The Effects of the MTHFR 677C>T (rs1801133) Genetic Variant on Susceptibility and Disability in Multiple Sclerosis Patients are Mediated by Homocysteine but Not Folate Levels

We investigated whether the MTHFR 677C>T (rs1801133) variant and plasma homocysteine and folate are associated with multiple sclerosis (MS), disability, disability progression, and inammatory biomarkers. We included 163 MS patients categorized using the Expanded Disability Status Scale (EDSS) as mild (EDSS<3) and moderate/high (EDSS ≥ 3) disability, and 226 healthy controls. Disability progression was evaluated using Multiple Sclerosis Severity Score (MSSS) and the MTHFR 677C>T was genotyped using real time polymerase chain reaction. The levels of some inammatory biomarkers and inammatory activity index (IAI) were determined. There was no association between the MTHFR 677 C>T genotypes and MS, EDSS, and MSSS (p>0.05). Plasma folate and homocysteine were higher and adiponectin was lower in MS patients than controls (p<0.001). Moreover, 21.8% of the EDSS variance was explained by age, IAI and C-reactive protein (CRP) (all positively associated); 10.9% of the MSSS variance was predicted by IAI and CRP (both positively) and vitamin D3 (negatively), whereas 54.4% of the MS-EDSS-MSSS score was explained by the regression on age, IAI, homocysteine, folate, and CRP (all positively) and adiponectin, body mass index, and vitamin D3 (all negatively), female sex and the MTHFR 677 TT genotype. In patients and controls, 16.6% of the variance in the homocysteine was explained by the MTHFR 677 TT genotype and age (both positively), folate (negatively) and male sex. In conclusion, the MTHFR 677C>T variant was not directly associated with MS, disability, and disability progression; however, the TT genotype showed indirect effects on MS susceptibility and disability mediated by homocysteine. subjects [8, 29, 30]. We previously demonstrated that homocysteine levels are higher in MS patients than in controls, as well as higher among those with moderate/high disability than those with mild disability [8]. Discrepancies on the correlations between MS and plasma levels of homocysteine, vitamin B12, and folate have been reported. While some studies showed higher levels of homocysteine and lower levels of vitamin B12 and folate in MS patients compared to controls [30, 31], others reported no differences between patients with MS and controls in the levels of homocysteine [32–34], vitamin B12 or folate [32]. the (rs1801133) variant plasma levels of and folate associated with MS susceptibility, disability, disability progression, and inammatory biomarkers. Behring, Deereld, IL, USA). C-reactive protein (CRP) determined with high sensitivity assay (hsCRP) using turbidimetry (Architect C8000, Abbott Abbott Park, IL, USA), and plasma levels of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, soluble TNF-α receptor (sTNFR)1 and sTNFR2 were determined using immunouorimetric method with microspheres multiplex immunoassay (Novex Life Technologies, Frederick, USA) for Luminex platform in MAGPIX® instrument (Luminex Corp., TX, USA). macrophage cytokines cytokines and IFN-γ) cytokines (IL-6 and regulatory (Treg) cytokines (IL-4 and IL-10); namely as TNF-α and its was as a latent from TNF-α sTNFR1+sTNFR2. while checking R 2 changes, homoscedasticity (using White and modied Breusch-Pagan tests for homoscedasticity), multicollinearity (using tolerance and VIF), and multivariate normality (Cook’s distance and leverage). Results of multiple comparisons were p-corrected for false discovery rate (FDR) [42]. Automatic binary logistic regression analysis was conducted with MS or MS subgroups as dependent variables and the biomarkers as input variables. Odds ratios (OR) with 95% condence intervals (CI) and the accuracy of classication (with sensitivity and specicity) were computed and Nagelkerke’s peudo-R 2 values were used as effect size measurement. The results of these regression presented mild disability (EDSS <3) and 83 (58.9%) presented moderate/high disability (EDSS ≥ 3). Univariate analysis showed that MS patients with moderate/high disability were older and showed a higher frequency of MetS than those with mild disability and HC (p<0.001). Patients with moderate/high disability showed a higher frequency of SAH and T2DM than HC (p=0.011 and p=0.049, respectively). There were no signicant differences regarding sex, BMI, ethnicity and smoking between the three study groups. Patients with moderate/high disability showed higher duration of disease and disability progression than those with mild disability (p<0.001). There were signicantly more patients with progressive clinical forms (SPMS+PPMS) in the patient group with EDSS ≥ 3 as compared with the EDSS<3 group. 677C>T methylenetetrahydrofolate reductase genetic variant with C as the major allele and T as the minor allele: recessive model: CC + CT vs. TT; additive model: CC=-1; CT=0; TT=+1 prediction-oriented segmentation with multi-group analysis. This gure shows only the signicant pathways. The overall t of this model was adequate with SRMR=0.019. Moreover, the construct reliabilities of both latent vectors were adequate, namely Cronbach α > 0.9, rho A and composite reliability > 0.936, and AVE > 0.78. The outer model loadings on both latent vectors were all > 0.82 with p<0.0001. Blindfolding showed that the construct cross-validated redundancies were adequate, namely the MS latent vector 0.099 and IAI 0.480. We found that 56.0% of the variance in MS-EDSS-MSSS latent vector was explained by the direct effects of adiponectin and uric acid (both negatively), homocysteine, folate, CRP, and age (all positively). Furthermore, 33.8% of the variance in uric acid was explained by age, sex and BMI, and 20.2% of the variance in CRP by sex and BMI. Our results are in agreement with previous studies which found no association between the MTHFR 677C>T variant and MS susceptibility [44–47]. The role of the MTHFR 677C>T variant in the pathophysiology of MS has been evaluated in some genetically specic worldwide populations with inconsistent results. While three case-control studies [48–50] demonstrated an association between the T allele with MS, others failed to conrm this association [44–47]. Tajouri et al. genotyped the MTHFR 677C>T variant among Australian MS patients and unaffected control subjects, matched for sex, age and ethnicity and, although the TT homozygous genotype was slightly over-represented in the MS group than controls, the difference failed to reach statistical signicance and, according to these authors, their result could not support a major role for this functional gene variation in MS susceptibility [47]. Further, Cevik et al. found that the T allele of MTHFR 677C>T variant was associated with MS susceptibility [48]. Naghibalhossaini et al. reported that the CT genotype of MTHFR 677C>T showed a higher risk of MS incidence for both cases with the recessive (TT vs. CT + CC) and codominant (CT vs. CC) pattern of inheritance in comparison with controls [49]. These results point to the need for additional studies involving individuals from other world populations. relapsing-remitting (RRMS) patients and controls but difference in homocysteine between patients with or PPMS and controls [51]. responses as a major factor in the pathophysiology of MS [83, 84, 85]. We also showed, in previous studies, increased TNF-α, IL-17, and IFN-γ in MS patients than in controls [86]. Another study showed that the change in EDSS during a follow-up of 16 months was associated with changes in IL-17 (positively) and IL-4 (negatively) independently from the clinical MS forms, treatment modalities, smoking, age and systemic arterial hypertension. This study also showed that, in addition to homocysteine, IL-6 and IL-4 levels were positively associated with progressive forms of MS vs RRMS while 25(OH)D was negatively associated [87].


