New Variations in Human OX40 Ligand (CD134L) Gene

Home , Chemokine, OX40 ligand

K Hikami, N Tsuchiya and K Tokunaga Department of Human Genetics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan 113–0033

We found three new variations in the OX40 ligand (OX40L, CD134L) gene and its 5Ј upstream region. −921 (G → A) at 5Ј upstream region, −19 (C → G) at 5Ј untranslated region, and 202+25 (G → T) at intron 2 were identiﬁed. −921 (G → A) and 202+25 (G → T) were detected in a substantial proportion of healthy Japanese individuals and were considered to be single nucleotide polymorphisms (SNPs). −19 (C → G) was detected in only one healthy individual. There was no association between these variations and the presence of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Genes and Immunity (2000) 1, 521–522.

Keywords: OX40L (CD134L); single nucleotide polymorphism; Japanese; rheumatoid arthritis; systemic lupus erythematosus

OX40 ligand (OX40L, CD134L, TNFSF4) is a member of For the fragments including intron 2: the TNF superfamily that is expressed on activated B cells and dendritic cells.1,2 OX40L interacts with its receptor Forward: 5Ј-CAGGCTACTGCATTTCTGCT-3Ј OX40 (CD134), which is expressed on activated T cells.3 Reverse: 5Ј-GCAGGCTACAACAATTCTGG-3Ј OX40L/OX40 is considered to act as a costimulator of T cells.4,5 Signaling through this pathway has been reported PCR was performed as follows: an initial denaturation to be involved in autoimmune diseases such as experi- at 96°C for 10 min, followed by 35 cycles of denaturation mental allergic encephalomyelitis (EAE) and inflamma- at 96°C for 30 sec, annealing at 60°C for 30 sec, and exten- tory bowel disease (IBD).6,7 Human OX40L gene is sion at 72°C for 1 min. The annealing temperature of the located at 1q25, which is close to the Fc␥ receptor cluster, primer pair for the fragment including 5Ј upstream one of the candidate susceptibility regions to systemic region was 56°C. After the PCR reaction, 1 ␮l each of the lupus erythematosus (SLE).8 Thus far, no information has PCR product was mixed with 7 ␮l of denaturing solution been published regarding the variations of OX40L gene. (95% formamide, 20 mm EDTA, 0.05% bromphenol blue, We carried out variation screening of the entire coding 0.05% xylene cyanol FF). The mixtures were denatured Ј region and 5 upstream region of human OX40L using at 96°C for 5 min and immediately cooled on ice. One ␮l polymerase chain reaction-single strand conformation of the mixtures was applied to polyacrylamide gel polymorphism (PCR-SSCP) analysis and nucleotide = − → Ј (acrylamide : bis 49:1). Electrophoresis was carried out sequencing. Three new variations, 921 (G A) at 5 in 0.5 × TBE constant current of 20 mA/gel. After electro- upstream region, −19 (C → G) at 5Ј untranslated region + → phoresis, single strand DNA fragments in the gel were and 202 25 (G T) at intron 2, were detected (Figure 1). visualized by silver staining. No variations were detected Nucleotide positions were counted from the ATG trans- within the coding sequence. lation initiation. The PCR primers used were as follows: We next examined the frequencies of these variations For the fragments including 5Ј upstream region: among 107 healthy Japanese individuals, 98 patients with Forward: 5Ј-CATTTCCCACATTCTCTTCC-3Ј rheumatoid arthritis (RA) and 72 patients with systemic Reverse: 5Ј-CCTTGGTTGTAGTCTATTGC-3Ј lupus erythematosus (SLE) (Table 1). The patients were For the fragments including 5Ј untranslated region: diagnosed according to the classification criteria of the American College of Rheumatology.9,10 −921 (G → A) and Forward: 5Ј-ATCGCACGTTCCCCTTTTCC-3Ј 202+25 (G → T) were detected in a substantial proportion Ј Ј Reverse: 5 -AGCTGCCATCAGCATGAGCT-3 of healthy individuals as well as patients, and were considered to be single nucleotide polymorphisms (SNPs). These SNPs were compatible with Hardy-Weinberg equi- Correspondence: Dr Naoyuki Tsuchiya, Department of Human Genetics, − → Graduate School of Medicine, University of Tokyo, Japan 113–0033. librium (data not shown). 19 (C G) was detected in E-mail: tsuchiya-tkyȰumin.ac.jp only one healthy individual. None of these variations Sequence accession numbers: The nucleotide sequence data were found to be associated with RA or SLE. reported in this paper have been submitted to the These results indicated that OX40L coding sequence is DDBJ/EMBL/GenBank nucleotide sequence databases and highly conserved. Although the variations were detected assigned the accession numbers AB042986–AB042988. This study was supported by the Grant-in-Aid for Scientific within the noncoding region, these variations were not Research (B) and Grant-in-Aid for Scientific Research on Priority associated with susceptibility to RA or SLE in Japanese. Areas (C). New variations in human OX40 ligand (CD134L) gene K Hikami et al 522

