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An assessment of the association between IL-2 gene polymorphisms and Japanese patients with multiple sclerosis
Journal of the Neurological Sciences 205 (2002) 47 – 50 www.elsevier.com/locate/jns
An assessment of the association between IL-2 gene polymorphisms and Japanese patients with multiple sclerosis Seiji Kikuchi a, Masaaki Niino a,*, Toshiyuki Fukazawa b, Ichiro Yabe a, Kunio Tashiro a a
Department of Neurology, Hokkaido University Graduate School of Medicine, Kita-15 Nishi-7, Kita-ku, 060-8638 Sapporo, Japan b Hokuyukai Neurology Hospital, Sapporo, Japan Received 18 March 2002; received in revised form 30 July 2002; accepted 5 August 2002
Abstract Interleukin-2 (IL-2) is a cytokine intimately involved with both the function and regulation of the immune system. Genetic analysis of experimental autoimmune encephalomyelitis (EAE) provides strong evidence supporting the candidacy of IL-2 as a susceptibility gene. We investigated the association of two single nucleotide polymorphisms (SNPs) at position 384 in the promoter region and +114 in the first exon of the IL-2 gene through a case-control study involving 113 Japanese patients with multiple sclerosis (MS) and 118 healthy controls. Our results showed no significant differences in the distribution of the two polymorphisms between MS patients and controls. Furthermore, no association was observed between IL-2 gene polymorphisms and clinical characteristics, such as clinical course and age at disease onset. Together, our findings suggest that IL-2 gene polymorphisms do not influence the susceptibility to MS or the clinical characteristics of MS in Japanese patients. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Genetics; Multiple sclerosis; Polymorphism; Japanese; Interleukin-2
1. Introduction Cytokines are potent immunomodulatory molecules that act as mediators of inflammation and the immune response. Multiple sclerosis (MS) is a major inflammatory and demyelinating disease in the central nervous system (CNS), and many cytokines have been associated with the pathology of MS. Interleukin-2 (IL-2) is one of the crucial immunoregulatory cytokines, and it is the principle product of naive resting T-cells upon primary T-cell receptor (TCR) stimulation. IL-2 has both pro-inflammatory and antiinflammatory actions, promoting T-cell proliferation during all mediated responses [1]. In experimental autoimmune encephalomyelitis (EAE), the levels of IL-2 and IL-2 mRNA were found to be elevated in the CNS during the induction and acute phases of disease, and declined during recovery [2,3]. Furthermore, IL-2 gene deletion markedly reduces EAE susceptibility in C57BL/6 mice [4]. The mouse IL-2 gene, located in a genome fragment of chromosome 3, has been associated with susceptibility to *
Corresponding author. Tel.: +81-11-700-5375; fax: +81-11-700-5356. E-mail address: [email protected] (M. Niino).
EAE [5]. The gene for human IL-2 is encoded on chromosome 4q26. In the human IL-2 gene, two polymorphisms have been identified: one located in the promoter region at nucleotide 384 [6], and another in the first exon at position +114 [7]. Genetic variability of key regulators leads to wide diversity within the immune response. This is demonstrated by the identification of polymorphisms in cytokine gene regulatory regions that correlate with intraindividual variations in actual cytokine production [8]. In transcriptional and posttranscriptional events, the presence of polymorphisms within nonstructural parts of the relevant gene might affect expression levels and such polymorphisms may be useful chromosomal markers in genetic studies of MS. In in vitro studies, IL-2 gene polymorphisms influence cytokine production levels in peripheral blood lymphocytes (PBL) after anti-CD3/CD28 stimulation [9]. Recently, it was reported that IL-2 gene polymorphisms might be associated with the susceptibility to secondary progressive type MS (SPMS) in a Spanish population [10]. Previously, we have reported an association between MS in Japanese patients and polymorphisms of the vitamin D receptor gene and IL-1h or IL-1 receptor antagonist (RA)
0022-510X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 5 1 0 X ( 0 2 ) 0 0 3 0 7 - 6
48
S. Kikuchi et al. / Journal of the Neurological Sciences 205 (2002) 47–50
genes [11,12]. Our results were not always consistent with prior reports on MS patients of other ethnicities, and this inconsistency may be due, in part, to differences in the polymorphism background of different ethnic groups. Therefore, in the present study, we have investigated whether IL-2 gene polymorphism plays a role in the clinical characteristics of Japanese MS patients and their potential genetic susceptibility to MS.
