region of human TNFR2: association with systemic ... We recently reported the association of the allele coding for Arg at the position 196 (196R: nucleotide [nt] ...
Genes and Immunity (2000) 1, 501–503 2000 Macmillan Publishers Ltd All rights reserved 1466-4879/00 $15.00 www.nature.com/gene
BRIEF COMMUNICATION
New single nucleotide polymorphisms in the coding region of human TNFR2: association with systemic lupus erythematosus N Tsuchiya, T Komata, M Matsushita, J Ohashi and K Tokunaga Department of Human Genetics, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan 113-0033
We recently reported the association of the allele coding for Arg at the position 196 (196R: nucleotide [nt] 587G) of tumor necrosis factor receptor 2 (TNFR2, TNF-R75) with systemic lupus erythematosus (SLE) in Japanese. In the present study, we completed the variation screening of the entire coding region of TNFR2. Three new single nucleotide polymorphisms within the coding sequence (cSNPs), as well as several variations within the promoter, introns and 3⬘-untranslated region (3⬘UTR), were identified. Among the new SNPs, nt168G, a synonymous substitution (K56K), was in tight linkage disequilibrium with nt587G. Two other cSNPs, nt543 (C →T) (P181P) and nt694 (G →A) (E232K), were not significantly associated with SLE. Thus, among the non-synonymous cSNPs, only nt587 (T→G) (M196R) was found to be significantly associated with SLE in Japanese. Genes and Immunity (2000) 1, 501–503. Keywords: TNFR2; polymorphism; SLE; genetics; susceptibility
In spite of extensive studies by a number of investigators, the susceptibility genes to systemic lupus erythematosus (SLE) largely remain to be determined.1 Previous reports suggested the role for TNF␣ in the pathogenesis of human2 and murine3 SLE, and the results from genomewide screening indicated possible linkage of chromosome 1p36, where TNFR2 gene is located, with SLE.4,5 Based on such findings, we considered TNFR2 as a candidate for a susceptibility gene to SLE. Through the initial screening of three exons containing the previously reported variation sites, we detected a significant association of the allele coding for a non-conservative change of Arg (196R) for Met (196M) at codon 196 (nucleotide [nt] 587 [T→G]) with SLE.6 However, it was still unclear whether the codon 196 polymorphism bears primary significance, or it represents linkage disequilibrium with other undefined polymorphism(s) of primary significance. In this study, we completed the variation screening of the entire TNFR2 coding region in 81 Japanese patients with SLE and 258 healthy individuals, and examined the possibility that any of the other variations may be associated with the susceptibility to SLE. The characteristics of the patients and controls are described in the previous paper.6 All of them are
Correspondence: Naoyuki Tsuchiya, MD, PhD, Department of Human Genetics, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan 113-0033. E-mail: tsuchiya-tky얀umin.ac.jp This study was supported by the Grant-in-Aid for Scientific Research (B) (11470505) from the Ministry of Education, Science, Sports and Culture. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB030949-53.
Japanese, unrelated individuals living in Tokyo area. Variation screening was performed using PCR-single strand conformation polymorphism (SSCP)6 and PCRpreferential homoduplex formation assay (PHFA).7 Both methods can detect any gene variations, including single nucleotide polymorphisms (SNPs). The human TNFR2 gene consists of 10 exons, among which exons 4, 9 and 5⬘-portion of exon 6 were already analyzed.6 Each exon was amplified using flanking intronic primer sets, except for exons 6 and 10, which were divided into two fragments due to the length of the exons (Table 1). Thus, among the introns, only 10–80 nucleotides flanking each exon were screened. Nucleotide sequences of detected variations were determined by direct sequencing.6 New SNPs were identified at nt168 (A→G) within exon 2, nt543 (C→T) within exon 5 and nt694 (G→A) within exon 6. Among these SNPs, nt694 (G→A) leads to the non-synonymous substitution within the extracellular domain, E232K. We next examined whether any of these coding sequence SNPs (cSNPs) is associated with the susceptibility to SLE, using the case-control association analysis. As shown in Table 2(a), 28 of 81 patients (35%) possessed at least one nt168G allele, as compared with 47 of 258 controls (18%). This difference was statistically significant (P = 0.002, odds ratio [OR] = 2.37, 95% confidence interval [CI] : 1.37–4.09). No significant association was observed in the other two SNPs (Table 2(b)). To obtain information on the relationship between nt168G/A and the previously reported nt587T/G,6 the same sets of controls and patients were genotyped for the two cSNPs. Thirty of 81 patients (37%) with SLE possessed nt587G, which was significantly increased as compared with 53 of 258 controls (21%) (P = 0.0026,
New SNPs in TNFR2 N Tsuchiya et al
502