Introduction
Multiple sclerosis (MS) is a chronic in ammatory disease characterized by the demyelination of the central nervous system (CNS), often followed by progressive and irreversible neurological dysfunction. The etiology of MS has been not yet well de ned but interactions between genetic and environmental factors have been recognized to contribute to the autoimmune in ammatory process [1]. De ciency of vitamin D, folate and vitamin All MS patients were in the remission clinical phase, de ned as the period of recovery with no relapse episodes within the last three months prior to the time of enrollment in the study. As controls, 226 healthy individuals (HC) were selected among blood donors of the Regional Blood Bank of Londrina, from the same geographic region of the MS patients. None of the participants of the study presented clinical symptoms or laboratory biomarkers of heart, thyroid, kidney, hepatic, gastrointestinal, or oncologic diseases, as well as other in ammatory and autoimmune diseases.
Demographic, epidemiological and anthropometric data (for MS patients and HC), as well as clinical history and the use of therapy for MS before the inclusion in this study (for MS patients) were obtained using a standard questionnaire at the admission of the individuals. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared and the ethnicity was classi ed according to individual's self-perception of skin color as Caucasian and non-Caucasian [39]. Other data were obtained including waist circumference, current smoking, systemic arterial hypertension (SAH), metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured twice and the mean of these two measurements was used in the analysis. Moreover, use of antihypertensive medication was an indication of SAH [40]. T2DM was de ned as a fasting serum glucose ≥126 mg/dL and/or use of hypoglycemic medication [41].

