Citation for published version (APA): Martens, H. A. (2009). Genetic and prognostic factors in lupus Groningen: s.n.

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1 University of Groningen Genetic and prognostic factors in lupus Martens, Henk Allard IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2009 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Martens, H. A. (2009). Genetic and prognostic factors in lupus Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Receptor for advanced glycation end products (RAGE) polymorphisms are associated with SLE, RA, and disease severity in lupus nephritis H.A. Martens 1, H.L.A. Nienhuis 1, S. Gross 2, G. van der Steege 3, E. Brouwer 1, J.H.M. Berden 4, R. de Sevaux 4, R.H.W.M. Derksen 5, A.E. Voskuyl 6, S.P. Berger 7, G.J. Navis 2, I.M. Nolte 8, C.G.M. Kallenberg 1, M. Bijl 1 1 Department of Rheumatology and Clinical Immunology, 2 Department of Nephrology, 3 Department of Medical Genetics, University Medical Center Groningen, University of Groningen, 4 Department of Nephrology, Radboud University Nijmegen Medical Center, Nijmegen, 5 Department of Rheumatology and Clinical Immunology, University Medical Center, Utrecht, 6 Department of Rheumatology, VU University Medical Center, Amsterdam, 7 Department of Nephrology, Leiden University Medical Center, Leiden, 8 Department of Epidemiology, University Medical Center Groningen, Groningen The Netherlands In preparation 83

3 Abstract Objective. Interaction of advanced glycation end products (AGE s) with their receptor (RAGE) plays an important role in inflammation in autoimmune diseases. Several functional polymorphisms of RAGE have been described. In this study, we analyzed the role of RAGE polymorphisms in disease susceptibility for systemic lupus erythematosus (SLE). Rheumatoid arthritis (RA) patients served as disease controls. In addition, we investigated whether these polymorphisms in SLE are associated with renal involvement (LN) and its outcome. Methods. DNA samples of 97 SLE patients, 114 LN patients, 408 RA patients and 429 healthy controls (HC) were genotyped for four RAGE polymorphisms: -429 T/C, -374 T/A, 2184 A/G and Gly82Ser. Differences in genotype frequencies and allele frequencies were tested between patients and HC. In addition, association of genotypes with disease severity in LN was analyzed. Results. The C allele in -429 T/C, the T allele in -374 T/A and the G allele in 2184 A/G were more prevalent in SLE and LN compared with HC (SLE: p=0.0007, p=0.004, p=0.0001; LN: p=0.0001, p=0.001, p=0.0004, respectively). RA was most significantly associated with the Ser allele in Gly82Ser (p= ). In LN, the C allele in RAGE -429 T/C, the A allele in -374 T/A and the G allele in RAGE 2184 A/G polymorphism were associated with more proteinuria and poorer renal function during the first two years of treatment. Conclusion. RAGE polymorphisms are associated with disease susceptibility in SLE and RA. In addition, some of these polymorphisms are associated with disease severity and response to treatment in LN. 84

4 RAGE polymorphisms in SLE, RA and lupus nephritis Introduction Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are autoimmune diseases characterized by chronic inflammation. Several factors have been shown to play a role in the pathogenesis of this chronic inflammation, amongst them advanced glycation end products (AGE s). AGE s are a class of compounds resulting from glycation of proteins, lipids or nuclear acids, partly under influence of oxidative stress. In both RA and SLE patients, an increased accumulation of AGE s has been found. 1;2 AGE s may contribute to inflammatory and destructive processes 3 by ligation to their receptor (RAGE). AGE s are also involved in the accelerated atherosclerosis observed in SLE and RA. 4;5 Besides AGE s, other proinflammatory ligands like HMGB-1 and S100 bind to RAGE, resulting in an increased inflammatory response. 6 The AGE-RAGE interaction can be disrupted by one of the isoforms of RAGE: soluble RAGE (srage). The function of srage is not fully understood, but it has been suggested that srage may act as a decoy receptor, blocking the proinflammatory AGE-RAGE interaction. This blockade stabilizes established atherosclerotic lesions and suppresses vascular inflammation. 7;8 In addition, higher levels of srage have been associated with longevity, suggesting a protective effect of srage on age-related disorders such as coronary heart disease, hypertension and Alzheimer s disease. 9 In line with this protective effect of high srage levels, low levels have been associated with the presence of coronary artery disease. 10 On the other hand, srage has also been linked with proinflammatory conditions. Increased levels of srage have been found in SLE patients with quiescent disease compared with healthy controls. These srage levels increased even further during active disease. 11 Thus, srage may have anti-inflammatory properties, particularly in atherosclerosis, but it has also been associated with proinflammatory conditions. In the latter case, srage may reflect active inflammation. The gene encoding for RAGE is situated on chromosome 6 in the locus of the major histocompatibility complex in the class III region. 12 Several functional polymorphisms for RAGE have been described. Some of them, e.g. the Ser allele in the Gly82Ser polymorphism and the A allele in the 2184 A/G polymorphism, seem to have a proinflammatory effect and are associated with progression of renal disease and cardiovascular disease in diabetes. 13;14 Other polymorphisms, e.g. the A allele in the -374 T/A polymorphism, are associated with a protective effect for cardiovascular disease. 14 In addition, some RAGE polymorphisms (Gly allele in Gly82 Ser, G allele in 2184 A/G and the C allele in -429 T/C polymorphism) have been associated with higher srage levels 85

