Tumor necrosis factor receptor 2 polymorphism in systemic lupus erythematosus: no association with disease

Tumor Necrosis Factor Receptor 2 Polymorphism in Systemic Lupus Erythematosus: No Association With Disease E. B. Lee, J. E. Yoo, Y. J. Lee, Y. J. Choi, K. S. Park, and Y. W. Song ABSTRACT: Genetic factors and immune dysregulation play important roles in the development of systemic lupus erythematosus (SLE). Tumor necrosis factor receptor 2 (TNFR2) is suggested to be involved in the development of SLE because its genetic locus (1p36) encompasses one of the susceptible loci for SLE and its ligand (TNF) is associated with SLE. To investigate the role of TNFR2 in the pathogenesis of SLE, 139 Korean patients were genotyped with SLE, 137 healthy control subjects were genotyped for TNFR2 196 R/M polymorphism in exon 6 with PCR-SSCP, and the clinical characteristics of SLE were analyzed according to the genotypes. The genotype frequencies of 196 R/R, 196 R/M, and 196 M/M were 3.6%, 30.9%, and 65.5% in SLE patients and 4.4%, 26.3%, and 69.3% in healthy controls (p ⫽ 0.676). The allelic frequency of 196 R was 19.1% in SLE patients and 17.5%

ABBREVIATIONS ACR American College of Rheumatology PCR-SSCP polymerase chain reaction–singlestrand conformation polymorphism SLE systemic lupus erythematosus

INTRODUCTION Tumor necrosis factor (TNF) has been suggested to be protective against systemic lupus erythematosus (SLE) [1, 2]. TNF functions via two distinct receptors: 55 kDa-TNFR1 and 75 kDa-TNFR2. Although TNFR1 mediates most of the activities of TNF, TNFR2 also

From the Department of Internal Medicine (E.B.L., Y.J.L, Y.J.C., Y.W.S.), Seoul National University College of Medicine, and the Department of Biology (J.E.Y., K.S.P.), Sungshin Women’s University, Seoul, Korea. Address reprint requests to: Yeong Wook Song, MD, Department of Internal Medicine, Seoul National University College of Medicine, 28 Yungon-dong, Chongno-gu, Seoul 110-744, Korea; Tel: ⫹82 (2) 7602234; Fax: ⫹82 (2) 762-9662; E-mail: [email protected]. Received December 11, 2000; revised February 12, 2001; accepted May 23, 2001. Human Immunology 62, 1148 –1152 (2001) © American Society for Histocompatibility and Immunogenetics, 2001 Published by Elsevier Science Inc.

in healthy controls (p ⫽ 0.638, odds ratio ⫽ 1.109, and the 95% confidence interval ⫽ 0.720 –1.708). The clinical characteristics were not different according to the genotypes. In conclusion, no skewed distribution of TNFR2 196 R/M polymorphism was found in Korean patients with SLE compared with healthy controls. Further studies in other populations will be needed to elucidate the role of the TNFR2 polymorphism in the development of SLE. Human Immunology 62, 1148 –1152 (2001). © American Society for Histocompatibility and Immunogenetics, 2001. Published by Elsevier Science Inc. KEYWORDS: systemic lupus erythematosus; TNFR2 196 R/M; polymorphism

SLICC TNF TNFR1 TNFR2

Systemic Lupus International Collaborating Clinics tumor necrosis factor tumor necrosis factor receptor 1 tumor necrosis factor receptor 2

mediates a wide spectrum of cellular responses ranging from cellular proliferation to apoptosis [3,4]. In SLE patients, the level of soluble TNFR2 has been found to correlate with the disease activity [5]. The gene that encodes TNFR2 lies in human chromosome 1p36, which is one of the probable susceptibility loci of SLE [6, 7]. Furthermore, the gene has been suggested as a candidate gene within the susceptibility loci in murine SLE [8, 9]. The strategic location of the gene and the role of TNF and TNFR2 in the pathogenesis of SLE suggest that TNFR2 polymorphism might be associated with SLE. Biallelic polymorphisms leading to amino acid substitutions have been found within exon 4, 6, and 9 of 0198-8859/01/$–see front matter PII S0198-8859(01)00280-4

