CTLA-4 and CD28 gene polymorphisms in susceptibility, clinical course and progression of multiple sclerosis

Journal of Neuroimmunology 140 (2003) 188 – 193 www.elsevier.com/locate/jneuroim

CTLA-4 and CD28 gene polymorphisms in susceptibility, clinical course and progression of multiple sclerosis Tineke van Veen a,b,*, J. Bart A. Crusius c, Lisa van Winsen a, Bing Xia d, Frederik Barkhof e, A. Salvador Pen˜a c, Chris H. Polman a, Bernard M.J. Uitdehaag a,b b

a Department of Neurology, VU University Medical Centre, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands Department of Clinical Epidemiology and Biostatistics, VU University Medical Centre, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands c Laboratory of Immunogenetics, VU University Medical Centre, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands d Department of Gastroenterology, Wuhan University Zhongnan Hospital, Wuhan 430071, PR China e MS-MRI Centre, VU University Medical Centre, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands

Received 21 October 2002; received in revised form 12 May 2003; accepted 12 May 2003

Abstract The balance between CD28 and CTLA-4 signalling is important for regulation of the immune response. We were interested whether a genetically mediated disturbance of this balance could be related to susceptibility or severity of multiple sclerosis (MS). We examined three polymorphisms in these genes, CTLA-4-318, CTLA-4 + 49 and CD28-I3 + 17, in 514 patients with MS and 181 controls. As the loci cannot be assumed independent of each other, we analysed the effects of each of the three polymorphisms corrected for the presence of the other two. We found no association between carriership of any of the alleles either with susceptibility to MS or with clinical features. For a subgroup of patients, longitudinal magnetic resonance imaging (MRI) data were available. We observed no effects of the polymorphisms on brain and lesion volumes. These data suggest that the polymorphisms under investigation do not affect the risk of developing MS and have no influence on the course of disease. D 2003 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; Susceptibility; CTLA-4; CD28; Severity; MRI

1. Introduction Multiple sclerosis (MS) is an autoimmune disorder, in which T cells infiltrate the central nervous system (CNS) white matter and initiate an inflammatory response that leads to demyelination (Martino and Hartung, 1999). In order to become activated, proliferate and secrete cytokines, T cells require two signals. The first signal is antigen-specific and based on recognition of a peptide-MHC complex on the antigen-presenting cell (APC), and the second signal is delivered by interaction of the T cell CD28 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) surface molecules with B7 costimulatory molecules on the APC. CTLA4 (CD152) shares homology with CD28 and binds to the same ligands, B7-1 (CD80) and B7-2 (CD86). However, * Corresponding author. Department of Clinical Epidemiology and Biostatistics, VU University Medical Centre, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. Tel.: +31-20-4442384; fax: +31-20-4444475. E-mail address: [email protected] (T. van Veen). 0165-5728/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-5728(03)00184-X

whereas CD28 provides a signal essential for the initiation and progression of a T cell response, CTLA-4 may actually down regulate T cell function (Krummel and Allison, 1995). In CD28-deficient mice, T cell unresponsiveness has been described (Shahinian et al., 1993), whereas CTLA-4 knockout mice develop early lethal autoimmunity (Waterhouse et al., 1995). Further, CD28/CTLA-4-mediated costimulation plays an essential role in the establishment and progression of experimental allergic encephalomyelitis (EAE), an animal model for MS. For example, mice transgenic for an MBPspecific T cell receptor develop EAE spontaneously. When these mice are crossed with CD28 knockout mice, spontaneous disease is abrogated, but this can be reestablished by increasing the dosage of MBP (Oliveira-dos-Santos et al., 1999). This shows that CD28 costimulation is necessary for EAE induction and that it acts by adjusting the threshold for stimulation. In addition, blockade of CTLA-4 with specific monoclonal antibodies at onset of clinical signs results in increased clinical severity and mortality (Karandikar et al., 1996; Perrin et al., 1996), whereas the development of EAE

