Combined influence of genetic and environmental factors in age of rheumatoid arthritis onset.

To study the combined effect of both genetic and environmental factors in the age of rheumatoid arthritis onset. Patients (n = 507). Shared epitope characterization was performed using Lifecodes HLA-SSO. Genotyping of protein tyrosine phosphatase non-receptor 22 (PTPN22) rs2476601 and signal transducers and activators of transcription 4 (STAT4) rs7574865 polymorphism was performed using fast real-time PCR System. Shared epitope, antibodies directed against cyclic citrulinated peptide (anti-CCP) antibodies and a higher level of education were associated with a younger age at disease onset (P = 0.033, P = 0.004 and P < 0.0001, respectively). Neither carriers of the minor allele of PTPN22 rs2476601 nor STAT4 rs7574 polymorphisms showed a significant association with a younger age at disease onset (P = 0.355, P = 0.065, respectively). We found an additive effect of the three genetic markers in the age at onset: subjects with three markers were associated with a disease onset 9.56, 8.61, and 6.41 years before than those with none, one, or two genetic markers (P = 0.004, P = 0.006 and P = 0.043, respectively). We also described the additive effect of shared epitope, anti-CCP antibodies, educational level, PTPN22, and STAT4 polymorphisms in age at onset. Patients with two, three, four, or five variables were associated with a significant younger age of disease onset (4.72 [0.05-9.38] years (P = 0.048), 9.56 [4.72-14.40] years (P < 0.0001), 12.74 [6.84-18.64] years (P < 0.0001), and 20.87 [10.40-37.17] years (P < 0.0001)). Risk factors for the development of rheumatoid arthritis are also associated, with an additive effect, with a younger age at disease onset.

A polymorphism in PTPN2 gene is associated with an earlier onset of type 1 diabetes.

The Wellcome Trust Case Control Consortium (WTCCC) genome-wide study found association of PTPN2 with three autoimmune diseases, among them is type 1 diabetes (T1D). This result was confirmed by a follow-up study that pointed to new independent signals within the region. However, both studies were performed in patients with an early-onset T1D. We aimed at replicating the previous results and studying the influence of these polymorphisms in the age at T1D debut. We genotyped 439 T1D Spanish subjects (age at onset, 1 to 65 years) and 861 controls for two PTPN2 single nucleotide polymorphisms (SNPs), rs2542151 and rs478582, and studied the effect of both polymorphisms in age at onset through stratified and continuous analyses. The frequency of rs2542151*G carriers was significantly higher in the early-onset group compared with late-onset patients (p = 0.023) and with controls (OR = 1.61 [1.14-2.26]; p = 0.005). No significant differences were found between controls and late-onset patients. The log-rank chi-square test for the Kaplan-Meier plots (carriers of susceptibility allele vs non carriers) was statistically significant (χ (1df) (2) = 4.485; p = 0.034), yielding an earlier disease debut for G carriers. The analysis of the SNP rs478582 did not reach statistical significance. In summary, we replicate the association detected by the WTCCC and propose that the rs2542151*G allele confers risk to an earlier onset of T1D.

CD46 in a Spanish cohort of multiple sclerosis patients: genetics, mRNA expression and response to interferon-beta treatment.

BACKGROUND: In a prior study of our group we found an up-regulation of CD46 expression in a cohort of Spanish multiple sclerosis (MS) patients. OBJECTIVE: To evaluate the potential role of CD46 in the response to interferon-beta treatment in MS patients through the analysis of five tagging single nucleotide polymorphisms (SNPs) and measurement of mRNA. METHODS: A total of 406 MS patients and 513 control patients were analysed for five SNPs at the CD46 locus. Furthermore, 163 MS patients and 163 matched control patients were analysed by RT-PCR for the CD46 mRNA expression in three blood samples (basal, and at 6 and 12 months of interferon-beta treatment) collected in the course of a 1-year follow-up. RESULTS: Two genotypes of rs2724385 polymorphism (AT and TT) could be markers of response to interferon-beta therapy in MS patients (p=0.007 and p=0.006, respectively). Furthermore, the frequency of interferon-beta responders was 44.4% (32/72) in MS patients with an increased CD46 mRNA expression, vs. 65.9% (60/91) in patients with a decreased CD46 mRNA expression (p=0.006). CONCLUSION: The present study shows that CD46 could be associated with the response to interferon-beta therapy; however, the genetic results should be replicated in an independent cohort and further studies are needed to confirm the role of CD46.

Evidence of epistasis between TNFRSF14 and TNFRSF6B polymorphisms in patients with rheumatoid arthritis.

