A significant association between CXCL10 -1447 A > G and IL18 -607 C > A gene polymorphism with human T-cell lymphotropic virus type 1 associated myelopathy/tropical spastic paraparesis (HAM-TSP), a case-control report from city of Mashhad, Iran

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. A non-significant association was observed between FAS-670 A > G, Foxp3-3279 C > A, and IL-18 -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. These polymorphisms might be recommended as a valuable prediction criterion for the 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.


Introduction
Human T-cell lymphotropic virus type 1 (HTLV-1) is a RNA virus from the retroviridae family and the Delta Virus species. HTLV-1 is the first isolated retrovirus from humans (Poiesz et al. 1980;Trevino et al. 2012). The HTLV-1 virus infection is worldwide (Trevino et al. 2012), and it is assessed that 15 to 20 million persons are infected with this virus globally (Futsch et al. 2018;Mozhgani et al. 2018). The infection exists in some parts of the world, including southwest Japan, some Caribbean countries, some sub-Saharan countries, Melanesia and Solomon Islands in the oceanic continent, South American, and northeast Iran (Eusebio-Ponce et al., 2019;Gessain and Cassar 2012;Salehi et al. 2017).
In most cases, HTLV-1 patients stay asymptomatic during their life, and only a minor percentage of them may develop illnesses caused by the virus. In fact, many infected people are not aware of their infection (Futsch et al. 2018;Mozhgani et al. 2018).
Studies indicated that HTLV-1 infection could lead to adult T cell leukemia/lymphoma (ATL), HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), uveitis, and infantile dermatitis in children (Eusebio-Ponce et al. 2019;Goncalves et al. 2010;Saito et al. 2012). Studies also revealed that 2-3% of people infected with HTLV-1 virus suffered from HAM-TSP (Rafatpanah et al. 2013), 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 (Araya et al. 2014;Enose-Akahata et al. 2017;Yamano and Coler-Reilly 2017). 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 (Nagai et al. 2001a(Nagai et al. , 2001b. In addition, studies have shown that HTLV-1-infected cells, through their cytokines, alter the immune system's performance. A large number of these cytokines, such as interleukins (ILs), platelet activating factors (PAFs), and tumor necrosis factors (TNFs), are identified. Depending on the cytokines function, the demyelinating procedure occurs in HAM/TSP cells (Azimi et al. 2001;Enose-Akahata et al. 2017;Rafatpanah et al. 2013;Shoeibi et al. 2013;Vallinoto et al. 2015). 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 (Nagai et al. 1995).
Moreover, the data of numerous studies demonstrate that the potential for HTLV-1 infection can be associated with the genetic differences of people (Assone et al. 2016;Jeffery et al. 2000Jeffery et al. , 1999, 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
In total, genotypes of 232 samples were assessed by various polymerase chain reaction (PCR) techniques in a case-control study. The Ethics Committee of Mashhad University of Medical sciences approved our study and the code was 931,203.

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, HCV, 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 ethylene di-amine tetra acetic acid (EDTA) for DNA extraction. DNA was extracted by employing a genomic DNA isolation kit (Genomic DNA kit; QIAGEN, Valencia, CA) in accordance with the manufacturer's directions and kept at − 20 °C for PCR reactions. The Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) technique was used to determine genotypes of FAS-670 A > G, CXCL10-1447A > G, and Foxp3-3279 C > A. The Polymerase Chain Reaction Single Specific Primer (PCR-SSP) method was selected to detect genotypes of IL-18 -137 C > G and IL-18 -607 C > A polymorphisms. A 20-ml PCR mixture was prepared by employing the Pre-Master Mix (Pishgam, Iran) and other components as follows: 100 µg DNA, 0.5 μΜ of each primer, and 10 μΜ of Pre-Master Mix in 1 × ammonium sulfate PCR reaction buffer. Table 1 presents the PCR condition, primer sequences, restriction enzymes, and the product size for each genotype. The PCR products were loaded 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 allele frequency of polymorphisms were 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 < 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 gel images, for each targeted gene polymorphism, are shown in Figs. 1, 2, 3, 4, and 5. The genotyping results for all gene polymorphisms are as follows: the CXCL10 -1447 A > G gene polymorphism frequencies were 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 frequencies were 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 frequencies for the HAM-TSP group were 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 frequencies for the Foxp3 -3279 C > A polymorphism were 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 frequencies for the IL-18 -137 C > G gene polymorphism were 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 nonsignificant association between the IL-18 -137 C > G gene polymorphism and HAM/TSP (P = 0.106) ( Table 2). The genotype frequencies for the IL18-607 C > A polymorphism were 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 IL-18 -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.
We analyzed the correlation between gender and genotype frequency of CXCL10 -1447 A > G, FAS -670 A > G, Foxp3 -3279 C > A, IL-18 -137 C > G, and IL-18 -607 C > A gene polymorphisms in all three groups including HAM-TSP patients, HTLV-1 carriers, and controls. A significant association was observed between Foxp3 -3279 C > A and IL-18 -607 C > A gene polymorphism with gender in the HTLV-1 carrier group (P = 0.048) and HAM/TSP patients (P = 0.03), respectively. Data of this analysis were presented in Table 3.