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and relapses can be partially prevented by treatment with CTLA-4-Ig (Khoury et al., 1995; Miller et al., 1995). In the CNS of MS patients, both HLA class II and B7-1 are expressed (Windhagen et al., 1995). This makes the patients vulnerable to activated T cells directed against CNS autoantigens, which have been demonstrated in the majority of MS patients (Lovett-Racke et al., 1998). The human genes encoding CTLA-4 and CD28 are located only about 100 kb apart on chromosome 2q33. In this region, two of the MS genome surveys found evidence for linkage with MS (Ebers et al., 1996; Kuokkanen et al., 1997). In the CTLA-4 gene, three polymorphisms have been identified. A dinucleotide repeat in the 3Vuntranslated region (Polymeropoulos et al., 1991) was shown to correlate with the T cell proliferative response, the 86 bp allele being more stable and therefore more abundant compared to the larger alleles (Huang et al., 2000). In addition, the 106 bp allele was associated with insulin-dependent diabetes mellitus (IDDM) and Myasthenia Gravis (Huang et al., 2000). A G/A transition at CTLA-4 + 49 leads to an amino acid substitution in the leader peptide (Harper et al., 1991). The GG genotype at this locus was demonstrated to be associated with less CTLA-4 surface expression after stimulation and increased T cell proliferation (Kouki et al., 2000). The CTLA-4 + 49*G allele has been reported to be associated with IDDM, Graves disease and autoimmune thyroiditis (Donner et al., 1997; Kotsa et al., 1997; Kouki et al., 2000). At a C/T transition at CTLA-4-318 in the promotor sequence (Deichmann et al., 1996), the T allele has been reported to be associated with higher promotor activity than the CTLA-4-318*C allele (Wang et al., 2002). In CD28, a C/T transition was identified in intron 3 at position 17 (CD28-I3 + 17)(Tomer et al., 2001). A genetic predisposition to high production of CD28 or to low production of CTLA-4 may increase the risk to develop MS, and several studies have reported on the role of polymorphisms in the CTLA-4 gene in multiple sclerosis. Increased homozygosity for allele CTLA-4 + 49*G was observed in 378 Swedish MS patients (Ligers et al., 1999) although this observation was not confirmed in an extension of this study (Masterman et al., 2002). Among Shanghai Chinese, evidence of interaction with HLA-DR2 was observed (Rasmussen et al., 2001), allele CTLA-4 + 49*G was associated with PP MS in two European populations (Masterman et al., 2002; Maurer et al., 2002); in Japanese MS patients, it has been suggested that CTLA-4 polymorphisms may modulate the disease (Fukazawa et al., 1999). However, other studies reported no association of MS with CTLA4(AT)n (Ligers et al., 1999; Dyment et al., 2002), CTLA-4318 (Harbo et al., 1999) or CTLA-4 + 49 (Harbo et al., 1999; Dyment et al., 2002). In the present study, we analysed the role of CTLA-4 and CD28 in disease susceptibility and clinical disease course. For a subgroup of patients, serial magnetic resonance imaging (MRI) data were available, which provide a very sensitive measure of disease progression. Furthermore, as the loci are located closely together, we assessed

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the effect of each of the three loci, corrected for the presence of the other two. Previous studies have looked at one locus at a time, and given the presence of linkage disequilibrium (LD) (Holopainen and Partanen, 2001), it is possible that associations have been missed as a consequence of confounding by the other loci. In addition, as the gene products have related functions, we investigated whether we could detect interaction between alleles (epistasis). Epistasis is believed to play a role in MS, based on the observation that the risk of family members of an MS patient decreases more rapidly with decreasing kinship than expected under inheritance due to an additive effect of genetic factors.