5 and higher RAGE expression. 10;15-18 Thus, RAGE and RAGE polymorphisms are associated with inflammatory conditions as well as with progression of renal disease and the development of proteinuria. As RAGE plays an important role in inflammation, we evaluated the association of 4 functional RAGE polymorphisms with susceptibility for SLE and RA, the latter serving as a disease control. In addition, we determined the role of RAGE polymorphisms in disease severity in SLE, by analyzing patients with biopsy proven, proliferative lupus nephritis (LN), being one of the most severe manifestations of SLE. We also determined whether RAGE polymorphisms are associated with disease severity (proteinuria, renal function) in LN. Furthermore, we were able to measure the srage levels in our cohort of SLE patients during quiescent disease and to correlate these levels with the RAGE polymorphisms. Our study is the first that analyzed the involvement of RAGE polymorphisms in such a large population of patients with an autoimmune disease. Methods Patient selection SLE patients In the period November 2000 to November 2001 all SLE patients who were treated at the University Medical Center Groningen (UMCG), were contacted whether they were willing to participate in a study on the genetic predisposition for SLE. All patients met the criteria for SLE according to the American College of Rheumatology (ACR). 19 In total, 106 patients were included. Nine of these patients, however, were already included in the lupus nephritis studies (see below), so 97 SLE patients were included for further analysis. Lupus nephritis patients From September 1995 until November 2006, patients with biopsy proven proliferative lupus nephritis (ISN/RPS class III or IV) were included in the first and second Dutch lupus nephritis studies. Patients included in this study were treated according to a protocol (first study: azathioprine combined with methylprednisolone and prednisone vs. cyclophosphamide with prednisone 20 ; second study: prednisone with cyclophosphamide followed by mycophenolate mophetil). From these patients, 114 subjects gave consent for genetic analysis of blood samples. In LN, proteinuria and creatinine clearance (as measured by the Cockcroft-Gault formula) measured at the time of inclusion and after 5, 24 86

6 RAGE polymorphisms in SLE, RA and lupus nephritis and 31 months of therapy, were used as markers for outcome. If the follow-up was shorter than 31 months, the last available follow-up data were included. Disease controls: RA patients As disease controls, RA patients were included. In 2002, all RA patients who fulfilled the ACR criteria for RA 21 treated at the University Medical Center Groningen were asked to participate in a study on genetic predisposition of RA. In total, 405 patients were included. In a subgroup of 107 patients, prospective information on disease severity was available. This included cumulative CRP levels over a period of 24 months and progression of erosions after 24 months of follow-up (measured by the modified Sharp-van der Heijden score 22 ). In addition, disease activity score (DAS) according to Van der Heijde with 3 variables (number of swollen joints, erythrocyte sedimentation rate, Ritchie articular index) 23 and Ritchie articular index 24, both at disease onset, were available. Also rheumatoid factor and anti-ccp levels were available. Healthy controls As healthy controls, partners of the patients included in the study of the RA and SLE patients of the cohort of the University Medical Center Groningen, were included. Neither of these controls had RA or SLE. Additional healthy controls were included from a study of risk for development of colorectal cancer as described previously. 25 These latter controls also had no RA or SLE. All patients and controls gave written informed consent to participate in this study. The study was approved by the local medical ethics committee. Genotyping DNA was extracted from whole blood samples following standard extraction procedures. For the determination of the SNP alleles, the TaqMan Allelic Discrimination Assay by Applied Biosystems was used. Information about DNA sequences adjacent to the SNPs was retrieved by accessing the NCBI SNP database. SNP-IDs are: rs (-429T/C), rs (-374T/A), rs (Gly82Ser), rs (2184A/G). The sequence information retrieved online was used to order Primers and TaqMan probes from Applera, Nieuwekerk a/d IJssel, The Netherlands, by using the probes and primer interface which is provided by Applera. PCR-reactions were performed in a total volume of 10µl using a minimum of 5 ng of patient DNA, 1x TaqMan Universal PCR Master Mix (Applied Biosystems) and 0.5x Primer-Probe-Mix (Applied Biosystems) 87

