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CTLA-4 -1661A/G and -1772T/C Dimorphisms in Japanese Patients with Systemic Lupus Erythematosus To the Editor: Genetic predisposition has been implicated in the pathogenesis of systemic lupus erythematosus (SLE) by various studies, although the etiology of SLE remains unclear. Associations with the complement C4AQ0, HLA-DR, HLA-DQ, tumor necrosis factor, interleukin 10 (IL-10), bcl-2, and Fas-L have been reported, but genetic susceptibility has not yet been confirmed1,2 . Cytotoxic T lymphocyte associated-4 (CTLA-4) and CD28 on T cells bind to CD80 and CD86, with CTLA-4 being a negative regulator of T cell activation3 . The ligation of CTLA-4 blocks CD28-dependent T cell activation and IL-2 accumulation. The CTLA-4 molecule is thought to terminate the immune response by CD28 and to keep the homeostatic balance of the immune system, so CTLA-4 would therefore be an important negative regulator of autoimmune diseases4 . Moreover, an increased level of soluble CTLA-4 in sera has been reported in SLE5 . The CTLA-4 gene is located on chromosome 2q33, and dimorphisms are reported to be at positions -1661 and -1772 in the promoter region 6 . The former is a substitution of adenine to guanine (-1661A/G), and the latter is a substitution of thymine to cytosine (-1772T/C). Recently, significant associations of the CTLA-4 -1772TT genotype and the -1772T allele with SLE were reported among Koreans6 . Fernandez-Blanco, et al also showed the involvement of CTLA-4 (-1772T/C) dimorphisms in SLE sus-ceptibility7 . Controversially, Aguilar, et al observed no association in Spanish patients with SLE 8 . Considering the immune-regulatory function of CTLA-4, the CTLA-4 gene is an interesting candidate as a disease-susceptible gene or genetic marker. Sixty randomly selected unrelated Japanese patients with SLE (57 women and 3 men; age 38.4 ± 12.5 yrs) diagnosed according to the criteria of the American Rheumatism Association 9 were examined. The control population consisted of 104 unrelated healthy volunteers. The dimorphisms at positions -1661 and -1772 were detected by the polymerase chain reaction (PCR)-restriction fragment length polymorphism method of Hudson et al 6 , using the specific oligonucleotide primers 5'-CTAAGAGCATCC GCT TGC ACC T-3' and 5'-TTG GTG TGATGC ACA GAA GCC TTT T-3'. PCR was performed under the following conditions: initial denaturation for 4 min at 94°C, annealing for 1 min at 58°C, extension for 1 min at 72°C, denaturation for 1 min at 94°C (30 cycles), and a final extension for 10 min at 72°C. The PCR product was digested using MseI for -1661 or Bbv1 for -1772 at 37°C for 4 h. Fisher's exact test was used for comparisons10.Genotype frequencies of the -1661A/G and -1772T/C dimorphisms are shown in Table 1. The frequency of the -1772TT genotype was not increased in patients with SLE (31.7%). No CTLA-4 -1661A/G or -1772T/C genotypes were found to be significantly associated with SLE. The frequencies of alleles in patients and controls are also shown in Table 1. In the controls, the -1661A allele (89.9%) and the -1772T allele (57.7%) are predominant among Japanese. The allele frequency of -1772C was slightly increased in patients with SLE compared to the controls (47.5% vs 42.3%), but the difference was not significant. No -1661A/G or -1772T/C alleles were found to be significantly associated with SLE.