TNFR2 196 R/M Polymorphism in SLE

the TNFR2 gene. Among these polymorphisms, exon 6 polymorphism, which consists of either methionine (M) or arginine (R) at amino acid position 196, revealed association with SLE in Japanese patients [10]. However, the association was not confirmed in Spanish or British patients with SLE [11]. The results of polymorphism studies can differ according to the study population or clinical subsets of the patients. In this study, the TNFR2 196 R/M polymorphism in Korean patients with SLE was genotyped to investigate the association between the polymorphism and SLE in Koreans, and to evaluate the effect of the polymorphism on clinical features. MATERIALS AND METHODS Patients A total of 139 Korean patients with SLE, seen at the Rheumatology Clinic of Seoul National University Hospital, were enrolled consecutively between January 1998 and May 1999 (12 males and 127 females, mean age 34.1 years old, SD ⫽ 11.9, range 16 – 64 years old). All patients were diagnosed as SLE according to the revised criteria of the American College of Rheumatology (ACR) [12]. Kidney biopsy was performed in 33 patients who had lupus nephritis not responding to high dose prednisolone. WHO classification was applied based on the pattern of the nephritis determined by a pathologist who did not know TNFR2 genotype of the patient [13]. The anti-dsDNA antibody was measured by radioimmunoassay (Johnson & Johnson Clinical Diagnostics, Rochester, NY, USA). The titer of anti-dsDNA antibody and the complement levels were measured every 3 months, and the 24-h urine protein was measured every 6 months. The highest values for the anti-dsDNA antibody titer and proteinuria were used as quantitative traits in this analysis. The amount of proteinuria in a 24-h period, creatinine clearance, titer of serum anti-dsDNA antibodies, number of ACR criteria fulfilled for SLE, and the Systemic Lupus International Collaborating Clinics (SLICC)/ ACR damage index [14] were determined for each patient. Every clinical feature in the ACR criteria for SLE was also determined in each patient [14]. A total of 137 healthy Korean people were enrolled as control subjects. Control subjects were randomly collected from blood donors. Polymerase Chain Reaction–Single-Strand Conformation Polymorphism Genomic DNA was extracted from peripheral blood of the patients and healthy controls using the QIAamp blood kit (Qiagen, Valencia, CA, USA), and polymerase

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chain reaction–single-strand conformation polymorphism (PCR-SSCP) was performed on each extracted DNA. The primers used to amplify the fragment, including a single nucleotide substitution within the exon 6 of TNFR2 gene, were the following: TR2A, 5⬘ACTCTCCTATCCTGCCTGCT-3⬘; and TR2B, 5⬘TTCTGGAGTTGGCTGCGTGT-3⬘ [10]. The total volume of 25-␮l reaction mixtures contained 7 ng of genomic DNA, 10 pmol of each primer, 150 ␮M of dNTPs, 10⫻ reaction buffer (10-mM Tris-HCL, pH 8.3, 40-mM KCl, 15-mM MgCl2), and 0.5 unit of Taq DNA polymerase (Bioneer, Chungju, Korea). The PCR amplification was carried out in a Thermal Cycler 9600 (Perkin Elmer, Norwalk, CT, USA). The conditions for amplification consisted of initial denaturation at 96 °C for 10 min, followed by 35 cycles of denaturation of 96 °C for 30 seconds, 62 °C for 30 seconds, and extension at 72 °C for 15 seconds with a final cycle of 72 °C for 8 min. The amplified products were diluted to a ratio of 1:7 with denaturing solution (95% formamide, 20-mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol), denatured at 96 °C for 6 min and immediately cooled on ice; 3-␮l aliquots of the reaction mixture were applied to 10% polyacrylamide gel (acrylamide:bis-acrylamide ⫽ 49:1) containing 5% glycerol. Electrophoresis was carried out in 0.5⫻ TBE (45-mM Tris-borate [pH 8.0], 1-mM EDTA) under 20 mA/gel for 2 h at 15 °C. Single-strand DNA fragments in the gel were visualized by silver staining. The nucleotide sequences of the samples revealing different SSCP patterns were confirmed by direct sequencing. Fluorescence-based automated cycle sequencing of the PCR products was performed on an ABI 377 DNA sequencer (PE Applied Biosystems, Norwalk, CT, USA). Statistical Analysis The Chi-square test was applied to compare the genotype and allele frequencies of TNFR2 196 R/M in patients and healthy controls. The ANOVA or Kruskal-Wallis test was used to compare the following clinical features according to genotype: disease duration, creatinine clearance, serum anti-dsDNA antibody titer, number of ACR criteria for SLE met, and SLICC/ ACR damage index. All calculations were performed with SAS 6.12 version for Windows (SAS Institute, Cary, NC, USA). RESULTS AND DISCUSSION This study revealed no significant differences in genotype or allele frequencies between SLE patients and healthy controls (for genotype: ␹2 ⫽ 0.783, p ⫽ 0.676, for allele frequencies: ␹2 ⫽ 0.221, p ⫽ 0.638, odds ratio ⫽ 1.109,

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TABLE 1 Distribution of TNFR2 196 R/M polymorphism in Korean patients with SLE and healthy controls

Genotype frequency, number (%) 196 R/R 196 R/M 196 M/M Allelic frequency, number (%) 196 R 196 M

SLE patients (n ⫽ 139)

Healthy controls (n ⫽ 137)

p Value

Odds ratio

95% Confidence interval

5 (3.6) 43 (30.9) 91 (65.5)

6 (4.4) 36 (26.3) 95 (69.3)

0.676

— 0.698 0.870

— 0.197–2.476 0.257–2.950

53 (19.1) 225 (80.9)

48 (17.5) 226 (82.5)

0.638

— 1.109

— 0.720–1.708

Abbreviations: SLE ⫽ systemic lupus erythematosus; TNFR2 ⫽ tumor necrosis factor receptor 2.

95% confidence interval ⫽ 0.720 –1.708, Table 1). The distribution of genotype or allele frequencies were similar between biopsy-proven lupus nephritis patients and healthy controls (for genotype: ␹2 ⫽ 0.542, p ⫽ 0.762, for allele frequencies, ␹2 ⫽ 0.574, p ⫽ 0.449, Table 2). When the patients were separated into those with or without nephritis, based on the amount of proteinuria (500 mg/d), there was no difference between patients with nephritis and healthy controls or patients with and without nephritis. The creatinine clearance, proteinuria, number of ACR criteria met, SLICC/ACR damage index, and titer of the anti-ds DNA antibodies were not different according to the TNFR2 196 R/M polymorphisms (Table 3). Furthermore, the distribution of genotype or allele frequencies was not different according to the other clinical features: malar rash, discoid rash, photosensitivity, oral ulcer, arthritis, serositis, central nervous system manifestations, hematologic manifestations, and autoantibodies (data not shown). To the best of our knowledge there have been three different studies on the association of TNFR2 polymorphism with SLE [10, 11, 15]. Komata et al. [10] reported a significant increase in the frequency of TNFR2 196 R allele in Japanese SLE patients compared with healthy controls, using the PCR-SSCP method. In their study, 30 of 81 patients (37.0%) with SLE were positive for the 196 R allele, compared with 39 of 207 healthy individ-