2. Patients and methods 2.1. Subjects A total of 514 Dutch MS patients were recruited from the Outpatient Department of Neurology at the VU University Medical Centre (VUMC), Amsterdam. The mean age of the patients was 46 years (S.D. 11 years), and there were 195 men and 319 women. All patients fulfilled the criteria for clinically definite MS as proposed by Poser et al. (1983), and the course of disease was classified based on clinical data (Lublin and Reingold, 1996). Of the patients, 20% had a primary progressive (PP) form of the disease, 45% relapsing – remitting (RR) MS and 35% were in the secondary progressive (SP) phase. Mean age at onset was 32 years (S.D. 9 years). 40% of the patients had reached an EDSS score of 6.0, and mean observation duration was 11 years (S.D. 8 years). All patients were unrelated and Dutch Caucasians. The control group consisted of 181 unrelated ethnically matched healthy volunteers. Of these, 54% were women, and the mean age was 37 years. This study was carried out with the approval of the Medical Ethical Committee of the VUMC; informed consent was obtained from all subjects. 2.2. Magnetic resonance imaging MRI examinations were performed as described previously (Kalkers et al., 2001). For measuring T2-weighted lesion load, hyperintense lesions (compared to the surrounding white matter) were marked, whereas for measuring T1weighted lesion load, lesions hypointense compared to grey matter were marked. Subsequently, T2 (T2LV) and T1 lesion volumes (T1LV) were calculated. To assess the rate of lesion development, DT1LV and DT2LV were calculated by dividing the difference in lesion volume by the time between the scans. Further, severity of the lesions was assessed by the black hole ratio (BHR), defined as T1LV/T2LV. Parenchymal and ventricular volumes were measured on T1-weighted images, and intracranial volume was measured on the corresponding slices of the heavily T2-weighted

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images. Two ratios were calculated: (1) the parenchymal fraction (PF), defined as whole brain parenchyma/intracranial volume as a measure of global brain atrophy; and (2) the ventricular fraction (VF), defined as ventricular volume/ intracranial volume to assess central atrophy. The progression of atrophy was assessed by dividing the difference in PF and VF by the time between the scans.

or progressive onset of disease (chi-square analysis). P values were considered significant when < 0.05.

2.3. Genotyping

Table 1 shows several clinical characteristics. Two partially overlapping study groups are shown, representing patients for which longitudinal MRI data were available. In 96 patients typed for all three polymorphisms, we had serial data on lesion volumes on T2- and T1-weighted images. In the other group, consisting of 99 patients, we performed serial analysis of atrophy in relation to the studied gene polymorphisms. The clinical parameters of the subgroups are similar to those of the total MS population with the exception of the proportion of patients having PP MS, which is higher in both subgroups.

DNA was extracted from peripheral blood by standard proteinase-K digestion and phenol/chloroform extraction. Haplotypes and genotypes for the CTLA-4-318 and CTLA-4 + 49 polymorphisms were determined by PCR amplification with allele-specific primers as described before (Ligers et al., 1999), with minor modifications. Control primers were 5V-TGC CAA GTG GAG CAC CCA A-3Vand 5V-GCA TCT TGC TCT GTG CAG AT3V and yielded a product of 796 bp. Genotyping of CD28I3 + 17 was performed as described previously (Tomer et al., 2001). PCR products were separated on a 3% agarose gel stained with ethidiumbromide and visualised by UV transillumination. 2.4. Statistical analysis Primary data were the genotype frequencies for the polymorphisms at positions CTLA-4-318, CTLA-4 + 49 and CD28-I3 + 17 as observed in patients and healthy controls. We analysed whether the distributions of genotypes deviated from Hardy –Weinberg equilibrium. As the three loci are located closely to each other on the same chromosome, we analysed pairwise LD in cases and controls (z statistic). In addition, as the loci cannot be assumed independent of each other, we analysed the effects of each of the three polymorphisms corrected for the presence of the other two loci. For the sake of comparison with previous reports, we analysed the polymorphisms separately as well. We investigated the effects of allele carriership of the three polymorphisms studied on susceptibility to MS and disease characteristics. To model susceptibility to MS, we carried out an analysis of interaction between allele carriership of all polymorphisms. Regression analysis was used to investigate the relationship between the combination of all three carrierships and disease type and the age at onset of disease. To simultaneously study the effect of all three polymorphisms on the time to reach EDSS 6.0, we performed Cox regression. Linear regression analysis was performed with each of the MRI parameters as the outcome measure and carriership of alleles as the determinants. We corrected for gender, disease duration and onset of disease; log transformation was carried out where necessary. For each locus, separately, we analysed allele carriership in patients and controls and among patients with relapsing

