Association of Polymorphisms in IL-10, TGF-β 1, IFN-γ , and TNF-α Genes with the Susceptibility to Chronic Obstructive Pulmonary Disease in Kerman, Iran

Background: One of the principal cigarette smokes (CS) mediated diseases is chronic obstructive pulmonary disease (COPD). Methods: In the current case-control study, the relationship between the polymorphisms of interleukin-10 ( IL-10 ), transforming growth factor-β 1 ( TGF-β 1 ) codon 10, TGF-β 1 codon 25, interferon-γ ( IFN-γ ), and tumor necrosis factor-α ( TNF-α ) in 213 individuals with COPD and susceptibility to the disease, with 100 healthy age and gender-matched people as a control group, was investigated using PCR-ARMS (polymerase chain reaction-amplification refractory mutation system). Moreover, the combination of the polymorphisms of TGF-β 1 codon 10.25 regarding this susceptibility was studied in the same condition. Results: There was a significant difference between polymorphism of TGF-β 1 codon 10 ( + 869 T/C), codon 25 (G + 915C), and susceptibility to the disease (OR = 0.50; (95 %CI = 0.24-1.07, p = 0.05), OR CC = 5.31; (95% CI: 1.22-23.2); p = 0.02), thus polymorphism of IL-10 and TGF-β 1 increased the risk of susceptibility to COPD but the polymorphisms of TNF-α (G-308A) and IFN-γ ( + 847 T/A) did not show any association. Conclusion: All in all, it is recommended that the patients carrying the above-said genotypes should be paid proper attention, especially

increases the transcription of the gene and production of TNF-α six to seven times. Individuals homozygous for the G allele are considered low manufacturers of TNF-α (TNF-α Lo), and those with genotype A as high manufacturers of TNF-α (TNF-α Hi) (14). TGF-β 1 is a factor with different effects on the propagation and differentiation of inflammatory cells. The human TGF-β1 gene is mapped in chromosome 19q13.1-3 (15). Although TGF-β 1 is an anti-inflammatory factor, it can cause fibrosis (16). In the lungs, the secretion of TGF-β 1 by bronchial epithelial cells stimulates fibroblast propagation (17). Moreover, TGF-β 1 production is under genetic control, and several polymorphisms in the TGF-β 1 gene have been identified (18), of which two of the most important SNPs were examined in the present study: the + 869 T/C (Leu/ Pro) at codon 10 (rs1982073) and + 915 G/C (Arg/ Pro) at codon 25 (rs1800471). For TGF-β1 at codon 10 individuals homozygous for the C allele are considered low manufacturers and those with genotype T as high manufacturers of TGF-β1 at codon 10 and for TGF-β1 at codon 25 individuals homozygous for the C allele are considered low manufacturers and those with genotype G as high manufacturers of TGF-β1 at codon 25. Interferon gamma (IFN-γ) has been demonstrated to play a key role in pathogen clearance and tumor surveillance (19). IFN-γ, a pro-inflammatory factor produced by activated CD4 + T cells and NK cells, defines the development of Th1 response and promotes cell-mediated immunity. The gene encoding IFN-γ is located on chromosome 12q24 and has four exons spanning around 5.4 kb (20)(21)(22)(23). It has been reported that a novel SNP, T to A, at the 5' end of the CA repeat region in the first intron of the human IFN-γ gene ( + 874T/A at rs62559044) is considered the most important gene. This SNP in the first intron of the IFN-γ gene + 874T/A can putatively influence the secretion of IFN-γ. The analysis of the biological role of this SNP suggested that + 874A carriers were low IFN-γ producers (24). Therefore, the T to A polymorphism could directly influence the level of IFN-γ production (25).
According to the nature of the gene coding, these cytokines, such as IL-10, TGF-β 1 , IFN-γ, TNF-α, and the results obtained from different populations cannot be applied to other populations. The current study aimed to investigate the relationship between these variants and COPD susceptibility in Iranian patients.

