Correlations of IL-6, IL-6R, IL-10 and IL-17 gene polymorphisms with the prevalence of COVID-2019 infection and its mortality rate


 The pathogenesis of COVID-19 implicates a potent inflammatory response resulting in cytokine storm. We aimed to evaluate association between polymorphisms in IL-6 gene at rs1800796/rs1800795, IL-6R at rs2228145, IL-10 at rs1800896 and rs1800871, IL-17 at rs2275913 and rs76378, and the prevalence (per million) and mortality rates (per million) of COVID-19 among populations of China, Japan, India, Iran, Spain, Italy, Mexico, Netherlands, Sweden, Turkey, Finland, Brazil, Czechia, Russia, Poland. AG and GG genotypes of rs2275913 in IL-17A was found to be correlated with prevalence and mortality rates, especially in Spain and Brazil populations (p<0.05) while TT genotype of rs763780 in IL-17F was not dependent on the high frequencies in all populations. However, the polymorphisms in IL-6, IL-6R and IL-10 appear not to be correlated with prevalence and mortality rates. The variations in the prevalence of COVID-19 and its mortality rates among countries may be explained by cytokine storm differed by the polymorphisms of rs2275913 locus in IL-17A gene. However, the prevalence of infection differs from severity of COVID-19, based on many factors such as public awareness, behaviors and antiviral policy of countries. Yet, the severity of disease induced by viral infection might be associated with genetic host factors including immune profiling.


Introduction
Since December 2019, coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has given rise to emerging respiratory infections with a pandemical diffusion. 1 By 7th September 2020, the global number of con rmed cases of COVID-19 reached 27,066,438 with a mortality of 885,480 2 In Turkey, 279,806 cases and 6673 deaths have been con rmed until 7 th September 2020. 2 Most patients present with mild symptoms that are not lifethreatening, however, the mortality rates are in such considerable amount among large population base. 2 The pathogenesis of COVID-19 implicates a potent in ammatory response, involving a complex group of mediators including interleukins. [3][4][5][6][7][8][9][10] These pleiotropic cytokines are secreted at the region of in ammation and released into the circulation by a variety of different cell types, including macrophages, lymphocytes, endothelial cells, epithelial cells and broblasts during sepsis and acute organ injuries. 10 There has recently found a number of studies reporting that the cytokine storms take part in the course of COVID-19. [3][4][5] Principally, SARS-CoV-2 can rapidly activate pathogenic Th1 cells to secrete pro-in ammatory cytokines, such as interleukin-6 (IL-6) and IL-17. 6,7 IL-6 acts as a key pro-in ammatory mediator for the induction of the acute phase response, 11 resulting in a variety of local and systemic uctuations including fever, leucocytes recruitment and activation and hemodynamic changes. Considering the key role of interleukins in mediating the acute phase response, they have been nominated as prognostic biomarkers in sepsis and various acute organ injuries in clinical and experimental studies. Some studies have also reported that patients with severe COVID-19 have higher levels of IL-2, IL-6, IL-7, IL-10 than patients with mild and moderate infections. 4,8,9 In viral infections, IL-10 is highly abundant, especially during the adaptive immune response. 12 IL-17 was found to be increased in intensive-care COVID-19 patients compared to controls. 4 Therefore, these key in ammatory factors in COVİD-19 are of great interest in understanding the cytokine storm-related mortality in severe cases. 3 The basis and value of single nucleotide polymorphisms (SNPs) relation to immune responses are called "immunogenetic pro ling". 13 SARS-CoV-2 infection is claimed to be correlated with IL-6 polymorphism. Recently, it was reported that IL6 rs1800795 G allele could act as a protective factor while IL10 rs1800896 A allele could act as a risk indicator in Pneumoniainduced sepsis in Chinese Han patients. In addition, these IL6 polymorphisms were associated with clinical stage of sepsis and have crucial effects on the secretion of IL-6 and IL-10 in the patients. 14 Therapies by IL6 receptor antagonists are proposed to be a therapeutic option for the therapy of cytokine storm syndrome in SARS-CoV-2 infected patients. 15 Therefore, we hypothesized that single nucleotide polymorphisms (SNPs) in IL-6, IL-6R, IL-10 and IL-17 genes may participate in the clinical course of COVID-19 infection.
The main goal of this study was to evaluate if there is any association between the IL-6 gene polymorphism at rs1800796/rs1800795 locus, IL-6R at rs2228145 locus, IL-10 at rs1800896 and rs1800871 loci, IL-17 at rs2275913 and rs76378 loci, and the prevalence of COVID-19 and the mortality rates among populations of 15 countries including Turkey.

