Polymorphism of the tumour necrosis factor-{alpha} gene at position –308 and renal manifestations of primary Sjögren's syndrome

M. Pertovaara1, J. Hulkkonen2, J. Antonen1,2, P. Holopainen3, P. Laippala1,4, A. Pasternack2 and M. Hurme1,2

1Tampere University Hospital, Tampere, 2University of Tampere Medical School, Tampere, 3Finnish Red Cross Blood Transfusion Service, Department of Tissue Typing, Helsinki and 4Tampere School of Public Health, University of Tampere, Tampere, Finland

Correspondence to: M. Pertovaara, Department of Internal Medicine, Section of Rheumatology, Tampere University Hospital, P.O. Box 2000, FIN-33521 Tampere, Finland. E-mail: marja.pertovaara{at}pshp.fi

SIR, Tumour necrosis factor-{alpha} (TNF-{alpha}) is a pro-inflammatory cytokine which plays an important role in the pathogenesis of many inflammatory and infectious diseases, including primary Sjögren's syndrome (pSS). TNF-{alpha} is expressed in minor salivary gland duct cells in patients with pSS [1]. It promotes the influx of mononuclear cells not only into the salivary glands but also, for example, to the renal epithelia [2], and has been shown to be involved in the pathogenesis of experimental tubulointerstitial nephritis. The roles of TNF-{alpha} in the tubulointerstitium are undefined, but may include infiltration of the interstitium by inflammatory cells and alteration in the tubular transport of fluid and electrolytes [3]. A base exchange polymorphism (G to A) at position -308 of the gene exists, designated TNF1 (G) and TNF2 (A) alleles. This polymorphism has an effect on susceptibility to and severity of several autoimmune diseases. The rarer TNF2 allele has been found to be associated with enhanced TNF-{alpha} production and higher transcriptional activity compared with TNF1 [4].

Renal manifestations, mostly due to tubulointerstitial inflammation, are among the commonest extraglandular findings in pSS. To investigate the possible role of the TNF-{alpha} (-308) gene polymorphism in the development of renal manifestations in pSS, we analysed this polymorphism after informed consent in 65 pSS patients (63 female, 2 male, mean age 60 ± 12 yr) selected by modified Californian criteria as described previously [5] and in 66 healthy Finnish Red Cross Blood Transfusion Service blood donors matched for sex, ethnic origin and area of residence. Sixty-two of the patients fulfilled the revised American–European consensus group criteria for SS [6]. The presence of the HLA-DR3 allele among the patients was also examined because of strong linkage disequilibrium with the TNF-{alpha} (-308) gene in northern European populations. The presence of distal renal tubular acidosis (dRTA) was ascertained by ammonium chloride loading tests in 45 of the patients and urinary total protein excretion was measured in 61 [7]. The study protocol was approved by the Ethical Committee of Tampere University Hospital.

Genomic DNA was isolated from blood samples using the salting-out method [8]. The base exchange polymorphism at position -308 of the TNF-{alpha} gene was analysed by polymerase chain reaction (PCR)-based genotyping [9]. HLA-DRB1 typing was performed with a commercial kit, LiPA HLA-DRB1 (Innogenetics, Murex Biotech Ltd, UK) or using the PCR method as previously described [10]. TNF-{alpha} plasma levels were measured using an enzyme-linked immunosorbent assay (Pelikine Compact human TNF-{alpha} ELISA kit, CLB, Amsterdam, The Netherlands), following the manufacturer's instructions. The optical density in individual wells was determined with a Multiscan Bichromatic 348 (Labsystems, Helsinki, Finland) spectrophotometer. The detection limit of the assay was 1.4 pg/ml.

The frequency of the TNF2 allele was higher in pSS patients compared with controls (52 vs 29%, P = 0.006, odds ratio 2.71, 95% confidence interval 1.32–5.58). A total of 48 and 71% of the patients and controls, respectively, were homozygous for TNF1, 51 and 26% heterozygous and 1.5 and 3% homozygous for the TNF2 genotype (P = 0.013, {chi}2-test for 3 x 2 table). The TNF2 allele was encountered more frequently in pSS patients with proteinuria than in those without (65 vs 40%, P = 0.050, n = 61) and in patients with dRTA compared with those with normal acidification capacity (69 vs 41%, P = 0.079, n = 45). The pSS patients with dRTA had significantly higher plasma TNF-{alpha} levels than those with normal renal acidification capacity (14.8 ± 21.7 vs 5.1 ± 10.1 pg/ml, P = 0.003). However, plasma TNF-{alpha} levels did not differ significantly between pSS patients with proteinuria and those with normal urinary protein excretion and were unaffected by the presence of TNF2 allele in pSS patients.

The pSS patients with the TNF2 allele yielded several laboratory findings suggestive of a more active form of the disease. Their mean levels of serum IgG (20.2 ± 6.8 vs 16.7 ± 7.1 g/l, P = 0.032) and the titres of anti-SSA or anti-SSB antibodies (84 ± 47 vs 48 ± 51 U/l, P = 0.005 and 98 ± 75 vs 24 ± 48 U/l, P < 0.0001, respectively) were higher than those of patients without this allele. The minor salivary gland histological findings were not related to TNF genotypes. The independent effect of the TNF2 allele on the clinical and immunological characteristics of the patients could, however, not be definitely judged as TNF2 allele and HLA-DR3 were highly significantly associated with each other (P < 0.0001) and there were no patients carrying TNF2 allele who were negative for DR3 (Table 1).


View this table:
[in this window]
[in a new window]
 
TABLE 1. Demographic, clinical and immunological characteristics of patients with primary Sjögren's syndrome (pSS) grouped by the combined presence of TNF2 allele positivity or negativity and HLA-DR3 positivity or negativity

 
In conclusion, these preliminary results suggest that the TNF-{alpha} gene polymorphism at position -308 might have an impact on susceptibility to renal manifestations of pSS, but the influence of HLA-DR3 cannot be ruled out owing to the limited number of patients. A power analysis showed that to reach a 30% difference in the presence of dRTA would necessitate a study of 35 patients with TNF2 allele but negative for HLA-DR3 and a comparable control group of other pSS patients.

This study was supported by grants from the Medical Research Fund of Tampere University Hospital, Tampere, Finland and the Finnish Medical Foundation.

The authors have declared no conflicts of interest.

References

  1. Fox RI, Kang HI, Ando D et al. Cytokine mRNA expression in salivary gland biopsies of Sjögren's syndrome. J Immunol 1994;152:5532–9.[Abstract/Free Full Text]
  2. Brennan FM, Feldmann M. Cytokines in autoimmunity. Curr Opin Immunol 1996;8:872–7.[CrossRef][ISI][Medline]
  3. Baud L, Ardaillou R. Tumor necrosis factor in renal injury. Miner Electrolyte Metab 1995;21:336–41.[ISI][Medline]
  4. Wilson AG, Symons JA, McDowell TL et al. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci USA 1997;94:3195–9.[Abstract/Free Full Text]
  5. Hulkkonen J, Pertovaara M, Antonen J, Pasternack A, Hurme M. Elevated interleukin-6 plasma levels are regulated by the promoter region polymorphism of IL-6 gene in primary Sjögren's syndrome (pSS) and correlate with clinical manifestations of the disease. Rheumatology 2001;40:656–61.[Abstract/Free Full Text]
  6. Vitali C, Bombardieri S, Jonsson R et al. Classification criteria for Sjögren's syndrome: a revised version of the European criteria proposed by the American–European consensus group. Ann Rheum Dis 2002;61:554–9.[Abstract/Free Full Text]
  7. Pertovaara M, Korpela M, Kouri T, Pasternack A. The occurrence of renal involvement in primary Sjögren's syndrome: a study of 78 patients. Rheumatology 1999;38:1113–20.[Abstract/Free Full Text]
  8. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215.[ISI][Medline]
  9. Wilson AG, di Giovine FS, Blakemore AI, Duff GW. Single base polymorphism in the human tumour necrosis factor alpha (TNF alpha) gene detectable by NcoI restriction of PCR product. Hum Mol Genet 1992;1:353.[Medline]
  10. Westman P, Kuismin T, Partanen J, Koskimies S. An HLA-DR typing protocol using group-specific PCR-amplification followed by restriction enzyme digests. Eur J Immunogenet 1993;20:103–9.[ISI][Medline]
Accepted 21 May 2003