2. Patients and methods
Table 2 IL-2 gene polymorphisms in MS patients and controls (A) The position Genotype
R-Ra (n=78)
S-Pb (n=35)
Our cases G/G G/T T/T
3.5 15.9 80.5
1.3 19.2 79.5
8.6 8.6 82.9
Genotype
MS patients (%) Total (n=173)
Selected for this study were 118 unrelated patients with relapsing – remitting type MS (RRMS) or SPMS who, after having been observed for at least 1 year, were diagnosed as having MS according to the criteria of McDonald et al. [13] (Table 1). They were ‘‘conventional’’ MS patients, as previously described [14], and were similar to Western MS patients. The control group was composed of 26 unrelated healthy men and 92 unrelated healthy women ranging from 20 to 58 years old (mean=33.1; S.D.=9.4). All of the patients and controls were Japanese and were residents of Hokkaido, the northernmost island of Japan. The differences in sex ratio and age between the patients and controls were not significant ( p=0.423, 0.108).
Matesanz et al. [10] G/G 9.2 G/T 45.1 T/T 45.7
Genotype
Table 1 Clinical profiles of MS patients Total (n=113) 83:30 35.2F10.8 26.5F9.0 78 (69.0%) 35 (31.0%) 8.7F8.5 3.0F2.5
S-Pb (n=58)
10.4 40.0 49.6
6.9 55.2 37.9
MS patients (%) a
b
Total (n=113)
R-R (n=78)
S-P (n=35)
Our cases G/G G/T T/T
23.0 51.3 25.7
22.4 50.0 27.6
22.9 54.3 22.9
Genotype
MS patients (%) Total (n=173)
After obtaining the informed consent of each of the subjects, high-molecular-weight DNA was extracted from their peripheral blood cells. IL-2 gene polymorphisms in the promoter ( 384) and exon one (+114) regions were determined by PCR restriction fragment length polymorphisms (RFLP) using DNA, according to previously described methods [7]. The restriction enzyme used to detect polymorphism at position 384 was XspI (Takara, Japan) instead of BfaI, as used in the studies of Matesanz. Polymorphism at position +114 was detected using MwoI (BioLabs) as described by Matesanz et al. [7].
R-Ra (n=115)
Control (%) (n=118)
3.4 14.4 82.2 Control (%) (n=153)
9.1 32.7 58.2
(B) The position +114 in the first exon
2.2. Analysis of IL-2 gene polymorphism
EDSS: Expanded Disability Status Scale of Kurtzke. a Mean (years)FS.D. b Mean EDSSFS.D.
MS patients (%) Total (n=113)
2.1. Patients and controls
Female/male Agea Age at onseta Course Relapsing – remitting type Secondary progressive type Duration (years)a EDSSb
384 in the promoter region
Matesanz et al. [10] G/G 50.9 G/T 38.2 T/T 11.0
a
b
R-R (n=115)
S-P (n=58)
51.3 35.7 13.0
50.0 43.1 6.9
Control (%) (n=118)
22.0 55.9 22.0 Control (%) (n=153)
46.4 41.2 12.4
Overall, no significant difference was detected ( p > 0.05). a R-R: relapsing – remitting. b S-P: secondary progressive.
2.3. Statistical analysis Comparisons between the various alleles of patients with MS and controls were made using the Chi-square test for 22 or 23 tables. The statistical analysis between the genotypes of each IL-2 gene polymorphism and the onset age of MS patients was tested by analysis of variance (ANOVA) followed by Fisher’s protected least significant different difference (PLSD).
3. Results 3.1. IL-2 genotype frequencies The genotype proportions at position 384 of the promoter region (G/G, G/T, T/T) and at position +114 of the first exon (G/G, G/T, T/T) in MS patients and controls
S. Kikuchi et al. / Journal of the Neurological Sciences 205 (2002) 47–50
are shown in Table 2A and B. In control subjects, the genotype frequencies of each polymorphism conformed to Hardy – Weinberg expectations. No significant difference in the distributions of the 384 promoter polymorphism or the +114 first exon polymorphism was observed between MS patients and controls. 3.2. Clinical characteristics and IL-2 gene polymorphism Associations between IL-2 gene polymorphisms and MS clinical characteristics were studied in 113 patients. In clinical course, there were no significant differences between patients with RRMS and with SPMS, or between patients with RRMS or SPMS and controls (Table 2A and B). Furthermore, no associations were found between IL-2 gene polymorphisms and age at disease onset (data not shown).
