ORIGINAL RESEARCH published: 05 May 2020 doi: 10.3389/fimmu.2020.00771 A Polymorphism Within the MBP Gene Is Associated With a Higher Relapse Number in Male Patients of Multiple Sclerosis Laura Espino-Paisán 1* , Teresa Agudo-Jiménez 1 , Isabel Rosales-Martínez 1 , Pilar López-Cotarelo 1 , María Ángel García-Martínez 2 , María Inmaculada Domínguez-Mozo 2 , Silvia Pérez-Pérez 2 , Romina Dieli-Crimi 3 , Manuel Comabella 4 , Elena Urcelay 1† and Roberto Álvarez-Lafuente 2† 1 Laboratorio de Investigación en Genética de Enfermedades Complejas, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Red Española de Esclerosis Múltiple (REEM), Madrid, Spain, 2 Grupo de Investigación de Factores Ambientales en Enfermedades Degenerativas, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos Edited by: (IdISSC), Red Española de Esclerosis Múltiple (REEM), Madrid, Spain, 3 Servicio de Inmunología, Hospital Universitari Vall Amy Lovett-Racke, d’Hebron (HUVH), Diagnostic Immunology, Vall d’Hebron Research Institute (VHIR), Barcelona, Spain, 4 Servei The Ohio State University, de Neurologia-Neuroimmunologia, Centre d’Esclerosi Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d’Hebron United States (VHIR), Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Red Española de Esclerosis Múltiple (REEM), Reviewed by: Barcelona, Spain Bruce V Taylor, University of Tasmania, Australia Bonaventura Casanova, Myelin basic protein (MBP) is thought to be one of the key autoantigens in multiple University and Polytechnic Hospital sclerosis (MS) development. A recent study described the association of the single of La Fe, Spain nucleotide polymorphism (SNP) rs12959006, within the MBP gene, with a higher *Correspondence: Laura Espino-Paisán risk of relapse and worse prognosis. We aim at studying potential associations of lauraep80@gmail.com this SNP to MS in an independent population. Clinical data of the first 5 years of † These authors have contributed the disease were collected retrospectively from 291 MS confirmed patients. MBP equally to this work polymorphism rs12959006 was genotyped in all patients. Associations with EDSS, Specialty section: number of relapses and serology for Herpesvirus 6 (HHV-6) and Epstein Barr (EBV) This article was submitted to viruses were studied. Lymphocyte activation measured by CD69 expression was also Multiple Sclerosis and Neuroimmunology, analyzed according to sex and rs12959006 genotype. The rs12959006 polymorphism a section of the journal contributed significantly to a higher number of relapses at 5 years after onset only in Frontiers in Immunology male patients (rs12959006∗ TT β = 0.74 [0.36–1.09]; p = 7 × 10−5 ). Titers of anti-HHV6 Received: 21 January 2020 Accepted: 06 April 2020 IgG antibodies showed also a mild association with relapses, both in male and female Published: 05 May 2020 patients (β = 0.01 [0.01–0.02]; p = 3.7 × 10−8 ). Both the genetic variation in MBP and Citation: HHV-6 infection aid in predicting a higher number of relapses during the first years of Espino-Paisán L, MS. The association described in MBP rs12959006∗ T is exclusive to male patients. Agudo-Jiménez T, Rosales-Martínez I, López-Cotarelo P, Keywords: multiple sclerosis, SNP, HHV-6, myelin basic protein, relapse, sex differences, genetic marker García-Martínez MÁ, Domínguez-Mozo MI, Pérez-Pérez S, Dieli-Crimi R, Comabella M, Urcelay E and Álvarez-Lafuente R (2020) A INTRODUCTION Polymorphism Within the MBP Gene Is Associated With a Higher Relapse Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease affecting the central Number in Male Patients of Multiple nervous system (CNS) and is one of the primary neurological causes of physical disability in Sclerosis. Front. Immunol. 