uals (18.8%) (p ⫽ 0.0001). However, a skewed distribution was not confirmed in the other studies. D’Alfonso et al. [15] found no association of TNFR2 with SLE in Italian patients using microsatellite polymorphism in TNFR2 intron 4. Al-Ansari et al. [11] did not describe any significant difference in the TNFR2 196 R/M polymorphism in Spanish and British SLE patients populations using the PCR-restriction fragment length polymorphism method. Contrary to our expectation that the polymorphism in Korean patients would be more similar to that in Japanese than European patients, this study did not find any skewed distribution of TNFR2 196 R/M polymorphism in Korean SLE patients. The difference between the current results and those of Komata et al. [10] could be explained in several ways. First, SLE is a complex disease and the difference in the disease phenotype between Korean and Japanese patients might produce different results. Second, there may be a genetic difference between the Koreans and Japanese. The distribution of TNFR2 196 R/M polymorphism was considerably different in the Korean and Japanese healthy controls, where the frequencies of TNFR2 196 R/R, R/M, and M/M were 0.9%, 17.9%, and 81.2% in Japanese controls [10] and 4.4%, 26.3%, and 69.3% in the Korean controls. Third, TNF receptor polymorphisms might be associated with the development of SLE indirectly. If a gene is the direct cause of a disease, it tends

TABLE 2 Distribution of TNFR2 polymorphism in SLE patients with biopsy-proven nephritis compared with healthy controls SLE patients with nephritis (n ⫽ 33)* Genotype frequency (%) 196 R/R 196 R/M 196 M/M

I

II 1 2

III

IV

1

1 5 20

V

UD

Sum

Healthy controls (n ⫽ 137)

1

1 1

1 (3.0) 7 (21.2) 25 (75.8)

6 (4.4) 36 (26.3) 95 (69.3)

SLE patients versus healthy controls, p ⫽ 0.390 by chi-squared test. * WHO classification of lupus nephritis [16]. Abbreviations: SLE ⫽ systemic lupus erythematosus; TNFR2 ⫽ tumor necrosis factor receptor 2; UD ⫽ undetermined.

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TABLE 3 Clinical analysis of the lupus patients according to the TNFR2-196 R/M genotype

Disease duration, months Creatinine clearance, ml/min Proteinuria, mg/24 h Number of ACR criteria met SLICC/ACR damage index Anti-dsDNA antibody, IU/ml

196 R/R (n ⫽ 5)

196 R/M (n ⫽ 43)

196 M/M (n ⫽ 91)

46.8 ⫾ 28.4 85.0 ⫾ 18.6 592.8 ⫾ 505.6 5.6 ⫾ 0.9 0.4 ⫾ 0.9 792.1 ⫾ 842.0

52.4 ⫾ 31.4 90.5 ⫾ 34.2 1827.9 ⫾ 3952.3 5.6 ⫾ 1.4 1.2 ⫾ 1.4 393.2 ⫾ 506.9

67.6 ⫾ 50.1 84.6 ⫾ 33.8 2399.6 ⫾ 3426.9 5.5 ⫾ 1.4 1.5 ⫾ 1.5 381.5 ⫾ 879.6

Values are the mean ⫾ SD. There is no significant difference in the clinical manifestations according to the genotypes. Abbreviations: Anti-dsDNA ⫽ antidouble-stranded DNA; ACR ⫽ American College of Rheumatology; SLICC ⫽ Systemic Lupus International Collaboratory Clinics; TNFR2 ⫽ tumor necrosis factor receptor 2.

to exhibit consistent skewing regardless of the ethnic group. However, if a gene is a simple genetic marker associated with a true causative gene, the results of the polymorphism studies will vary depending on the study populations because of different patterns of linkage disequilibrium. Therefore, the different results between Korean and Japanese patients suggest that the TNFR2 gene might be an indirect marker gene rather than a direct causative gene. Assuming the ␣ level of 0.1, the statistical power of this study is 9.6% with double-sided testing and 17.3% with one-sided testing. In order to reach the adequate power of 80%, a study of over 7300 persons in each group would be necessary to illustrate the difference of 1.6%, if the ratio does not change, which is a figure of the total expected number of Korean patients with SLE. In conclusion, no association of TNFR2 196 R/M polymorphisms with SLE in Koreans was evident. In addition, there was no difference between the lupus nephritis patients and healthy controls, and the clinical characteristics were not different according to the TNFR2 196 R/M genotypes. Further studies in the other ethnic groups will be needed to elucidate the association of TNFR2 196 R/M polymorphisms with SLE. ACKNOWLEDGMENT

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This work was supported by a grant from Seoul National University.

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