1 Deutsches Rheuma Forschungs Zentrum Berlin,
2 Department of Medicine, Division of Nephrology and Rheumatology, Klinikum Benjamin Franklin, Free University, Berlin and
3 Immanuel-Krankenhaus Berlin, Germany
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Abstract |
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Methods. Combining single-stranded oligo or sequence-specific primer typing procedures, 139 randomly selected controls and 106 OA patients were typed for their HLA-DRB1 alleles.
Results. The OA cohort showed statistically significant differences in the frequencies of the DR2 and DR5 alleles compared with the controls. While the frequency of the DR2 allele was elevated among the OA patients, the DR5 allele was negatively associated with the disease. The P values for differences from the controls were 0.0431 for DR2 and 0.0386 for DR5 and the odds ratios for the two alleles were 1.58 and 0.54 respectively.
Conclusion. The association of DR2 and DR5 with OA hints at linkage disequilibrium between HLA-DRB1 genes and genes involved in the pathogenesis of OA. Alternatively, DR2 has a direct role in restricting immunological responses to the low-grade inflammation characteristic of OA.
KEY WORDS: Osteoarthritis, Cytokines, HLA-DRB1 haplotypes.
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Introduction |
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The joint destruction of OA comprises loss of articular cartilage resulting from imbalance between cartilage breakdown and regeneration [5, 6]. We have shown previously that cytokines and growth factors involved in cartilage metabolism are up-regulated in the articular cartilage of OA patients [7]. This up-regulation resulted in two different phenotypes, which we called tumour necrosis factor (TNF)-Hi and TNF-
Lo. The two phenotypes differed significantly in the expression of a number of cytokines and growth factors: high percentages of TNF-
+ and interleukin (IL)-6+ and low percentages of IL-1ß+, TGF-ß+, IL-4+ and IL-10+ chondrocytes are characteristic of the TNF-
Hi phenotype while the TNF-
Lo phenotype shows the opposite expression pattern, with low percentages of TNF-
+ and IL-6+ and elevated percentages of IL-1ß+, TGF-ß+, IL-4+ and IL-10+ chondrocytes [7]. The combination of low-grade inflammation and elevated levels of inflammatory cytokines associated with OA led us to investigate the involvement of the immune system in balancing inflammatory vs degenerative reactions. As disease-associated and protective HLA-DRB1 alleles have been described for the chronic inflammatory disease rheumatoid arthritis (RA) [8, 9], we set out to investigate the association between HLA-DRB1 alleles and OA. To this end, 106 OA patients and 139 controls were analysed for their DRB1 haplotypes.
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Patients and methods |
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HLA typing procedures
DNA was extracted from peripheral blood mononuclear cells (PBMC) from the controls and the second OA cohort and from chondrocytes from the first cohort using standard procedures. Briefly, cartilage was digested with 1 mg/ml collagenase type IV (Sigma) at 37°C overnight. Both isolated chondrocytes and PBMC were lysed in NP-40-containing buffer (0.2% NP-40, 0.9% NaCl in distilled water) and the nuclei were pelleted, resuspended in 5 ml SET buffer [150 mM NaCl, 5 mM EDTA (pH 8), 50 mM Tris, pH 7.5, 1% sodium dodecyl sulphate] and incubated with 0.1 mg/ml proteinase K for 12 h at 37°C. The genomic DNA was phenol-extracted and precipitated with ethanol. Alternatively, the Wizard DNA Purification Kit (Promega, Madison, WI, USA) was used.
Polymorphism at the HLA-DRB1 locus was typed using genomic DNA, the polymerase chain reaction (PCR) and biotinylated single-stranded oligo (SSO) [10]. To distinguish between the alleles HLA-DRB1*05 and *06, additional PCRs with sequence-specific primer (SSP) were performed. For the identification of amino acids in position 713 within the third hypervariable region, we used the 5' primers TTTCTTGGAGTACTCTACGTC and TTTCTTGGAGTACTCTAC GGG in combination with the 3' primer GH50 [10]. For the identification of amino acids in positions 5260 and 6571, the 5' primer GAGTACTCTACGTCTGAG was used in combination with the 3' primers TGGCTGTTCCAGTACTCCTCATC, GGCTGTTCCAGTACTCGGCGCT, TGGCTGTTCCAGGACTCGGCGA, TGGCTGTTCC AGTGCTCCGCAG, CTTCTGGCTGTTCCAGTACTCGG (5260) and TGTCCACCGCGG CCCGCCTG, GTGTCCACCGCGGCCCGCTC, CCACCGCGGCCCGCCTCCGC, GTGTC CACCGCGGCCCGCTT and GTCCACCGCGGCCCGCCTCT (6571). PCR conditions were 32 cycles, an annealing temperature of 57°C and 2.5 mM MgCl2 (amino acids 713) and 39 cycles, an annealing temperature of 69°C and 1.7 mM MgCl2 (amino acids 5260 and 6571).