7 which consisted of 700 nm Primer and 100 nm probe. PCR was performed on a GeneAmp PCR System 9700 (Applied Biosystems); cycle conditions were: 15 min. enzyme activation at 95ºC followed by 40 cycles with 15 sec. at 95ºC for denaturation and 1 min. at 60ºC for annealing and elongation. Endpoint measurement of the fluorescence signal was performed on an ABI7900HT machine. The SNP-alleles were finally assigned visually using the SDS 2.0 software (Applied Biosystems). Statistical analysis The genotype frequencies of each group (SLE, RA, LN and controls) were tested for Hardy-Weinberg equilibrium using the chi-square test on the observed frequencies versus the expected frequencies according to the Hardy- Weinberg formula. Differences in genotype frequencies and allele frequencies were tested between patients and controls also using the chi-square test. For the difference in disease parameters in LN patients, Mann-Whitney or Student s t-test was used, where appropriate. Due to the explorative design of the study a power calculation was not performed. However, the well characterized and homogeneous cohort strengthens the power of the study as other genetic studies in complex diseases are often hampered by phenotypic heterogeneity. Linkage analysis studies in SLE have already proven that stratification by clinical symptoms increases power to detect genetic association. 26;27 Results In total, 97 SLE patients (median age 44; range 23-78, 83 females), 114 LN patients (median age 36; range 16-69, 91 females), 408 RA patients (median age 61; range 25-86, 295 females) and 431 healthy controls were included in this study. For additional characteristics of the SLE, LN and RA patients, table 1. The genotype frequencies of all patient groups and the controls were in Hardy- Weinberg equilibrium, assuring quality of the markers used (table 2). Linkage disequilibrium (LD) was determined for the RAGE polymorphisms. There was a strong disequilibrium only between the RAGE -429 T/C and 2184 A/G polymorphisms (r 2 =0.99). In order to determine whether RAGE polymorphisms are associated with SLE, we analyzed the genotype frequencies of four RAGE polymorphisms in these groups. For SLE, there was a significant association with all RAGE polymorphisms (p<0.001), except for the RAGE Gly82Ser polymorphism. These associations were present in the genotype analysis (table 2) as well as in 88

8 RAGE polymorphisms in SLE, RA and lupus nephritis the allele frequency analysis (table 3). SLE was more often associated with the C allele in the -429 T/C, the T allele in the -374T/A and the G allele in the 2184 A/G polymorphism in comparison to both healthy and disease controls (RA patients). Table 1: baseline characteristics of SLE and RA patients SLE UMCG cohort (n=97) Median age, years (range) 44 (23-78) Sex female, n (%) 83 (86%) ACR criteria, n (%) Malar rash 36 (37) Discoid rash 30 (31) Photosensitivity 50 (52) Oral ulcers 13 (13) Arthritis 61 (63) Serositis 38 (40) Renal disorder 38 (40) Neurologic disorder 7 (7) Hematologic disorder 68 (70) Immunologic disorder 79 (81) anti-dsdna 75 (77) anti-sm 13 (13) anti-phospholipid antibodies 16 (16) Anti-nuclear antibody 97 (100) LN (n=114) Median age, years (range) 36 (16-69) Sex female, n (%) 91 (81) RA (n=408) Median age, years (range) 61 (25-86) Sex female, n (%) 295 (72) Rheumatoid Factor positive (%) 379 (93) Erosive disease (%) 379 (93) 89