11:771. young adults (1). The underlying mechanism seems to be an autoimmune response to unidentified doi: 10.3389/fimmu.2020.00771 antigens in the CNS. One of these proposed antigens is the myelin basic protein (MBP), Frontiers in Immunology | www.frontiersin.org 1 May 2020 | Volume 11 | Article 771 Espino-Paisán et al. MBP and Relapse in MS the second most abundant component of the myelin sheath, Clinical data collected included EDSS at 2 and 5 years after and a protein that plays an important role in the myelination onset, relapse in the 30 days prior to EDSS measurement, process. Interestingly, MBP is part of the peptide cocktail used number of relapses at 2 and 5 years after onset, age at disease for inducing experimental autoimmune encephalomyelitis (EAE) onset and disease modifying treatments employed during the in mice, along with proteolipid protein (PLP) and myelin evaluated period. Titer of anti-HHV-6 (IgG and IgM) and anti- olygodendrocyte glycoprotein (MOG) (2). Although MBP has EBV (EBNA-1 and VCA) antibodies, against two viral agents not been proved to be the key autoantigen in MS, MBP-specific related to MS that have been previously associated to disease autoreactive T-cells have been found in blood of MS patients at a course, were also determined. higher rate than in healthy individuals (3). Seventy-one percent of patients received disease modifying Several studies have focused on finding new markers for treatments during the 5 year follow-up, with 28.3% (82 patients) MS diagnosis and prognosis that are cost-effective, reliable, remaining without treatment; among the treated patients, 85.6% easy to implement and as minimally invasive as possible. Many (178) received only first line treatments (interferon-β and/or candidates have been singled out in individual studies (4), but a glatiramer acetate) during the period of study. The remaining necessity exists to replicate these findings in independent cohorts 14.4% (30) patients received a combination of first line and to ascertain the validity and reproducibility of these new potential second line treatments (natalizumab or mitoxantrone, among biomarkers. Genetic markers are especially interesting due to others).There were no statistically significant differences in the their easy determination, stability through time and accessibility groups studied concerning the distribution of treatment options of the sample. Genome-wide association studies (GWAS) (no treatment, first line therapies, combined use of first line have identified more than 200 polymorphisms associated with and second line therapies). To adjust for treatment effect, all susceptibility to MS (5). Additional studies with carefully selected multivariable regression models included two variables: “DMT patients are warranted to find genetic markers that can be used as use,” defined as no treatment versus use of any disease modifying predictive and prognostic biomarkers for MS. treatment in the first 5 years of the disease, and “Onset before A recent study pointed to polymorphism rs12959006 within 2002,” to account for the reduced treatment options and different the MBP gene as a risk modifier of conversion to definite MS therapy approaches before the availability of natalizumab and and progression of the disease (6). The authors studied genetic other second line treatments. The year 2002 was selected because markers in a prospective cohort after a first demyelinating event. a Spanish patient with onset of disease in that year would have the They found that the rs12959006 minor allele T was associated option to be treated with natalizumab at least 1 year in the 5 year with a higher risk of conversion to MS, and a greater hazard follow-up considered for the study. of relapse (HR = 1.74 [1.19–2.56]; p = 0.005) and annualized Viral antibody data were available for a maximum of 227 disability progression (β = 0.18 [0.06–0.30]; p = 0.004). Moreover, patients. In several cases, viral antibody measurement was in this study, the risk genotype carrying the T allele showed performed after the 5-year period considered for evaluation an interaction with baseline titers of IgG antibodies against (mean duration of MS until antibody determination was Human Herpesvirus 6 (HHV-6). We aimed at studying this 7.47 ± 4.78 years). Statistical tests were performed to compare polymorphism and its putative interaction with viral antibodies antibody titers from patients evaluated close to diagnosis and against viruses