4. Discussion In this study, we examined whether IL-2 gene polymorphism was associated with susceptibility to MS in Japanese patients. The distributions of genotypes at position 384 in the promoter region and position +114 in the first exon did not differ significantly between patients and controls. Furthermore, no association was observed between IL-2 gene polymorphisms and the clinical course of MS or the age at disease onset. To date, there are some reports about the association of the IL-2 gene and MS. In a gene association study of a Caucasian population, using separate polymorphic microsatellite markers for the IL-2 gene, no significant differences existed in allele frequencies between the MS groups and controls, nor were statistically significant differences observed in primary progressive MS (PPMS) vs. RRMS or SPMS for the IL-2 microsatellite marker [15]. Transmission disequilibrium testing (TDT) of a closely encoded IL-2 microsatellite marker in 502 trio families (both parents and a single affected offspring) in the UK disclosed no significant evidence for linkage disequilibrium [16]. In the IL-2 gene, two novel polymorphisms have been identified recently. One is a silent mutation (+114) in the leader peptide and therefore of no functional significance. In fact, Matesanz’s and our data showed that +114 polymorphism was not associated with susceptibility to MS. On the other hand, the second polymorphism ( 384) occurs within the promoter region and may therefore have an influence on levels of IL-2 production. In a prior study on cytokine production in anti-CD3/CD28-stimulated peripheral blood lymphocytes (PBL), individuals that were homozygous for the G allele (G/G) at site 384 in the promoter region of the IL-2 gene produced more than three times more IL-2 than their G/T and T/T counterparts [9]. These studies indicate that the polymorphism at position 384 in the promoter region has a different effect on the immune
49
system than the +114 polymorphism. In a Spanish population study, it was demonstrated that two of the 384 genotypes (G/T, T/T) were associated with susceptibility to SPMS [10]. However, our study showed no significant associations of that polymorphism in Japanese patients with MS. The association between genetic polymorphisms and MS susceptibility may also vary with ethnicity. Racial and ethnic differences may affect not only susceptibility, but also the phenotypic expression of MS, including clinical manifestations, site of lesions, disease course and prognosis. In summary, we analyzed IL-2 gene polymorphisms in Japanese MS patients, using PCR – RFLP. Our findings suggest that IL-2 gene polymorphisms do not influence the susceptibility to MS or the clinical characteristics of MS in Japanese patients. However, a case-control study design may give spurious results attributable to population stratification, including negative findings such as were found in this study. Additional studies may be necessary to exclude the possibility of type 2 errors.
Acknowledgements Our research has been supported, in part, by a Research fund from the Ministry of Health, Labor and Welfare in Japan.
