Molecular analysis of genetic susceptibility, sensitivity and oxidative stress markers in asthma patients

Asthma, which is a chronic respiratory disease leading acute attacks, is a serious health problem for people of all ages. One in every 12-13 adults and 7-8 children in Turkey are suffering from asthma. It is pointed out that the total number of asthma patients reached to 300 million in the World and 3.5 million in Turkey, and these numbers are predicted to increase very rapidly in the future. Allergic asthma develops depending on an IgE-mediated response, activating the mast cells and Th2 cells, increasing the number of eosinophils in the tissue, and trigerring the secretion of cytokines, such as IL-13, IL-4 and TSLP. Consequently, airway inflammation occurs as a result of the complex interactions of immune cells, structural cells, and cytokines. Various individual (genetics, obesity, gender) and environmental (house dust mite, mold fungus, pollens, some drugs etc.) factors contribute to the development of the disease. Several studies have confirmed the relationship between gene polymorphisms and asthma susceptibility. Therefore, studies on the possible polymorphic genes in allergic asthma patients and their roles in progression of asthma are important. The association of interleukin 13 (IL-13) and forkhead box protein 3 (FOXP3) with some allergic and immunological diseases has been documented in a number of studies. Single nucleotide polymorphisms (SNPs) on these genes have been studied in different populations and some of them have been shown to be important in the progression of asthma. As a transcription factor, FOXP3 acts as the main regulator of the development and function of T-regulatory (Treg) cells and is the most specific Treg biomarker. Some scientific papers have shown decreased FOXP3 mRNA level and protein expression in asthmatic patients. After a signal, the FOXP3 protein suppresses expression of some genes whereas increases of some others. One of the genes that FOXP3 affects its expression is interlukin 35 gene (IL35). The protein product of this gene, IL-35, mediates the suppression of effector T cells, and its deficiency correlates with the pathogenesis of several diseases, such as inflammatory and autoimmune diseases. In allergic and chronic inflammatory diseases, oxidative stress occurs as a result of immune system activation. During inflammation and immune response, excessive reactive oxygen species (ROS) formation can induce oxidative stress, ultimately leading to cell damage. Reactive oxygen species damage cell components, such as lipid, DNA, RNA, and proteins. Oxidative stress is known to contribute to asthma progression by damaging surrounding tissues in the respiratory system, and also there are some evidences of its correlation with asthma severity. In this study, single nucleotide polimorfisms on FOXP3 and IL13 genes, and some markers of oxidative stress and antioxidant status were analyzed with the aim of understanding their roles in asthma progression and control. Blood samples were obtained from total 120 allergic asthma patients (92 females + 28 males) whose sensitivity tests were completed, and 120 healthy subjects (90 females + 30 males). Polymorphism analyses were performed on FOXP3 (for SNPs rs3761548 and rs3761549) and IL13 (for SNPs rs20541 and rs1881457) genes by PCR-RFLP method. The serum IL-35 level was measured by ELISA in the patient and healthy control groups. Besides, plasma protein carbonyl (PC), malondialdehyde (MDA), reduced glutathione (GSH) levels, ferric reducing antioxidant power (FRAP) and serum catalase activity were detected using spectrophotometric methods in both groups. Since the FOXP3 gene is located on the X chromosome, the results were evaluated as male and female individually. Statistical analysis of the genetic variations at the position -3279A/C on FOXP3 gene (SNP rs3761548) revealed that CC genotype in female and C genotype in male were more prevalent in patients with allergic asthma, and increased the susceptibility to the disease. Polymorphism status at the position -2383C/T (SNP rs3761549) on FOXP3 gene exhibited that female with CT genotype had high risk of allergic asthma. For this SNP, TT genotype in female and T genotype in male were not encountered in none of the study groups (patients and healthy). No significant differences were observed for the variations at the positions +2044G/A (SNP rs20541) and -1512A/C (SNP rs1881457) on IL13 gene between allergic asthma patients and healthy subjects. Although serum IL-35 level was higher in allergic asthma patients compared to healthy subjects, this difference was not statistically significant (17.74 ± 5.76 pg/mL and 15.62 ± 4.9 pg/mL, p>0.05, respectively). There was a positively weak correlation between serum IL-35 and IgE levels in patients (r = + 0.365, p<0.001). Besides, serum IL-35 levels in female who have AC (both patient and healthy) or AA (healthy female subjects) genotypes at SNP rs3761548 on FOXP3 gene were higher in comparison to other genotypes. Plasma protein carbonyl (PC) level in patients (1.47 ± 0.74 nmol/mg protein) was detected as significantly (p<0.001) higher than that of healthy subjects (1.01 ± 0.43 nmol/mg protein). Plasma malondialdehyde (MDA) level was also significantly higher in patients (3.39 ± 1.1 nmol/mL) compared to healthy subjects (2.31 ± 0.6 nmol/mL) (p<0.001). Plasma reduced glutathione (GSH) level (16.41 ± 6 nmol/mL) in the patients was significantly lower than that of healthy subjects (23.74 ± 9.3) (p<0.001). Similarly, ferric reducing total antioxidant power (FRAP) was lower in patient group (371.7 ± 105 nmol/mL) in comparison to healthy group (413.7 ± 133 nmol/mL) (p<0.01). Serum catalase activity in patients (0.353 ± 0.25 U/mg protein) was lower than that of healthy subjects (0.425 ± 0.185 U/mg protein) (p<0.05). In conclusion, rs3761548 and rs3761549 SNPs seem to be associated with allergic asthma susceptibility. Besides, our findings revealed the presence of increased oxidative stress and decreased antioxidant status in patients with allergic asthma, even under controlled conditions. Thus, treatment strategies reducing oxidative stress and/or supporting antioxidant defence may be beneficial for allergic asthma patients. In the future, studies in expanded patient and control groups, by regarding the SNPs reported here, and the others, will definitively bring out the utility of these genetic variations to predict allergic asthma susceptibility. Besides, further clinical trials are required in this manner to indicate the supporting capacity of antioxidants in allergic asthma treatment.