Table 1 Primers used in this study Name promoter region TNFR2proF TNFR2proR exon1 TNFR2E1-F TNFR2E1-R exon2 TNFR2E2-F TNFR2E2-R exon3 TNFR2E3-F TNFR2E3-R exon5 TNFR2E5-F TNFR2E5-R exon6 TNFR2E6-F TNFR2E6-R exon7 TNFR2E7-F TNFR2E7-R exon8 TNFR2E8-F TNFR2E8-R exon10 TNFR2E10–1F TNFR2E10–1R TNFR2E10–2F TNFR2E10–2R
Primer sequence
Fragment size
5⬘-AAGCACCATCCTTGCAGGCT-3⬘ 5⬘-AGATGAACAGAGGCGGGAG-3⬘
385 bp
5⬘-AGCGGAGCCTGGAGAGAAGG-3⬘ 5⬘-ACATGCGGGGCGGCTGTC-3⬘
202 bp
5⬘-ATCAGGCATGGCAGAACCCA-3⬘ 5⬘-TGTACACACACGCTCCTCCA-3⬘
219 bp
5⬘-ATGAGCCAGGGTCCTGGCA-3⬘ 5⬘-AAGTTGGAGGCAGGGGTGTA-3⬘
235 bp
5⬘-CGCAGAGTGTCTGAGTGGTT-3⬘ 5⬘-CTCCCTGCTCCTCCAGAAC-3⬘
178 bp
5⬘-CAGCCAGTGTCCACACGAT-3⬘ 5⬘-GACAGGCAGACAGAAGGAGT-3⬘
182 bp
5⬘-TGGCCCCTGGTACATTTGA-3⬘ 5⬘-CCTGAACAAGTGGATGAAGG-3⬘
179 bp
5⬘-CAGATGTGCCTGAGGAAGTC-3⬘ 5⬘-ACTGCTTCCTCTGTGACAGC-3⬘
165 bp
5⬘-GAATCTGCATCTTGGGCAGG-3⬘ 5⬘-GTCTCCAGCTGTGACCGAAA-3⬘ 5⬘-ATTCCAGCCCCTCGGAGT-3⬘ 5⬘-TTGGCCCAGAAAGAGCCTCA-3⬘
278 bp 283 bp
Exons 4, 9 and 5⬘-portion of exon 6 were analyzed in the previous paper.6
OR = 2.28). In addition, it was revealed that almost all individuals possessing nt168G had nt587G. We therefore carried out haplotype estimation based on the EH program.8 Relative linkage disequilibrium values were estimated from the genotypes of the healthy Japanese individuals using the difference between observed and expected frequencies of allele combinations, standardized by the maximum possible value.9 As shown in Table 3, these two SNPs were in tight linkage disequilibrium; nt168A was positively associated with nt587T, while nt168G was positively associated with nt587G. Such linkage disequilibrium was also observed in the patients. Two variations, −1413 (A→C) and −1120 (G→C), were previously reported in the promoter region of TNFR2.10 However, population-based studies to estimate the allele frequencies have not been reported. In order to test the possibility that the association of nt168G–nt587G allele with SLE derived from linkage disequilibrium with promoter polymorphisms, we carried out PCR-SSCP analysis of the region encompassing the two known variation sites (−1458 to −1073) in 81 Japanese patients with SLE and 129 healthy individuals randomly selected from 258 controls. Fifteen individuals were also examined by direct sequencing. A new single nucleotide substitution was detected at position −1194 (G→A) in one patient with SLE. On the other hand, all samples were homozygous for both of the previously reported −1413C and −1120C variations. Thus, at least in the Japanese population, these nucleotides are considered to constitute the common TNFR2 promoter allele. Furthermore, single nucleotide substitutions were detected in intron 2 [178+13 (A→G)] and intron 5 [552−28 Genes and Immunity
(C→T)] while the samples containing exon 2 and exon 6 SNPs were sequenced. Among the sequenced eight alleles, all three alleles possessing nt168G and nt587G were found to possess these intronic nucleotide substitutions. In addition, nt1409 (G→A) was detected within the 3⬘-untranslated region (3⬘UTR) in only one patient with SLE, but not in healthy individuals. The association of SLE with TNFR2-196R11 or another SNP within the 3⬘UTR12 was not observed in the Caucasian populations. It is therefore possible that this SNP might possess a significant effect in the Japanese, but not in the Caucasian populations. Further studies on other populations, especially other Asian populations, will be of particular interest. It is interesting to note that, although located within the same exon 6, SNP at nt587 is associated with SLE, while SNP at nt694 is not. This is because nt694A (232K) seems to be present almost exclusively in the allele carrying nt587T (196M) (data not shown), suggesting that nt694A originated from the allele carrying nt587T after the divergence of nt587G allele. Thus, it will be important to take the evolutionary pathway of SNPs into consideration when any disease association is examined using a huge number of SNPs in the future. In conclusion, we identified several new variations in the coding region, promoter and introns of TNFR2. Association with SLE was observed only in nt168G and nt587G SNPs, among which the latter results in amino acid substitution (M196R). Although sufficient experimental data is not available concerning the biological effects of M196R substitution, it is possible that this nonconservative substitution within the fourth cysteine-rich
New SNPs in TNFR2 N Tsuchiya et al
Table 2 New single nucleotide polymorphism of TNFR2 in the Japanese patients with SLE and controls (a) Positivity and genotype frequency of TNFR2-nt168A/G (56K/K) in SLE and controls SLE (n = 81) allele positivity nt168G+ 28 nt168A+ 80 genotype frequency nt168G/G 1 nt168A/G 27 nt168A/A 53
2
Controls (n = 258)
(35%) 47 (18%) (99%) 253 (98%)
P
Odds ratio
9.57a 0.002 NS
2.37
(b) Frequencies of other TNFR2-SNPs in SLE and controls Controls (n = 91)
503
Acknowledgements
( 1%) 5 ( 2%) 11.10b 0.004 (33%) 42 (16%) (66%) 211 (82%)
SLE (n = 80)
variations in other genes linked with TNFR2 nt587T cannot be excluded. Further studies should focus on the effect of the M196R substitution as well as on the screening of other genes located close to TNFR2.