3. Results 3.1. Patients

3.2. Genotypes in cases and controls The genotype frequencies at the CTLA-4-318, CTLA4 + 49 and CD28-I3 + 17 loci in healthy controls and MS patients did not deviate from Hardy –Weinberg equilibrium (see Table 2). In controls, significant LD was present between all pairs of loci, except for the combination CTLA-4 + 49/CD28. In patients, statistically significant LD was observed between all pairs of loci (data not shown). 3.3. Allele carriership and susceptibility to MS We did not have an a priori hypothesis on which alleles were disease-predisposing alleles and, therefore, tested carriership of both alleles at all three polymorphisms. Separate analysis revealed no significant association of

Table 1 Characteristics of patients of whom MR images were analysed Characteristics

Gender (% women) Age at January 1, 2001a Age at onseta Disease durationb Type MSc RR SP PP EDSS z 6.0c Time to EDSS 6.0b Time between scansb (months) a

Total MS

Lesion volumes

Atrophy

N = 514

N = 95

N = 99

62 46 (11) 32 (9) 11 (7 to 17)

54 51 (10) 35 (10) 13 (10 to 19)

56 49 (12) 35 (11) 12 (8 to 16)

45 35 20 40 9 (5 to 14) –

19 50 31 57 9 (6 to 15) 35 (23 to 37)

28 34 38 44 7 (5 to 12) 34 (24 to 38)

Expressed as mean (S.D.). Expressed as median (interquartile range). c Expressed as %. b

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carriership of any of the six alleles with susceptibility to MS (Table 3). However, the loci are located close to one another and cannot be assumed to be independent of each other because of LD. The effect of one locus may mask that of another; thus, ideally, the loci should not be analysed in isolation. Simultaneous analysis at the three loci in this manner consisted of eight regression models. Controlled for the effect of the other polymorphisms, none of the alleles appeared to be a susceptibility factor.

Table 3 The effects of carriership for each allele separately on susceptibility to MS

3.4. Combined effect of all three loci and disease characteristics

3.6. Combined effect of all three loci and MRI parameters

Using regression analysis, we studied the eight possible combinations of allele carriership at the three loci. None of the loci had significant effect on onset type, age at onset or on progression rate, as measured by the time to reach EDSS 6.0. 3.5. Analysis of interaction We analysed for all two-way and three-way interaction terms if they had significant effect on susceptibility to MS or on time to reach EDSS 6.0. However, no significant interactions were observed.

Table 2 Genotype frequencies at the CD28-I3 + 17, CTLA-4-318 and CTLA-4 + 49 loci in controls and in relapsing – remitting (RR), secondary progressive (SP) and primary progressive (PP) MS patients Genotype

Controls: N (%)

MS: N (%)

RR: N (%)

SP: N (%)

PP: N (%)

N = 181

N = 514

N = 226

N = 177

N = 98

CD28-I3 + 17 CC 8 (5) CT 53 (29) TT 120 (66)

11 (2) 153 (30) 350 (68)

5 (2) 75 (33) 146 (65)

3 (2) 41 (23) 133 (75)

3 (3) 33 (34) 62 (63)

CTLA-4 haplotypes 11 49 (27) 12 75 (41) 13 19 (11) 22 24 (13) 23 10 (6) 33 4 (2)

160 202 51 70 27 4

CTLA-4-318 CC 148 (82) CT 29 (16) TT 4 (2)

432 (84) 78 (15) 4 (1)

CTLA-4 + 49 AA 72 (40) AG 85 (47) GG 24 (13)

215 (42) 229 (45) 70 (13)

(31) (39) (10) (14) (5) (1)

80 86 21 28 10 1

(36) (38) (9) (12) (4) (1)

(27) (39) (9) (17) (7) (1)

30 (31) 39 (40) 13 (13) 11 (11) 4 (4) 1 (1)

194 (86) 31 (14) 1 (0)

147 (83) 29 (16) 1 (1)

80 (82) 17 (17) 1 (1)

102 (45) 96 (43) 28 (12)

64 (36) 83 (47) 30 (17)

44 (45) 43 (44) 11 (11)

For 13 patients, the type of disease was not known. Haplotype 4 was not detected in this study.

