A Significant association between CXCL10 -1447 A>G and IL18 -607 C>A gene polymorphisms with Human T-cell lymphotropic virus type 1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM-TSP), a case-control report from city of Mashhad, Iran CURRENT STATUS:

Background: Human T-cell lymphotropic virus type 1 (HTLV-1) is the first isolated retrovirus from humans, and 2-3% of infected individuals suffer from HTLV-1 associated myelopathy tropical spastic paraparesis (HAM-TSP). Previous studies indicated that the risk of HAM-TSP could be correlated with the individuals’ genetic alterations. Mashhad is one of the areas infected with HTLV-1 in Iran. This study designed to examine the association between several important gene polymorphisms and HAM-TSP. Genotypes of 232 samples from controls, HTLV-1 carriers and HAM-TSP patients were examined for FAS-670 (A> G), CXCL10-1447 (A> G), Foxp3-3279 (C> A), IL-18 -137 (C> G) and IL-18 -607 (C> A) gene polymorphisms by different polymerase chain reaction (PCR) techniques. Results: A non-significant association was observed between FAS-670 A> G, Foxp3-3279 C> A and IL18 -137 C> G gene polymorphisms and HAM-TSP. Nevertheless, a significant (P <0.001) association between CXCL10-1447 A> G and IL-18 -607 C>A gene polymorphisms with HAM-TSP was observed in our study population. As previous studies revealed that the CXCL10 level in the cerebrospinal fluid of HAM-TSP patients was associated with the disease progression, and as we noticed, a direct association was observed between CXCL10-1447 A> G polymorphism and HAM-TSP. Conclusion: This polymorphism might be recommended as a valuable prediction criterion for severity of the disease. The contradiction between our findings and other studies regarding IL-18 -607 C>A gene polymorphism might be associated with various factors such as genotypes frequency in diverse races and population heterogeneity in the city of Mashhad. variations to the effects of to on heterogeneity in Interferon Lambda Family along with HTLV-1 Proviral Load, Tax, and HBZ Implicated in the Pathogenesis of Myelopathy/Tropical Spastic Paraparesis.

the virus. In fact, many infected people are not aware of their infection [3,4].
Studies indicated that HTLV-1 infection could lead to Adult T cell leukemia/lymphoma (ATL), HAM/TSP, uveitis, and infantile dermatitis in children [5,8,9]. Studies also revealed that 2-3% of people infected with HTLV-1 virus suffered from HAM-TSP [10], and they proposed variations in host-to-host immune responses against the virus. Most studies recommend that the disease be initiated by the immune system responses to eliminate the HTLV-1 infection. In HAM / TSP patients, there are records of spinal cord infection, along with a high frequency of CD4 + T lymphocytes moving to the infection site [11][12][13]. These lymphocytes, in addition to CD8 + T lymphocytes as part of the immune response to the viral infection, release several cytokines that some of these cytokines cause damage to nerve cells [14,15]. In addition, studies have shown that HTLV-1infected cells, through their cytokines, alter the immune system's performance. A large number of these cytokines, such as interleukins, Platelet Activating Factors (PAFs) and Tumor Necrosis Factors (TNFs), are identified. Depending on the cytokines function, the demyelinating procedure occurs in HAM / TSP cells [10,11,[16][17][18]. One study revealed that the proliferative response of the CD8 + T cells to the CD4 + T cells helping plasma cells to produce anti-HTLV-1 antibody was clearly inhibited by the Transforming Growth Factor-β1 (TGF-β1).
Furthermore, it is supposed that this cytokine plays a destructive role by the virus localization in the CNS of patients with HAM / TSP. Therefore, it can be mentioned that HTLV-1 causes the glial cells to be infected and leads to a pathological response by the immune system, and eventually an axonal damage occurs [19].
Moreover, the data of numerous studies demonstrate that the potential for HTLV-1 infection can be associated with the genetic differences of people [20][21][22], but its exact mechanism is not yet completely understood. Thus far, several studies have focused on the candidate gene polymorphisms and their association with the risk of HAM-TSP. However, there are still several important genes that have not been examined in terms of their association with the disease. Since the city of Mashhad in Iran is one of the infected area with HTLV-1 in the world, this study designed to evaluate the effect of multiple gene polymorphisms with HAM-TSP.
Material And Methods techniques in a case-control study. The Ethics Committee of Mashhad University of Medical sciences approved our study and the code was 931203.