References [1] Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996;383:787 – 93. [2] Jensen MA, Arnason BG, Toscas A, Noronha A. Global inhibition of IL-2 and IFN-gamma secreting T cells precedes recovery from acute monophasic experimental autoimmune encephalomyelitis. J Autoimmun 1996;9:587 – 97. [3] Kennedy MK, Torrance DS, Picha KS, Mohler KM. Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery. J Immunol 1992;149: 2496 – 505. [4] Petitto JM, Streit WJ, Huang Z, Butfiloski E, Schiffenbauer J. Interleukin-2 gene deletion produces a robust reduction in susceptibility to experimental autoimmune encephalomyelitis in C57BL/6 mice. Neurosci Lett 2000;285:66 – 70. [5] Encinas JA, Wicker LS, Peterson LB, et al. QTL influencing autoimmune diabetes and encephalomyelitis map to a 0.15-cM region containing IL2. Nat Genet 1999;21:158 – 60. [6] John S, Turner D, Donn R, et al. Two novel biallelic polymorphisms in the IL-2 gene. Eur J Immunogenet 1998;25:419 – 20. [7] Matesanz F, Delgado C, Fresno M, Alcina A. Allelic selection of human IL-2 gene. Eur J Immunol 2000;30:3516 – 21. [8] Hutchinson IV, Pravica V, Perrey C, Sinnott P. Cytokine gene polymorphisms and relevance to forms of rejection. Transplant Proc 1999; 31:734 – 6. [9] Hoffmann SC, Stanley EM, Darrin CE, et al. Association of cytokine polymorphic inheritance and in vitro cytokine production in anti-CD3/ CD28-stimulated peripheral blood lymphocytes. Transplantation 2001;72:1444 – 50. [10] Matesanz F, Fedetz M, Collado-Romero M, et al. Allelic expression and
50
S. Kikuchi et al. / Journal of the Neurological Sciences 205 (2002) 47–50
interleukin-2 polymorphisms in multiple sclerosis. J Neuroimmunol 2001;119:101 – 5. [11] Niino M, Fukazawa T, Yabe I, Kikuchi S, Sasaki H, Tashiro K. Vitamin D receptor gene polymorphism in multiple sclerosis and the association with HLA class II alleles. J Neurol Sci 2000;177:65 – 71. [12] Niino M, Kikuchi S, Fukazawa T, Yabe I, Sasaki H, Tashiro K. Genetic polymorphisms of IL-1beta and IL-1 receptor antagonist in association with multiple sclerosis in Japanese patients. J Neuroimmunol 2001;118:295 – 9. [13] McDonald WI, Compston A, Edan G, et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001;50:121 – 7.
[14] Fukazawa T, Miyasaka K, Tashiro K, et al. MRI findings of multiple sclerosis with acute transverse myelopathy. J Neurol Sci 1992;110: 27 – 31. [15] McDonnell GV, Kirk CW, Hawkins SA, Graham CA. An evaluation of interleukin genes fails to identify clear susceptibility loci for multiple sclerosis. J Neurol Sci 2000;176:4 – 12. [16] Feakes R, Sawcer S, Smillie B, Chataway J, Broadley S, Compston A. No evidence for the involvement of interleukin 2 or the immunoglobulin heavy chain gene cluster in determining genetic susceptibility to multiple sclerosis. J Neurol Neurosurg Psychiatry 2000;68:679.
An assessment of the association between IL-2 gene polymorphisms and Japanese patients with multiple sclerosis Seiji Kikuchi a, Masaaki Niino a,*, Toshiyuki Fukazawa b, Ichiro Yabe a, Kunio Tashiro a a
Department of Neurology, Hokkaido University Graduate School of Medicine, Kita-15 Nishi-7, Kita-ku, 060-8638 Sapporo, Japan b Hokuyukai Neurology Hospital, Sapporo, Japan Received 18 March 2002; received in revised form 30 July 2002; accepted 5 August 2002
Abstract Interleukin-2 (IL-2) is a cytokine intimately involved with both the function and regulation of the immune system. Genetic analysis of experimental autoimmune encephalomyelitis (EAE) provides strong evidence supporting the candidacy of IL-2 as a susceptibility gene. We investigated the association of two single nucleotide polymorphisms (SNPs) at position 384 in the promoter region and +114 in the first exon of the IL-2 gene through a case-control study involving 113 Japanese patients with multiple sclerosis (MS) and 118 healthy controls. Our results showed no significant differences in the distribution of the two polymorphisms between MS patients and controls. Furthermore, no association was observed between IL-2 gene polymorphisms and clinical characteristics, such as clinical course and age at disease onset. Together, our findings suggest that IL-2 gene polymorphisms do not influence the susceptibility to MS or the clinical characteristics of MS in Japanese patients. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Genetics; Multiple sclerosis; Polymorphism; Japanese; Interleukin-2
1. Introduction Cytokines are potent immunomodulatory molecules that act as mediators of inflammation and the immune response. Multiple sclerosis (MS) is a major inflammatory and demyelinating disease in the central nervous system (CNS), and many cytokines have been associated with the pathology of MS. Interleukin-2 (IL-2) is one of the crucial immunoregulatory cytokines, and it is the principle product of naive resting T-cells upon primary T-cell receptor (TCR) stimulation. IL-2 has both pro-inflammatory and antiinflammatory actions, promoting T-cell proliferation during all mediated responses [1]. In experimental autoimmune encephalomyelitis (EAE), the levels of IL-2 and IL-2 mRNA were found to be elevated in the CNS during the induction and acute phases of disease, and declined during recovery [2,3]. Furthermore, IL-2 gene deletion markedly reduces EAE susceptibility in C57BL/6 mice [4]. The mouse IL-2 gene, located in a genome fragment of chromosome 3, has been associated with susceptibility to *
Corresponding author. Tel.: +81-11-700-5375; fax: +81-11-700-5356. E-mail address: [email protected] (M. Niino).