47 70 16 30 13 1

Allele Carriership Carriership Carriership Carriership Carriership Carriership

CD28-I3 + 17*C CD28-I3 + 17*T CTLA-4-318*C CTLA-4-318*T CTLA-4 + 49*A CTLA-4 + 49*G

Odds ratio (95% CI)

p Value

0.92 (0.64 to 1.3) 2.1 (0.84 to 5.3) 2.9 (0.71 to 11.6) 0.88 (0.56 to 1.39) 0.97 (0.59 to 1.6) 0.92 (0.65 to 1.3)

0.657 0.113 0.137 0.590 0.930 0.630

Data on (changes in) LV on T2- and T1-weighted MR images were available for 96 patients. We performed linear regression analysis to assess the combined effects of allele carriership of all three loci on MRI parameters, corrected for gender, duration of disease and type of MS. No forms of (severe) multicollinearity occurred. No significant associations were found between allele carriership and T2LV, T1LV, BHR, DT2LV or DT1LV. 3.7. Allele carriership and lesion volumes Data on (changes in) PF and VF were available for 99 MS patients. Simultaneous analysis of all three loci revealed no significant effect on brain volume parameters PF, VF, DPF, or DVF.

4. Discussion The study of the role of candidate genes in complex diseases typically yields poorly replicable results. This is probably due to the modest role each of these genes plays in combination with the fact that none of the disease-predisposing alleles is necessary for disease. For the study of disease course, it has been recommended to approach candidate studies in MS using both clinical and MRI data (Kantarci et al., 2002). We carried out this thorough investigation and found no evidence in support of a role for the CTLA-4-318, CTLA-4 + 49 and CD28-I3 + 17 gene polymorphisms in MS. One explanation is that allele CTLA4 + 49*G is in LD with a disease predisposing allele in some populations, but less so in others. Most likely, several genetic susceptibility factors common to different autoimmune diseases exist (Becker et al., 1998). Among these seem to be CD28 and CTLA-4, as these have been implicated in coeliac disease, rheumatoid arthritis, systemic lupus erythematosus and MS. Several investigators have studied the role of polymorphisms in these genes in the pathogenesis of these diseases. Not all studies observed association of CTLA-4 polymorphisms with disease, but in those that did, the association often involved the same alleles. Specifically, at the CTLA-4 + 49 polymorphism, allele G is associated with Graves’ disease,

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IDDM and autoimmune thyroiditis (Donner et al., 1997; Kotsa et al., 1997; Kouki et al., 2000). At the CTLA-4(AT)n locus, the larger allele is associated with Graves’ disease, IDDM and myasthenia gravis (Yanagawa et al., 1995; Nistico` et al., 1996; Huang et al., 2000). The only study regarding CD28-I3 + 17 reported no association with the production of thyroid autoantibodies (Tomer et al., 2001). Of relevance to the present study, studies of CTLA-4 polymorphisms in MS have yielded similar conflicting results (Fukazawa et al., 1999; Harbo et al., 1999; Ligers et al., 1999; Rasmussen et al., 2001; Masterman et al., 2002; Maurer et al., 2002). We hypothesised that with simultaneous analysis of the loci, we might be able to detect associations with disease characteristics in our large patient population. However, the age at onset, time to EDSS 6.0 and type of MS were not influenced by the polymorphisms in CTLA-4 and CD28. Given these observations in a large cohort of patients with considerable follow-up data, it seems unlikely that these polymorphisms influence the severity of disease. In a previous study, CTLA-4 + 49*G was shown to be associated with relapsing – remitting MS (Harbo et al., 1999); in two other studies, this allele was associated with PP MS (Masterman et al., 2002; Maurer et al., 2002). In the latter two studies, the definition of PP MS was similar to ours, but the number of PP MS patients was very small in one study (Maurer et al., 2002); in the other study, a twostage design was chosen in which the observation of the association between CTLA-4 haplotype frequency and PP MS could not be confirmed in the second stage (Masterman et al., 2002). In an investigation of Japanese patients, carriers of allele CTLA-4 + 49*A were shown to experience more severe disability (Fukazawa et al., 1999). We are the first to also investigate the CTLA-4 and CD28 genes using MRI parameters as a measure of disease progression. T2LV is a very sensitive marker for the total burden of disease as many pathological changes associated with MS are visible on T2-weighted images (Miller et al., 1998). However, we did not observe an effect of allele carriership on (changes in) T2LV. T1 lesions reflect severe tissue destruction and axonal damage (Van Walderveen et al., 1998). In addition, the black hole ratio (T1LV/T2LV) is regarded as a measure for repair of lesions. In our patients, the yearly increase in T1LV as well as the black hole ratio were unaffected by the loci studied. Atrophy is another measure of disease progression. In this respect, we assessed the parenchymal (PF) and ventricular volumes (VF) relative to the skull size. In our patients, the yearly increase in VF or decrease in PF were not associated with allele carriership at the polymorphisms studied. Our data suggest that the CTLA-4-318, CTLA-4 + 49 and CD28-I3 + 17 polymorphisms do not influence the risk of developing MS and do not influence the course of disease. This is in contrast to several studies that report an association of autoimmune disorders with CTLA-4 + 49*G. However, the alleles CTLA-4-318*C and CTLA-4 + 49*G, which were shown to be associated with decreased CTLA-