9 We performed an additional analysis to determine whether RAGE polymorphisms are associated with different disease manifestations in SLE (according to the ACR criteria). All SLE patients with neurologic disorder (n=7) had the TT phenotype in -429 T/C and the AA phenotype in 2184 A/G (p=0.002 and p=0.003, respectively). For all other disease manifestations, no significant differences were found. Table 2: RAGE genotype frequencies and Hardy-Weinberg equilibrium 2a: RAGE -429 T/C RAGE -429T/C Total P HW P χ2 TT TC CC Systemic lupus erythematosus 44 (45.8%) 44 (45.8%) 8 (8.3%) 96(100.0%) Rheumatoid arthritis 294 (73.3%) 102 (25.4%) 5 (1.2%) 401(100.0%) Lupus nephritis 56 (50.0%) 45 (40.2%) 11 (9.8%) 112(100.0%) Controls 279 (65.3%) 139 (32.6%) 9 (2.1%) 427(100.0%) 0.22 Total 673 (65.0%) 330 (31.8%) 33 (3.2%) 1036(100.0%) 2b: RAGE -374 T/A RAGE 374 T/A Total P HW P χ2 TT TA AA Systemic lupus erythematosus 68 (72.3%) 23 (24.5%) 3 (3.2%) 94 (100.0%) Rheumatoid arthritis 211 (54.1%) 146 (37.4%) 33 (8.5%) 390 (100.0%) Lupus nephritis 80 (74.8%) 24 (22.4%) 3 (2.8%) 107 (100.0%) Controls 239 (55.7%) 164 (37.5%) 26 (6.1%) 429 (100.0%) 0.95 Total 598 (58.6%) 357 (35.0%) 65 (6.4%) 1020 (100.0%) 2c: RAGE Gly82Ser RAGE Gly82Ser Total P HW P χ2 Ser/ Gly/Gly Gly/Ser Ser Systemic lupus erythematosus 91 (94.8%) 5 (5.2%) 0 96(100.0%) Rheumatoid arthritis 330 (81.7%) 70 (17.3%) 4 (1.0%) 404(100.0%) Lupus nephritis 110 (96.5%) 4 (3.5%) 0 114(100.0%) Controls 392 (91.8%) 35 (8.2%) 0 427(100.0%) 0.67 Total 923 (88.6%) 114 (11.0%) 4 (0.4%) 1041(100.0%) 90

10 RAGE polymorphisms in SLE, RA and lupus nephritis 2d: RAGE 2184 A/G RAGE 2184 A/G Total P HW P χ2 AA AG GG Systemic lupus erythematosus 42 (44.7%) 44 (46.8%) 8 (8.5%) 94 (100.0%) Rheumatoid arthritis 295 (72.3%) 107 (26.2%) 6 (1.5%) 408 (100.0%) Lupus nephritis 56 (49.6%) 46 (40.7%) 11 (9.7%) 113 (100.0%) Controls 275 (64.4%) 140 (32.8%) 12 (2.8%) 427 (100.0%) Total 668 (64.1%) 337 (32.3%) 37 (3.6%) 1042 (100.0%) P HW =p-value Hardy-Weinberg equilibrium; P χ2 =p-value genotype differences between patient groups and controls For RA, in contrast to SLE, a strong association with the Gly82Ser polymorphism was found. The Ser allele was more often present in RA subjects (p= ), compared with healthy controls. Table 3: RAGE polymorphisms allele frequencies 3a: RAGE -429 T/C Systemic lupus erythematosus T RAGE -429T/C Total P χ2 C 132 (68.8%) 60 (31.2%) 192 (100.0%) Rheumatoid arthritis 690 (86.0%) 112 (14.0%) 802 (100.0%) Lupus nephritis 157 (70.1%) 67 (29.6%) 224 (100.0%) Controls 697 (81.6%) 157 (18.4%) 854 (100.0%) Total 1676 (80.9%) 396 (19.1%) 2072 (100.0%) 3b: RAGE -374 T/A RAGE 374 T/A Total P χ2 T A Systemic lupus erythematosus 159 (84.6%) 29 (15.4%) 188 (100.0%) Rheumatoid arthritis 568 (72.8%) 212 (27.2%) 780 (100.0%) Lupus nephritis 184 (86.0%) 30 (14.0%) 214 (100.0%) Controls 642 (74.8%) 216 (25.2%) 858 (100.0%) Total 1553 (76.1%) 487 (23.9%) 2040 (100.0%) 91

