Multiple sclerosis susceptibility may be associated with the coding rs20541 (R130Q) IL-13 gene polymorphism in the Polish population

Some of the multiple autoimmune diseases have been already associated with IL-13 single-nucleotide polymorphisms (SNPs). However, there are only few studies regarding multiple sclerosis (MS) risk and IL-13 rs20541 (R130Q) polymorphism, and their results are conflicting. Therefore, the aim of our study was to investigate the frequency of the IL-13 gene rs20541 (R130Q) polymorphism in MS participants and its association with MS clinical subsets in the Polish population. We conducted a case‒control study including 94 relapsing remitting MS patients and 160 healthy volunteers. We genotyped the rs20541 polymorphism in the IL-13 gene and analysed the genotype frequency, age of MS onset and clinical condition (EDSS values) of the MS participants. Fisher’s exact test was used for statistical analysis, and the log-linear model was applied to test for associations. Allele A, as well as the AA and AG genotypes, was observed to be significantly more common in the MS subjects. The OR (odds ratio) for the A compared to the G allele was 1.71 (1.14–2.56), whereas OR 2.33 (0.86–6.26) and OR 1.92 (1.11–3.30) were obtained for the AA and AG genotypes, respectively. We did not identify any significant associations of the studied IL-13 SNP with the investigated clinical parameters of the MS participants. Our results suggest that the rs20541 polymorphism in the IL-13 gene may play an important role in MS predisposition but not in investigated clinical parameters in MS subjects of the Polish population.

may have not only immunomodulatory effects but also neuroprotective benefits for these patients.Nevertheless, results related to the role of IL-13 in MS have been inconsistent among studies.Some findings suggest a pathogenic role for T-cell-derived IL-13 production in the central nervous system (CNS) in MS 5,6 .
Overall, MS is a chronic immune-mediated disease; however, it is also known that genetic predisposition contributes to its development.Genome-wide association studies (GWASs) have identified more than 200 genetic loci that show association with MS risk 7 , and molecular pathomechanisms may include effects on pre-mRNA splicing 8 .An impaired immune system response also includes a strong heritable component due to effects on the expression or biological activity of cytokines 9 .IL-13 is encoded by the IL-13 gene located on the long arm of chromosome 5q31-33 in the cluster of genes encoding Th2 cytokines and contains four exons encoding a 146 amino acid protein 10 .Several single-nucleotide polymorphisms (SNPs) associated with immunological diseases have been identified in coding and noncoding regions of the IL-13 gene 11 .Some of them (C-1112T, A-1512C) may influence the level of cytokine expression, whereas some (G+2044A) may have an impact on the affinity of IL-13 for its receptor 12,13 .Some SNPs (including rs20541 G+2044A (R130Q)) in the IL-13 gene have been associated with chronic inflammatory and autoimmunological diseases, such as elderly associated chronic inflammatory diseases 14 , asthma 15 , autoimmune thyroid disease 16 , idiopathic nephrotic syndrome 17 , and food allergies 18 .
The association between the IL-13 gene polymorphism rs20541 (R130Q) and MS has been investigated, to the best of our knowledge, by two other studies but only in the Iranian population 19,20 , which indicates that further investigations are needed, especially in different ethnicities.In the study by Mirahmadi et al. 20 no relation between G+2044A (R130Q) and MS was observed, but associations of some IL-13 gene SNPs were reported by Seyfizadeh et al. 19 .To our knowledge, there are no other studies in which the relationship between the rs20541 SNP and MS have been investigated.Overall, the studies to date suggest that ethnic differences cannot be excluded.In our study, we aimed to determine the frequency of the rs20541 (R130Q) polymorphism in MS and healthy control individuals in the Polish population and to analyse associations of genotype and allele with MS susceptibility and clinical disease manifestation.

Results
The clinical characteristics of the MS study population are presented in Table 1.The frequencies of the investigated genotypes in the studied population are presented in Table 2.We did not observe any deviation from Hardy-Weinberg equilibrium (chi-square test p value = 0.866).We noted that the AA and AG genotypes were more common in the MS population than in the healthy subjects (9.57% vs. 5.62% and 44.68% vs. 31.87%,respectively) but that the frequency of the GG genotype was higher in the control subjects than in the MS individuals (62.50% vs. 45.74%,respectively).These differences in genotype distributions were statistically significant, with a Fisher's test p value of 0.031.Allele A was more common in the MS subjects (31.91% vs. 21.56%,p = 0.01), while allele G was more common in the control group (78.44% vs. 68.09%,Fisher's test p value 0.011).When comparing the test statistic from Fisher's exact test and the generalized linear model, the results were almost the same but not equal.The results of the generalized linear model, which uses the likelihood ratio statistic, confirmed the MS-rs20541 (R130Q) polymorphism association (p value 0.032).Genotypes AA and AG were significantly associated with an increased risk of MS compared to the GG genotype (OR 2.33 (0.86-6.26) and OR 1.92 (1.11-3.30),respectively).Compared to the G allele, the allele A was significantly associated with an increased risk of MS (OR 1.71 (1.14-2.56)).We did not identify any statistically significant differences in clinical data, such as age of onset or EDSS score, between the studied genotypes.The ages of MS onset for the AA, AG and GG genotype carriers were 45.0 (± 9.0), 43.6 (± 8.8) and 38.8 (± 10.0) years, respectively.Dunn's test adjusted p values for the AA vs. AG, AA vs. GG, and AG vs. GG genotype comparisons were 0.60, 0.14 and 0.07, respectively.EDSS values for AA, AG and GG genotype carriers were 1.88 (± 1.09), 2.01 (± 1.29) and 1.93 (± 1.33), respectively, and we did not observe any statistically significant differences.