4 expression, often occur together on a common haplotype. It is tempting to speculate that in those populations, this allele is in LD with the true disease susceptibility allele and that we did not study the polymorphism with the diseasepredisposing allele. This would explain the results of the MS genome surveys that found evidence for linkage with MS on the long arm of chromosome 2. In that case, additional polymorphisms in the region will have to be studied to find the allele that contributes to the pathogenesis of MS.

Acknowledgements The authors would like to thank Dr. D. Knol for statistical assistance. This work was supported by the Netherlands Foundation for the Support of MS Research (Stichting Vrienden MS Research).

References Becker, K., Simon, R., Bailey Wilson, J., Freidlin, B., Biddison, W., McFarland, H., Trent, J., 1998. Clustering of non-major histocompatibility complex susceptibility candidate loci in human autoimmune diseases. Proc. Natl. Acad. Sci. U. S. A. 95, 9979 – 9984. Deichmann, K., Heinzmann, A., Bruggenolte, E., Forster, J., Kuehr, J., 1996. An MseI RFLP in the human CTLA4 promotor. Biochem. Biophys. Res. Commun. 225, 817 – 818. Donner, H., Rau, H., Walfish, P.G., Braun, J., Siegmund, T., Finke, R., Herwig, J., Usadel, K.H., Badenhoop, K., 1997. CTLA4 alanine-17 confers genetic susceptibility to Graves’ disease and to type 1 diabetes mellitus. J. Clin. Endocrinol. Metab. 82, 143 – 146. Dyment, D.A., Steckley, J.L., Willer, C.J., Armstrong, H., Sadovnick, A.D., Risch, N., Ebers, G.C., 2002. No evidence to support CTLA-4 as a susceptibility gene in MS families: the Canadian Collaborative Study. J. Neuroimmunol. 123, 193 – 198. Ebers, G., Kukay, K., Bulman, D., Sadovnick, A., Rice, G., Anderson, C., Armstrong, H., Cousin, K., Bell, R., Hader, W., Paty, D.W., Hashimoto, S., Oger, J., Duquette, P., Warren, S., Gray, T., O’Connor, P., Nath, A., Auty, A., Metz, L., Francis, G., Paulseth, J., Murray, T., Pryse-Phillips, W., Nelson, R., Freedman, M., Brunet, D., Bouchard, J.-P., Hinds, D., Risch, N., 1996. A full genome search in multiple sclerosis. Nat. Genet. 13, 472 – 476. Fukazawa, T., Yanagawa, T., Kikuchi, S., Yabe, I., Sasaki, H., Hamada, T., Miyasaka, K., Gomi, K., Tashiro, K., 1999. CTLA-4 gene polymorphism may modulate disease in Japanese multiple sclerosis patients. J. Neurol. Sci. 171, 49 – 55. Harbo, H.F., Celius, E.G., Vartdal, F., Spurkland, A., 1999. CTLA4 promoter and exon 1 dimorphisms in multiple sclerosis. Tissue Antigens 53, 106 – 110. Harper, K., Balzano, C., Rouvier, E., Mattei, M.G., Luciani, M.F., Golstein, P., 1991. CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location. J. Immunol. 147, 1037 – 1044. Holopainen, P.M., Partanen, J.A., 2001. Technical note: linkage disequilibrium and disease-associated CTLA4 gene polymorphisms. J. Immunol. 167, 2457 – 2458. Huang, D., Giscombe, R., Zhou, Y., Pirskanen, R., Lefvert, A.K., 2000. Dinucleotide repeat expansion in the CTLA-4 gene leads to T cell hyperreactivity via the CD28 pathway in myasthenia gravis. J. Neuroimmunol. 105, 69 – 77.

T. van Veen et al. / Journal of Neuroimmunology 140 (2003) 188–193 Kalkers, N.F., Bergers, E., Castelijns, J.A., Van Walderveen, M.A.A., Bot, J.C.J., Ade`r, H.J., Polman, C.H., Barkhof, F., 2001. Optimizing the association between disability and biological markers in MS. Neurology 57, 1253 – 1258. Kantarci, O., de Andrade, M., Weinshenker, B., 2002. Identifying disease modifying genes in multiple sclerosis. J. Neuroimmunol. 123, 144 – 159. Karandikar, N.J., Vanderlugt, C.L., Walunas, T.L., Miller, S.D., Bluestone, J.A., 1996. CTLA-4: a negative regulator of autoimmune disease. J. Exp. Med. 184, 783 – 788. Khoury, S.J., Akalin, E., Chandraker, A., Turka, L.A., Linsley, P.S., Sayegh, M.H., Hancock, W.W., 1995. CD28-B7 costimulatory blockade by CTLA4-Ig prevents actively induced experimental autoimmune encephalomyelitis and inhibits Th1 but spares Th2 cytokines in the central nervous system. J. Immunol. 155, 4521 – 4524. Kotsa, K., Watson, P., Weetman, A., 1997. A CTLA-4 gene polymorphism is associated with both Graves disease and autoimmune thyroiditis. Clin. Endocrinol. 46, 551 – 554. Kouki, T., Sawai, Y., Gardine, C.A., Fisfalen, M.E., Alegre, M.L., DeGroot, L.J., 2000. CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves’ disease. J. Immunol. 165, 6606 – 6611. Krummel, M.F., Allison, J.P., 1995. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J. Exp. Med. 182, 459 – 465. Kuokkanen, S., Gschwend, M., Rioux, J., Daly, M., Terwilliger, J., Tienari, P., Wikstro¨m, J., Palo, J., Stein, L., Hudson, T., Lander, E., Peltonen, L., 1997. Genomewide scan of multiple sclerosis in Finnish multiplex families. Am. J. Hum. Genet. 61, 1379 – 1387. Ligers, A., Xu, C., Saarinen, S., Hillert, J., Olerup, O., 1999. The CTLA4 gene is associated with multiple sclerosis. J. Neuroimmunol. 97, 182 – 190. Lovett-Racke, A.E., Trotter, J.L., Lauber, J., Perrin, P.J., June, C.H., Racke, M.K., 1998. Decreased dependence of myelin basic protein-reactive T cells on CD28-mediated costimulation in multiple sclerosis patients. A marker of activated/memory T cells. J. Clin. Invest. 101, 725 – 730. Lublin, F.D., Reingold, S.C., 1996. Defining the clinical course of multiple sclerosis: results of an international survey. National Multiple Sclerosis Society (USA) Advisory Committee on Clinical Trials of New Agents in Multiple Sclerosis. Neurology 46, 907 – 911. Martino, G., Hartung, H., 1999. Immunopathogenesis of multiple sclerosis: the role of T cells. Curr. Opin. Neurol. 12, 309 – 321. Masterman, T., Ligers, A., Zhang, Z., Hellgren, D., Salter, H., Anvret, M., Hillert, J., 2002. CTLA4 dimorphisms and the multiple sclerosis phenotype. J. Neuroimmunol. 131, 208 – 212. Maurer, M., Ponath, A., Kruse, N., Rieckmann, P., 2002. CTLA4 exon 1 dimorphism is associated with primary progressive multiple sclerosis. J. Neuroimmunol. 131, 213 – 215. Miller, S.D., Vanderlugt, C.L., Lenschow, D.J., Pope, J.G., Karandikar, N.J., Dal Canto, M.C., Bluestone, J.A., 1995. Blockade of CD28/B71 interaction prevents epitope spreading and clinical relapses of murine EAE. Immunity 3, 739 – 745. Miller, D., Grossman, R., Reingold, S., McFarland, H., 1998. The role of