11 3c: RAGE Gly82Ser RAGE Gly82Ser Total P χ2 Gly Ser Systemic lupus erythematosus 187 (97.4%) 5 (2.6%) 192 (100.0%) Rheumatoid arthritis 730 (90.3%) 78 (9.7%) 808 (100.0%) Lupus nephritis 224 (98.2%) 4 (1.8%) 228 (100.0%) Controls 819 (95.9%) 35 (4.1%) 854 (100.0%) Total 1960 (94.1%) 122 (5.9%) 2082 (100.0%) 3d: RAGE 2184 A/G Systemic lupus erythematosus A RAGE 2184 A/G Total P χ2 G 128 (68.2%) 60 (31.9%) 188 (100.0%) Rheumatoid arthritis 697 (85.4%) 119 (14.6%) 816 (100.0%) Lupus nephritis 158 (69.9%) 68 (30.1%) 226 (100.0%) Controls 690 (80.8%) 164 (19.2%) 854 (100.0%) Total 1673 (80.3%) 411 (19.7%) 2084 (100.0%) P χ2 =p-value differences between patient groups and controls For the -429 T/C and the 2184 A/G polymorphisms, there was a clear difference in genotypes in RA subjects compared to HC but also compared to SLE patients. Compared with both HC and SLE patients, there was a significant lower frequency of the C allele in the -429 T/C and the G allele in the 2184 A/G polymorphism in RA patients. In a subgroup of 107 RA patients, RAGE polymorphisms were not significantly associated with markers of disease severity. We also wanted to determine whether RAGE polymorphisms are associated with disease severity in SLE. As LN is a severe manifestation of SLE, we chose to analyze RAGE polymorphisms in a group of 114 patients with biopsy proven, proliferative LN from the Netherlands. The genotype frequencies and allele frequencies were comparable to those from the SLE patients from the UMCG cohort. All polymorphisms were associated with LN except the Gly82Ser polymorphism, although there was a trend for a higher frequency of the Gly/Gly phenotype in LN (p=0.068), compared with HC. Since 40% of 92

12 RAGE polymorphisms in SLE, RA and lupus nephritis the SLE patients from our UMCG cohort fulfilled the ACR criterion for renal involvement, we compared those patients with the SLE patients without renal involvement. For all RAGE polymorphisms, there were no differences in genotype or allele frequencies between these groups (RAGE -429 T/C p=0.6; -374 T/A p=0.4; Gly82Ser p=1.0; 2184 A/G p=0.6). Fig. 1a Urinary protein excretion (g/24hr) Fig. 1a: Urinary protein excretion in relation to RAGE polymorphism status T0: time of inclusion; T1: after 5 months; T2: after 24 months; T3: after 31 months of treatment or latest available follow-up data if follow-up was less than 1 months; * p<0.05 ** p<

13 Fig. 1b Fig. 1b: Creatinine Clearance in relation to RAGE polymorphism status T0: time of inclusion; T1: after 5 months; T2: after 24 months; T3: after 31 months of treatment or latest available follow-up data if follow-up was less than 1 months; * p<0.05 ** p<0.01 There was neither a difference in genotype nor in allele frequencies between the SLE patients without renal involvement from our cohort and the LN patients (RAGE -429 T/C p=0.6; -374 T/A p=0.8; Gly82Ser p=0.6; 2184 A/G p=0.6). 94