Discussion
The rs20541 SNP (G+2044A, R130Q) of the IL-13 gene has been associated with autoimmunological diseases.We sought to examine whether this variation in the IL-13 gene may be associated with the susceptibility and clinical condition of individuals with MS of the Polish population.We noted significantly higher AA and AG genotypes, as well as A allele frequencies, in the individuals with MS, whereas the G allele and GG genotype were more frequent in the control group.Our analysis of ORs for the A allele and AA and AG genotypes suggests that the A allele may contribute to MS incidence in the studied population.
Our results are in contrast with results from a study by Mirahmadi et al. 20 , who did not observe any relation between G+2044A (R130Q) and MS.However, ethnic differences should be considered because a significant association for the investigated SNP polymorphism in patients of the Turkmen ethnic group of the Iranian population was noted.The association of some SNPs of the IL-13 gene (including rs20541 (R130Q)) was observed in the Iranian population by Seyfizadeh et al. 19 .
The coding SNP rs20541 in exon 4 leads to a nonconservative guanine to adenine transition at nucleotide + 2044, leading to replacement of Arg 130 with Gln in α-helix D. This is a critical region and a significant amino acid for interactions between IL-13 and its receptors, and therefore, it may have an impact on IL-13-mediated signalling, resulting in increased biological activity and biological affinity between IL-13 and its receptor and may lead to inhibitory effects on immune responses 21 .However, the high frequency of the A allele in MS patients, as Seyfizadeh et al. 19 pointed out, was not anticipated because the activity of IL-13 is decreased in MS patients.The protective role that is generally ascribed to Th2 cells has been questioned because a positive correlation has been observed between IL-13-producing cerebrospinal fluid (CSF) T cells and disability measured by the EDSS in RRMS patients 5 .Furthermore, a pathogenic phenotype with high production of IL-13 and other Th2 cytokines by brain-infiltrating T cells in demyelinating lesions has been identified 6 .
It is also worth emphasizing that IL-13 is involved in the maturation and differentiation of B cells, and an emerging role for B cells and the IgE pathway in triggering MS and other autoimmune diseases has been described 22 .In the study by Graves et al. 23 carriers of rs20541 allele A had significantly higher total serum IgE levels than individuals carrying the G allele.A significant association between the rs20541 SNP and IgE serum concentrations was also noted in a study by Imraish et al. 24 , in which minor alleles were associated with a significant, approximately 5 times lower, total serum IgE level compared with the major alleles in individuals with asthma.All of these observations need further investigation with regard to the possible mechanisms to better understand the associations of IL-13 gene polymorphisms with MS.We did not observe any association between the studied SNP and the age of MS onset or the clinical condition of MS subjects, and these results are in line with results from the study of Seyfizadeh et al. 19 , who did not observe associations between any investigated IL-13 genotypes or alleles and the expanded disability status scale (EDSS) scores of Iranian participants.
When interpreting our results, the following limitations should be considered.The most important limitation is that we did not measure IL-13 or IgE concentrations in the study participants, which might provide more information about the observed associations.Nevertheless, this is the first pilot study in a Polish population investigating associations of MS with IL-13 gene polymorphisms, and in future studies, more factors involved in the possible pathways should be included and analysed.Moreover, we focused only on the IL-13 rs20541 variant, and it was already found that rs20541 is in strong linkage disequilibrium (LD) with, for example, another IL-13 SNP, rs1295685, and that its haplotypes are associated with serum IgE levels 25 .Therefore, the possible impact of other SNPs being in strong LD or haplotypes should be considered in further analysis.

Conclusions
In conclusion, our results suggest that the IL-13 gene rs20541 polymorphism may play an important role in MS predisposition.We noted that allele A, as well as AA and AG genotypes, are significantly more common in MS participants of our study, with the ORs indicating the strength of the observed associations.However, investigated rs20541 SNP seems to be not associated with investigated clinical parameters, in MS subjects of the Polish population.Considering results from the other studies to date we can not exclude the impact of ethnic differences.An association observed in our study may be related to both mechanisms of SM development, T-cells and B-cells pathways as well, what we have discussed above.Nevertheless, further replication and functional studies are needed to investigate how common IL-13 genetic variants may contribute to biological processes related to MS.More studies should also investigate other IL-13 and IL-13 receptor SNPs, as well as cytokine and other protein concentrations, to explore their clinical effects and roles in protecting individuals from or predisposing them towards MS development and progression.

Ethics
The study was approved by the Bioethical Committee of Medical University of Bialystok, Poland (R-I-002/334/2018).The study was conducted in accordance with the Helsinki Declaration, and all of the participants signed an informed consent form before enrolment in the study.

Table 1 .
Clinical characteristics of the MS group.Data are presented as the mean ± SD.MS multiple sclerosis, EDSS expanded disability status scale, IQR interquartile range.*For EDSS, median values and IQRs are also presented.