Materials And Methods
To test this hypothesis and to limit confounding bias (latitude, etc.), we focused on the countries whose IL-6 gene polymorphisms at rs1800796 and rs1800795 loci, IL-6R at rs2228145 locus, IL-10 at rs1800896 and rs1800871 loci, IL-17 at rs2275913 and rs76378 loci were de ned and the allele frequencies were reported in 37 studies.  We searched the literature for interleukin genes polymorphisms in China, Japan, India, Iran, Spain, Italy, Mexico, Netherlands, Sweden, Turkey, Finland, Brazil, Czechia, Russia, Poland. We recorded the total number of cases of COVID-19 and per million population in each of the countries to nd the prevalence, and the mortality rates caused by the Coronavirus infection recorded at 7 th September 2020 according to WHO COVID-19 Weekly Epidemiological Update . 2 All data were analyzed by SPSS (statistical package for social sciences) for Windows 22 program. In the analysis of the data, rst the assumptions that must be met were tested to decide which tests (parametric / nonparametric tests) to apply. Shapiro Wilk test, kurtosis and skewness values which are other assumptions of normal distribution, and histogram graph were used to decide the normality of the distribution. Considering the insu cient number of data in each group, it was decided that the data did not exhibit normal distribution. The relationship between independent variables was examined with Spearman correlation coe cient (rho). In the interpretation of whether the obtained values are signi cant or not, 0.05 signi cance level was used as a criterion.

Results
Population diversities of IL-6 gene polymorphisms at rs1800796/rs1800795 loci showed that the populations of China, Spain, Sweden and Poland mostly have GC genotype while the populations of India, Mexico, Turkey, Brazil and Russia mostly have GG genotype. Japanese population mostly showed CC genotype for rs1800796 polymorphism (Table 1).
Population diversities of IL-6R gene polymorphisms at rs2228145 locus revealed that the populations of all countries except India and Sweden mostly have AC genotype while Indian and Swedish populations have AA genotype at rs2228145 locus. The prevalence of COVID-19 infection and relevant mortality rates per country recorded at 7th September 2020 showed that Spain and Brazil had the highest number of COVID-19 cases and mortality rates per million of the populations of countries involved in the study (Table 1).
Correlation between the frequencies of rs1800796/rs1800795 and rs2228145 polymorphisms, and the prevalence of COVID-19 and mortality rates per country demonstrated that there was no signi cant correlation between the prevalence (per million), mortality rates (per million), and these polymorphisms found in IL-6 and IL-6R genes (  (Table 3).
Correlation between the frequencies of rs1800896 and rs1800871 polymorphisms, and the prevalence of COVID-19 and mortality rates per country demonstrated that there was no signi cant correlation between the prevalence (per million), mortality rates (per million), and these polymorphisms found in IL-10 gene (Table 4).
Population diversities of IL-17A gene polymorphism at rs2275913 revealed that the populations of China, Japan, Iran, Finland and Czechia mostly have AG genotype while Spain, Mexico, Netherlands, Turkey and Brazil populations have GG genotype at rs2275913 locus (Table 5). Population diversities of IL-17F gene polymorphism at rs763780 locus revealed that all populations generally have TT genotype (Table 5).
Correlation analysis between the frequencies of rs2275913 polymorphism in IL-17A gene and prevalence of COVID-19 and mortality rates per country demonstrated that there was positive signi cant correlation between the prevalence (per million), mortality rates (per million), and GG genotype (p < 0.05) while there was negative correlation between those rates and AG genotype (p < 0.05). For AA genotype, a signi cant negative correlation was present between the prevalence and the frequency of rs2275913 polymorphism among countries (Table 6).
Correlation analysis between the frequencies of rs763780 polymorphism in IL-17F gene and prevalence of COVID-19 and mortality rates per country demonstrated that there was negative signi cant correlation between the prevalence (per million) and mortality rates (per million), and CC genotype (p < 0.05). However, no signi cant correlation was found between the prevalence (per million) and mortality rates (per million), and the frequencies TT and CC genotypes at rs763780 locus (Table  6).