Blood samples
Peripheral blood samples were drawn after 12 h of fasting using vacuo system tubes (Vacutainer System, Becton-Dickinson, New Jersey, U.S) with and without ethylenediamine tetra-acetic acid (EDTA) as anticoagulant. The specimens were centrifuged at 2,500 rpm for 10 minutes within 2 hours after sampling. The buffy-coat, plasma and sera were frozen at -80 o C until analysis.

MTHFR 677C>G Genetic Variant Genotyping
Genomic DNA was extracted from the buffy-coat of peripheral blood cells using a resin column procedure (Biopur, Biometrix Diagnostika, Curitiba, Brazil) following the manufacturer's instructions. DNA concentration was measured using a spectrophotometer at 260 nm (NanoDrop 2000c™, ThermoScienti c, Waltman, MA, USA) and the DNA purity was assessed by 260/280 nm ratio. The MTHFR 677C>T (rs1801133) variant was determined using TaqMan® allelic discrimination validated assay on real-time polymerase chain reaction (qPCR) system (StepOne, Applied Biosystems by Life Technologies, Carlsbad, CA, USA) with the allele-speci c uorogenic oligonucleotide probe (C___1202883_20). The reaction was performed using 5 ng of genomic DNA, 0.25 µL TaqMan SNP Genotyping Assay 40x (Applied Biosystems, Foster City, CA, USA) containing two sequence-speci c primers and two allele-speci c TaqMan® MGB probes with a reporter dye at its 5' end (VIC® or FAM™), 5 µL TaqMan Universal Genotyping Master Mix (Applied Biosystems, Foster City, CA, USA). Negative and positive controls were also included in the reactions.
analyses were always bootstrapped using 5.000 bootstrap samples and the latter results are shown if the results are not concordant. All tests are two-tailed and a p-value of 0.05 was employed to determine statistical signi cance. Statistical analyses were carried out using IBM SPSS Windows version 25, 2017.
Partial Least Squares (SmartPLS) analysis [43] was used to measure the multi-step multiple mediation associations between biomarkers (input variables) and the MS-severity index. The latter was entered as a latent vector extracted from the EDSS and MSSS scores and the diagnosis MS. All biomarkers were entered as single indicators except IAI, which was entered as a latent vector extracted from different immune pro les. Both latent vectors were entered as re ective models. Consequently, complete PLS path analysis using 5.000 bootstrap samples was performed only when the outer and inner models complied with pre-speci ed quality data: a) all outer model loadings on both latent vectors are > 0.7 at p<0.001 and all latent vectors show good construct validity as indicated by Cronbach's alpha (> 0.7), composite reliability (> 0.7), rho A (> 0.8), and average variance extracted (AVE) > 0.5; b) the overall t of the model is adequate as indicated by Standardized Root Mean Squared Residual (SRMR) < 0.08; and c) Con rmatory Tetrad analysis indicates that both latent vectors models are not mis-speci ed as re ective models. PLS predict with 10-fold crossvalidation was used to assess the predictive performance when analyzing new data. Predicted-Oriented Segmentation analysis, Multi-Group Analysis and Measurement Invariance Assessment were employed to examine compositional invariance.