EAE [5]. The gene for human IL-2 is encoded on chromosome 4q26. In the human IL-2 gene, two polymorphisms have been identified: one located in the promoter region at nucleotide 384 [6], and another in the first exon at position +114 [7]. Genetic variability of key regulators leads to wide diversity within the immune response. This is demonstrated by the identification of polymorphisms in cytokine gene regulatory regions that correlate with intraindividual variations in actual cytokine production [8]. In transcriptional and posttranscriptional events, the presence of polymorphisms within nonstructural parts of the relevant gene might affect expression levels and such polymorphisms may be useful chromosomal markers in genetic studies of MS. In in vitro studies, IL-2 gene polymorphisms influence cytokine production levels in peripheral blood lymphocytes (PBL) after anti-CD3/CD28 stimulation [9]. Recently, it was reported that IL-2 gene polymorphisms might be associated with the susceptibility to secondary progressive type MS (SPMS) in a Spanish population [10]. Previously, we have reported an association between MS in Japanese patients and polymorphisms of the vitamin D receptor gene and IL-1h or IL-1 receptor antagonist (RA)
0022-510X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 5 1 0 X ( 0 2 ) 0 0 3 0 7 - 6
48
S. Kikuchi et al. / Journal of the Neurological Sciences 205 (2002) 47–50
genes [11,12]. Our results were not always consistent with prior reports on MS patients of other ethnicities, and this inconsistency may be due, in part, to differences in the polymorphism background of different ethnic groups. Therefore, in the present study, we have investigated whether IL-2 gene polymorphism plays a role in the clinical characteristics of Japanese MS patients and their potential genetic susceptibility to MS.
2. Patients and methods
Table 2 IL-2 gene polymorphisms in MS patients and controls (A) The position Genotype
R-Ra (n=78)
S-Pb (n=35)
Our cases G/G G/T T/T
3.5 15.9 80.5
1.3 19.2 79.5
8.6 8.6 82.9
Genotype
MS patients (%) Total (n=173)
Selected for this study were 118 unrelated patients with relapsing – remitting type MS (RRMS) or SPMS who, after having been observed for at least 1 year, were diagnosed as having MS according to the criteria of McDonald et al. [13] (Table 1). They were ‘‘conventional’’ MS patients, as previously described [14], and were similar to Western MS patients. The control group was composed of 26 unrelated healthy men and 92 unrelated healthy women ranging from 20 to 58 years old (mean=33.1; S.D.=9.4). All of the patients and controls were Japanese and were residents of Hokkaido, the northernmost island of Japan. The differences in sex ratio and age between the patients and controls were not significant ( p=0.423, 0.108).
Matesanz et al. [10] G/G 9.2 G/T 45.1 T/T 45.7
Genotype
Table 1 Clinical profiles of MS patients Total (n=113) 83:30 35.2F10.8 26.5F9.0 78 (69.0%) 35 (31.0%) 8.7F8.5 3.0F2.5
S-Pb (n=58)
10.4 40.0 49.6
6.9 55.2 37.9
MS patients (%) a
b
Total (n=113)
R-R (n=78)
S-P (n=35)
Our cases G/G G/T T/T
23.0 51.3 25.7
22.4 50.0 27.6
22.9 54.3 22.9
Genotype
MS patients (%) Total (n=173)
After obtaining the informed consent of each of the subjects, high-molecular-weight DNA was extracted from their peripheral blood cells. IL-2 gene polymorphisms in the promoter ( 384) and exon one (+114) regions were determined by PCR restriction fragment length polymorphisms (RFLP) using DNA, according to previously described methods [7]. The restriction enzyme used to detect polymorphism at position 384 was XspI (Takara, Japan) instead of BfaI, as used in the studies of Matesanz. Polymorphism at position +114 was detected using MwoI (BioLabs) as described by Matesanz et al. [7].