Discussion
In the present study, AG and GG genotypes of rs2275913 locus in IL-17A gene were found to confer COVID-19 susceptibility to the populations of Spain and Brazil. TT genotype of rs763780 locus in IL-17F gene was not found to confer any susceptibility to any severe COVID-19, which is an important reason of death. 55 On the other hand, polymorphisms in the IL6 was linked to certain viral infections like hepatitis C (HCV), in uenza virus, and hepatitis B virus (HBV). 56 Mao et al. revealed that IL6 rs1800795 G allele could act as a protective factor while IL10 rs1800896 A allele could act as a risk indicator in pneumonia-induced sepsis in Chinese Han patients. Furthermore, these IL6 polymorphisms were associated with clinical stage of sepsis and affected the secretion of IL-6 and IL-10. 14 A very recent meta-analysis reported a relation between the IL6 gene polymorphism and predisposition as well as disease severity of pneumonia, suggested the carrier status of IL6 allele with higher IL-6 production and pneumonia severity. 57 However, in the present study, we could not nd a signi cant correlation between the frequencies of rs1800796/rs1800795 and rs2228145 polymorphisms in IL-6 and IL-6R genes, probably due to the variations among the genetic immune pro ling of populations.
IL-10 is one of the complex group of mediators participating in the pathogenesis of COVID-19. In in uenza infection, IL-10 is highly abundant, especially during the adaptive immune response. 58 Serum IL-10 levels with IL-6 were found to be signi cantly higher in critical COVID-19 patients, than in moderate and severe patients. The levels of IL-10 were also reported to be positively correlated with CRP amount, and IL-6 and IL-10 were found to be predictive of disease severity. 59 The rst study for IL-10 polymorphism in SARS did not show any signi cant association of this SNP with SARS. 60 A case-control study for cytokine genotyped the SNP IL-10 also did not nd a signi cant association between the genotype and allele frequencies of IL-10 polymorphisms among the SARS patients in terms the death and survival ratio. 61 This result is consistent with our present nding showing no signi cant correlation between two polymorphisms of IL-10 gene (rs1800896 and rs1800871 loci) and the prevalence of SARS-CoV-2 and mortality rates due to its infection. However, we cannot exclude the role of IL-10, IL-6 and IL-6R as the susceptibility genes for COVID-19, since other SNPs in these genes may also be involved in gene expression regulation. Further association studies on other SNPs, which could alter the gene expression level are required to ascertain the relationship of IL-6, IL-6R and IL-10 in COVID-19 infection.
IL-17 is the most well-known and multifunctional cytokine of a cytokine family. Its predominant either based on the gene expression or the trigger of precipitation. These two phenomena seem to affect the dominant effect of its expression as a protective cytokine or lead to a damaging hyper-in ammatory state. 62 IL-17 and other T helper 17 cell-related pro-in ammatory cytokines, such as IL-1, IL-6, IL-15, TNF and IFNγ were found to be positively correlate with the severity of MERS-CoV, SARS-CoV and SARS-CoV-2. 63,64 A retrospective analysis of IL-17 gene polymorphisms in patients with acute respiratory distress syndrome (ARDS) revealed that patients with a polymorphism that resulted in attenuated IL-17 production had an increased 30-day survival, whereas a genetic polymorphism that resulted in producing more IL-17 correlated with decreased survival. 65 Mikacenic et al. measured circulating IL-17A in ARDS and showed that elevated circulating and alveolar levels of IL-17A are associated with increased percentage of alveolar neutrophils, alveolar permeability and organ dysfunction in ARDS. 66 However, there is no detailed information about the association between frequency of polymorphisms of IL-17A and IL-17F genes of COVID patients among the populations. This is the rst study showing the positive signi cant correlation between the prevalence and mortality rates in COVID-19 and GG genotype of rs2275913 SNP in IL-17A gene, additionally, a negative correlation between those prevalence and mortality rates and AG genotype. AA genotype also gave a signi cant negative correlation with the prevalence of disease among countries. We found another negative signi cant correlation between the prevalence and mortality rates, and the frequencies of CC genotype for rs763780 SNP in IL-17F gene, suggesting that the genetic variations in IL-17 gene may be linked to the distribution of COVID-19 infection among nations.
The pathology of COVID-19 involves a complex interaction between the SARS-CoV2 and the body immune system. Vitamin D plays a major role regulating the immune system, including immune responses to viral infection. 67 Vitamin D has been reported to modulate macrophages' response, preventing them from releasing too many in ammatory cytokines and chemokines, which are frequently observed in COVID-19 cases. 68 Recently, we reported the genetic polymorphism especially in the gene of vitamin D binding protein is associated with the increased risk of COVID-19 infection and its mortality among populations with white race. 69