Results
Sociodemographic and clinical data Table 1 shows the sociodemographic and clinical data of the HC and MS patients divided into those with and without an increased EDSS score (≥3 as threshold value). This table presents the data on all HC and the selected group of MS patients who were not treated with folic acid (n=141) because all computations (except the genotypic associations) are calculated using this data set. Among these 141 MS patients, 58 (41.1%) presented mild disability (EDSS <3) and 83 (58.9%) presented moderate/high disability (EDSS ≥ 3). Univariate analysis showed that MS patients with moderate/high disability were older and showed a higher frequency of MetS than those with mild disability and HC (p<0.001). Patients with moderate/high disability showed a higher frequency of SAH and T2DM than HC (p=0.011 and p=0.049, respectively). There were no signi cant differences regarding sex, BMI, ethnicity and smoking between the three study groups. Patients with moderate/high disability showed higher duration of disease and disability progression than those with mild disability (p<0.001). There were signi cantly more patients with progressive clinical forms (SPMS+PPMS) in the patient group with EDSS≥3 as compared with the EDSS<3 group. Biomarker data in MS subgroups Table 2 shows the biomarker assessment in HC and MS patients divided into two subgroups using the EDSS score. Both the HC group (χ 2 = 0.01, df=1, p=0.907) and total study group (χ 2 = 0.15, df=1, p=0.698) were in Hardy-Weinberg equilibrium regarding the MTHFR allelic frequencies. The rst part of the table (computed on the total sample) shows that there were no signi cant associations between these diagnostic groups and the MTHFR phenotypes using different genetic models. The second part of the table (calculated on the selected study groups) shows the measurements of the non-genetic biomarkers in the study groups. We found that folate and homocysteine were signi cantly higher in MS patients than in HC, whereas adiponectin was reduced in both patient groups. There were no signi cant differences in vitamin D3 and uric acid between the three groups. CRP was signi cantly higher in patients with an EDSS score ≥3 versus those with EDSS <3. The M1, Th1, Th17, and Th2 Treg cytokine values, named IAI, were signi cantly different between the three study groups and increased from HC to MS patients with EDSS<3 and to MS patients with EDSS≥3. The composite score of the levels of TNF-α and their soluble receptors sTNFR1 and sTNFR2 was signi cantly higher in MS patients with EDSS≥3 than in the two other groups (EDSS<3 and HC). These differences remained signi cant after FDR p-correction.   Table 4 shows the results of multiple regression analyses with the MS-EDSS-MSSS scores as dependent variables and all biomarkers as explanatory variables while allowing for the age, sex, BMI, SAH, and T2DM. We found that 21.8% of the variance in the EDSS score in MS patients was explained by age, IAI and CRP (all positively associated). IAI and CRP (both positively) and vitamin D3 (negatively) predicted 10.9% of the variance in the MSSS score. Not one of the genotypic MTHFR 677C>T models was signi cant in this regression model. The third multiple regression in Table 4 shows that 54.4% of the variance in the MS-EDSS-MSSS score was explained by the regression on age, IAI, homocysteine, folate, and CRP (all positively associated) and adiponectin, BMI, and vitamin D3 (all negatively associated), female sex and the MTHFR TT genotype. Prediction of homocysteine and folate using MTHFR 677C>T genotypes Table 5 shows the results of multiple regression analyses with homocysteine as dependent variable and folate and MTHFR genotypes as explanatory variables while allowing for the effects of age, sex, and BMI. In the total study group, we found that 16.6% of the variance in homocysteine was explained by the MTHFR TT genotype and age (both positively) and folate (negatively) and male sex. In the HC group, the same variables predicted 15.9% of the variance in homocysteine levels. In the total study group, 4.5% of the variance in folate levels was predicted by the MTHFR additive model (negatively) and age (positively). We found that 11.7% of the variance in the IAI was explained by the regression on adiponectin and age (both negatively) and folate (positively). The MTHFR genotypic models were not signi cant in this regression.

Results of PLS analysis
The associations between the MTHFR genotypic models and homocysteine with folate as putative mediator (while allowing for the effects of age, sex, and BMI) were rstly examined in the HC group. Figure 1 shows the results of complete PLS path analysis conducted on 5.000 bootstrap samples. We found that 15.9% of the variance in homocysteine was explained by the TT genotype, age, and folate (all positively) and male sex. In addition, 5.7% of the variance in folate was predicted by the additive MTHFR genotype. Most importantly, the speci c indirect effect of this additive model on homocysteine mediated by folate was not signi cant (t=1.64, p=0.14). Folate partly mediated the effects of age on homocysteine (t=2.45, p=0.014). All in all, there were signi cant direct effects of the TT genotype, but not any of the other genotypes, on homocysteine.
Consequently, we have examined the same associations in the total study group after entering the other biomarkers of MS, namely IAI (entered as a latent vector extracted from the M1, Th1, Th17 and Th2Treg cytokine pro les), adiponectin, uric acid, age, sex, CRP, BMI (all entered as single indicators). The nal outcome variable, named MS-EDSS-MSSS, was a latent vector extracted from MS, EDSS, and MSSS, which thus is an index of MS and its severity. We used a multi-step multiple mediation model whereby the effects of the MTHFR genotypes (recessive and additive models), age, sex and BMI could be explained by the biomarkers. Figure 2 shows the PLS path model using complete PLS path analysis on 5.000 bootstrap samples and after feature selection, PLS predict analysis, prediction-oriented segmentation with multi-group analysis. Predict values of all endogenous construct indicators were positive indicating that they outperform the most naïve benchmark (the prediction error is smaller than the error of the most naïve benchmark).

Discussion
The main result of the present study is that the MTHFR 677C>T variant exerts indirect effects on MS susceptibility through the increased levels of homocysteine (but not folate) associated with the TT genotype in a recessive model. In fact, homocysteine levels exert a direct effect on the in ammatory response expressed as the IAI index and thus on the MS disability progression. Another important result is that the MTHRF 677C>T genotypes did not show a direct effect on the disability as well as disability progression of the MS patients. Other biomarkers showed an effect on the EDSS and MSSS scores, such as age, IAI, and CRP. These biomarkers, together, exerted a strong effect on the disability, as assessed with the EDSS. Moreover, a panel of the IAI, CRP, and vitamin D3 biomarkers exerted a modest effect on disability progression. On the other hand, the MTHFR 677C>T genotype had a partial effect on the MS diagnosis, disability and disability progression. In fact, other variables were also involved in these clinical biomarkers of the MS.

MTHFR C677T and multiple sclerosis and homocysteine
Our results are in agreement with previous studies which found no association between the MTHFR 677C>T variant and MS susceptibility [44][45][46][47]. Different factors may account for these apparent con icting results. First, the heterogenic clinical characteristics of the MS as different groups of patients may have various genetic factors which predispose to the disease. Second, some alleles of candidate genes may be strongly associated with the disease in one population, whereas in another this association may be weak or not observed due to the presence of other genetic factors or genetic by environment interactions, including smoking, diet, and lifestyle habits [51]. Third, MS development cannot be predicted based on genotype alone because even the strongest major histocompatibility complex (MHC) class II-linked risk genes for MS are incompletely penetrant [52]. These authors a rm that the incomplete penetrance of MS susceptibility alleles probably re ects interactions with other genes, post transcriptional regulatory mechanisms, and signi cant nutritional and environmental in uences. Therefore, modi able environmental exposures may determine whether MS develops in individuals who carry risk genes. Fourth, con icting results about the association between MTHFR and MS could be explained by the study design, the sample size of the study, the time points of testing the biomarkers and, technically, by different laboratory methods for the measurement of the homocysteine and folate, as well as for the MTHFR genotyping analysis.
In the present study, our results showed that in all subjects 16.6% of the variance of homocysteine was explained by the TT genotype independently from age, folate, and sex. Therefore, higher homocysteine levels were observed among the patients carrying the TT genotype than those carrying the CC + TT genotype (recessive model). The association between the MTHFR 677C>T variant and high levels of homocysteine is well established in the general population [53,54,55]

Homocysteine and folate levels in multiple sclerosis
Our results of the present study showed that MS patients had higher levels of homocysteine and folate than controls. Several studies have focused on the role of homocysteine, vitamin B12, and folate as possible participants in the neurodegenerative process [59]. Previous studies have reported increased plasma levels of homocysteine in MS patients when compared to controls [8, 29,30]. We previously demonstrated that homocysteine levels are increased in MS patients, and that hyperhomocysteinemia was associated with disease progression evaluated by the MSSS [8]. Li et al., in a meta-analysis, showed that patients with MS had higher levels of homocysteine compared with controls and that there were no signi cant differences for vitamin B12 or folate levels between MS and controls. In a subgroup analysis, these authors demonstrated that there was a signi cant difference in homocysteine between relapsing-remitting MS (RRMS) patients and controls but no signi cant difference in homocysteine between patients with SPMS or PPMS and controls [51].
In agreement with previous studies, we found that high levels of homocysteine were positively associated with MS and these ndings underscore that hyperhomocysteinemia may be a risk factor of this neuroin ammatory disease [7,10]. Studies have shown that high levels of homocysteine are toxic to neural cells and cause neuronal damage by several mechanisms. First, homocysteine may sensitize neurons to oxidative stress via oxidation of sulfhydryl groups subsequently resulting in generation of ROS, such as superoxide and hydrogen peroxide [11,60]. Another major nding of this study is that adiponectin, age, sex, BMI, MTHFR 677C>T variant, homocysteine, vitamin D3, folate, and CRP explain 54.4% of the variance in MS and its severity. The latter index combinates the diagnosis of MS, EDSS, and MSSS into a composite score, which re ects MS and its severity, including disabilities and disability progression. Previous papers showed inconsistent results regarding folate levels in MS. Some studies showed decreased levels of folate in MS patients compared to controls [29,30] while other studies reported no differences between patients with MS and controls [32,67,68]. Meta-analysis studies showed no signi cant difference in folate levels between MS patients and controls [7,51,59]. However, two of these meta-analysis [7,59] failed to take some critical factors into account, including sex, age, disease phase and/or severity, and/or ethnicity of study populations. A case-control and meta-analysis showed no signi cant difference in levels of folate and in the frequency of folate de ciency in MS patients compared to controls [9].
Several previous studies have investigated the roles of homocysteine, vitamin B12, and folate in MS since myelin replacement requires normal function of folate-vitamin B12 methylation pathway, which is vital to provide methyl groups for myelin regeneration [69]. Folate and vitamin B12 are needed in the process of methionine-synthase mediating the conversion of homocysteine to methionine. Both 5-methyltetrahydrofolate and methylvitamin B12 are essential factors for methionine synthesis of homocysteine [70]. It is well established that the de ciency of vitamin B12 and folate may cause increased homocysteine. Previous studies included in the meta-analysis [51] showed high heterogeneity, which could have a certain impact on the apparent discrepant results regarding the folate and MS. In addition, some studies have explicitly restricted that subjects had not received folic acid supplementation, while others did not control the effects of confounding factors such as diet and drugs, which may interfere with the association between folate and MS.
In contrast with some previous studies, we observed high levels of folate in MS as compared with controls [29,30]. There are three possible explanations. First, MS patients could have a lower expression of the folate receptor (FR)-β than controls. Healthy cells acquire their folate (or folic acid as supplement) using reduced folate carriers and/or the proton-coupled folate transporter, which are needed for normal cell survival and proliferation. However, during in ammation, folate uptake by activated macrophages is mediated primarily by the beta isoform of FR (FR-β) which exhibits approximately 1000 higher a nity for folate than the reduced folate carrier [71,72]. A recent study [73] showed that macrophages express FR-β during the active phase of MS, according to animal experiments and tissue autopsy from MS patients versus controls. Because all our MS patients were in the remission phase of the disease, there would be a lower expression of FR-β in the cells and, therefore, greater availability of folate in the circulation than controls. In addition, macrophages expressing functional FR-ß are present in both CNS and peripheral sites of in ammation [74].
The second hypothesis is that increased homocysteine may reduce the expression of human FR-α. These authors propose that low levels of homocysteine increase FR-α expression and that high doses have an opposite effect. Since our MS patients show high levels of homocysteine, we may assume that they have a decreased expression of FR-α, with less internalization of folate into the cells, resulting in increased levels in the circulation. The third hypothesis is that the MTHFR 677C>T variant may be associated with the production of autoantibodies against FR [75]. The latter authors observed that plasma levels of FR autoantibodies in women with the TT genotype at MTHFR 677C>T were signi cantly higher than those of women with the CC genotype [76,77]. By inference, since the frequency of the TT genotype was higher among the MS patients than controls, folate transport could be impaired. Although these three aforementioned hypotheses may have some reasonableness, the present study did not allow us to elucidate the precise mechanism of high levels of folate were found in patients with MS compared to controls.
In ammation, homocysteine, MS disability and disability progression Regarding the combination of a panel of biomarkers associated with MS, our results showed that adiponectin levels and sex male were negatively associated with MS, while folate and homocysteine, IAI and age were positively associated with MS. Studies have identi ed a strong association between high levels of homocysteine and in ammation in both human and experimental models [78,79]. An experimental study showed that high levels of homocysteine induce in ammatory responses in mouse brain through of the activation of microglial cells and increased expression of proin ammatory cytokines such as IL-1β and TNF-α [80]. Previous study reported association between homocysteine and in ammation that include mechanisms the expression of adhesion molecules, leukocyte adhesion, endothelial dysfunction, oxidative stress, and reduced nitric oxide bioavailability [81].
Human and experimental studies showed that MS is not governed by a particular cytokine but instead involves a complex interplay between proand anti-in ammatory cytokines [82]. Taken this into account, our study evaluated a broad in ammatory and anti-in ammatory cytokine pro le, expressed as the IAI, a score computed as rst principal component extracted from the main cytokines produced by M1, Th1, Th17, Th2, and Treg Adiponectin is the most abundant anti-in ammatory adipokine in the plasma and regulates the pro-in ammatory nuclear factor kappa B (NF-kB) signaling pathway [88], decreases the expression of the pro-in ammatory cytokines TNF-α, IL-6, and IFN-γ, and increases the expression of antiin ammatory molecules, such as IL-10 and IL-1 receptor antagonist (IL-1Ra) [89], underscoring its role in the modulation of the in ammatory response in MS [90].
We found that age, IAI, and CRP were associated with moderate/high disability in MS (EDSS ≥ 3). Moreover, IAI and CRP were positively associated with disability progression (MSSS), whereas vitamin D3 was negatively associated. These results are in agreement with previous studies [87, 91,92] showing that vitamin D3 de ciency is associated with progression of disability in MS patients. Vitamin D3 exerts important immunomodulatory effects and has been associated with the regulation of in ammatory response [91,93], for example, by inhibiting the NF-kB pathway [94], downregulating pro-in ammatory cytokines, such as TNF-α, IL-6, IL-12, and IFN-γ, and upregulating anti-in ammatory Treg and Th2 cells and their cytokines [95]. These ndings underscored that several mechanisms underlie disease progression independent of relapses. Although all the MS patients were in remission clinical phase of the disease, we observed that the disease progression is occurring. Although are available at least 12 drugs approved as disease-modifying treatments for MS, the major challenge for the clinicians is the identi cation, at the disease onset, of the subjects most likely to develop an aggressive, quickly progressing form of the disease in order to start with high-impact treatments before severe disability builds up. At the same time, patients with mild forms should avoid overtreatment, with substantial bene ts in terms of safety, quality of life and overall allocation of resources [96,97].