Sampling
The samples in the control group included 100 healthy persons from the National Blood Transfusion, Mashhad, Iran. Control subjects were obtained from individuals who did not suffer from any of the disorders, such as malignancies, allergic, inflammatory and autoimmune diseases. Individuals with viral infections such as HIV, HVC and HTLV were excluded. Samples in the carrier group were 70 specimens that HTLV-1 virus in their blood samples was first confirmed by Enzyme-Linked Immunosorbent assay (ELISA) and then by Western Blotting. Carrier group samples did not have any underlying disease. Samples in HAM/TSP patients group were 62, and HTLV-1 virus in their blood were first confirmed by ELISA and then by Western blotting, and the HAM / TSP disease was confirmed by an experienced neurologist. The range of age for all individuals was between 18 and 55 years. All individuals filled consent forms before participating in the study.

Genomic DNA extraction and genotyping
After selecting the samples, 5 ml peripheral blood was obtained and kept in tubes containing EDTA for  Table 1 presents the PCR condition, primer sequences, restriction enzymes, and the product size for each genotype. The PCR products were parted on 2.5 % agarose gel containing ethidium bromide and visualized under a UV trans-illuminator.

Statistical analysis
The data were evaluated by the SPSS software Ver16. The genotypes and alleles frequency of polymorphisms was calculated by direct counting. The Hardy-Weinberg equilibrium was analyzed via 2χ by comparing the frequencies of genotypes in the studied groups. In addition, odds ratios (OR), confidence intervals (CI), Chi square and P values were calculated to assess the relationship between the genotypes, allotypes frequencies, and the disease. P-value less than 0.05 was considered statistically significant.

Results
In our study, the participants with HAM / TSP were 12 males and 50 females with an average age of 48.16 ± 11.15 years, the HTLV-1 carriers were 22 males and 48 females with the mean age of 43.77 ± 11.38 years, and the individuals in the control group were 50 males and 50 women with the average age of 45.6 ± 10.07. There was no significant difference in age (P = 0.067) between the three groups of patient, carrier and control. The genotype frequencies were in Hardy-Weinberg equilibrium for all gene polymorphisms except CXCL10 -1447 A > G. The genotyping results for all gene polymorphisms are as follows: the CXCL10 -1447 A > G gene polymorphism frequency was 17%, 83%, and 0% for AA, AG and GG genotypes in the control group, respectively, and 22.9%, 68.6% and 8.6% for AA, AG and GG genotypes in the carrier group, respectively, and 35.5%, 51.6% and 12.9% for AA, AG and GG genotypes in the HAM/TSP group, respectively. The results showed a significant association between the risk of HAM/TSP and the inheritance of the GG genotype in the CXCL10-1447A > G polymorphism as the risk of HAM / TSP with the GG genotype was 1.377 times higher than that of AA (P < 0.001, OR: 1.377; 95% CI: 0.00-24.008) ( Table 2). The FAS-670 A > G gene polymorphism frequency was 23%, 61% and 16% for AA, AG and GG genotypes in the control group, respectively, and 30%, 57.1% and 12.9% for AA, AG and GG genotypes in the carrier group, respectively. The genotype frequency for the HAM-TSP group was 27.4% for the AA, 46.8% for the AG and 25.8% for the GG. Although the AG genotype in the HAM/TSP group was greater than that of carrier and control groups, a non-significant association was observed between this gene polymorphism and HAM/TSP (p = 0.23) ( Table 2). The genotype frequency for the Foxp3 -3279 C > A polymorphism was 19%, 52% and 29% for AA, AC and CC genotypes in the control group, respectively, and 28.6%, 44.3% and 27.1% for AA, AC and CC genotypes in the carrier group, respectively, and 21%, 50% and 29% for AA, AC and CC genotypes in the HAM/TSP group, respectively. There was a non-significant association between the Foxp3 -3279 C > A gene polymorphism and HAM/TSP (p = 0.675) ( Table 2).The genotype frequency for the IL18-137 C > G gene polymorphism was 31%, 49% and 20% for CC, CG and GG genotypes in the control group, respectively, and 31.4%, 54.3% and 14.3% for CC, CG and GG genotypes in the carrier group, respectively, and 16.1%, 54.8% and 29% for CC, CG and GG genotypes in the Ham/Tsp group, respectively. There was a non-significant association between the IL18-137 C > G gene polymorphism and HAM/TSP (p = 0.106) ( Table 2).The genotype frequency for the IL18-607 C > A polymorphism was 38%, 52% and 10% for CC, CA and AA genotypes in the control group, respectively, and 27.1%, 47.1% and 25.7% for CC, CA and AA genotypes in the carrier group, respectively, and 22.6%, 38.7% and 38.7% for CC, CA and AA genotypes in the HAM/TSP group, respectively. A significant association was detected between the IL18-137 C > G gene polymorphism and HAM/TSP (p = 0.001) ( Table 2). The risk of HAM / TSP with the CA genotype was 1.146 times higher than that of CC (P < 0.001, OR: 1.146; 95% CI: 0.474-2.773), and the risk of this disease with the AA genotype was 6.697 times higher than that of the CC genotype (P < 0.001, OR: 6.697; 95% CI: 2.220-20.203) ( Table 2). Furthermore, the CG and GG genotypes in the IL-18-137 C > G polymorphism supported the risk of HAM / TSP disease by 2.835 (P < 0.106, OR: 2.835, 95% CI: 0.979-8.211) and 3.667 (P < 0.106, OR: 3.667; 95% CI: 1.132-11.882), respectively. Additionally, the GG genotype FAS-670 A > G polymorphism supported the risk of HAM / TSP by 1.334 (P < 0.23, OR: 1.334, 95% CI: 0.427-4.165). Table 2 shows the other relationships between the genotypes.

