Analysis of the functional NFKB1 promoter polymorphism in rheumatoid arthritis and systemic lupus erythematosus

Nuclear factor (NF)-kappaB plays an important role in inflammatory diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). A functional insertion/deletion polymorphism (-94ins/delATTG) has been identified in the promoter of the NFKB1 gene. In addition, a polymorphic dinucleotide repeat (CA) has been identified in proximity to the coding region of the human NFKB1 gene. The aim of the present study was to investigate the influence of both the -94ins/delATTG and the (CA) microsatellite NFKB1 polymorphisms in the susceptibility/severity of RA and SLE. We analyzed the distribution of -94ins/delATTG and the multiallelic (CA)(n) repeat in 272 RA patients, 181 SLE patients, and 264 healthy controls from Southern Spain, in both cases using a polymerase chain reaction-fluorescent method. No statistically significant difference in the distribution of the -94delATTG NFKB1 genotypes and alleles between RA patients, SLE patients, and control subjects was observed. Similarly, we found no statistically significant differences in the (CA)(n) microsatellite allele frequency between controls and RA patients or SLE patients. In addition, no association was found between the above mentioned NFKB1 polymorphisms with any of the demographic and clinical parameters tested either in RA or in SLE patients. From these results, it seems that the -94ins/delATTG and the (CA)(n) repeat of NFKB1 gene may not play a relevant role in RA and/or SLE in our population.     

(GT)N dinucleotide repeat polymorphism of haem oxygenase-1 promotor region is not associated with inflammatory bowel disease risk or disease course

Haem oxygenase-1 (HO-1) up-regulation was suggested to reduce mucosal tissue damage in inflammatory bowel disease (IBD) and an up-regulation of HO-1 expression in patients with Crohn's disease (CD) and ulcerative colitis (UC) was demonstrated. A HO-1 gene promoter microsatellite (GT)(n) dinucleotide repeat polymorphism was associated with regulation of HO-1 in response to inflammatory stimuli. We therefore hypothesized that IBD patients might segregate into phenotypes with high or low HO-1 inducibility. Ethylenediamine tetraacetic acid blood samples were obtained from 179 CD patients, 110 UC patients and 56 control patients without inflammation. Genomic DNA was purified and the 5'-flanking region of the HO-1 gene containing the (GT)(n) dinucleotide repeat was amplified. Polymerase chain reaction (PCR) products were purified and the length of the PCR fragments was analysed. The number of (GT)(n) repeats in the population studied ranged from 13 to 42. The distribution of the allele frequencies was comparable in patients and controls for both the short and the long alleles. The frequencies of short-, middle- and long-sized alleles were not changed among the groups studied. No correlation was found between IBD and microsatellite instability detected in five individals. Our data indicate that (GT)(n) dinucleotide repeats of the HO-1 promotor region have no significance for the pathophysiology and disease course of IBD.     

Analysis of a GT microsatellite in the promoter of the foxp3/scurfin gene in autoimmune diseases

The aim of this study was to assess the possible association of the functional (GT)(n) microsatellite polymorphism in the FOXP3 gene with predisposition to several autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ulcerative colitis (UC), Crohn's disease, and celiac disease. We analyzed a case-control cohort composed of 231 SLE patients, 293 RA patients, 528 inflammatory bowel disease (354 Crohn's disease patients and 260 UC patients) patients, 103 celiac disease patients, and 274 healthy controls ethnically matched. Genotyping of (GT)(n) microsatellite was performed by polymerase chain reaction (PCR)-based method combined with fluorescent technology. We found no evidence for association of this polymorphism between controls and these autoimmune disease patients. Additionally, no differences in the genotype and allele distribution were found when patients were stratified according to clinical manifestation. The (GT)(n) microsatellite of the FOXP3 gene may not play a relevant role in the susceptibility to SLE, RA, inflammatory bowel disease, and celiac disease in our population.     

Analysis on the association of human BLYS (BAFF,TNFSF13B) polymorphisms with systemic lupus erythematosus and rheumatoid arthritis

Recent studies indicated a substantial role of BLyS (BAFF, TNFSF13B) in the pathogenesis of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in humans and in animal models. This study was conducted to screen for polymorphisms of human BLYS, and to examine whether they are involved in the genetic susceptibility to human SLE and RA. A systematic polymorphism screening was performed in the coding region, 5' and 3' untranslated regions, and promoter region of human BLYS. Association of the detected polymorphisms with SLE and RA was analyzed in 221 Japanese patients with RA, 156 with SLE, and 227 healthy individuals, using the case-control approach. Four single nucleotide polymorphisms (SNPs) in the promoter, one SNP in intron 1, and one rare nonsynonymous substitution (Ala105Thr) in the coding region were detected. The BLYS SNPs were found to form three common haplotypes. Significant association with the susceptibility to SLE or RA was not observed. However, a tendency for the increase of -871T/T genotype was observed in SLE patients with anti-Sm antibody (P=0.082). BLYS mRNA level was significantly elevated in the monocytes from individuals carrying -871T (P=0.010). In addition, although statistically not significant, 105Thr allele was slightly increased in patients with RA compared with controls (P=0.058). Characterizing the functional and clinical significance of these new SNPs requires further study.     