relevant to MS in an independent cohort of those with a longer evolution of disease and no differences MS-confirmed patients. were found. Despite these results, antibody titers were always adjusted by duration of disease until measurement when used in regression studies. Viral antibodies were determined in serum MATERIALS AND METHODS samples with ELISA commercial kits: ELISA-VIDATEST anti- HHV-6 IgG and IgM (Vidia), Captia EBNA-1 IgG and VCA-EBV Patients IgG (Trinity Biotech), according to the manufacturer’s protocols. A total of 291 patients (66.7% female) were included in the study. Patients were diagnosed with relapsing remitting Genotyping multiple sclerosis (RRMS) between 1991 and 2014 according Patients were genotyped for the MBP polymorphism to the Poser or McDonald criteria (7–10), and followed in rs12959006∗ C > T with Taqman assay C___3079229_20 in the Multiple Sclerosis Unit at Hospital Clinico San Carlos a 7900HT Fast Real-Time PCR system (Life Technologies, (Madrid, Spain). Mean age at onset was 29.40 + 7.78 years. United States). The genotyping success rate was 100%. Medical records of each patient were reviewed and data from the first 5 years after a first demyelinating event were extracted. Patients were selected based on two criteria: the patient was In vitro Activation Analysis and Flow followed in the unit from MS onset and clinical data such Cytometry as relapse and EDSS for the first 5 years of the disease were Peripheral blood mononuclear cells (PBMCs) from 75 patients available. Due to a slightly higher availability of relapse data, were selected and divided in 4 groups according to sex and this variable was chosen as inclusion criterion over EDSS. genotype at rs12959006 (approximately 19 patients per group). Written informed consent was obtained from each patient and Due to the low frequency of the minor homozygote, only major the study was approved by the Ethics Committee of Hospital homozygote and heterozygote patients were included. Treatment Clínico San Carlos. effect was accounted for by including patients with the same Frontiers in Immunology | www.frontiersin.org 2 May 2020 | Volume 11 | Article 771 Espino-Paisán et al. MBP and Relapse in MS treatment in every group. Samples were cultured in RPMI using Poisson regression. The rs12959006∗ TT genotype showed supplemented with 10% FBS, 1% penicillin-streptomycin and a statistically significant association with higher number of 1% L-glutamine (Sigma-Aldrich, Bremen, Germany), without relapses (β = 0.32 [0.07–0.55]; p = 0.008) in all patients. stimulus or treated with 5 µg/ml Concanavalin A (Con A, Surprisingly, when stratified by sex, both rs12959006 genotypes Sigma-Aldrich, Bremen, Germany). Afterward, they were kept carrying the minor allele T showed association only in male at 37◦ C and 5% CO2 for 24 h. Cells were stained with patients (Table 1). anti-CD69-FITC, anti-CD3-PE, and 7-AAD to exclude non- We investigated other variables that correlated significantly viable cells (Biolegend, San Diego, CA, United States). Samples with relapse to build a multivariable model. Out of the four were processed and analyzed in a Cytomics FC500 flow viral serology studies (IgG and IgM for HHV-6 virus and cytometer with Kaluza 2.0 software (Beckman Coulter, Brea, EBNA-1 and VCA for EBV) only IgG anti-HHV-6 antibodies CA, United States) and percentage of CD69+ cells and median showed correlation and were included in further statistical fluorescence intensity (MFI) of CD69+ cells in CD3+ and CD3− analyses, corrected by duration of disease until antibody populations were measured. Final values were calculated by measurement. Use of DMT during the 5 years evaluated extracting the basal CD69 measures (without stimulus) to the was also included, and adjusted for “Onset before 2002” (as samples treated with Con A. detailed in the Methods section), to account for treatment effect. When analyses were re-run, the rs12959006∗ TT genotype Statistical Analysis showed a slightly stronger statistical association with relapse EDSS and number of relapses at 5 years after onset were at 5 years after