Statistical evaluation
Odds ratios and P values for individual allele distributions were calculated, using the software SPSS 6.1 (SPSS, Chicago, IL, USA) [11]. P-values were obtained by the use of the 2 test (2x2 contingency tables). Continuity (Yates') correction was applied if the expected frequency in a cell was below 5, resulting in the corrected P value (Pcorr). Global
2 tests were performed to compare HLA-DR haplotype distributions in our controls with those in patient cohorts and ethnically matched controls published elsewhere [12, 13].
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Results |
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For DR2, the allele frequency was statistically significantly increased in the first cohort, and this increase was independent of the TNF-Hi or TNF-
Lo phenotype described previously [2]. The frequency was 26.5% (31/117 alleles) compared with only 17% in the controls. In the second cohort, the allele frequency for DR2 was 21.7% (20/92 alleles) and was not different enough from that of the controls to reach statistical significance. However, the trend towards an elevated DR2 frequency in the first cohort was repeated in the second. The combined allele frequency of 24.4% for DR2 for both cohorts is again statistically significantly different from that for the controls, resulting in a P value of 0.0431. The odds ratio for the development of OA conferred by the DR2 allele was 1.58 (Table 1
). In addition, 9.4% of the OA patients and only 2.9% of the controls were homozygous for DR2, raising the odds ratio conferred by the double dose of OA-associated alleles to 3.5 (data not shown). In most instances, our typing method of combining SSO with SSP allows only the determination of groups of alleles rather than single alleles. Thus, 34 out of 51 DR2 alleles were *1501, *1502, *1504 or *1602. The increase in the allele frequency for DR6 observed in the first cohort was not detected in the second cohort.
DR5 is negatively associated with OA
Comparing the combined allele frequencies of both patient cohorts with the control frequencies revealed a statistically significant association with the DR5 allele, resulting in a P value of 0.0386 (Table 1). This association was negative and the odds ratio for protection from OA conferred by the DR5 allele was 0.542. Taken alone, neither of the two cohorts showed any significant difference from the controls.
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Discussion |
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While the DR4 haplotype is associated with the chronic inflammatory joint disease RA, DR2 in RA has been described as neutral if not protective [8, 9]. The interesting observation here is that DR2 is positively associated with OA (Table 1). It is intriguing to speculate that DR2 as a haplotype allows the development of a degenerate joint disease with only low-grade inflammatory episodes while DR24 favours chronic inflammation.
The OA patients analysed here for their DRB1 haplotypes have previously been described phenotypically for their cytokine expression pattern [2]. However, the observed increase in the frequency of DR2 does not segregate with either the TNF-Hi or the TNF-
Lo phenotype. We thus speculate that the association of OA with a certain HLA-DRB1 haplotype is greater than that of additional cytokine gene polymorphisms.
An association between DR2 and Heberden's nodes has been shown previously in a group of Ashkenazi patients [15] and a similar trend was described for Mexican mestizos [16]. However, it has been shown for autoimmune diseases that HLA class II associations are different for the various ethnic groups and the same seems to apply to OA [17]. It cannot be ruled out that an association with DR2 or DR5 is only indicative of an association with a polymorphic gene linked to the DRB1 locus. Indeed, HLA and the gene encoding the 2 chain of type XI collagen (COL11A2) are tightly linked on the short arm of chromosome 6 (6p21.3) and evidence of linkage of female hip OA to HLA/COL11A2 has been shown [18]. It remains to be determined whether a specific COL11A2 gene polymorphism segregates with the DR2 and/or DR5 haplotypes.
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Acknowledgments |
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Notes |
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References |
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