Genes and Immunity (2001) 2, 373–380 2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00 www.nature.com/gene Six microsatellite markers on the short arm of chromosome 6 in relation to HLA-DR3 and TNF−308A in systemic lupus erythematosus MW van der Linden1,2, AR van der Slik3, E Zanelli3, MJ Giphart3, E Pieterman2, GMTh Schreuder3, RGJ Westendorp4 and TWJ Huizinga2 Departments of 1Clinical Epidemiology, 2Rheumatology, 3Immunohaematology and Blood Transfusion, 4General Internal Medicine, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands Differences in allelic distribution at loci surrounding the human HLA-DRB1 and tumor necrosis factor (TNF) genes have been observed in association with systemic lupus erythematosus (SLE). We investigated whether the association of HLA- DRB1*0301 (HLA-DR3) and TNF−308A with SLE could be attributed to polymorphic markers in the chromosomal region encompassed by HLA-DRB1 and HLA-C. Ninety-one consecutive Caucasian patients with SLE and 253 controls (organ donors) were typed for HLA-DRB1, microsatellites D6S1014, D6S273, TNFa, MIB, C1F2F5, and C1F3F2 and the single nucleotide polymorphism at position −308 in the promoter of TNF. The independent contribution of alleles to disease susceptibility was estimated by cross-tabulation and multivariate logistic regression. Possession of TNF−308A was associated with susceptibility to SLE (odds ratio [95% confidence interval], 3.70 [2.24–6.11]). This remained present after stratification on possession of HLA-DR3 (pooled odds ratio, 2.53 [1.37–4.70]). Stratification revealed a possible association of possession of C1F2F5*192 with protection from SLE beyond the effects of HLA-DR3 and TNF−308A. A gene dosage effect was observed for −308A only (homozygotes, 7.75 [3.01–20.0], heterozygotes, 3.15 [1.85–5.37]). In multivariate analysis, possession of HLA-DR3, TNF−308A, and C1F2F5*192 remained independently associated with susceptibility to SLE (2.58 [1.29–5.18], 2.76 [1.43–5.31], and 0.26 [0.10–0.66], respectively). The association of possession of TNF−308A with susceptibility to SLE cannot be attributed to linkage to HLA-DR3 alone, nor to other polymorphic markers in the vicinity of the TNF gene. Further loci that are independently associated with SLE might be in the vicinity of marker C1F2F5. Genes and Immunity (2001) 2, 373–380. Keywords: HLA-DRB1; TNF; genetics; susceptibility; systemic lupus erythematosus Introduction be associated with susceptibility to SLE among African- Americans.12 Estimates of the allele frequency of the The chromosomal region encompassing the human leu- allele TNF−308A range from 17% to 28% in different Cau- kocyte antigen (HLA) Class II to Class I genes has been casian control populations.8,13–17 In our study among Eur- implicated in susceptibility to systemic lupus ery- opean Caucasians, the association between possession of thematosus (SLE) and other autoimmune diseases, such TNF−308A and SLE was independent of the existing as rheumatoid arthritis, insulin-dependent diabetes mel- association between possession of the HLA-DRB1*0301 litus, atopic astma, multiple sclerosis, and Crohn’s dis- (HLA-DR3) allele and SLE, in spite of linkage disequilib- 1–7 ease. Alleles of the HLA-Class II gene, HLA-DRB1, are rium between alleles of these loci.18 in linkage disequilibrium with genetic variations in the This previously detected association between presence 8,9 tumor necrosis factor (TNF) gene. of a TNF−308A allele and susceptibility to SLE might be Involvement of TNF in SLE was suggested by animal attributed to genetic variations in the region surrounding models, in which development of glomerulonephritis the TNF locus, apart from HLA-DR3. To investigate this 10 was delayed upon administration of recombinant TNF. possibility, we typed six microsatellite loci in a 1.8 million Disease activity in patients with SLE correlates with the base-pair region encompassed by HLA-DRB1 and HLA- 11 levels of circulating TNF. Recently, possession of the C in patients with SLE and controls. rare allele of a single nucleotide polymorphism located − at 308 base pairs from the transcription initiation site in Results the promoter of TNF (TNF2, TNF−308A), was shown to Demographic characteristics The mean age of the patients was similar to that of the Correspondence: Dr TWJ Huizinga, Department of Rheumatology, Build- controls (39.6 ± 1.5 vs 38.6 ± 1.1 years, mean ± standard ing 1, C4-R, Leiden University Medical Center, P.O. Box 9600, 2300 RC error). The patients were more often female than the con- Leiden, The Netherlands. E-mail: T.W.J.Huizinga.reumaȰlumc.nl MW van der Linden is supported by a grant of the Dutch Organiza- trols (92% and 46% females, respectively), but this did tion for Scientific Research (N.W.O.), grant