorphisms with the occurrence of CVD was estimated by calculation of the OR and its 95%CI by exact method. P values less than 0.05 were considered significant. Logistic regression analysis was applied to estimate the risk in a multivariable predictive model with dependent variable (case/control) and all independent variables significant in the bivariate analysis. SPSS 9.0 was used for the statistical analysis.
Results: Genotype analysis showed a significant higher prevalence of the G/A and A/A genotypes of −238 G/A TNFα in patients (p&lt; 0.01;OR= 2.16;95%CI= 1.40–3.34). The prevalence of the A allele was also significantly increased in the group of CVD patients than in the controls (13.6% vs 7.0%; p&lt;0.01;OR=2.10;95%CI=1.40–3.17). By contrast, the distribution of −308 genotypes for patients did not differ from their control group (84.9% vs 81.1% and 14.4% vs 16.9%, respectively; p=0.21;OR=0.76;95%CI=0.48–1.20) or in the A allele frequencies (7.9% vs 10.4%; p=0.12;OR=0.73;95%CI=0.48–1.11). When analysis was performed for the two more frequently subtypes of CVD, atherotrombotic subtype showed an higher prevalence of the A allele of -238 G/A polymorphism, as compared with the undetermined etiology (16.5% vs 11.2%;p=0.06;OR=1.86;95%CI= 0.91–3.82). By contrast, the variant −308 G/A did not differ from the different subtypes. Logistic regression analysis showed a independent association of A haplotype of −238 G/A TNFα with CVD. Also hypertension, diabetes mellitus and current smoking status were statistically associated with CVD.
Conclusions: Our findings suggest that the A allele of −238 G/A TNFα promoter polymorphism is a genetic risk factor for ischemic CVD in Spanish population. Data on the distribution of genotypes and corresponding allelic frequencies of −238 G/A and −308 G/A TNFα polymorphisms in CVD are scant. The present study is the first to analyses these variants in CVD patients from this geographic area.