Discussion
In our current study, the frequency of five gene polymorphisms, including Foxp3-3279 C > A FAS-670 A > G, IL-18 -137 C > G, IL-18 -607 C > A, and CXCL10 -1447 A > G was assessed in 100 healthy controls, 100 HTLV-1 carriers and 100 patients with HAM-TSP in a residential population in the city of Mashhad, center of Khorasan Razavi Province, Iran. Our findings for the first time showed that the GG genotype in the CXCL10-1447 A > G polymorphism was a risk factor for the HAM-TSP susceptibility, which could be considered a predisposing genetic factor of the incidence of HAM-TSP in the carrier of the HTLV-1 virus. In addition, our results regarding the IL-18 gene − 607 C > A presented a noticeable association between this gene polymorphism and the risk of HAM-TSP disease (P < 0.001), as it was observed that the AC genotype could be a considered a protective factor against the disease, whereas the CC genotype was a risk factor for HAM-TSP.
CXCL10, known as IP10 (IFN-γ inducible protein 10), is one of the important chemokines in inflammatory procedures in the central nervous system. The receptor of this chemokine is CXCR3, and the signal generated in this path regulates immune cells migration, cell activation and cellular differentiation [23]. Moreover, CXCL10 is expressed by astrocytes, and by binding with the receptor of CXCR3, which is expressed on the surface of NK cells and T cells, causes to attract these cells, and it is likely to degenerate myelin sheath. Consequently, it develops symptoms of the disease, and supports the inflammation process in the central nervous system [3,24]. The results of Tomoo Sato et al. (2013) revealed that the CXCL10 chemokine level in the CSF of HAM-TSP patients was directly associated with the disease development, and as a biomarker, it could play an essential role in timely diagnosis of high-risk patients [25].
IL-18 is an essential cytokine in the human immune system responses and is a member of the IL-1 family. IL-18 secretion is performed by active monocytes and macrophages, and it plays an essential role in stimulation of innate and adaptive immune responses. This cytokine is well-known as an interferon gamma-stimulating factor in Th1 cells, acting in the existence of IL-12 and playing a proinflammatory role [26,27]. IL-18, along with IFN-γ, as two pro-inflammatory cytokines, is needed to activate CTL and NK cells, and thus plays a vital role in the viral agent's clearance like HTLV1.
Moreover, these cytokines plays an essential role in the incidence and symptoms severity associated with the HTLV-1infection [11,26].
Considering the essential functions of IL-18, in the current study, we analyzed the role of two polymorphisms in the IL-18 gene. The results of the IL-18-137 C > G gene polymorphism showed a non-meaningful difference in the frequency of the genotypes of this polymorphism among the three groups of patients (P = 016).