Conclusion
Taken all together, these ndings provide important insights into the role of the MTHFR 677C>T, homocysteine, folate and in ammatory response in the underlying pathophysiological mechanisms of MS. Although the MTHFR 677C>T variant was not directly associated with MS, disability, and disability progression, the TT genotype exert indirect effects on MS susceptibility and disability mediated by homocysteine but not folate these results underscore the complex interaction between the genetic factors, with in ammatory and anti-in ammatory mechanisms in the susceptibility, disability and disability progression of MS and suggest other possible new targets that may contribute to outline the concept of precision medicine to the MS management and improved care of the patients.

Declarations Funding
The study was supported by Coordination for the Improvement of Higher Level of Education Personnel (CAPES) of Brazilian Ministry of Education: Finance Code 001.

Con ict of interest
There is no con ict of interest to declare. None of the authors are involved in the publication process or have a nancial or other bene cial interest in the products or concepts mentioned in the submitted manuscript.
Availability of data and material The data and materials are available.

Standards for reporting
The manuscript was prepared taken into account the recommendations of the guidelines hosted by the Strengthening the Reporting of Observational studies in Epidemiology (STROBE). STROBE is used for observational studies (cohort, case-control, or cross-sectional designs) according to the STROBE statement (www.strobe-statement.org)

Consent to participate
Informed consent was obtained from all individual participants included in the study.

Consent for publication
The studied participants were informed about the present research, and a written consent form was taken from all of them before their enrollment.
Moreover, all the authors and co-authors participated and contributed su ciently in the research, and all of them concur with the submission. The manuscript has been approved by the responsible authorities where the work was carried out. The authors also concur that, if accepted, the manuscript shall not be published elsewhere in the same form in either the same or any other language, without the consent of the Editor-in Chief of Molecular Neurobiology.