onset in the general population (β = 0.36 evaluated. Data at 2 years after onset were used to confirm [0.08–0.63]; p = 0.009) (Table 2). In patients stratified by sex, the obtained results at a different time point. For multivariable both rs12959006∗ CT and TT genotypes showed statistically analysis, all variables were analyzed in a correlation matrix with significant association with a higher number of relapses Pearson and Spearman coefficients. Relevant clinical features only in male patients (Table 2), with a stronger effect of that showed significant correlation with the dependent variable the rs12959006 minor homozygote (β = 0.74 [0.36–1.09]; were thus selected, and models were built to check for joint p = 7 × 10−5 ). contribution and to adjust for potential confounding variables. Interestingly, titers of IgG anti-HHV6 antibodies were Influence of rs12959006 in EDSS was analyzed with linear independently associated with higher number of relapses in both regression; influence in relapses, with Poisson regression. Linear male and female MS patients (Table 2). regression analyses were performed with SPSS v15.0.1, Poisson We attempted at replicating these observations with relapse regression analyses with RStudio v1.2.5019, and graphical data of the first 2 years of the disease. Although the representations were carried out with GraphPad Prism V5.01. effect of rs12959006 was weaker, when evaluating carriers of rs12959006∗ T it was observed that the presence of the minor allele in male patients was associated with a higher number of RESULTS relapses (β = 0.30 [0.02–0.57]; p = 0.03) while it showed no effect in female patients (β = −0.05 [−0.12 to 0.80]; p = 0.78) or in the All participants were genotyped for rs12959006 (minor allele general population (β = 0.11 [−0.07 to 0.68]; p = 0.23). T frequency = 0.18). Allele frequencies were concordant For EDSS we built multivariable linear regression models with with those reported for the Iberian population in the 1000 rs12959006 and the other selected variables and no statistically Genomes database. significant contributions were found. We aimed at studying the contribution of rs12959006 To evaluate whether potential alterations in immune cells genotype in relapse and EDSS at 5 years after onset. For correlated with the presence of the rs12959006∗ T allele, we relapse, we analyzed the number of relapses by genotype analyzed activation in PBMCs of MS patients through stimulation TABLE 1 | Poisson regression to test for influence of rs12959006 genotype in number of relapses at 5 years after MS onset. Variables included β(95%CI) p MS overall rs12959006*CC Reference Reference N = 291 rs12959006*CT 0.07 (−0.04 to 0.19) 0.24 rs12959006*TT 0.32 (0.07–0.55) 0.008 MS males rs12959006*CC Reference Reference N = 97 rs12959006*CT 0.21 (0.00–0.41) 0.03 rs12959006*TT 0.68 (0.35–0.98) 1.8 × 10−5 MS females rs12959006*CC Reference Reference N = 194 rs12959006*CT −6.55 × 10−5 (−0.15 to 0.15) 0.99 rs12959006*TT −9.99 × 10−2 (−0.53 to 0.28) 0.63 Coefficients (β) and 95% confidence intervals of the variables included are presented. Major homozygote is set as a reference in each analysis. N: number of patients in each group. Frontiers in Immunology | www.frontiersin.org 3 May 2020 | Volume 11 | Article 771 Espino-Paisán et al. MBP and Relapse in MS TABLE 2 | Poisson regression models for number of relapses at 5 years after MS onset. Variables included β(95%CI) p MS overall rs12959006*CC Reference Reference N = 224 rs12959006*CT 0.08 (−0.06 to 0.21) 0.27 rs12959006*TT 0.36 (0.08–0.63) 0.009 Anti-HHV-6 IgG antibodies 0.01 (0.01–0.02) 3.7 × 10−8 MS duration until antibody measurement 0.02 (0.00–0.03) 0.009 DMT use 0.25 (0.02–0.48) 0.04 Onset of MS before 2002 0.03 (−0.11 to 0.16) 0.68 Males MS rs12959006*CC Reference Reference N = 77 rs12959006*CT 0.26 (0.03–0.48) 0.02 rs12959006*TT 0.74 (0.36–1.09) 7 × 10−5 Anti-HHV-6 IgG antibodies 0.01 (0.00–0.02) 0.02 MS duration until antibody measurement 0.01 (−0.01 to 0.03) 0.44 DMT use 0.63 (0.20–1.07) 0.004 Onset of MS before 2002 −0.15 (−0.40 to 0.09) 0.23 Females MS rs12959006*CC