Study population
In this case-control study, 100 healthy subjects (control group) and 213 patients with COPD (study group) were selected from Afzalipoor hospital and Besat clinic in Kerman. Participants were selected by convenience sampling. The number of samples was determined according to a previous similar study, in which the eligibility criteria for the cases were as follows (26): male, COPD diagnosis by pulmonologist based on GOLD (global initiative for obstructive lung disease) guideline and confirmed by performing two steps spirometry ( Table 1). The inclusion criteria for healthy subjects were: male, smoking (the same number of cigarettes for more than 10 years), no symptoms of pulmonary involvement, and filling out the informed consent form. Then, the research objectives, study phases, and follow-up process were explained to all patients (27). The demographic data, general health conditions, lifestyle, and smoking habits were registered through a questionnaire. The written informed consent form was obtained from the subjects after describing the aim of the study.

Sampling
For genotyping, 5 mL of blood was drawn into an EDTA tube, and after centrifugation, stored at -70°C until DNA extraction was carried out. DNA was extracted using a standard salt precipitation technique (28) and quantified by measuring the optical density (OD) at λ = 260 nm. The 260/280 ratio was used to assess the quality of DNA, being close to 1.8. The polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) method was used for genotyping. Then demographic data and disease susceptibility were recorded according to GOLD criteria and based on the result of spirometry in the previous year.

Detection and genotyping
IL-10 -1082 (G/A), TGF-β 1 codon 10 + 869 (T/C), TGF-β1 codon 25 + 915 (G/C), IFN-γ + 874 (T/A), and TNF-α -308 (G /A) SNPs were genotyped by ARMS-PCR technique using specific primers as described . ARMS method is an application of PCR in which DNA is amplified by allele-specific primers. This is due to the absence of 3' to 5' exonuclease checking activity of Taq polymerase. High-reliability DNA polymerases, that have this activity, cannot be used in ARMS. It is an extremely valuable method for the identification of point mutations or polymorphisms. Technical factors for genotyping these SNPs, which could affect sensitivity and specificity, included replications and sample retesting. As a result, all relevant measurements were made to ensure the study's The fragments and products of PCR were analysed on ethidium bromide-stained agarose gel, and visualized under ultraviolet light (Table 2). Finally, the resolute patterns of bands in the gel electrophoresis were obtained for the genotypes of each sample. The primer sequences of genes and demographic data for COPD and the healthy controls are shown in Tables 3 and 4.

Statistical analysis
Data analysis was done using IBM SPSS Statistics version 23. For the comparison of continuous variables, first, we checked the assumption that they were normally distributed. Logistic regression was used to determine the independent effect of each polymorphism on COPD risk. Additionally, the 95% confidence interval (CI) and odds ratio (OR) were calculated. Clinical and demographic differences between the two groups were tested using an independent student's t test or Fisher's exact test whenever   appropriate. The P value < 0.05 was considered significant.

Evaluation of gene polymorphisms frequency in the study and control groups Evaluation of IL-10 gene polymorphisms
The frequency of IL-10 gene polymorphisms including A allele carriers and wild-type genotype (GG) in the two groups was listed in Table 5. According to the results, there was a significant relationship between IL-10 gene polymorphisms and the disease (OR A carrier = 0.44; 95% CI: 0.24-0.80; P = 0.00). The frequency of wild-type (GG), the heterozygous genotype (GA), and the homozygous genotype (AA) in the codominant IL-10 gene were reported in Table 5, indicating a significant relationship between the GA genotype and the disease. A comparison of the G allele between the controls and COPD cases showed that the genotypes carrying the G allele are more common in the study group.