Figure 1 Representative SSCP patterns of ampliﬁed fragments of the OX40L gene. Genotypes are shown beneath each lane. (a) −921 (G → A) at 5Ј upstream region. Electrophoresis was carried out in 10% polyacrylamide gels with 5% glycerol at 12°C for 90 min. (b) −19 (C → G) at 5Ј untranslated region. Electrophoresis was carried out in 10% polyacrylamide gels at 20°C for 90 min. (c) 202+25(G → T) at intron 2. Electrophoresis was carried out in 12.5% polyacrylamide gels with 5% glycerol at 7°C for 180 min.

Table 1 Allele positivity and allele frequency of OX40L gene vari- Delespesse G. Expression and function of OX40 ligand on ations human dendritic cells. J Immunol 1997; 159: 3838–3848. 3 Al-Shamkhani A, Mallett S, Brown MH, James W, Barclay AN. RA SLE controls Affinity and kinetics of the interaction between soluble trimeric (n = 98) (n = 72) (n=107) OX40 ligand, a member of the tumor necrosis factor superfamily, and its receptor OX40 on activated T cells. J Biol Chem allele positivity* 1997; 272: 5275–5282. −921 (G → A) 20 (20.4) 13 (18.1) 21 (19.6) 4 Stuber E, Strober W. The T cell-B cell interaction via OX40- −19 (C → G) 0 (0) 0 (0) 1 (0.9) OX40L is necessary for the T cell-dependent humoral immune 202+25 (G → T) 10 (10.2) 9 (12.5) 12 (11.2) response. J Exp Med 1996; 183: 979–989. 5 Flynn S, Toellner KM, Raykundalia C, Goodall M, Lane P. CD4 allele frequency T cell cytokine differentiation: the B cell activation molecule, −921 (G → A) 22 (11.2) 14 (9.7) 21 (9.8) OX40 ligand, instructs CD4 T cells to express interleukin 4 and −19 (C → G) 0 (0) 0 (0) 1 (0.5) J Exp 202+25 (G → T) 10 (5.1) 9 (6.3) 12 (5.6) upregulates expression of the chemokine receptor, Bir-1. Med 1998; 188: 297–304. 6 Weinberg AD, Wegmann KW, Funatake C, Whitham RH. Block- Percentages are shown in parentheses. None of the variations was ing OX-40/OX-40 ligand interaction in vitro and in vivo leads significantly associated with RA or SLE. *Allele positivity is defined to decreased T cell function and amelioration of experimental as the percentage of individuals carrying at least one allele. allergic encephalomyelitis. J Immunol 1999; 162: 1818–1826. 7 Higgins LM, McDonald SA, Whittle N, Crockett N, Shields JG, MacDonald TT. Regulation of T cell activation in vitro and in Acknowledgements vivo by targeting the OX40-OX40 ligand interaction: amelior- The authors were indebted to Masaki Matsushita ation of ongoing inflammatory bowel disease with an OX40-IgG (Wakunaga Pharmaceutical Co. Ltd.) and Tae Komata fusion protein, but not with an OX40 ligand – IgG fusion protein. J Immunol 1999; 162: 486–493. (Department of Human Genetics, University of Tokyo) 8 Moser KL, Neas BR, Salmon JE et al. Genome scan of human for helpful suggestions. systemic lupus erythematosus: evidence for linkage on chromo- some 1q in African-American pedigrees. Proc Natl Acad Sci USA References 1998; 95: 14869–14874. 9 Arnett FC, Edworthy S, Bloch DA et al. The American Rheuma- 1 Godfrey WR, Fagnoni FF, Harara MA, Buck D, Engleman EG. tism Association 1987 revised criteria for the classification of Identification of a human OX-40 ligand, a costimulator of CD4+ rheumatoid arthritis. Arthritis Rheum 1988; 31: 315–324. T cells with homology to tumor necrosis factor. J Exp Med 1994; 10 Tan EM, Cohen AS, Fries JF et al. The 1982 revised criteria for 180: 757–762. the classification of systemic lupus erythematosus. Arthritis 2 Ohshima Y, Tanaka Y, Tozawa H, Takahashi Y, Maliszewski C, Rheum 1982; 25: 1271–1277.

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