14 RAGE polymorphisms in SLE, RA and lupus nephritis Since several RAGE polymorphisms were associated with disease susceptibility for SLE, we also analyzed whether RAGE polymorphisms are associated with disease severity of proliferative LN. The T allele in the -374 T/A polymorphism was associated with susceptibility for SLE. In LN, A allele carriers had more severe proteinuria and lower creatinine clearance at the time of inclusion (figure 1). This association was no longer present after five months of treatment. The C allele in RAGE -429 T/C and the G allele in RAGE 2184 A/G polymorphism were associated with poorer renal function during the first two years of treatment. This association was no longer present later at 31 months of follow-up. Finally, we analyzed whether RAGE polymorphisms are associated with srage levels. For this purpose and to avoid influence of disease activity, we measured srage levels in SLE patients during quiescent disease (SLEDAI < 4). None of the RAGE polymorphisms were associated with srage levels (table 4). Table 4: srage levels in relation to RAGE polymorphisms RAGE 374 T/A TT (n=64) TA & AA (n=25) p srage levels (pg/ml) 1414± ± RAGE 2184 A/G AA (n=41) AG & GG (n=48) p srage levels (pg/ml) 1433± ± RAGE Gly32Ser Gly/Gly (n=86) Gly/Ser & Ser/Ser (n=5) p srage levels (pg/ml) 1478± ± RAGE 429 T/C TT (n=64) TA & AA (n=25) p srage levels (pg/ml) 1414± ± srage: soluble receptor for advanced glycation end products T0: time of inclusion; T1: after 5 months; T2: after 24 months; T3: after 31 months of treatment or latest available follow-up data if follow-up was less than 1 months 95

15 Discussion This study is the first to describe RAGE polymorphisms in large, well defined populations of patients with SLE, and LN. In addition, we were able to perform the same analysis in a large group of RA patients, serving as disease controls. We found that RAGE polymorphisms were not only associated with disease susceptibility in SLE and RA, but also with disease severity and response to treatment in LN. In our study, three polymorphisms were associated with SLE. First, the C allele in -429 T/C was more frequent in SLE patients compared with both HC and RA patients. Also the T allele in the RAGE -374 T/A and the G allele in 2184 A/G were more often present in SLE patients. Thus, these polymorphisms might be susceptibility loci for SLE. Both the T/C and the -374 T/A polymorphisms have been shown to exert significant effects on the transcription of the RAGE gene. It has been hypothesized that the A allele in -374 T/A leads to less RAGE expression, thereby diminishing the inflammatory response. 16;28 This diminished inflammatory response is in agreement with the protective effect of the A allele on CVD in non-diabetic and on proteinuria in diabetic subjects. 14;29 The C allele in -429 T/C was shown to be associated with retinopathy in diabetes. 16 The G allele in 2184 A/G has been associated with lower antioxidant levels in diabetic subjects. This suggests that the G allele causes increased oxidative stress, possibly leading to inflammation. 30 Based on these observations, it seems plausible that RAGE polymorphisms are leading to increased or altered RAGE expression, which at least in part is responsible for the inflammatory state in SLE. In order to determine whether RAGE polymorphisms are associated with disease severity in SLE, we analyzed these polymorphisms in 114 patients with biopsy proven, proliferative LN. Compared with HC and RA patients, we found the same differences in genotype and allele frequencies for the LN patients as for the SLE patients. In addition, we found no difference in genotype and allele frequencies between LN patients and SLE patients without renal involvement. Thus, we can conclude that the polymorphisms mentioned above are associated with disease susceptibility for SLE in general, but not with renal involvement in particular. As for other disease manifestations in SLE, we found only an association of the RAGE -429 T/C and the 2184 A/G polymorphisms with neurologic disorder. Until now, no studies have been performed focussing on RAGE polymorphisms as disease susceptibility loci in SLE or LN. The gene encoding for the RAGE molecule is situated on the short arm of chromosome 6 in 96