Evaluation of TGF-β 1 codon10 gene polymorphisms
The gene polymorphism frequency of the C allele carriers for TGF-β 1 codon10 was shown in Table 6 for case and control groups. Based on the results, there was not a significant relationship between TGF-β1 codon10 gene polymorphisms and the disease (OR C carrier = 0.71; 95% CI: 0.41-1.24; P = 0.28).
According to the results in Table 6, Wild-type (TT) and heterozygous (GC) genotypes in the codominant TGF-β 1 codon 10 gene did not show any association, but there was a significant relation between the homozygous genotype (CC) of the two groups (OR TC = 0.78; 95% CI: 0.44-1.40; P = 0.40 and OR CC = 0.50; 95% CI: 0.24-1.07; P = 0.05). Table 7 shows the frequency of C allele carriers and Wild-type genotypes (GG) of TGF-β 1 codon 25 gene polymorphisms in the case and control groups. The results indicated no significant relationship between TGF-β 1 codon 25 gene polymorphisms and the disease (OR C carrier = 0.71; 95% CI: 0.41-1.24; P = 0.28).

Evaluation of TGF-β 1 codon 25 gene polymorphisms
The frequency of wild-type (TT) and heterozygous a Data are shown as mean ± SD.

Evaluation of TGF-β1 codon 10.25 gene polymorphisms
The frequency of TT/GG and the rest of the genotypes in the TGF-β1 C10.25 genes are shown in Table 8 for the two groups. Based on the results, there was no significant relationship between TGF-β1 C10.25 gene polymorphisms and the disease (OR C carrier = 0.78; 95% CI: 0.43-1.40; P = 0.46).

Evaluation of IFN-γ gene polymorphisms
The frequency of the A allele carriers and wild-type (TT) genotype for IFN-γ gene polymorphisms revealed that there was no significant correlation between the study and control groups (OR A carrier = 0.69; 95% CI: 0.37-1.26; P = 0.30). The frequency of wild-type (TT), heterozygous (TA), and homozygous (AA) genotypes in the codominant IFN-γ gene, as shown in Table 9, indicated no significant relationship between the genotype of controls and the case group (OR TA = 0.53; 95% CI: 0.28-1.02; P = 0.05 and OR AA = 0.90; 95% CI: 0.46-1.80; P = 0.77). Table 10 shows that there was no significant relationship between the genotype of the two groups.

Discussion
Since genetic agents are proposed as risk factors for COPD, many studies have examined the role of genetic polymorphisms and various diseases, including respiratory    diseases. However, sometimes conflicting results have been achieved. On the other hand, investigating the effects of polymorphisms of TNF-α, IL-10, TGF-β 1 codon 10, TGF-β 1 codon 25, and IFN-γ on COPD has not been done in Iran. Accordingly, in the present study, the relationship and susceptibility between COPD and polymorphisms of TNF-α, IL-10, TGF-β 1 codon 10, TGF-β 1 codon 25, and IFN-γ were investigated. IL-10 is an anti-inflammatory factor (32). The results of the current study showed that there was a significant relationship between IL-10 gene polymorphisms and the disease. The existence of one or two A alleles increases the anti-inflammatory factor production in the IL-10 G-1082A polymorphism. Since the heterozygous genotype (GA) was a high producer, polymorphism in COPD was higher than in the control group (84% vs. 69%). As the frequency of the AA genotype in our population was very low, and no significant difference was found among GA, AA, and anti-inflammatory activity, these two groups were merged (Table 5). In the current study, A allele carriers were less susceptible to COPD, and this significance remained despite the presence of confounding factors, which means this allele has a protective role. In other words, the risk of COPD in individuals with the GA genotype was 0.44 times more than in normal people. These results are incompatible with the result achieved by Huang et al (33) reporting that the IL-10 genotypes are associated with COPD, and a significant relationship between the IL-10 gene polymorphism and the disease was reported by Sangil et al (34). In 2015, Larocca et al reported that IL-10 (-1082G/A) genotypes were associated with COPD (35).
The TGF-β 1 has various effects on cell proliferation, differentiation, and inflammation; it also has antiinflammatory properties but can improve pulmonary fibrosis (36). The existence of one or two C alleles provokes the anti-inflammatory cytokine production in the TGF-β 1 codon 10 (T + 869C) polymorphism. This polymorphism in the control group was higher than in the COPD group (78% vs. 71%, Table 6). In the current study, C allele carriers were less likely to develop COPD, and there was also a trend relationship in the presence of confounding factors, demonstrating the protective role of TGF-β 1 codon 10 (T + 869 C). In other words, individuals with the CT genotype were likely to develop COPD 0.7 times more than normal people. In this study, there was a possible significant relationship found between TGF-β 1 gene polymorphisms and COPD. Liao et al reported that the TGF-β 1 polymorphisms were not associated with COPD risk (37). In addition, studies by Gong et al and Zhang et al could not find any association between TGF-B 1 rs1800470 polymorphism and COPD (38,39).
The presence of one or two C alleles enhances the anti-inflammatory cytokine production in the TGF-β 1 codon 25 (G + 915C) polymorphism. This polymorphism in the control group was lower than that in COPD patients (Table 7). In the present study, C allele carriers were more susceptible to COPD even in the presence of confounding factors. In other words, the risk of COPD in the individuals with the GC genotype was 1.26 times more compared to normal people, depicting codon 25 of the TGF-β 1 genotype as a risk factor in susceptibility to COPD. In line with our results, Celedón et al (40) and Ogawa et al (41) reported the TGF-β 1 genotypes were associated with COPD, which was also congruent with the results obtained by Ito et al (42).
The genotype frequency in TGF-β 1 codon 10.25 in the patient group was 23%, while it was 18% in the control group. The frequency of the rest of the genotypes in the patient group was 78%, whereas it was 83% in the control group. According to the results, there was no significant relationship between TGF-β 1 C10.25 gene polymorphisms and the disease (OR C carrier = 0.78; 95% CI: 0.43-1.40; P = 0.46). The only study in Iran on TGF-β 1 C10.25 polymorphism has been carried out by Mandegary et al (43). The evidence shows that TGF-β 1 may have a dual role in the lungs; on one side, it reduces the production of inflammatory cytokines, and on the other side, it induces pulmonary fibrosis (44,45).
One of the productions of Th1 lymphocytes and the crucial factor of the host immune responses to pathogens is IFN-γ. In the current study, the A allele carrier people were less susceptible to COPD even in the presence of confounding factors, which means this allele has a protective role. In other words, the risk of COPD in individuals with the TA genotype was 0.69 times more than in normal people. Since the frequencies of the AA genotype in the Iranian population were very low, and there was no significant difference between AT, AA, and inflammatory activities, these two groups were merged. In line with Di Stefano et al (46), we confirmed that the IFN-γ gene as an inflammatory factor was not a risk factor in susceptibility to COPD.
Several studies have demonstrated that TNF-α is relevant to the pathogenesis of COPD, including involvement in the neutrophil release from the bone marrow and neutrophil activation. Increased levels of TNF-α have been found in the sputum, bronchoalveolar lavage fluid, bronchial biopsies, and circulation of COPD patients. The outcomes of the current study showed that there was no significant relationship between TNF-α G-308A gene polymorphisms and susceptibility to COPD (OR = 0.81, CI = 0.42-1.5). Zhang et al concluded that there was a significant relationship between the above-said gene polymorphisms and COPD in Asian populations, but not in the Caucasian population (39).

Conclusion
Altogether, this is the first report demonstrating that the TGF-β 1 (rs1982073) and (rs1800471) SNPs are related to the progression of COPD in the Kerman population. The IL-10 (rs1800896) polymorphism may be less susceptible to COPD, with the genotypes carrying the G allele more common in COPD cases. There was no association between TNF-α (rs1800629) and IFN-γ (rs62559044) with COPD risk. Further studies with larger various populations are needed for definitive associations and results, future applications, and the pathways involved in the susceptibility of COPD. All in all, it is substantial to give special care to carriers of such genotypes, especially those who are exposed to chemicals at work, pollution, and cigarette smoke.