Junior et al. (2012) in a Brazilian population reported that the IL-18-137 C > G polymorphism was not
associated with the risk of HAM-TSP and was not an index of susceptibility to the disease. The G allele frequency of this polymorphism in the HTLV1-infected group was more than that of the control group and therefore it was supposed as a risk factor for the disease. On the contrary, the C allele frequency in the control group was greater than that of the infected group and suggested the protective role of this allele against HTLV1 [27].
However, similar to our results, the data of the study by Vagatsuma et al. (2011) showed a lack of correlation between IL-18-137 C > G and HAM-TSP disease [28].
Our results regarding another polymorphism in the IL-18 gene, namely − 607 C > A demonstrated a significant difference between patients with HAM-TSP, HTLV-1 carriers and healthy controls. The CC genotypes inheritance presented a significant risk for vulnerability to HAM-TSP disease, whereas the CA genotype had a protective role against the disease.
The study by Rocha et al. indicated that the CC genotype frequency of the IL-18 -607 C > A polymorphism was lower in HTLV-1 carriers and patients with HAM-TSP than in the healthy control group and it could be considered a protective genotype for the virus. Along with this finding, they reported that CA genotype was a risk factor forinfection with HTLV-1 [26], which was not the same with the results found in our study. Similarly, Vagatsuma et al. showed a predisposing and protective effect for CC and CA genotypes, respectively [28].
Our results regarding the FAS − 670 A > G gene polymorphism revealed a non-significant difference between HAM-TSP, HTLV-1 carriers and healthy controls (P = 0.23). FAS or CD95 is a 48-KDa protein and is a member of the TNFR family. The ligand of this molecule is FASL. The interaction between FAS and FASL causes to activate caspase 8, triggering the external pathway of apoptosis. Moreover, FASL prompts granulocyte cells, Th-1, cytotoxic T and Th-17, and persuades an inflammatory process activating myeloid differentiation signaling pathway factor 88 / interlukin-1 receptor-associated kinase-4 (MyD88 / IRAK4) [11,28]. Expression of FAS on the surface of CD4 + T cells, which is one of the main targets of infection with the HTLV1 virus, can be associated with FASL binding with CD8 + T cells as a cell death factor in the course of virus infection [29]. Inconsistent with our results, Vallinoto et al. (2012) demonstrated a meaningful difference in the GG genotype of the FAS − 670 A > G polymorphism between the HTLV-1 carrier and healthy controls. Moreover, they reported that the AA genotype of this polymorphism in patients with HAM-TSP was greater than that of the control group, which was involved not only in the ability to develop HTLV1, but also as an indicator of disease progression toward HAM / TSP [30]. Additionally, Rosado et al. (2016) reported that the AA genotype of the FAS − 670 A > G polymorphism could be considered an indicator of increased proviral load (PVL) in progression of HAM-TSP disease [31].
The study results regarding the FOXP3 -3279 C > A gene polymorphism indicated a non-significant difference between the HAM-TSP group, the HTLV-1 carriers and the healthy controls. As far as we know, this is the first time that the association between this polymorphism and HAM -TSP disease is evaluated. Immune system responses are always controlled and adjusted with high precision. One of the important factors regulating immune responses are regulatory T cells (Treg), which are characterized by the expression of FOXP3 as the unique transcription factor [32]. FOXP3 can attach to more than 2,800 genetic sites directly or through cofactors, thereby inducing or inhibiting the function of Treg cells. For example, the connection of FOXP3 to factors such as Nuclear Factor of Activated T Cells (NFAT) and Runt-related factor 1 (RUNX1) activates Treg cells. Similarly, FOXP3 binding with interferon regulatory factor 4 (IRF4) stimulates the expression of the gene in Treg cells, and the continuation of this binding will inhibit the Th-17 cell function [33]. Studies have indicated that the gene expression of the HTLV-1 basic leucine zipper (HBZ) causes instability in the FOXP3 gene expression, as a result of reduced Treg cells' function, and leads to a chronic inflammatory pathway in the pathogenesis of the disease [34]. Previous reports revealed that the number of regulatory T cells was changed by the HTLV1 virus, and the virus employed this change for further pathogenesis [35,36]. Considering that FOXP3 polymorphisms can play a role in the function of Treg cells and modify the regulatory activities of the immune system against the viral infection [37,38], it is recommended that further studies be conducted on the association between FOXP3 gene polymorphisms, including Consent for publication: All authors of the manuscript have read and agreed to its content and are accountable for all aspects of the accuracy and integrity of the manuscript in accordance with ICMJE criteria. All authors agreed to the terms of the BioMed Central Copyright and License Agreement. All authors approved that the current article is original and has not already been published in another journal, and is not currently under consideration by a journal.

Availability of data and materials:
The data obtained in this study are accessible from the corresponding author after well-reasoned and valid request.
Competing interests: Authors affirm that there is no competing interest related with this article.  Tables   Table 1: