pd 1 gene polymorphic variation is linked with first symptom of disease and severity of relapsing remitting form of ms

Accepted Manuscript PD-1 gene polymorphic variation is linked with first symptom of disease and severity of relapsing-remitting form of MS Edyta Pawlak-Adamska, Oskar Nowak, Lidia Karabon, Anna Pokryszko-Dragan, Anna Partyka, Anna Tomkiewicz, Jakub Ptaszkowski, Irena Frydecka, Ryszard Podemski, Jaroslaw Dybko, Malgorzata Bilinska PII: DOI: Reference: S0165-5728(16)30316-2 doi: 10.1016/j.jneuroim.2017.02.006 JNI 476522 To appear in: Journal of Neuroimmunology Received date: Revised date: Accepted date: 14 October 2016 12 January 2017 February 2017 Please cite this article as: Edyta Pawlak-Adamska, Oskar Nowak, Lidia Karabon, Anna Pokryszko-Dragan, Anna Partyka, Anna Tomkiewicz, Jakub Ptaszkowski, Irena Frydecka, Ryszard Podemski, Jaroslaw Dybko, Malgorzata Bilinska , PD-1 gene polymorphic variation is linked with first symptom of disease and severity of relapsing-remitting form of MS The address for the corresponding author was captured as affiliation for all authors Please check if appropriate Jni(2017), doi: 10.1016/j.jneuroim.2017.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT PD-1 gene polymorphic variation is linked with first symptom of disease and severity of relapsing-remitting form of MS Edyta Pawlak-Adamska1,*,#, Oskar Nowak2,*, Lidia Karabon1, Anna Pokryszko-Dragan3, Anna Partyka1, Anna Tomkiewicz1, Jakub Ptaszkowski4, Irena Frydecka1, Ryszard PT Podemski3, Jaroslaw Dybko5, Malgorzata Bilinska3,* Department of Experimental Therapy, Hirszfeld Institute of Immunology and Experimental Department of Human Evolutionary Biology, Institute of Anthropology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland Department of Neurology, Wroclaw Medical University, Borowska 213, 50-566 Wroclaw, NU Poland SC RI Therapy, Polish Academy of Sciences, R Weigl 12, 53-114 Wroclaw, Poland Department of Clinical Biomechanics and Physiotherapy in Motor System Disorders, MA Faculty of Health Science, Wroclaw Medical University, Grunwaldzka 2, 50-355 Wroclaw, Poland PT E D Department of Hematology, Blood Neoplastic Diseases, and Bone Marrow Transplantation, Medical University, Pasteura 4, 50-367 Wroclaw, Poland *- these authors equally contributed to this work CE key words: multiple sclerosis, PD-1 gene, PD-1.5, diplopia, pyramidal signs, RRMS AC #Corresponding author: Edyta Pawlak-Adamska, Ph.D Department of Experimental Therapy Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences R Weigl Str 12, 53-114 Wroclaw, Poland tel (+48) 71 3371172; fax: (+48) 71 3371382; e-mail: epawlak@iitd.pan.wroc.pl ACCEPTED MANUSCRIPT Abbreviations CE PT E D MA NU SC RI PT Akaike Information Criteria alternative name runt-related transcription factor ankylosing spondylitis alternative name Programmed cell death-2 ligand alternative name Programmed cell death-1 ligand confidence intervals central nervous system delayed-type hypersensitivity experimental autoimmune encephalomyelitis expanded disability status score exonic splicing enhancer exonic splicing silencer Graves’ disease Hardy-Weinberg equilibrium interferon γ interleukin linkage disequilibrium myasthenia gravis myelin oligodendrocyte glycoprotein multiple sclerosis Multiple Sclerosis Severity Score National Center for Biotechnology Information neuromyelitis optica odds ratio polymerase chain reaction PCR-restriction fragment length polymorphism Programmed cell death-1 Programmed cell death-1 gene Programmed cell death-1 ligand Programmed cell death-2 ligand primary progressive MS rheumatoid arthritis relapsing-remitting MS runt-related transcription factor standard deviation systemic lupus erythematosus secondary progressive MS single nucleotide polymorphism type diabetes AC AIC AML1 AS B7-DC B7-H1 CI CNS DTH EAE EDSS ESE ESS GD HWE IFN-γ IL LD MG MOG MS MSSS NCBI NMO OR PCR PCR-RFLP PD-1 PDCD1 PD-L1 PDL-2 PPSM RA RRMS RUNX1 s.d SLE SPMS SNP T1D ACCEPTED MANUSCRIPT Abstract Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), where inflammation, demyelination together with the axonopathy are the cardinal features on pathologic ground, with a combined genetic and environmental background The associations of PD-1 single nucleotide polymorphisms (SNPs): PD-1.3 (in intron PT 4), PD-1.5 and PD-1.9 (both in exon 5) with clinical manifestation of MS in 479 south Polish individuals including 203 MS patients were analyzed RI Presence of PD-1.5 T allele was linked with the first manifestations of disease: SC diplopia and pyramidal signs - favored pyramidal signs but protected against of diplopia development Farther, PD-1.3G/PD-1.5C/PD-1.9C haplotype significantly favored whereas GTC protected NU against diplopia Besides, GTT haplotype strongly favored non-severe RRMS outcome and ATC haplotype was specific only for these MS patients MA Our population-based case-control study, investigating selected three PD-1 SNPs: PD1.3, PD-1.5 and PD-1.9, revealed that polymorphic variation may be rather disease-modifying AC CE PT E D than MS risk factor ACCEPTED MANUSCRIPT Introduction Multiple sclerosis (MS) is a chronic immune-mediated inflammatory disease of the central nervous system (CNS), where inflammation and demyelination of the white matter is a consequence of an autoimmune attack aimed at myelin sheath, with a highly heterogeneous course [1] The most common form of the disease (≈85%) is a relapsing-remitting MS (RRMS) with episodes of acute worsening of function followed by partial or complete PT recovery [2, 3], and, within 20 years, approximately 50% will go on to develop secondary progressive MS (SPMS) SPMS defined as a steady clinical deterioration, and independent of RI relapses [3, 4] Moreover, a higher female preponderance is consistently seen in relapsing- SC onset but not in progressive-onset of MS [4] The etiology of MS is not well understood but is likely a combination of both genetic NU and environmental factors One hypothesis on the etiology of MS is that autoreactive T cells play an essential role in inducing tissue inflammation in MS It has been proposed that autoreactive T cells are activated by chance in the peripheral immune compartment and MA transport to the CNS, where they get reactivated by self-antigen to induce inflammation and demyelination [1] One of the controller of peripheral tolerance regulation, by inhibition the D expansion of self-reactive T cells, is the PD-1 pathway, one of the immune checkpoint regulation pathway The PD-1/PD-L1 interactions influence reactivation, expansion and PT E effector function of autoreactive T cells in tissue, and what is important - loss of PD-1 on T cells compromises peripheral tolerance [5, 6] In EAE PD-1 blockade causes an increase of autoreactive (MOG-specific) T cells frequencies, CE and DTH responses to the autoantigen, as well as greater autoantibody production, and disease severity, which altogether pointed PD-1 pathway as regulator of the autoimmune, AC MOG-reactive T cells Moreover, inhibitions apply not only to antigen specific T cell expansion, but also T cell activation and both Th1 and Th2 cytokines production Altogether, this clearly indicates the PD-1 pathway as essential players in autoimmunity inhibition by regulating both central and peripheral tolerance Consequently, prevoiusly mentioned strong evidences clearly justify selection of PD-1 gene (PDCD1) as a candidate MS susceptibility gene Instead of candidate gene and genome wide association studies, single-nucleotide polymorphisms (SNPs) in the PD-1 gene have demonstrated relevant associations with a higher risk of developing autoimmune diseases in certain ethnic groups [7] In detail, observations from case-control association studies of polymorphic variation within PDCD1 and other autoimmune diseases, i.e systemic lupus ACCEPTED MANUSCRIPT erythematosus (SLE) [8-14], rheumatoid arthritis (RA) [10, 15-19], type diabetes (T1D) [20-24], ankylosing spondylitis (AS) [25-27], Graves’ disease (GD) [28], myasthenia gravis (MG) [29] and neuromyelitis optica (NMO) [30] pointed its connection with autoimmunity, which altogether may testify that the PD-1 gene is rather one of the good candidate overall autoimmune than disease-specific gene In context of multiple sclerosis, no matter how a connection of the PDCD1 genetic variability was not being observed, a relationship between PDCD1 variability and MS phenotype - a primary progressive MS (PPMS) was observed PT [31] Therefore we conducted a population-based case-control study to investigate the RI association of selected three SNPs within PDCD1: PD-1.3, PD-1.5 and PD-1.9 with MS as SC well as disease outcome NU Materials and Methods 2.1 Study population MA Patients characteristic 203 unrelated MS patients (129 (63.5%) female and 74 (36.5%) men), with clinically diagnosed MS according to the McDonald Criteria [32] classification, were enrolled in this D study All patients were recruited since 1995 at the MS Clinic, Department of Neurology, PT E Wroclaw Medical University, Poland Medical documentation was available from the records of the MS Clinic A neurological examination, together with an evaluation of severity using Kurtzke’s expanded disability status score (EDSS) [33], was performed on the day on blood CE sampling for genetic studies The mean age (±standard deviation (s.d.)) of the MS patients examined at the time of blood AC sampling was 41.4±10.4 years (range 20-74 years) The mean age at disease onset in MS group was 30.2±7.4 years (mean±s.d.) (range 16-50 years), and the mean duration of the disease (±s.d.) was 11.2±7.2 years (1.0-34.0 years) As a first symptom of disease, optic neuritis occurred in 45 (22.1%), diplopia in 42 (20.6%), ataxia/vertigo in 35 (17.2%), paresthesias in 29 (14.2%), pyramidal signs in 62 (30.4%), and vegetative signs in (0.98%) patients Based on criteria considering the clinical course of the disease proposed by Lublin and Reingold [34] there were one 123 (60.6%) MS patients with relapsing-remitting phenotype of disease (RRMS, 83 (67.5%) female and 40 (32.5%) male), while 80 (39.4%) patients developed secondary progressive MS (SPMS, 46 (57.5%) female and 34 (42.5%) male) ACCEPTED MANUSCRIPT The mean duration of RRMS was 7.58±4.95 years (mean±s.d.) (range 1.0-27.0 years) Disease severity according to the EDSS scale ranged between 1.0-8.0 (mean± s.d.: 4.20±2.01) in all MS patients, in RRMS ranged between 1.0-6.0 (mean 2.9±1.33 s.d.) an in SPMS ranged between 4.0-8.0 (mean 6.2±0.99 s.d.) The median value for interattack intervals (first two relapses) was 18 months RRMS group of patients was divided according to severity of disease by meaning of duration between I and II relapse (the cut off was 2xs.d., ≤60 months) to group of RRMS with severe (n=107, 87.7%) PT and non-severe (n=15, 12.3%) outcome The SPMS was defined when continual worsening of symptoms and signs was evident for a RI period of at least months without superimposed relapses This resulted in a change of score SC on the EDSS scale of at least 0.5 points In the later observation, periods of stability could be recorded but disease was characterized by continuous progression The median time of NU progression from the RRMS to the SPMS was years Multiple Sclerosis Severity Score (MSSS) [35] was determined for whole MS group, RRMS MA and SPMS subgroups and was 5.25±2.26, 4.34±2.04, 6.64±1.82 (mean±s.d.), respectively Control group The control group consisted of 276 apparently healthy volunteers from the same geographical D area (125 (45.3%) females and 151 (54.7%) males) PT E The local ethics committee approved the study protocol, and informed consent was obtained from each individual prior including into the study Ethical approvals CE The local ethics committee approved the study protocol 2.2.Genotyping of polymorphisms al [36] AC Genomic DNA was isolated from whole frozen blood as described previously by Karabon et The SNPs: PD-1.3 (rs11568821, c.627+189G>A, +7146G>A) located in intron 4, PD-1.5 (rs2227981, c.804T>C, +7785C>T) and PD-1.9 (rs2227982, c.644C>T, +7625C>T) both located in exon of the PDCD1 were examined by PCR-restriction fragment length polymorphism (PCR-RFLP) technique using PstI, PvuII, and DrDI enzymes (Fermentas, Burlington, Ontario, Canada) The primers were designed according to the complete PDCD1 (NC_000002.12) sequence derived from the NCBI Sequence Viewer (http://www.ncbi nlm.nih.gov/) The primer sequence and RFLP condition were listed in Table 2.3 Statistical analyses ACCEPTED MANUSCRIPT The Hardy-Weinberg equilibrium (HWE) was tested separately for the studied groups by comparing observed and expected frequencies of genotypes using χ2 analysis Categorical data between MS patients and controls as well as between patients with different disease phenotypes were compared using the χ2 test with appropriate correction: Yates' (when at least one cell value was not more than 5) or Bonferroni multiple adjustments (in case of the multiple comparisons) Genotype and allele comparison was performed using the SHEsis software PT (http://analysis.bio-x.cn/myAnalysis.php) and Simple Interactive Statistical Analysis platform coefficients were estimated using the SHEsis software RI (SISA, http://www.quantitativeskills.com/sisa/) Haplotype and linkage disequilibrium (LD) SC Comparison of the descriptive continuous variables of age, age at disease onset, duration between I and II relapse, EDSS (expressed by means or medians) in context of MS outcome NU as well as PDCD1 polymorphic variation was done using one-way analysis of variance (ANOVA) and Tukey's test (post-hoc test) or the Mann–Whitney rank test (M–W test) depending on normality of distribution (tested using the Kolmogorov–Smirnov test) MA The interattack interval and time to transition from the RRMS to the SPMS were estimated using the Kaplan–Meier method The log rank test was used to compare the interattack D interval and time to transition from the RRMS to the SPMS, and the F Cox test when comparison was made in the context of PDCD1 polymorphic variations PT E The combined effects of genetic markers (PD-1.3, PD-1.5, and PD-1.9) and non-genetic factors (age [year], gender and first symptom of disease analyzed as individual parameter: optic neuritis, diplopia, ataxia/vertigo, paresthesias, pyramidal signs) on the MS progression CE were analyzed by multivariate logistic regression analysis forward logistic regression analysis using the stepwise method in 191 MS patients with full genotyping results for all studied AC SNPs With respect to clinical value of severity of first MS symptoms the presence of the cerebellar symptoms: pyramidal signs and/or ataxia/vertigo was classified as prone severe first MS symptoms (coded as in stepwise multivariate logistic regression model), similarly as in the case of male gender [5] Odds ratios (ORs) are presented with 95% confidence intervals (CI) The model was selected on the basis of goodness-of-fit criteria such as determination coefficient R2 Cox-Snell and Akaike Information Criteria (AIC), where a greater R2 and a lower AIC value was an indicator for a the best statistical fit Goodness of fit of the regression model was assessed with the Hosmer and Lemeshow’s test All tests were performed with STATISTICA 10.0 software (StatSoft, Crakow, Poland) Differences were considered statistically significant if the p0.05, Table 2) 3.1 PD-1 gene polymorphisms and MS risk Univariate analysis did not show any association of studied PD-1 SNPs with the risk of MS Neither dominant nor recessive models adopted to the analysis showed association with MS PT risk (Table 2) A strong LD between all studied PD-1 SNPs in MS patients: PD-1.3 and PD-1.5 RI (D’=0.913, r2=0.077), PD-1.3 and PD-1.9 (D’=1.000, r2=0.002) and PD-1.5 and PD-1.9 (D’=0.822, r2=0.009) was seen, whereas the LD within control group was observed only exon 5: PD-1.5 and PD-1.9 (D’=0.993, r2=0.008) SC between PD-1.3 and PD-1.5 (D’=0.700, r2=0.040), and between two SNPs located within NU Haplotype evaluation of all polymorphic sites did not show any impact on MS risk (Supplementary Table 1) MA 3.2 PD-1 gene polymorphisms and clinical data in MS All selected PD-1 SNPs were subjected to analysis for correlations with clinical data regarding gender, age, age at disease onset, initial manifestation of disease (optic neuritis, D ataxia and/or vertigo, diplopia, pyramidal signs, paresthesias), first interattack interval, PT E transition into progression from the RRMS to the SPMS, and severity of disease by meaning of EDSS We found that PD-1.5 polymorphism correlated with the first manifestations of CE disease: diplopia and pyramidal signs (Table 3) In recessive model the presence of PD-1.5 T allele (TT+CT vs CC) favored pyramidal signs development (p=0.02, OR=2.20, 95%CI:1.13- AC 4.32), but protected against of diplopia as initial manifestation of MS (p=0.008, OR=0.40, 95%CI:0.20-0.80) (Table 3) At the haplotype level association was preserved only in case of diplopia PD1.3G/PD-1.5C/PD-1.9C haplotype significantly favored whereas GTC haplotype protected against diplopia development as an initial MS manifestation (p=0.03, OR=1.74, 95%CI:1.042.89, and p=0.02, OR=0.54, 95%CI:0.32-0.93, respectively) (Table 4) We did not find any association of studied SNPs with stage of MS (RRMS or SPMS) on univariate as well as on haplotype level (Table and Supplementary 1) However, when we analysed RRMS with regard to duration between I and II relapse, we observed an association at haplotype level PD-1.3A/PD-1.5T/PD-1.9C haplotype was observed only in ACCEPTED MANUSCRIPT patients with non-severe (χ2=11.42, p=0.0007), and GTT strongly favored non-severe outcome of RRMS (χ2=12.92, p=0.0003, OR=0.005, 95%CI:0.00-0.09) (Table 5) We did not found any connection of studied SNPs on age at disease onset as well as on transition into progression from the RRMS to the SPMS (data not showed) A different genotype distribution at site PD-1.3 within control group with respect to gender (χ2=10.03, pcorrected=0.01) was observed (Supplementary Table 2) Presence of the PD1.3 A allele (dominant model: GG vs AA+GA adopted) was more prevalent in healthy PT women (30.5% vs 16.2%, χ2=7.25, p=0.007) The combined univariate analysis of MS and control group stratified with respect to gender as RI well as with respect of form of disease, showed that this allele variant protected women SC against MS development (p=0.05, OR=0.55, 95%CI:0.30-1.01) (Supplementary Table 2) A similar effect was observed in case of SPMS in women, but in this case did not reach NU statistical significance (χ2=2.81, p=0.09) At the haplotype level the ACC haplotype was significantly differentially distributed within control group stratified according to gender (χ2=4.30, p=0.04) (Supplementary Table 3) MA Moreover, the same haplotype favored SPMS outcome in men, but it did not reach statistical significance (χ2=2.95, p=0.09) (Supplementary Table 3) Contrary, GCC haplotype was more frequent in healthy men than in women (χ2=3.49, p=0.06), and, what is important, its PT E p=0.09) (Supplementary Table 3) D frequency was lower in men with SPMS, but it did not reach significant difference (χ2=2.81, Kaplan–Meier analysis showed no correlation of PD-1 gene polymorphisms with the first interattack interval as well as on time to transition from the RRMS to the SPMS (data not CE showed) Logistic regression analysis showed that SPMS was associated with age, age at disease AC onset, and presence of cerebellar symptoms as first signs of MS (p=0.0000, OR=1.16, 95%CI:1.11-1.22, p=0.0412, OR=1.04, 95%CI:1.00-1.09, and p=0.01, OR=2.09, 95%CI:1.16-3.76, respectively) (Table 6) Farther, significant predictors of occurrence of SPMS results in the model were age, age at disease onset, and PD-1.5 marker The increase of age (by year) significantly increases the risk of MS progression of 33% (B=0.29, p=0.00, OR=1.33, 95%CI:1.23-1.44), and each year of early occurrence of MS increases the risk of progression (B=-0.21, p=0.0000, OR=0.814, 95%CI:0.75-0.89) Moreover, the presence of the PD-1.5 wild C allele over 3-fold increases risk of transition from RR to SPMS (p=0.05, OR=3.03, 95%CI:1.00-9.14) (Table 6) The goodness of fit statistics (Hosmerand Lemeshow's test: p=0.76) indicated a satisfactory fit for the model (Table 6) ACCEPTED MANUSCRIPT T 162 (40.3) 95 (39.3) 67 (41.9) 209 (38.7) CC 72 (35.8) 47 (38.8) 25 (31.2) 99 (36.7) Dominant TT + CT 129 (64.2) 74 (61.2) 55 (68.8) 171 (63.3) TT 33 (16.4) 21 (17.4) 12 (15.0) 38 (14.0) D E CC + CT 168 (83.6) 100 (82.6) 68 (85.0) CC + TT 105 (52.2) 68 (56.2) T P E 137 (50.7) 43 (53.7) 133 (49.3) CT 96 (47.8) A C C 53 (43.8) 37 (46.3) Recessive 232 (86.0) Over-dominant Additive PD-1.9 (rs2227982) Genotype 0.52 0.25 0.28 0.02 0.52 0.04 1.21 0.17 0.79 0.04 1.21 0.17 0.79 0.49 0.20 0.70 0.04 0.49 0.20 0.70 0.04 0.10 1.91 1.00 0.50 0.10 1.91 1.00 0.50 0.12 0.92 0.04 0.82 T P I R C S U N A M III:IV I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV 0.47 0.62 0.60 0.88 0.47 0.85 0.27 0.68 0.37 0.85 0.27 0.68 0.37 0.48 0.66 0.40 0.84 0.48 0.66 0.40 0.84 0.75 0.17 0.32 0.48 0.75 0.17 0.32 0.48 0.73 0.34 0.84 0.37 0.88 1.07 1.11 1.02 1.14 0.96 0.72 1.10 0.79 1.04 1.40 0.91 1.27 1.20 0.84 1.28 1.08 0.83 1.19 0.78 0.93 1.06 1.49 1.25 0.84 0.94 0.67 0.80 1.20 1.07 0.75 0.96 1.27 0.61 – 1.26 0.82 – 1.39 0.74 – 1.67 0.75 – 1.40 0.80 – 1.63 0.66 – 1.41 0.39 – 1.30 0.71 – 1.71 0.46 – 1.34 0.71 – 1.52 0.77 – 2.54 0.59 – 1.42 0.75 – 2.17 0.72 – 1.99 0.39 – 1.82 0.72 – 2.30 0.53 – 2.18 0.50 – 1.98 0.55 – 2.58 0.44 – 1.40 0.46 – 1.88 0.74 – 1.53 0.85 – 2.63 0.81 – 1.92 0.51 – 1.38 0.65 – 1.36 0.38 – 1.18 0.52 – 1.24 0.73 – 1.97 0.74 – 1.54 0.42 – 1.34 0.63 – 1.46 0.76 – 2.13 ACCEPTED MANUSCRIPT CC 187 (96.4) 111 (94.9) 76 (98.7) 249 (97.6) CT (3.6) (5.1) (1.3) (2.4) Codominant1 TT (0.0) (0.0) (0.0) (0.0) Codominant2 Allele C 381 (98.2) 228 (97.4) 153 (99.4) 504 (98.8) T (1.8) (2.6) (0.6) (1.2) 187 (96.4) 111 (94.9) 76 (98.7) TT + CT (3.6) (5.1) TT (0.0) CC + CT CC + TT CC T P E D E 249 (97.6) (1.3) (2.4) (0.0) A (0.0) (0.0) 194 (100.0) 117 (100.0) 77 (100.0) 255 (100.0) 187 (96.4) 111 (94.9) 76 (98.7) 249 (97.6) C C 0.62 1.01* 1.19* 0.01* – – – – reference 0.43 1.55 0.31* 0.24 0.28* 2.24 1.00* 0.55 – – – – – – – – 0.51 – 4.70 0.03 – 2.06 0.71 – 7.11 0.07 – 4.61 – – – – 0.61 0.99* 1.95 0.01* 0.61 0.99* 1.95 0.01* 0.62 1.01* 1.19* 0.01* 0.62 1.01* 1.19* 0.01* – – – – – – – – 0.62 0.44 0.32* 0.16 1.00* 0.44 0.32* 0.16 1.00* 0.43 0.31* 0.28* 1.00* 0.43 0.31* 0.28* 1.00* – – – – – – – – 0.43 0.22 – 1.94 0.48 – 33.78 0.14 – 1.42 0.22 – 15.25 0.51 – 4.63 0.03 – 2.08 0.71 – 6.93 0.07 – 4.60 0.21 – 1.95 0.49 – 34.81 0.14 – 1.41 0.22 – 15.45 0.51 – 4.70 0.03 – 2.06 0.71 – 7.11 0.07 – 4.61 – – – – – – – – 0.21 – 1.95 T P C S U I R I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV Dominant I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV Recessive I:IV II:III II:IV III:IV Over-dominant I:IV N A M I:IV II:III II:IV III:IV I:IV II:III II:IV III:IV 0.65 4.03 0.45 1.82 1.54 0.25 2.21 0.55 0.64 4.11 0.45 1.83 1.55 0.24 2.24 0.55 – – – – – – – – 0.64 ns11 ns12 ns13 ns14 ACCEPTED MANUSCRIPT CT (3.6) (5.1) (1.3) II:III 1.01* 0.31* 4.11 II:IV 1.19* 0.28* 0.45 III:IV 0.01* 1.00* 1.83 I:IV 0.62 0.43 1.55 II:III 1.01* 0.31* 0.24 (2.4) II:IV 1.19* 0.28* 2.24 III:IV 0.01* 1.00* 0.55 I:IV 0.62 0.43 1.55 II:III 1.01* 0.31* 0.24 Additive II:IV 1.19* 0.28* 2.24 III:IV 0.01* 1.00* 0.55 PD-1.5 (rs2227981) HWEMS: χ2=0.01, p=0.92 HWEControls: χ2=0.39, p=0.53 – I:IV: χ =0.50, df=2, p=0.78 – II:III: χ2=1.94, df=2, p=0.38 – II:IV: χ2=1.23, df=2, p=0.54 10 – III:IV: χ2=0.80, df=2, p=0.67 PD-1.9 (rs2227982) HWEMS: χ2=0.07, p=0.80 HWEControls: χ2=0.04, p=0.85 11 – I:IV: χ =0.62, df=1, p=0.43 12 – II:III: χ2=1.96, df=1, p=0.16 13 – II:IV: χ2=1.98, df=1, p=0.16 14 – III:IV: χ2=0.32, df=1, p=0.57 T P I R C S U *- after Yates’ correction – – PD-1.3 (rs11568821) HWEMS: χ2=2.08, p=0.15 HWEControls: χ2=0.39, p=0.73 – I:IV: χ =2.44, df=2, p=0.30 – II:III: χ2=0.005, df=2, p=1.00 – II:IV: χ2=1.98, df=2, p=0.37 – III:IV: χ2=1.68, df=2, p=0.43 N A T P E D E A C C M 0.49 – 34.81 0.14 – 1.41 0.22 – 15.45 0.51 – 4.70 0.03 – 2.06 0.71 – 7.11 0.07 – 4.61 0.51 – 4.70 0.03 – 2.06 0.71 – 7.11 0.07 – 4.61 ACCEPTED MANUSCRIPT Table PD-1.3 (rs11568821), PD-1.5 (rs2227981) and PD-1.9 (rs2227982) genotype and allele frequencies in patients with multiple sclerosis (MS) according to initial manifestation of disease: diplopia, paresthesias, ataxia and/or vertigo, pyramidal signs, optic neuritis diplopia paresthesias pyramidal signs ataxia/vertigo optic neuritis – + – + – + – + – + I n= 161 n (%) II n= 42 n (%) III = 174 n (%) IV n= 29 n (%) V n= 141 n (%) VI n= 62 n (%) VII n= 168 n (%) VIII n= 35 n (%) IX n= 158 n (%) X n= 45 n (%) 32 (76.2) 134 (77.9) 24 (82.8) 110 (78.6) 48 (78.7) 133 (79.6) 25 (73.5) 122 (78.2) GG 126 (79.2) GA 30 (18.9) (19.0) 34 (19.8) (13.8) 25 (17.9) AA (1.9) (4.8) (2.3) (3.4) (3.6) Allele G 282 (88.7) 72 (85.7) 302 (87.8) A 36 (11.3) 12 (14.3) 42 (12.2) 13 (21.3) 30 (18.0) D E T P E C C (0.0) (2.4) N A 36 (80.0) (23.5) 30 (19.2) (17.8) (2.9) (2.6) (2.2) M 52 (89.7) 245 (87.5) 109 (89.3) 296 (88.6) 58 (85.3) 274 (87.8) 80 (88.9) (10.3) 35 (12.5) 13 (10.7) 38 (11.4) 10 (14.7) 38 (12.2) 10 (11.1) A p OR 95%CI pglobal I R C S U PD-1.3 (rs11568821) Genotype T P χ2 reference I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 0.01 0.23* 0.21 0.60 0.05 0.26* 0.11* 0.94* 0.14* 0.16* 1.00 0.63* 0.65 0.44 0.82 0.61* 0.74* 0.33* 0.71* 0.69* 1.05 0.66 1.19 1.42 0.90 2.63 1.40 – 1.33 0.85 0.44 – 2.51 0.21 – 2.02 0.56 – 2.53 0.58 – 3.45 0.38 – 2.14 0.42 – 16.38 0.15 – 13.03 – 0.14 – 12.40 0.09 – 7.82 I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 0.56 0.16 0.28 0.60 0.08 0.56 0.16 0.28 0.60 0.08 0.46 0.69 0.60 0.44 0.78 0.46 0.69 0.60 0.44 0.78 0.77 1.21 1.20 0.74 1.11 1.31 0.83 0.83 1.34 0.90 0.38 – 1.55 0.49 – 2.98 0.61 – 2.35 0.35 – 1.58 0.53 – 2.33 0.65 – 2.64 0.34 – 2.05 0.43 – 1.64 0.63 – 2.85 0.43 – 1.89 ns1 ns2 ns3 ns4 ns5 ACCEPTED MANUSCRIPT Dominant model GG 126 (79.2) 32 (76.2) 134 (77.9) 24 (82.8) 110 (78.6) 48 (78.7) 133 (79.6) 25 (73.5) 122 (78.2) 36 (80.0) AA + GA 33 (20.8) 10 (23.8) 38 (22.1) (17.2) 30 (21.4) 13 (21.3) 34 (20.4) (26.5) 34 (21.8) (20.0) Recessive model AA GG + GA (1.9) 156 (98.1) (4.8) 40 (95.2) (2.3) 168 (97.7) (3.4) 28 (96.6) (3.6) 135 (96.4) GA 129 (81.1) 34 (81.0) 138 (80.2) 30 (18.9) (19.0) 34 (19.8) Additive model 61 (100.0) (2.4) D E T P E Over-dominant model GG + AA (0.0) C C 163 (87.6) N A (2.9) M 33 (87.1) (2.6) (2.2) 152 (87.4) 44 (87.8) 25 (86.2) 115 (82.1) 48 (78.7) 137 (82.0) 26 (76.5) 126 (80.8) 37 (82.2) (13.8) 25 (17.9) 13 (21.3) 30 (18.0) (23.5) 30 (19.2) (17.8) A I R 0.18 0.35 0.00 0.63 0.07 0.18 0.35 0.00 0.63 0.07 0.67 0.56 1.00 0.43 0.80 0.67 0.56 1.00 0.43 0.80 0.84 1.36 1.01 0.71 1.11 1.19 0.73 0.99 1.41 0.90 0.37 – 1.88 0.49 – 3.81 0.48 – 2.10 0.30 – 1.66 0.49 – 2.54 0.53 – 2.67 0.26 – 2.06 0.48 – 2.07 0.60 – 3.29 0.39 – 2.04 I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 0.26* 0.08* 1.00* 0.17* 0.17* 0.26* 0.08* 1.00* 0.17* 0.17* 0.61* 0.78* 0.32* 0.68* 0.68* 0.61* 0.78* 0.32* 0.68* 0.68* 2.60 1.50 – 1.23 0.86 0.38 0.67 – 0.81 1.16 0.42 – 16.09 0.16 – 13.92 – 0.13 – 11.40 0.09 – 7.93 0.06 – 2.38 0.07 – 6.19 – 0.09 – 7.50 0.13 – 10.63 I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 0.001 0.25* 0.33 0.57 0.05 0.001 0.25* 0.33 0.57 0.05 1.00 0.61* 0.57 0.45 0.83 1.00 0.61* 0.57 0.45 0.83 0.99 1.54 0.80 0.71 1.10 1.01 0.65 1.25 1.41 0.91 0.42 – 2.35 0.50 – 4.72 0.38 – 1.70 0.29 – 1.73 0.47 – 2.61 0.43 – 2.41 0.21 – 1.99 0.59 – 2.64 0.58 – 3.41 0.38 – 2.15 C S U I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X T P ACCEPTED MANUSCRIPT I:II III:IV V:VI VII:VIII IX:X PD-1.5 (rs2227981) Genotype CC 50 (31.4) 22 (53.7) 60 (35.1) 12 (41.4) 57 (41.3) 15 (24.2) 61 (36.5) 11 (33.3) CT 82 (51.6) 13 (31.7) 85 (49.7) 10 (34.5) 59 (42.8) 36 (58.1) 77 (46.1) 18 (54.5) TT 27 (17.0) (14.6) 26 (15.2) (24.1) 22 (15.9) 11 (17.7) 29 (17.4) (12.1) D E Allele C T 182 (57.2) 136 (42.8) 57 (69.5) 205 (59.9) 25 (30.5) 137 (40.1) 22 (53.7) 60 (35.1) 34 (58.6) T P E 173 (62.7) C C A 66 (53.2) 56 (36.1) C S U 74 (47.7) N A M 25 (16.1) 21 (46.7) (17.8) 199 (59.6) 40 (60.6) 186 (60.0) 53 (58.9) 24 (41.4) 103 (37.3) 58 (46.8) 135 (40.4) 26 (39.4) 124 (40.0) 37 (41.1) 12 (41.4) 57 (41.3) 15 (24.2) 61 (36.5) 11 (33.3) 56 (36.1) 16 (35.6) T P I R 16 (35.6) 0.49 0.02 0.19 0.66 0.08 0.48 1.00 0.66 0.42 0.78 1.31 1.06 0.85 1.40 0.89 0.61 – 2.81 0.45 – 2.54 0.41 – 1.75 0.62 – 3.17 0.41 – 1.96 reference I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 7.04 1.35 5.62 0.38 0.00 1.77 0.32 1.90 0.02* 0.05 0.008 0.25 0.02 0.54 1.00 0.18 0.57 0.17 1.00* 0.82 0.36 0.59 2.32 1.30 0.99 0.51 1.35 1.90 0.76 1.12 0.17 – 0.78 0.24 – 1.45 1.15 – 4.69 0.57 – 2.95 0.48 – 2.08 0.18 – 1.40 0.48 – 3.81 0.76 – 4.77 0.22 – 2.61 0.42 – 2.96 I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 4.09 0.04 3.18 0.02 0.04 4.09 0.04 3.18 0.02 0.04 0.04 0.85 0.07 0.88 0.85 0.04 0.85 0.07 0.88 0.85 1.70 0.95 0.68 1.04 0.95 0.59 1.06 1.48 0.96 1.05 1.01 – 2.87 0.54 – 1.67 0.44 – 1.04 0.61 – 1.79 0.59 – 1.54 0.35 – 0.99 0.60 – 1.86 0.96 – 2.27 0.56 – 1.64 0.65 – 1.69 I:II III:IV V:VI VII:VIII 6.98 0.43 5.44 0.12 0.008 0.51 0.02 0.73 2.52 1.31 0.45 0.87 1.26 – 5.08 0.59 – 2.92 0.23 – 0.89 0.40 – 1.91 Dominant model CC 50 (31.4) 0.066 ns7 ns8 ns9 ns10 ACCEPTED MANUSCRIPT TT + CT 109 (68.6) 19 (46.3) 111 (64.9) 17 (58.6) 81 (58.7) 47 (75.8) 106 (63.5) 22 (66.7) 99 (63.9) 0.005 6.98 0.43 5.44 0.12 0.005 1.00 0.008 0.51 0.02 0.73 1.00 0.98 0.40 0.77 2.20 1.15 1.03 0.49 – 1.95 0.20 – 0.80 0.34 – 1.71 1.13 – 4.32 0.52 – 2.53 0.51 – 2.05 I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X I R 0.13 1.44 0.10 0.24* 0.07 0.13 1.44 0.10 0.24* 0.07 0.72 0.23 0.75 0.63* 0.79 0.72 0.23 0.75 0.63* 0.79 0.84 1.77 1.14 0.66 1.12 1.19 0.56 0.88 1.52 0.89 0.32 – 2.19 0.69 – 4.58 0.51 – 2.52 0.21 – 2.01 0.47 – 2.70 0.46 – 3.12 0.22 – 1.45 0.40 – 1.95 0.50 – 4.67 0.37 – 2.14 I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 9.46 2.31 4.02 0.79 0.002 9.46 2.31 4.02 0.79 0.002 0.002 0.13 0.04 0.38 1.00 0.002 0.13 0.04 0.38 1.00 3.10 1.88 0.54 0.71 1.04 0.32 0.53 1.85 1.40 0.96 1.48 – 6.49 0.83 – 4.27 0.29 – 0.99 0.34 – 1.51 0.54 – 2.03 0.15 – 0.68 0.23 – 1.21 1.01 – 3.40 0.66 – 2.97 0.49 – 1.86 I:II III:IV V:VI VII:VIII IX:X 6.98 0.12 5.34 0.03 0.02 0.008 0.73 0.02 0.87 1.00 2.39 1.14 2.14 1.07 1.04 1.24 – 4.62 0.54 – 2.43 1.11 – 4.11 0.50 – 2.30 0.54 – 2.03 29 (64.4) IX:X I:II III:IV V:VI VII:VIII IX:X T P Recessive model TT 27 (17.0) (14.6) 26 (15.2) (24.1) 22 (15.9) 11 (17.7) 29 (17.4) (12.1) CC + CT 132 (83.0) 35 (85.4) 145 (84.8) 22 (75.9) 116 (84.1) 51 (82.3) 138 (82.6) 29 (87.9) Over-dominant model CC + TT 57 (48.4) 28 (68.3) 86 (50.3) 19 (65.5) CT 82 (51.6) 13 (31.7) 85 (49.7) 10 (34.5) Additive model PD-1.9 (rs2227982) 79 (57.2) D E T P E C C A 26 (41.9) 59 (42.8) 36 (58.1) 25 (16.1) (17.8) C S U N A 130 (83.9) 37 (82.2) M 90 (53.9) 15 (45.5) 81 (52.3) 24 (53.3) 77 (46.1) 18 (54.5) 74 (47.7) 21 (46.7) ACCEPTED MANUSCRIPT Genotype CC CT TT 147 (96.1) (3.9) (0.0) 39 (97.5) (2.5) (0.0) 159 (95.8) (4.2) (0.0) 27 (100.0) (0.0) (0.0) 129 (97.0) (3.0) (0.0) 57 (95.0) (5.0) 155 (95.7) (0.0) (4.3) (0.0) 31 (100.0) (0.0) (0.0) Allele C T 300 (98.0) (2.0) 79 (98.8) (1.3) 325 (97.9) (2.1) 54 (100.0) (0.0) 147 (96.1) 39 (97.5) 159 (95.8) TT + CT (3.9) (2.5) (4.2) A 117 (97.5) 317 (97.8) D E T P E (1.5) C C Dominant model CC 262 (98.5) (2.5) (2.2) 144 (97.3) (2.7) 42 (93.3) (6.7) reference N A (0.0) 62 (100.0) M 292 (98.6) 87 (96.7) (0.0) (1.4) (3.3) 27 (100.0) 129 (97.0) 57 (95.0) 155 (95.7) 31 (100.0) 144 (97.3) 42 (93.3) (0.0) (3.0) (5.0) (4.3) (0.0) (2.7) (06.7) 0.002* 0.28* 0.07* 0.43* 0.62* – – – – – 1.00* 0.59* 0.79* 0.51* 0.43* – – – – – 0.63 – 1.70 – 2.57 – – – – – 0.07 – 5.37 – 0.37 – 7.83 – 0.55 – 11.95 – – – – – I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 0.002* 0.28* 0.07* 0.42* 0.61* 0.002* 0.28* 0.07* 0.42* 0.61* 1.00* 0.60* 0.79* 0.52* 0.43* 1.00* 0.60* 0.79* 0.52* 0.43* 1.58 – 0.60 – 0.40 0.63 – 1.68 – 2.52 0.19 – 13.32 – 0.13 – 2.70 – 0.09 – 1.81 0.08 – 5.33 – 0.37 – 7.62 – 0.55 – 11.46 I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 0.002* 0.28* 0.07* 0.43* 0.62* 0.002* 0.28* 0.07* 0.43* 0.62* 1.00* 0.59* 0.79* 0.51* 0.43* 1.00* 0.59* 0.79* 0.51* 0.43* 1.59 – 0.59 – 0.39 0.63 – 1.70 – 2.57 0.19 – 13.61 – 0.13 – 2.72 – 0.08 – 1.81 0.07 – 5.37 – 0.37 – 7.83 – 0.55 – 11.95 T P I R C S U (0.0) I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X ns11 ns12 ns13 ns14 ns15 ACCEPTED MANUSCRIPT Recessive model TT CC + CT (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) 153 (100.0) 40 (100.0) 166 (100.0) 27 (100.0) 133 (100.0) 60 (100.0) 162 (100.0) 31 (100.0) 148 (100.0) 45 (100.0) CT 147 (96.1) (3.9) 39 (97.5) (2.5) 159 (95.8) (4.2) 27 (100.0) (0.0) 129 (97.0) (3.0) 57 (95.0) (5.0) 155 (95.7) D E T P E Additive model C C *- after Yates’ correction Bold indicates statistically significant association A PD-1.3 (rs11568821) – χ2=1.14, df=2, p=0.56 – χ2=0.67, df=2, p=0.72 – χ2=2.45, df=2, p=0.29 – χ2=0.63, df=2, p=0.73 – χ2=0.07, df=2, p=0.97 (4.3) N A 31 (100.0) M (0.0) I R – – – – – – – – – – – – – – – – – – – – I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 0.002* 0.28* 0.07* 0.43* 0.62* 0.002* 0.28* 0.07* 0.43* 0.62* I:II III:IV V:VI VII:VIII IX:X 0.002* 0.28* 0.07* 0.43* 0.62* T P C S U Over-dominant model CC + TT I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X 144 (97.3) 42 (93.3) (2.7) (6.7) PD-1.5 (rs2227981) – χ2=7.28, df=2, p=0.03 – χ2=2.68, df=2, p=0.26 – χ2=5.67, df=2, p=0.06 – χ2=0.95, df=2, p=0.62 10 – χ2=0.07, df=2, p=0.97 – – – – – – – – – – – – – – – – – – – – 1.00* 0.59* 0.79* 0.51* 0.43* 1.00* 0.59* 0.79* 0.51* 0.43* 1.59 – 0.59 – 0.39 0.63 – 1.70 – 2.57 0.19 – 13.61 – 0.13 – 2.72 – 0.08 – 1.81 0.07 – 5.37 – 0.37 – 7.83 – 0.55 – 11.95 1.00* 0.59* 0.79* 0.51* 0.43* 0.63 – 1.70 – 2.57 0.07 – 5.37 – 0.37 – 7.83 – 0.55 – 11.95 PD-1.9 (rs2227982) 11 – χ2=0.18, df=1, p=0.67 12 – χ2=1.18, df=1, p=0.28 13 – χ2=0.47, df=1, p=0.49 14 – χ2=1.39, df=1, p=0.24 15 – χ2=1.55, df=1, p=0.21 ACCEPTED MANUSCRIPT Table PD-1.3 (rs11568821)/ PD-1.5 (rs2227981) / PD-1.9 (rs2227982) haplotype frequency in patients with multiple sclerosis (MS) according to initial manifestation of disease: diplopia, paresthesias, ataxia and/or vertigo, pyramidal signs, optic neuritis diplopia – + I II n (freq) n (freq) paresthesias pyramidal signs ataxia/vertigo optic neuritis – + – + – + – + III n (freq) IV n (freq) V n (freq) VI n (freq) VII n (freq) VIII n (freq) IX n (freq) X n (freq) ACC 33.00 (0.109) 8.79 (0.113) 37.12 (0.113) 4.00 (0.074) 28.48 (0.109) 13.00 (0.108) 32.12 (0.100) GCC 133.02 (0.438) 44.21 (0.567) 150.96 (0.460) 26.00 (0.481) 128.78 (0.492) 48.01 (0.400) 149.94 (0.466) GCT 5.98 (0.020) 0.00 (0.000) 6.93 (0.021) 0.00 (0.000) 3.74 (0.014) 3.00 (0.025) GTC 131.98 (0.434) 22.79 (0.292) 131.04 (0.400) GTT 0.02 (0.000) 1.00 (0.013) 0.07 (0.000) C C A 31.38 (0.107) 10.00 (0.111) 27.00 (0.450) 136.70 (0.468) 40.01 (0.445) 6.94 (0.022) 0.00 (0.000) 3.92 (0.013) 2.99 (0.033) D E T P E U N 9.00 (0.150) I R SC PD-1.3 (rs11568821)/ PD-1.5 (rs2227981) / PD-1.9 (rs2227982) A M T P 24.00 (0.444) 99.22 (0.379) 55.99 (0.467) 131.06 (0.407) 24.00 (0.400) 118.30 (0.405) 36.99 (0.411) 0.00 (0.000) 0.26 (0.001) 0.00 (0.000) 0.06 (0.000) 0.00 (0.000) 0.08 (0.000) 0.01 (0.000) I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII IX:X I:II III:IV V:VI VII:VIII χ2 p OR 95%CI 0.02 0.84 0.00 1.15 0.006 4.54 0.01 2.72 0.17 0.19 – – – – 1.50 5.03 0.22 2.79 0.07 0.004 – – – – 0.90 0.36 1.00 0.28 0.94 0.03 0.91 0.10 0.68 0.66 – – – – 0.22 0.02 0.64 0.10 0.79 0.95 – – – – 1.05 0.61 1.00 1.54 1.03 1.74 1.03 0.69 0.89 0.89 – – – – 2.51 0.54 1.15 1.46 0.93 1.02 – – – – 0.48 – 2.32 0.21 – 1.78 0.50 – 2.01 0.79 – 3.43 0.49 – 2.19 1.04 – 2.89 0.58 – 1.84 0.44 – 1.07 0.51 – 1.55 0.56 – 1.45 – – – – 0.55 – 11.53 0.32 – 0.93 0.64 – 2.05 0.94 – 2.26 0.53 – 1.63 0.63 – 1.64 – – – – ACCEPTED MANUSCRIPT ATC 0.00 (0.000) 1.21 (0.016) 1.88 (0.006) 0.00 (0.000) 1.52 (0.006) 0.00 (0.000) 1.88 (0.006) 0.00 (0.000) 1.62 (0.006) 0.00 (0.000) IX:X I:II III:IV V:VI VII:VIII IX:X T P – – – – – – – – – – – – – – – – – – – – – – – – Global result (frequency