Reference Reference N = 147 rs12959006*CT −0.03 (−0.21 to 0.15) 0.73 rs12959006*TT 0.06 (−0.43 to 0.49) 0.80 Anti-HHV-6 IgG antibodies 0.02 (0.01–0.02) 1.4 × 10−6 MS duration until antibody measurement 0.03 (0.01–0.05) 0.01 DMT use 0.12 (−0.14 to 0.39) 0.37 Onset of MS before 2002 0.09 (−0.07 to 0.26) 0.29 MS duration until antibody measurement was included in each model to adjust for time until viral antibody determination. DMT use reflects the use of a disease modifying treatment in the 5 years studied versus no treatment. “Onset before 2002” was included to adjust “DMT use” for the availability of second line treatments. Coefficients (β) and 95% confidence intervals of the variables included are presented. Mayor homozygote is set as reference (with no effect) in each analysis. N: number of patients in each group. with Con A and study of CD69 expression in CD3+ and Recent studies have pointed to other genetic polymorphisms CD3− cells. Patients were stratified by sex and genotype at that affect MS only in male patients (13, 14) that could rs12959006, and the percentage of positive cells and MFI explain part of the difference in prevalence and course of of CD69 were studied. Due to the low frequency of the MS between sexes. rs12959006∗ TT genotype, only the heterozygote and major We observe a trend for a lower expression of CD69 in activated homozygote were analyzed. A trend for a lower expression of CD3+ cells in male patients carrying rs12959006∗ T allele. Several CD69 in male patients carrying rs12959006∗ T was observed, studies associate the blocking or lower expression of CD69 in although it was not statistically significant (female CT vs. male T cells with an exacerbation of autoimmune diseases (15, 16) CT p = 0.09; Figure 1). and a higher induction of pro-inflammatory Th17 cells in mice (17). Moreover, CD69+ T regulatory cells have been reported to have higher immunosuppressive potential in the mouse model DISCUSSION of colitis (18). A lower expression of CD69 in T cells could be related to a defective control of inflammation and a worse Our study aims at analyzing the putative associations of the evolution of autoimmune diseases. In the light of previous MBP polymorphism rs12959006 with MS. A previous study knowledge, it would be interesting to replicate the observed trend reported associations of this polymorphism with conversion to in rs12959006∗ CT patients and to extend it to the low-frequency MS and worse prognosis in these patients (6). The authors rs12959006∗ TT genotype. studied an Australian cohort of 127 patients with a first We observe that levels of anti-HHV6 IgG antibodies are demyelinating event compatible with MS. Patients were followed positively associated with number of relapses. Other studies for 5 years. Only 68 patients developed confirmed MS within (19, 20) have previously described associations between high or the duration of the study and they experienced a total of increasing anti-HHV-6 antibody titers and risk of relapse, and 152 relapses versus the 1292 relapses registered in our study even pointed to a possible predictive value of anti-HHV-6 IgG group of 291 MS-confirmed patients. Our analysis benefits from and IgM levels (19). An elevated antibody titer could suggest a higher statistical power. We describe an association between stronger immunological response to HHV-6 infection. Previous rs12959006 and risk of a higher number of relapses at 2 and studies compared the molecular structure of MBP and HHV-6 5 years after MS onset. Interestingly, we observe this association proteins. MBP and peptide U24 of the HHV-6 viral particle share only in male MS patients. MS presents sex differences, being an identical sequence of 6 aminoacids (MBP residues 96–102, more prevalent in females but more severe in males (11, 12). HHV-6 U24 residues 4–10), pointing at a possible contribution of Frontiers in Immunology | www.frontiersin.org 4 May 2020 | Volume 11 | Article 771 Espino-Paisán et al. MBP and Relapse in MS FIGURE 1 | Expression of CD69 in CD3+ cells measured as MFI according to sex and genotype at rs12959006. (A) Dot