R-Ra (n=115)
Control (%) (n=118)
3.4 14.4 82.2 Control (%) (n=153)
9.1 32.7 58.2
(B) The position +114 in the first exon
2.2. Analysis of IL-2 gene polymorphism
EDSS: Expanded Disability Status Scale of Kurtzke. a Mean (years)FS.D. b Mean EDSSFS.D.
MS patients (%) Total (n=113)
2.1. Patients and controls
Female/male Agea Age at onseta Course Relapsing – remitting type Secondary progressive type Duration (years)a EDSSb
384 in the promoter region
Matesanz et al. [10] G/G 50.9 G/T 38.2 T/T 11.0
a
b
R-R (n=115)
S-P (n=58)
51.3 35.7 13.0
50.0 43.1 6.9
Control (%) (n=118)
22.0 55.9 22.0 Control (%) (n=153)
46.4 41.2 12.4
Overall, no significant difference was detected ( p > 0.05). a R-R: relapsing – remitting. b S-P: secondary progressive.
2.3. Statistical analysis Comparisons between the various alleles of patients with MS and controls were made using the Chi-square test for 22 or 23 tables. The statistical analysis between the genotypes of each IL-2 gene polymorphism and the onset age of MS patients was tested by analysis of variance (ANOVA) followed by Fisher’s protected least significant different difference (PLSD).
3. Results 3.1. IL-2 genotype frequencies The genotype proportions at position 384 of the promoter region (G/G, G/T, T/T) and at position +114 of the first exon (G/G, G/T, T/T) in MS patients and controls
S. Kikuchi et al. / Journal of the Neurological Sciences 205 (2002) 47–50
are shown in Table 2A and B. In control subjects, the genotype frequencies of each polymorphism conformed to Hardy – Weinberg expectations. No significant difference in the distributions of the 384 promoter polymorphism or the +114 first exon polymorphism was observed between MS patients and controls. 3.2. Clinical characteristics and IL-2 gene polymorphism Associations between IL-2 gene polymorphisms and MS clinical characteristics were studied in 113 patients. In clinical course, there were no significant differences between patients with RRMS and with SPMS, or between patients with RRMS or SPMS and controls (Table 2A and B). Furthermore, no associations were found between IL-2 gene polymorphisms and age at disease onset (data not shown).
4. Discussion In this study, we examined whether IL-2 gene polymorphism was associated with susceptibility to MS in Japanese patients. The distributions of genotypes at position 384 in the promoter region and position +114 in the first exon did not differ significantly between patients and controls. Furthermore, no association was observed between IL-2 gene polymorphisms and the clinical course of MS or the age at disease onset. To date, there are some reports about the association of the IL-2 gene and MS. In a gene association study of a Caucasian population, using separate polymorphic microsatellite markers for the IL-2 gene, no significant differences existed in allele frequencies between the MS groups and controls, nor were statistically significant differences observed in primary progressive MS (PPMS) vs. RRMS or SPMS for the IL-2 microsatellite marker [15]. Transmission disequilibrium testing (TDT) of a closely encoded IL-2 microsatellite marker in 502 trio families (both parents and a single affected offspring) in the UK disclosed no significant evidence for linkage disequilibrium [16]. In the IL-2 gene, two novel polymorphisms have been identified recently. One is a silent mutation (+114) in the leader peptide and therefore of no functional significance. In fact, Matesanz’s and our data showed that +114 polymorphism was not associated with susceptibility to MS. On the other hand, the second polymorphism ( 384) occurs within the promoter region and may therefore have an influence on levels of IL-2 production. In a prior study on cytokine production in anti-CD3/CD28-stimulated peripheral blood lymphocytes (PBL), individuals that were homozygous for the G allele (G/G) at site 384 in the promoter region of the IL-2 gene produced more than three times more IL-2 than their G/T and T/T counterparts [9]. These studies indicate that the polymorphism at position 384 in the promoter region has a different effect on the immune
49
system than the +114 polymorphism. In a Spanish population study, it was demonstrated that two of the 384 genotypes (G/T, T/T) were associated with susceptibility to SPMS [10]. However, our study showed no significant associations of that polymorphism in Japanese patients with MS. The association between genetic polymorphisms and MS susceptibility may also vary with ethnicity. Racial and ethnic differences may affect not only susceptibility, but also the phenotypic expression of MS, including clinical manifestations, site of lesions, disease course and prognosis. In summary, we analyzed IL-2 gene polymorphisms in Japanese MS patients, using PCR – RFLP. Our findings suggest that IL-2 gene polymorphisms do not influence the susceptibility to MS or the clinical characteristics of MS in Japanese patients. However, a case-control study design may give spurious results attributable to population stratification, including negative findings such as were found in this study. Additional studies may be necessary to exclude the possibility of type 2 errors.