The size of a microsatellite polymorphism of the haem oxygenase 1 gene is associated with idiopathic recurrent miscarriage

Endothelial damage, impaired microvascularization and immune maladaptation have been described as aetiological factors in recurrent miscarriages. We investigated the relationship between idiopathic recurrent miscarriage (IRM) and a (GT)(n) repeat microsatellite polymorphism of the gene encoding haem oxygenase 1 (HO-1), known to modulate immune functions such as T-helper (TH) cell function and to be associated with cardiovascular disease. We investigated 162 women with IRM and 129 healthy, post-menopausal controls. The length of the HO-1 (GT)(n) microsatellite was assessed by PCR and direct sequencing in all women. Results were correlated with clinical data. The distribution of genotypes was in Hardy-Weinberg equilibrium. The HO-1 (GT)(n) microsatellite repeat numbers ranged from 13 to 37, with (GT)(23) and (GT)(30) being the most common alleles in both groups. We compared alleles consisting of < or =27 GT repeats, termed class S (short) alleles and alleles consisting of >28 GT repeats, termed class L (long) alleles. Seventy per cent of women with IRM had an S allele either in heterozygous (L/S) or homozygous (S/S) form, compared to 56% of controls (P = 0.02; OR 0.54; 95% CI 0.32-0.90). With respect to S allele frequencies, we found no significant difference among women with IRM and controls [P = 0.3; odds ratio (OR) 1.23, 95% confidence interval (CI) 0.86-1.76]. Comparing women with primary and secondary IRM, no difference with respect to the length of the HO-1 (GT)(n) microsatellite was ascertained. In summary, this is the first report on a HO-1 (GT)(n) microsatellite polymorphism among women with IRM, demonstrating that the investigated polymorphism is associated with IRM in a relatively large Caucasian population.     

Dinucleotide repeat polymorphism in intron II of human Toll-like receptor 2 gene and susceptibility to rheumatoid arthritis

Human Toll-like receptors (TLRs) participate in innate immune response and signal the activation of adaptive immunity. The presence of a functional intronic polymorphism consisting of guanine-thymine repeats in TLR2 gene was recently reported. Here, we investigated a dinucleotide repeat polymorphism in intron II of TLR2 in Korean patients with rheumatoid arthritis (RA). The numbers of guanine-thymine [(GT)(n)] repeats in intron II of the TLR 2 gene were counted in 183 patients with RA and in 148 healthy controls, using the gene scanning technique. We classified alleles into two subclasses for further analysis, 12-16 GT repeats (S allele) and 17-28 repeats (L allele). By subgroup analysis, we also examined whether the S allele is associated with the presence of shared epitope (SE), rheumatoid factor (RF), joint erosion and extra-articular complications. S-allele frequency was significantly increased in patients with RA than in healthy controls [30.3% vs. 23.0%, P = 0.03, or 1.46, 95% confidence interval (CI) 1.03-2.07], and genotypes containing S alleles were more frequent in patients with RA than in healthy controls (54.4% vs. 46.5%. P = 0.04, or 1.57, 95% CI 1.01-2.42). A skewed S-allele distribution was not found to be related to the presence of SE. Subgroup analysis showed no genotypic or allele frequency differences between patients with/without RF, joint erosion, or extra-articular complications. Genotype containing shorter GT repeats in intron II of the TLR2 gene may confer susceptibility to RA in Koreans.     