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, 70 HTLV-1 carriers, and 62 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.   220-20.203 CXCL10, known as IFN-γ inducible protein 10 (IP10), 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 (Tokunaga et al. 2018). 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 (Futsch et al. 2018;Liu et al. 2001). 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 (Sato et al. 2013).
IL-18 is an essential cytokine in the human immune system responses and is a member of the IL-1 family. This cytokine 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 pro-inflammatory role (Nakanishi 2018;Rocha-Junior et al. 2012). IL-18, along with IFN-γ, as two inflammatory cytokines, is needed to activate CTL and NK cells, and thus plays a vital role in the viral agent's clearance like HTLV-1. Moreover, these cytokines play an essential role in the incidence and symptoms severity associated with the HTLV-1infection (Enose-Akahata et al. 2017;Nakanishi 2018).
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 = 0.106). Rocha-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 HTLV-1-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 HTLV-1 (Rocha-Junior et al., 2012).
However, similar to our results, the data of the study by Wagatsuma et al. showed a lack of correlation between IL-18 -137 C > G and HAM-TSP disease (Virgínia M D Wagatsuma, 2011). Table 3 Genotype frequency of FAS-670 A > G, CXCL10-1447A > G, Foxp3-3279 C > A, IL-18 -137 C > G, and IL-18 -607 C > A polymorphisms in male and female, in three different groups including, healthy controls, HTLV-1 carriers, and patients with HAM/TSP 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 genotype 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 for infection with HTLV-1 (Nakanishi 2018), 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 (Virgínia M D Wagatsuma, 2011).
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 (member of the TNFR family), and the interaction between FAS and FAS ligand (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) (Enose-Akahata et al. 2017;Virgínia M D Wagatsuma, 2011). Expression of FAS on the surface of CD4 + T cells, which is one of the main targets of infection with the HTLV-1 virus, can be associated with FASL binding with CD8 + T cells as a cell death factor in the course of virus infection (Wang et al. 2010). 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 HTLV-1, but also as an indicator of disease progression toward HAM/TSP (Vallinoto et al. 2012). Additionally, Rosado et al. (2017) 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 (Rosado et al. 2017).
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 (Li et al. 2015). FOXP3 can attach to more than 2800 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 (Lu et al. 2017). 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 (Yamamoto-Taguchi et al. 2013). Previous reports revealed that the number of regulatory T cells was changed by the HTLV-1 virus, and the virus employed this change for further pathogenesis (Pinto et al. 2014;Satou et al. 2012). 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 (Wawrusiewicz-Kurylonek et al., 2018;Wu et al. 2012), it is recommended that further studies be conducted on the association between FOXP3 gene polymorphisms, including -3279 C > A, and infection with HTLV-1.
Interestingly, our data showed a significant association between gender and Foxp3 -3279 C > A and IL-18 -607 C > A gene polymorphism in HTLV-1 carrier group and HAM/ TSP patients, respectively. There are few studies showing the influence of gender on some gene polymorphisms and susceptibility to the diseases. For example, Bolufer et al. 2007, reported that males inherited NAD(P)H: quinone oxidoreductase 1 (NQO1) polymorphism associated with a higher risk of acute leukemia (AL) than females (Bolufer et al. 2007). Additionally, Mei.Bai et al. described that IL-7R rs6451231 was correlated with an increased risk of rheumatoid arthritis (RA) in males, whereas IL-7R rs969129 was reported to be correlated with a higher risk of RA in females (Bai et al. 2019). Due to the unknown reasons, prevalence of HAM/ TSP in women is more than men with more rapid progression in female. Moreover, Matsuura et al. reported that factors such as gender might affect on susceptibility to HAM/TSP disease (Matsuura et al. 2016). Our data regarding the higher frequency of IL-18 -607 C > A gene polymorphism in female patients with HAM/TSP than male is novel and might show the potential effects of gender on the gene polymorphisms and an association with increased susceptibility to the disease.

Conclusion
Overall, the result of our study demonstrated a significant (P < 0.001) association between the inheritance of the GG genotype in CXCL10-1447 A > G polymorphism and the CC genotype in IL-18 -607 C > A, and the risk of susceptibility to HAM-TSP disease. The contradiction between our results and other studies might be related to the sample size, frequency of the genotypes in different races, variations in host-to-host immune responses to the virus, effects of geneto-gene interactions on the disease, and heterogeneity of the population in the city of Mashhad. Additionally, finding of a significant association between gender and IL-18 -607 C > A gene polymorphism in our patients with HAM-TSP may suggest future studies to get a better conclusion regarding the potential effects of gender on the candidate gene polymorphisms in patients with HAM/TSP in Iran and other races in different countries.