P
The authors are indebted to Dr Tetsufumi Inoue, Dr Keiko Ishihara, Dr Shigeto Tohma, Dr Naoto Hirose, Dr Takeshi Suzuki, Dr Hiroshi Furukawa (Department of Allergy and Rheumatology, University of Tokyo) and Dr Kunio Matsuta (Matsuta Clinic) for the recruitment of the patients, to Michiko Shiota (Department of Human Genetics, University of Tokyo) for technical assistance, and to Dr Chika Morita and Dr Takahiko Horiuchi (The First Department of Internal Medicine, Kyushu University) for helpful discussions.
References exon5 nt543 (C→T) (P181P) genotype frequency nt543T/T nt543C/T nt543C/C
exon6 nt694 (G→A) (E232K) genotype frequency nt694A/A nt694G/A nt694G/G a
0 (0%) 4 (5%) 76 (95%)
0 (0%) 3 (3%) 88 (97%)
NS
SLE (n = 81)
Controls (n = 205)
P
0 (0%) 6 (7%) 75 (93%)
0 (0%) 16 (8%) 189 (92%)
NS
d.f. = 1, bd.f. = 2
Table 3 Linkage disequilibrium between nt168A/G (56K/K) and nt587T/G (196M/R) estimated from the genotypes of 258 healthy Japanese individuals Haplotype 168A-587T 168A-587G 168G-587T 168G-587G
HF
LD
RLD
2
P
0.886 0.014 0.006 0.095
+0.086 −0.086 −0.086 +0.086
+0.93 −0.93 −0.93 +0.93
414.8
⬍10−10
HF, haplotype frequency; LD, linkage disequilibrium parameter; RLD, relative linkage disequilibrium value.
domain in the extracellular region has some effect on the TNF binding affinity and/or signaling pathway. On the other hand, the possibility of the primary role of the
1 Tsao BP. Genetic susceptibility to lupus nephritis. Lupus 1998; 7: 585–590. 2 Elliott MJ, Maini RN, Feldmann M et al. Repeated therapy with monoclonal antibody to tumor necrosis factor ␣ (cA2) in patients with rheumatoid arthritis. Lancet 1994; 344: 1125–1127. 3 Jacob CO, Lee SK, Strassmann G. Mutational analysis of TNF␣ gene reveals a regulatory role for the 3⬘-untranslated region in the genetic predisposition to lupus-like autoimmune disease. J Immunol 1996; 156: 3043–3050. 4 Gaffney PM, Kearns GM, Shark KB et al. A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families. Proc Natl Acad Sci USA 1998; 95: 14875–14879. 5 Shai R, Quismorio FP Jr, Li L et al. Genome-wide screen for systemic lupus erythematosus susceptibility genes in multiplex families. Hum Mol Genet 1999; 8: 639–644. 6 Komata T, Tsuchiya N, Matsushita M, Hagiwara K, Tokunaga K. Association of tumor necrosis factor receptor 2 (TNFR2) polymorphism with susceptibility to systemic lupus erythematosus. Tissue Antigens 1999; 53: 527–533. 7 Matsushita M, Tsuchiya N, Oka T, Yamane A, Tokunaga K. New variations of human SHP-1. Immunogenetics 1999; 49: 577–579. 8 Terwilliger JD, Ott J. Handbook of Human Linkage Analysis. Johns Hopkins University Press: Baltimore, 1994, pp 188–193. 9 Lewontin RC. The interaction of selection and linkage. I. General considerations, heterotic models. Genetics 1964; 49: 49–67. 10 Santee SM, Owen-Schaub LB. Human tumor necrosis factor receptor p75/80 (CD120b) gene structure and promoter characterization. J Biol Chem 1996; 271: 21151–21159. 11 Al-Ansari AS, Ollier WER, Villarreal J, Ordi J, Teh L-S, Hajeer AH. Tumor necrosis factor receptor II (TNFRII) exon 6 polymorphism in systemic lupus erythematosus. Tissue Antigens 2000; 55: 97–99. 12 Sullivan KE, Piliero LM, Goldman D, Petri MA. A TNFR2 3⬘ flanking region polymorphism in systemic lupus erythematosus. Genes Immun 2000; 1: 225–227.
Genes and Immunity