no. 904–61–110. not influence genotype distributions among patients Received 8 February 2001; revised and accepted 6 August 2001 and controls. Microsatellite markers in SLE MW van der Linden et al 374 Allelic distribution of polymorphic markers contribution of each risk factor and thus to adjust for the Homozygosity of polymorphic markers was largest for effect of linkage disequilibrium, a stratified analysis was C1F3F2 with 39% in patients and 40% in controls and undertaken. The risk to develop SLE in the presence of lowest in MIB, with 15% and 11%. Four percent of all candidate alleles was studied in the presence and in the subjects were homozygous for HLA-DR3 (7% in patients, absence of HLA-DR3 separately. In Table 2 these odds 3% in controls) and 6% were homozygous for TNF−308A ratios are depicted separately for the HLA-DR3-positive (14% in the patients, 3% in the controls). and DR3-negative strata. To summarize the effect of each Individual alleles were studied. The relative risk to allele in both strata combined, the weighed estimate of develop disease, conferred by an extremely rare allele, the stratum-specificoddsratiosisgiven(Mantel–Haenszel may be larger than the relative risk conferred by a com- odds ratio). This pooled odds ratio is an estimate of the mon allele, merely as a consequence of small numbers. effect of each allele on susceptibility to (or protection from) To prevent the low background frequency of certain SLE, adjusted for its distribution over HLA-DR3-positive alleles from introducing spurious associations, the con- persons and HLA-DR3-negative persons. clusions were restricted to the effects of either relatively By this analysis of effects across the separate strata, the common alleles (more than 20% frequency of persons consequences of linkage disequilibrium between studied possessing at least of these alleles), or alleles with a clear alleles and HLA-DR3 became apparent. The odds ratios and distinctive impact on disease risk (unadjusted odds for presence of the alleles D6S1014*134 and MIB*350 ratio or unadjusted odds ratio−1 of 2.5 or greater). Also, became nonsignificant. This indicates that the effect of alleles that are bound to have such an impact merely on risk to develop SLE in the presence of these alleles is the basis of their low frequency, below 5%, are excluded. dependent on possession of HLA-DR3. For D6S273*139 Frequencies of persons possessing these 22 selected and TNFa*99, the stratum-specific odds ratios appeared alleles are depicted in Table 1, with odds ratios rep- to indicate opposite effects among HLA-DR3-positive resenting the risk to develop SLE among persons with and HLA-DR3-negative persons (test indicating hetero- the allele compared with that among persons without the geneity: P = 0.004 and P = 0.049, respectively). Therefore, allele. Frequencies of the presence of 10 alleles were sig- the pooled odds ratio is inconclusive for D6S237*139 and nificantly different among the patients compared with TNFa*99. Possession of the alleles D6S1014*143, controls (eight alleles present in increased frequencies, TNF−308A, C1F2F5*196 and C1F3F2*354 appeared to be two alleles present in decreased frequency). statistically significantly associated with susceptibility to SLE, independent of possession of HLA-DR3. The effect Stratified analysis was present and concordant in HLA-DR3-positives and Confounder effects arise, for example due to linkage dis- HLA-DR3-negatives. Possession of C1F2F5*192 appeared equilibrium with HLA-DR3. To estimate the individual protective, independent of possession of HLA-DR3. Table 1 Frequencies of selected polymorphic marker alleles in patients with systemic lupus erythematosus and controls Locus Allelea Patients (n = 91) Controls (n = 253) Odds ratiob (95% confidence interval) n (%) n (%) HLA-DR 3 45 (49) 57 (23) 3.36 (2.03–5.58)* D6S1014 134 28 (31) 113 (45) 0.55 (0.33–0.92)* 137 63 (69) 178 (70) 0.95 (0.56–1.60) 143 42 (46) 47 (19) 3.76 (2.23–6.32)* D6S273 131 26 (29) 60 (24) 1.29 (0.75–2.21) 133 37 (41) 131 (52) 0.64 (0.39–1.04) 135 43 (47) 140 (55) 0.72 (0.45–1.17) 139 27 (29) 45 (18) 1.95 (1.12–3.39)* TNF −308 A 52 (57) 67 (26) 3.70 (2.24–6.11)* TNFa 99 56 (65) 125 (49) 1.64 (1.01–2.67)* 107 20 (22) 71 (28) 0.72 (0.41–1.27) 115 17 (19) 53 (21) 0.87 (0.47–1.59) 117 29 (32) 78 (31) 1.05 (0.57–1.55) MIB 326 17 (19) 61 (24) 0.72 (0.40–1.32) 336 31 (34) 90 (36) 0.94 (0.57–1.55) 350 49 (54) 81 (32) 2.48 (1.52–4.04)* C1F2F5 192 6 (7) 41 (16) 0.37 (0.15–0.89)* 196 41 (45) 55 (22) 2.95 (1.77–4.91)* 199 28 (31) 94 (37) 0.75 (0.45–1.26) C1F3F2 342 33 (36) 100 (40) 0.87 (0.53–1.43) 354 54 (59) 95 (38) 2.43 (1.49–3.96)* 360 23 (25) 82 (32) 0.71 (0.41–1.21) aAlleles defined by fragment size (in base pairs), except HLA-DR3 and TNF-308A.