Presence of four major haplotypes in human BCMA gene: lack of association with systemic lupus erythematosus and rheumatoid arthritis

BCMA (TNFRSF17), along with TACI, has recently been demonstrated to be a receptor for BLyS (TNFSF13B). Recent studies indicated substantial role of BLyS signaling pathway for systemic lupus erythematosus (SLE). In the present study, we made an attempt to screen for polymorphisms of human BCMA, and to test their possible association with SLE and rheumatoid arthritis (RA). Two single nucleotide polymorphisms (SNPs) were detected within the coding sequence, both of which were synonymous substitutions. In addition, two SNPs within the promoter, two SNPs in the 5'-untranslated region (UTR), one SNP and one single nucleotide deletion in the 3'UTR and four rare variations were detected. From the combination of the polymorphisms, it was elucidated that four major haplotypes account for most of the genotypes in the Japanese population. Association with SLE and RA was not detected, although a slight tendency for the increase of BCMA.03 in SLE was observed (P = 0.089). These results indicated that human BCMA is conserved with respect to the amino acid sequence, and evidence for association with SLE and RA was not observed.     

New variations in human OX40 ligand (CD134L) gene

We found three new variations in the OX40 ligand (OX40L, CD134L) gene and its 5' upstream region. -921 (G-->A) at 5' upstream region, -19 (C-->G) at 5' untranslated region, and 202 + 25 (G-->T) at intron 2 were identified. -921 (G-->A) and 202 + 25 (G-->T) were detected in a substantial proportion of healthy Japanese individuals and were considered to be single nucleotide polymorphisms (SNPs). -19 (C-->G) was detected in only one healthy individual. There was no association between these variations and the presence of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).     

Matrix metalloproteinase-9 promoter polymorphisms in Korean patients with systemic lupus erythematosus

To investigate the association between functional promoter polymorphisms of matrix metalloproteinase-9 (MMP-9) and systemic lupus erythematosus (SLE), we analyzed MMP-9 promoter -1562 C>T and MMP-9 -90 (CA)(n) repeat polymorphisms in 135 Korean SLE patients (mean age, 34.7 years; 124 female and 11 male) and in 135 gender- and age-matched healthy controls (mean age, 35.4 years). Clinical and laboratory findings were collected during the follow-up period (mean, 63.5 months; range, 3-252 months), and Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Indexes were calculated. The levels of total MMP-9 were measured in sera of SLE patients and controls by enzyme-linked immunoabsorbent assay. The serum levels of MMP-9 in SLE patients were significantly lower than those of controls (mean +/- standard error of the mean, 1421.6+/-177.4 vs 3731.4+/-441.4 ng/ml, p=1.2 x 10(-5) by t test). Both functional polymorphisms were under the Hardy-Weinberg equilibrium state except (CA)(n) repeat polymorphisms in SLE patients (p=2.6 x 10(-5) by chi(2) goodness-of-fit test). The distribution of the MMP-9 promoter polymorphisms or haplotypes was not significantly different in SLE patients and controls. However the frequency of alleles with low numbers of CA repeats (n<21, 11.9% vs 7.0%, p=0.06 by the chi(2) test; odds ratio=1.78, 95% confidence interval=0.99-3.20) and the prevalence of low CA repeats homozygote tended to be higher in patients than in controls (5.2% vs 0.7%, p=0.07 by logistic regression, odds ratio=7.29, 95% confidence interval=0.88-60.10) in the recessive model. No relationship was found between MMP-9 polymorphisms and clinical features or damage as indicated by SLICC/ACR Damage Index in the study subjects. These results suggest that genetic polymorphisms of the MMP-9 promoter regions are not associated with the development of SLE in Korea.     

Inducible and endothelial nitric oxide synthase genes polymorphism in inflammatory bowel disease

To assess the influence of inducible and endothelial nitric oxide synthase gene (NOS2A and NOS3) polymorphisms in susceptibility to Crohn's disease (CD) and ulcerative colitis (UC). A total of 505 inflammatory bowel disease (IBD) patients (221 with UC and 284 with CD) and 332 ethnically matched controls were studied. Patients and controls were genotyped by polymerase chain reaction -based techniques for a multiallelic (CCTTT)(n) repeat and biallelic marker (TAAA)(n) in the promoter region of the NOS2A gene and for a T/C polymorphism at position -786 in the promoter region and a polymorphism in exon 7(298Glu/Asp) of the NOS3 gene. There was not association between NOS2A and NOS3 genotypes, alleles or haplotypes frequencies with UC, CD and controls. Our data suggest that NOS2A and NOS3 polymorphisms do not play a major role in IBD predisposition.     