Associations between the CTLA-4 (-1772T/C) dimorphism and SLE have been described in several reports; however, these results are controversial 6-8 . In our experiment, using Japanese SLE patients, no association of the -1772C/T dimorphism was observed. Our results are compatible with the observations by Aguilar, et al 8 . Lee, et al 10 also reported no association of the -1772C/T dimorphism with SLE using a metaanalysis, although they did observe a significant association of the +49A/G dimor-phism with SLE. The significant increase in the -1772C allele reported in Spanish patients with SLE 7 was different from the increase in the -1772T allele observed in Koreans with SLE 6 . Fernandez-Blanco, et al assumed that the different associations observed in Koreans and Spaniards with SLE would most likely be related to genetic differences in the pattern of haplo-types on the CTLA-4 locus between the Korean and Spanish populations 7 . The frequencies of the -1772T/C genotypes and alleles were different between Korean controls and Spanish controls 6-8 . The different observa-tions between Koreans and Spaniards with SLE could also indicate that CTLA-4 (-1772T/C) itself would not contribute directly to the pathogene-sis of SLE. Our results showed no association of the -1772T/C dimorphism in Japanese patients with SLE, although the distributions of the -1772T/C genotypes and alleles in Japanese controls were almost equal to the Korean controls 6 . These data strongly suggest that the -1772T/C is not the suscep-tibility gene in Japanese with SLE. There also was no association of the -1661A/G dimorphism. On the other hand, no association had previously been shown between SLE in Japanese and the CTLA-4 dimorphisms at positions -308 and +49 11 . This observation was compatible with reports by Heward, et al 12 , D'Alfonso, et al1 and Mehrian, et al 2 , although the results are still controversial. Taking our previous observations 11 into consideration, it is very likely that the CTLA-4 gene is not genetically involved in the pathogenesis of SLE in Japanese. FUJIO TAKEUCHI, MD, Department of Internal Medicine (Allergy and Rheumatology), Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; SHOJI KUWATA, MD, Associate Professor, 3rd Department of Internal Medicine, Faculty of Medicine, Teikyo University School of Medicine, Ichihara; MASAKI MORI, MD, Department of Internal Medicine (Allergy and Rheumatology), Faculty of Medicine, University of Tokyo. Address reprint requests to Dr. Takeuchi; E-mail: fujio-tky@umin.ac.jp Supported by grants from the Ministry of Education, Culture, Sport, Science and Technology of Japan and The Manabe Foundation. We thank Dr. Keiichiro Nakano and Dr. Kiyoaki Tanimoto, Tokyo University, for sampling; and Naoko Nakane and Natsuko Kobayashi for technical support. 1. D'Alfonso S, Rampi M, Bocchio D, Colombo G, Scorza-Smeraldi R, Momigliano-Richardi P. Systemic lupus erythematosus candidate genes in the Italian population: evidence for a significant association with interleukin-10. Arthritis Rheum 2000;43:120-8. 2. Mehrian R, Quismorio FP Jr, Strassmann G, et al. Synergistic effect between IL-10 and bcl-2 genotypes in determining susceptibility to systemic lupus erythematosus. Arthritis Rheum 1998;41:596-602. 3. Walunas TL, Bakker CY, Bluestone JA. CTLA-4 ligation blocks CD28-dependent T cell activation. J Exp Med 1996;183:2541-50. 4. Carreno BM, Collins M. The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu Rev Immunol 2002;20:29-53. 5. Liu MF, Wang CR, Chen PC, Fung LL. Increased expression of soluble cytotoxic T-lymphocyte-associated antigen-4 molecule in patients with systemic lupus erythematosus. Scand J Immunol 2003;57:568-72. 6. Hudson LL, Rocca K, Song YW, Pandey JP. CTLA-4 gene polymorphisms in systemic lupus erythematosus: a highly significant association with a determinant in the promoter region. Hum Genet 2002;111:452-5. 7. Fernandez-Blanco L, Perez-Pampin E, Gomez-Reino J, Gonzalez A. A CTLA-4 polymorphism associated with susceptibility to systemic lupus erythematosus [letter]. Arthritis Rheum 2004;50:328-9. 8. Aguilar F, Torres B, Sanchez-Roman J, Nunez-Roldan A, Gonzalez-Escribano MF. CTLA4 polymorphism in Spanish patients with systemic lupus erythematosus. Hum Immunol 2003;64:936-40. 9. Tan EM, Cohen AS, Fries JF, et al. The 1982 American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271-7. 10. Lee YH, Harley JB, Nath SK. CTLA-4 polymorphisms and systemic lupus erythematosus (SLE): a meta-analysis. Hum Genet 2005;116:361-7. 11.Takeuchi F, Kawasugi K, Nabeta H, Mori M, Tanimoto K. CTLA-4 dimorphisms in Japanese patients with systemic lupus erythematosus [letter]. Clin Exp Rheumatol 2003;21:527-8. 12. Heward J, Gordon C, Allahabedia A, Barnett AH, Franklyn JA, Gough SC. The A-G polymorphism in exon 1 of the CTLA-4 gene is not associated with systemic lupus erythematosus. Ann Rheum Dis 1999;58:193-5. |