16 RAGE polymorphisms in SLE, RA and lupus nephritis the HLA class III region. Many studies show a strong association between SLE and the HLA class II region in particular 31, but also associations of polymorphisms of the HLA class III region with SLE have been described. This concerned, however, polymorphisms other than RAGE genes, e.g. the TNF alpha promotor region, the MSH5 gene or the genes encoding for complement C2 and C4. 32;36 Our present study underlines that also the HLA class III region may still play an important role in the susceptibility for SLE. For the RA patients, a strong association with the Ser allele in the Gly82Ser polymorphism was found. This high frequency of the Ser allele in RA patients is in accordance with findings in literature. Hofmann et al. found an increased prevalence of the Ser allele in a group of 205 RA patients compared with HC. In addition, they were able to demonstrate an increase in the inflammatory response of human peripheral blood mononuclear phagocytes in the presence of the Ser allele. 13 Thus, in a considerably larger population of RA patients, we were able to confirm that the Ser allele, indeed, is a susceptibility allele for RA. In addition, we found an association of the T allele in -429 T/C and the A allele in 2184 A/G with RA. These associations are the opposite of the associations we found for these polymorphisms in SLE. This strongly suggests that in the pathogenesis of SLE and RA, different RAGE polymorphisms play a role. The associations of RA with -429 T/C and 2184 A/G, however, were not as strong as the association with the Gly82Ser polymorphism. As for disease severity in RA, we could not find a significant association of RAGE polymorphisms with disease severity parameters in a subgroup of 107 RA patients. As mentioned before, the gene encoding for the RAGE molecule is situated in the HLA III region. One has to take in mind that strong LD is present throughout the entire HLA region. So, although we found highly significant associations of several RAGE polymorphisms with RA, SLE and LN, it can not be excluded that association of these markers is due to LD with other markers within the HLA region. Between the RAGE polymorphisms that we analyzed, there was a strong LD between the RAGE -429 T/C and 2184 A/G polymorphisms only. The polymorphisms analyzed in this study, however, have been demonstrated to have clear functional effects 13;14;16;28 on a transcriptional level, as we mentioned before. Thus, although LD cannot be excluded, there is evidence that the association of SLE and RA with RAGE polymorphisms found in our study are true causal variants. In other to determine whether functionality of RAGE polymorphisms is reflected in levels of srage, we measured srage levels in our SLE patients during quiescent disease. Although in literature some RAGE polymorphisms have been 97

17 associated with srage levels 17;18, we could not confirm these associations in our study. The question arises whether srage levels reflect the true functional consequences of the RAGE polymorphisms. In addition, srage levels are influenced by factors such as renal function and use of medication, e.g. ACEinhibitors. 37;38 In addition, part of the total srage level is determined by truncation of membrane-bound RAGE. 39 As for disease severity in LN, we found that the A allele in -374 T/A polymorphism was associated with poorer renal function and proteinuria in LN patients. In addition, the C allele in -429T/C, and the G allele in 2184 A/G polymorphism were associated with poorer renal function after start of treatment, suggesting that these alleles are associated with poorer response to treatment. In literature, the -374 T/A polymorphism has been the most extensively investigated and it has been associated with proteinuria and CVD in diabetic patients. In contrast to our LN patients, in diabetic patients the A allele seemed to have a protective effect on proteinuria. This protective effect, however, was only found in diabetes patients with poor metabolic control (HbA1c > 9.5%), and could not be confirmed in other studies. 28;40 Thus, in literature, the protective effect of the A allele on proteinuria seems to be rather modest. Our findings show a difference between disease association (association with the T allele) and disease severity (more severe disease associated with A allele) in LN. This finding suggests that carriers of the A allele represent a subgroup of patients with more severe disease within the LN group. Also the C allele in -429 T/C and the G allele in have been associated with nephropathy in diabetic patients. 28 This suggests a role for these polymorphisms in the development and severity of renal disease. One might designate the C allele in -429 T/C, the A allele in -374 T/A and the G allele in 2184 A/G polymorphism as risk alleles representing LN patients with more severe disease or patients that respond less well to treatment. The clinical relevance of these associations is not clear as these associations were no more present after two years of treatment. One might hypothesize that LN patients carrying these alleles are prone to have more severe disease than patients who do not carry these alleles. This difference in disease severity between LN patients then might have consequences for the choice of treatment. This, however, needs to be investigated in further study. Until now, no genetic studies have been performed analyzing the RAGE polymorphisms focussing on LN patients. Our study is the first analyzing RAGE polymorphisms in a well-defined population of SLE patients, RA patients and HC. This gave us the opportunity not only to compare SLE 98

18 RAGE polymorphisms in SLE, RA and lupus nephritis and LN patients with HC, but also with RA patients, the latter serving as a disease control group. In addition, we analyzed RAGE polymorphisms in a homogenous, well-defined population of patients with biopsy proven, proliferative LN and were also able to assess disease severity in LN patients. Although the numbers of patients studied were relatively low, significant associations were found allowing firm conclusions. However, due to the relatively short duration of follow-up, effects of RAGE polymorphisms on outcome of disease in the long term might be missed. In conclusion, in a well-defined population of SLE, RA and LN patients we were able to demonstrate that these diseases are significantly associated with RAGE polymorphisms. Although the functional consequences of these polymorphisms in autoimmune diseases are still subject of further study, a causal association of RAGE polymorphisms with disease susceptibility in SLE and RA, as well as with disease severity in LN, may exist. 99

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