193

magnetic resonance techniques in understanding and managing multiple sclerosis. Brain 121, 3 – 24. Nistico`, L., Buzzetti, R., Pritchard, L.E., Van der Auwera, B., Giovannini, C., Bosi, E., Larrad, M.T.M., Rios, M.S., Chow, C.C., Cockram, C.S., Jacobs, K., Mijovic, C., Bain, S.C., Barnett, A.H., Vandewalle, C.L., Schuit, F., Gorus, F.K., Registry, B.D., Tosi, R., Pozzilli, P., Todd, JA., 1996. The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Hum. Mol. Genet. 5, 1075 – 1080. Oliveira-dos-Santos, A.J., Ho, A., Tada, Y., Lafaille, J.J., Tonegawa, S., Mak, T.W., Penninger, J.M., 1999. CD28 costimulation is crucial for the development of spontaneous autoimmune encephalomyelitis. J. Immunol. 162, 4490 – 4495. Perrin, P.J., Maldonado, J.H., Davis, T.A., June, C.H., Racke, M.K., 1996. CTLA-4 blockade enhances clinical disease and cytokine production during experimental allergic encephalomyelitis. J. Immunol. 157, 1333 – 1336. Polymeropoulos, M.H., Xiao, H., Rath, D.S., Merril, C.R., 1991. Dinucleotide repeat polymorphism at the human CTLA4 gene. Nucleic Acids Res. 19, 4018. Poser, C.M., Paty, D.W., Scheinberg, L., McDonald, W.I., Davis, F.A., Ebers, G.C., Johnson, K.P., Sibley, W.A., Silberberg, D.H., Tourtellotte, W.W., 1983. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann. Neurol. 13, 227 – 231. Rasmussen, H.B., Kelly, M.A., Francis, D.A., Clausen, J., 2001. CTLA4 in multiple sclerosis. Lack of genetic association in a European Caucasian population but evidence of interaction with HLA-DR2 among Shanghai Chinese. J. Neurol. Sci. 184, 143 – 147. Shahinian, A., Pfeffer, K., Lee, K.P., Kundig, T.M., Kishihara, K., Wakeham, A., Kawai, K., Ohashi, P.S., Thompson, C., Mak, T.W., 1993. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261, 609 – 612. Tomer, Y., Greenberg, D.A., Barbesino, G., Concepcion, E., Davies, T.F., 2001. CTLA-4 and not CD28 is a susceptibility gene for thyroid autoantibody production. J. Clin. Endocrinol. Metab. 86, 1687 – 1693. Van Walderveen, M., Kamphorst, W., Scheltens, P., Van Waesberge, J., Ravid, R., Valk, J., Polman, C., Barkhof, F., 1998. Histopathologic correlate of hypointense lesions on T1-weighted spin-echo MRI in multiple sclerosis. Neurology 50, 1282 – 1288. Wang, X.B., Zhao, X., Giscombe, R., Lefvert, A.K., 2002. A CTLA-4 gene polymorphism at position 318 in the promoter region affects the expression of protein. Genes Immun. 3, 233 – 234. Waterhouse, P., Penninger, J.M., Timms, E., Wakeham, A., Shahinian, A., Lee, K.P., Thompson, C.B., Griesser, H., Mak, T.W., 1995. Lymphoproliferative disorders with early lethality in mice deficient in CTLA-4. Science 270, 985 – 988. Windhagen, A., Newcombe, J., Dangond, F., Strand, C., Woodroofe, M., Cuzner, M., Hafler, D., 1995. Expression of costimulatory molecules B7-1 (CD80), B7-2 (CD86) and interleukin 12 cytokine in multiple sclerosis lesions. J. Exp. Med. 182, 1985 – 1996. Yanagawa, T., Hidaka, Y., Guimaraes, V., Soliman, M., DeGroot, L.J., 1995. CTLA-4 gene polymorphism associated with Graves’ disease in a Caucasian population. J. Clin. Endocrinol. Metab. 80, 41 – 45.