plot showing the selection of CD3+ CD69+ lymphocytes. (B) Histogram showing the selection of CD3+ CD69+ population for MFI measurement. (C,D) Statistical comparison of CD69 expression in CD3+ cells, measured as median fluorescence intensity (MFI), between the four groups studied (C) and all groups vs. male CT (D). Median of each group is represented. viral infections to MS risk through molecular mimicry. A classic ETHICS STATEMENT study (3) showed that T-cells cross-reactive to both MBP and HHV-6 were more abundant in MS patients than in healthy The studies involving human participants were reviewed controls, an observation that was replicated in a more recent and approved by the Comité Ético de Investigación study in Chinese population (21). These studies point to the idea Clínica, Hospital Clínico San Carlos, Madrid (Spain). The that the joint effect of HHV-6 infection and its molecular mimicry patients/participants provided their written informed consent to with MBP could be important in MS pathogenesis. participate in this study. For future research, it would be interesting to replicate these observations in a group with baseline anti-HHV-6 IgG antibody measurements and in the context of a prospective study to AUTHOR CONTRIBUTIONS determine the usefulness of rs12959006 in clinical practice. LE-P, TA-J, IR-M, PL-C, MG-M, MD-M, SP-P, and RD-C Our study, and those from others, consolidates the performed the experiments and collected the clinical data. LE-P, contribution of rs12959006, within the MBP gene, as a marker MC, EU and RÁ-L designed the experiments, analyzed the data, to predict MS activity. Furthermore, according to our results, and drafted the manuscript. there seems to be an influence of both the genetic polymorphism in MBP and HHV-6 infection in predicting a higher relapse rate that is exclusive to male MS patients. FUNDING This work was supported by the projects PI15/00821 and DATA AVAILABILITY STATEMENT PI16/01259, integrated in the Plan National de I+D+I, AES 2013-2016; funded by the ISCIII and co-funded by the All datasets generated for this study are included in the European Regional Development Fund (ERDF) “A way to article/supplementary material. make Europe”; “Fundación Ramón Areces” (CIVP18A3860) Frontiers in Immunology | www.frontiersin.org 5 May 2020 | Volume 11 | Article 771 Espino-Paisán et al. MBP and Relapse in MS and “Fundación LAIR”. LE-P and MG-M are recipients ACKNOWLEDGMENTS of a contract from “REEM: Red Española de Esclerosis Múltiple” (RETICS-REEM RD16/0015/0013; www.reem.es). SP- The authors wish to thank the “Unidad de apoyo metodológico P holds a research contract from the program “Formación de a la investigación” (UAMI) of the Instituto de Investigaciones Profesorado Universitario” – Ministerio de Educación, Cultura Sanitarias San Carlos (IdISSC) for their expert assistance in y Deporte” (FPU16/00969). statistical analysis. REFERENCES 14. Benedek G, Meza-Romero R, Jordan K, Zhang Y, Nguyen H, Kent G, et al. MIF and D-DT are potential disease severity modifiers in male MS subjects. 1. Reich DS, Lucchinetti CF, Calabresi PA. Multiple sclerosis. N Engl J Med. Proc Natl Acad Sci USA. (2017) 114:E8421–9. doi: 10.1073/pnas.1712288114 (2018) 378:169–80. 15. Gonzalez-Amaro R, Cortes JR, Sanchez-Madrid F, Martin P. Is CD69 an 2. Rangachari M, Kuchroo VK. Using EAE to better understand principles of effective brake to control inflammatory diseases? Trends Mol Med. (2013) immune function and autoimmune pathology. J Autoimmun. 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Genetic variant rs755622 regulates expression or reproduction in other forums is permitted, provided the original author(s) and of the multiple sclerosis severity modifier D-Dopachrome tautomerase in a the copyright owner(s) are credited and that the original publication in this journal sex-specific way. Biomed Res Int. (2018) 2018:8285653. doi: 10.1155/2018/ is cited, in accordance with accepted academic practice. No use, distribution or 8285653 reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 6 May 2020 | Volume 11 | Article 771