Association of Fc gamma receptor IIIB, but not of Fc gamma receptor IIA and IIIA polymorphisms with systemic lupus erythematosus in Japanese

Human Fc gamma receptor (Fc gamma R) genes form a clustered gene family on chromosome 1q21-24. Although the association of Fc gamma R polymorphisms with systemic lupus erythematosus (SLE) has been extensively studied, the results are often contradictory. In this study, Fc gamma RIIA-131H/R, Fc gamma RIIIA-176F/V and Fc gamma RIIIB-NA1/2 genotypes were determined in the Japanese patients with SLE (n = 81) or rheumatoid arthritis (RA, n = 115) as well as in healthy individuals (n = 217), and possible association with the disease was tested using case-control analysis. Unlike in other populations, significant difference was not observed in the frequencies of Fc gamma RIIA and Fc gamma RIIIA genotypes between patients with SLE and healthy individuals. However, significant difference was detected in the frequencies of Fc gamma RIIIB genotypes between SLE and healthy individuals (P = 0.008). The odds ratio [OR] of the Fc gamma RIIIB-NA2/NA2 homozygotes for the development of SLE was 2.52 (95% confidence interval [CI]: 1.33-4.79). Among the patients with SLE, individuals with NA2/2 were significantly more likely to have lupus nephritis (P = 0.007). No association was observed between any of the Fc gamma R polymorphisms and RA. Significant linkage disequilibrium was detected between Fc gamma RIIIA and IIIB, but neither between IIA and IIIA, nor between IIA and IIIB. These observations may underscore the relevance of defective immune complex handling in the pathogenesis of SLE, or may suggest the presence of primarily associated gene(s) in linkage disequilibrium with Fc gamma R genes.     

Association between SLC11A1 (formerly NRAMP1) and the risk of sarcoidosis in Poland

Sarcoidosis (SA) is a systemic granulomatous disorder of unknown etiology characterized by T helper 1-type inflammatory responses at sites of disease with signs of B cell hyperactivity. Like rheumatoid arthritis and diabetes, an infectious etiology has frequently been postulated but no single infectious trigger definitively identified. Polymorphic alleles at SLC11A1 have previously been associated with susceptibility to both the putative infectious agents and to these autoimmune disorders. We therefore investigated its candidacy as a genetic determinant of SA in Poland in an association-based study comparing 86 SA patients with 85 tuberculosis (TB) patients and 93 control subjects. The functional promoter (GT)(n) polymorphism and four of 10 other single nucleotide or insertion/deletion polymorphisms genotyped across SLC11A1 were informative in our sample. Consistent with previous autoimmune disease studies, allele 3 at the functional (GT)(n) promoter region repeat polymorphism was significantly associated with SA when compared with healthy controls (odds ratio 1.68; 95% CI: 1.01-2.81; P=0.04) or with TB patients (odds ratio 1.69; 95% CI: 1.042-0.78; P=0.03).     

Association of apoptosis-related microsatellite polymorphisms on chromosome 1q in Taiwanese systemic lupus erythematosus patients

Apoptosis is important in the pathogenesis of systemic lupus erythematosus (SLE). Several genome-wide scan studies have suggested chromosome 1q as a genetic susceptibility locus for SLE. This study investigated the association of apoptosis-related genes on chromosome 1q, Fas ligand (FasL), interleukin (IL)-10 and poly(ADP-ribose) polymerase (PARP), promoter microsatellite multi-allelic polymorphisms with SLE susceptibility and clinical characteristics in Taiwan. This study recruited 237 SLE patients and 304 healthy controls. FasL, IL-10 and PARP promoter microsatellite polymorphisms were genotyped employing gene scan. IL-10, located on 1q31-32, emerged as a significant susceptibility gene locus in Taiwanese SLE (T4 statistic = 0.01). IL-10 CA21 allele was the most common allele of 15 identified in Taiwanese, displaying skewed distribution of susceptibility in Taiwanese SLE patients. Conversely, the IL-10 CA20 allele showed a protective effect of SLE susceptibility. Additionally, the IL-10 CA26 allele displayed a negative significant association with ascites and IL-10 CA25 allele increased the occurrence of the anti-cardiolipin IgM antibody. This study identified five alleles of FasL and nine alleles of PARP of microsatellite polymorphisms in Taiwanese patients. FasL and PARP alleles displayed no skewing distribution between Taiwanese SLE patients and controls. However, FasL GT15 and PARP CA17 allele demonstrated a high discoid rash presentation (T4 statistic 0.01 and 0.03, respectively) and PARP CA12 allele displayed a significant association with anti-cardiolipin IgM antibody production (T4 statistic 0.02). IL-10, FasL and PARP microsatellite polymorphisms exhibited significant associations with SLE susceptibility and/or clinical characteristics in Taiwanese patients. Thus, SLE is a complex and multiple genetics determined autoimmune disease. Chromosome 1q23-42 is an important genetic locus for further SLE subphenotype susceptibility study.     