Acknowledgements Our research has been supported, in part, by a Research fund from the Ministry of Health, Labor and Welfare in Japan.
References [1] Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996;383:787 – 93. [2] Jensen MA, Arnason BG, Toscas A, Noronha A. Global inhibition of IL-2 and IFN-gamma secreting T cells precedes recovery from acute monophasic experimental autoimmune encephalomyelitis. J Autoimmun 1996;9:587 – 97. [3] Kennedy MK, Torrance DS, Picha KS, Mohler KM. Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery. J Immunol 1992;149: 2496 – 505. [4] Petitto JM, Streit WJ, Huang Z, Butfiloski E, Schiffenbauer J. Interleukin-2 gene deletion produces a robust reduction in susceptibility to experimental autoimmune encephalomyelitis in C57BL/6 mice. Neurosci Lett 2000;285:66 – 70. [5] Encinas JA, Wicker LS, Peterson LB, et al. QTL influencing autoimmune diabetes and encephalomyelitis map to a 0.15-cM region containing IL2. Nat Genet 1999;21:158 – 60. [6] John S, Turner D, Donn R, et al. Two novel biallelic polymorphisms in the IL-2 gene. Eur J Immunogenet 1998;25:419 – 20. [7] Matesanz F, Delgado C, Fresno M, Alcina A. Allelic selection of human IL-2 gene. Eur J Immunol 2000;30:3516 – 21. [8] Hutchinson IV, Pravica V, Perrey C, Sinnott P. Cytokine gene polymorphisms and relevance to forms of rejection. Transplant Proc 1999; 31:734 – 6. [9] Hoffmann SC, Stanley EM, Darrin CE, et al. Association of cytokine polymorphic inheritance and in vitro cytokine production in anti-CD3/ CD28-stimulated peripheral blood lymphocytes. Transplantation 2001;72:1444 – 50. [10] Matesanz F, Fedetz M, Collado-Romero M, et al. Allelic expression and
50
S. Kikuchi et al. / Journal of the Neurological Sciences 205 (2002) 47–50
interleukin-2 polymorphisms in multiple sclerosis. J Neuroimmunol 2001;119:101 – 5. [11] Niino M, Fukazawa T, Yabe I, Kikuchi S, Sasaki H, Tashiro K. Vitamin D receptor gene polymorphism in multiple sclerosis and the association with HLA class II alleles. J Neurol Sci 2000;177:65 – 71. [12] Niino M, Kikuchi S, Fukazawa T, Yabe I, Sasaki H, Tashiro K. Genetic polymorphisms of IL-1beta and IL-1 receptor antagonist in association with multiple sclerosis in Japanese patients. J Neuroimmunol 2001;118:295 – 9. [13] McDonald WI, Compston A, Edan G, et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001;50:121 – 7.
[14] Fukazawa T, Miyasaka K, Tashiro K, et al. MRI findings of multiple sclerosis with acute transverse myelopathy. J Neurol Sci 1992;110: 27 – 31. [15] McDonnell GV, Kirk CW, Hawkins SA, Graham CA. An evaluation of interleukin genes fails to identify clear susceptibility loci for multiple sclerosis. J Neurol Sci 2000;176:4 – 12. [16] Feakes R, Sawcer S, Smillie B, Chataway J, Broadley S, Compston A. No evidence for the involvement of interleukin 2 or the immunoglobulin heavy chain gene cluster in determining genetic susceptibility to multiple sclerosis. J Neurol Neurosurg Psychiatry 2000;68:679.