Butyrophilin-like 2 gene is associated with ulcerative colitis in the Japanese under strong linkage disequilibrium with HLA-DRB1*1502

The human leukocyte antigen (HLA) region has been implicated in the pathogenesis of inflammatory bowel disease (IBD), which is classified into Crohn's disease (CD) and ulcerative colitis (UC). Recently, an association between sarcoidosis and the butyrophilin-like 2 (BTNL2) gene was reported. BTNL2 is located in the HLA region and its messenger RNA is expressed most abundantly in the intestine. In this study, we performed a case-control association study of BTNL2 in the Japanese patients with IBD and performed linkage disequilibrium (LD) analysis between BTNL2 and HLA-DRB1. We analyzed eight polymorphisms selected after direct sequencing and found that none of the polymorphisms were associated with the Japanese CD cohort. In contrast, five polymorphisms were significantly associated with UC, especially three single nucleotide polymorphisms (BTNL2_19, BTNL2_22 and BTNL2_23) were associated as a haplotype. The most frequent haplotype (GGC haplotype) was a low-risk haplotype (P= 0.000052), whereas the other TCT haplotype was a high-risk haplotype (P= 0.0000085). Among the eight polymorphisms, the strongest association with UC was found in BTNL2_19 (OR = 1.92, P= 0.0000035). As expected, the BTNL2_19-T allele showed strong LD with DRB1*1502 (D'= 0.92). When BTNL2_19 was tested as conditional on the DRB1*1502 carrier status, the significant association disappeared, suggesting that the association was because of its strong LD with DRB1*1502. We conclude that BTNL2 does not contribute to the susceptibility to Japanese CD but is associated with Japanese UC because of the strong LD with HLA-DRB1*1502. The strong LD between BTNL2 and HLA-DRB1 raises another issue about the potential role of BTNL2 in other diseases associated with HLA-DRB1.     

Association of the RAVER2 gene with increased susceptibility for ulcerative colitis

Crohn's disease (CD), ulcerative colitis (UC), systemic lupus erythematosus (SLE) and autoimmune polyglandular syndromes (APS) are autoimmune diseases (ADs) that may share common susceptibility pathways. We examined ribonucleo-protein, polypyrimidine tract-binding protein (PTB)-binding 2 (RAVER2) loci for these diseases in a cohort of 39 CD cases, 67 UC cases, 93 SLE cases, 60 APS cases and 162 healthy control subjects of Tunisian origin. We genotyped 3 SNPs of RAVER2 (rs2780814, rs1333739 and rs2780889) and evaluated it genetic association with each ADs, using X2-test. For each association, odds ratio (OR) and 95% CI were calculated. We show that rs2780814 is significantly associated with UC (P = 0.00016, P(corr) = 0.00048, OR = 3.66 (1.82; 7.34)). We also observed a trend of possible association to SLE (P = 0.023, P(corr) = 0.69, OR = 2.19 (1.1; 4.36)). None of these RAVER2 SNPs were associated with CD and APS susceptibility. These findings establish RAVER2 as a new UC genetic susceptibility factor and reveal a genetic heterogeneity of the associated polymorphisms and risk alleles between ADs suggesting different immunopathological roles of RAVER2 in these diseases.     

Cumulative association of eight susceptibility genes with systemic lupus erythematosus in a Japanese female population

Although large-scale studies established many susceptibility genes to systemic lupus erythematosus (SLE), effect of each gene is not sufficiently large to be used alone to identify individuals with strong genetic predisposition. In this study, we analyzed the cumulative number of risk alleles at eight established susceptibility loci, HLA-DRB1, IRF5, STAT4, BLK, TNFAIP3, TNIP1, FCGR2B and TNFSF13, in 282 Japanese female SLE and 222 healthy female controls. The average number of risk alleles was significantly increased in SLE (8.07±1.60) than healthy controls (7.02±1.64) (P=1.63 × 10(-12)). Significant gene-gene interaction was not detected. When the subjects carrying seven risk alleles were used as a reference, the odds ratio (OR) for individuals carrying 10 and 11-13 risk alleles were 4.17 (95% confidence interval (CI) 1.89-9.19, P=0.0002) and 8.77 (95% CI 1.92-40.0, P=0.0016), respectively. In contrast, subjects with ≤4 risk alleles were significantly decreased in SLE (OR 0.15, CI 0.03-0.67, P=0.007). The proportion of the patients with neurologic disorder was significantly increased in those carrying ≥10 risk alleles than those with <10 (OR 2.30, CI 1.09-4.83, P=0.025). This study suggested that the cumulative number of risk alleles may efficiently distinguish groups with high and low genetic predisposition to SLE and its severe manifestation.