OBJECTIVE: Genetic variants located close to 2 genes codifying for members of the tumor necrosis factor receptor superfamily (TNFRSF), TNFRSF14 and TNFRSF6B, have recently been associated with rheumatoid arthritis (RA) and with inflammatory bowel disease susceptibility, respectively. The TNFRSF6B protein has been related to osteoclastic activity, apoptosis inhibition, and modulation of T cell activation and differentiation. Interestingly, peptides encoded by both genes bind a common ligand called LIGHT, which is overexpressed in RA synovium. The aim of this study was to investigate the combined effect of the TNFRSF14 rs6684865 and TNFRSF6B rs4809330 polymorphisms in RA predisposition. METHODS: TaqMan genotyping of these polymorphisms was conducted in 649 patients with RA and 553 ethnically matched control subjects (first study). To validate the results, an independent replication cohort with 211 patients and 255 control subjects was additionally studied (replication study). RESULTS: The frequency of the rs6684865 G allele in the RA subgroup with the rs4809330 GG susceptibility genotype was significantly higher than that in the other patients with RA (74% versus 65%; P = 0.002) or in control subjects (74% versus 67%; P = 0.003). Because no significant differences between the control and patient groups in the first and replication studies were observed, the data were pooled. When compared with control subjects overall, the effect of the rs6684865 G allele in the group with the rs4809330 GG genotype (odds ratio [OR] 1.49) was significantly different from the effect observed in the group carrying the rs4809330 A allele (OR 0.97; P = 0.0015 by Breslow-Day test of homogeneity). CONCLUSION: We have identified and replicated a novel gene-gene interaction between 2 polymorphisms of TNFRSF members in Spanish patients with RA, based on the hypothesis of shared pathogenic pathways in complex diseases.

Plasma soluble IL-6 receptor concentration in rheumatoid arthritis: associations with the rs8192284 IL6R polymorphism and with disease activity.

Soluble interleukin-6 receptor α subunit (sIL-6R) is primarily generated by shedding of the membrane-bound form. This process is influenced by the single nucleotide polymorphism rs8192284 (A > C) resulting in an aspartic acid to alanine substitution (D358A) at the proteolytic cleavage site. The aim of this study was to determine whether plasma levels of sIL6R are influenced by the rs8192284 polymorphism in patients with rheumatoid arthritis and to assess the association between plasma sIL-6R levels and disease activity as reflected by anti-CCP status. Thirty-nine patients were randomly selected from a cohort of patients with RA of Spanish descent. Plasma sIL-6R concentrations were measured using sandwich ELISA. Genotyping of the rs8192284 (A > C) polymorphism was done using a Fast Real-Time PCR System. DAS 28 scores were used to assess disease activity. Plasma sIL-6R levels were positively associated with the number of C alleles (AA: 35.27 (3.50) ng/ml, AC: 45.50 (4.58) ng/ml, CC: 52.55 (3.18) ng/ml, P = 0.0001). DAS28 and plasma sIL-6R levels were positively associated in the anti-CCP-positive subgroup (r (2) = 0.45, P = 0.0336) and negatively associated in the anti-CCP-negative subgroup (r (2) = -0.45, P = 0.0825). No association between anti-CCP status and sIL-6R level was found. Our findings show that the rs8192284 polymorphism is operative in patients with RA. The presence of anti-CCP antibodies determines the relationship between sIL-6R concentration and disease activity.

Immunological mechanisms that associate with oligoclonal IgM band synthesis in multiple sclerosis.

We described previously that multiple sclerosis (MS) patients with oligoclonal IgM against myelin lipids (M+) develop an aggressive disease. Our aim was to assess possible mechanisms regulating the production of these antibodies. We studied B cell subsets in 180 patients with MS, and 69 with other neurological diseases. M+ MS patients showed a moderate increase of CD5(+) B-cell percentage in peripheral blood and a considerable augment of these cells in cerebrospinal fluid (CSF) that correlated with intrathecal IgM production. The appearance of CD5(+) B cells into the central nervous system (CNS) was related to increased CXCL13 and TNF-alpha levels in CSF. Moreover, the presence of oligoclonal IgM associated with a SNP at position -376 of the TNF-alpha promoter. These results help to elucidate the B lymphocytes responsible for intrathecal IgM secretion in MS and the origin of this abnormal B-cell response in patients with aggressive MS.

Localization of Type 1 Diabetes susceptibility in the ancestral haplotype 18.2 by high density SNP mapping.

Previous studies have suggested that the ancestral haplotype 18.2 (AH18.2) carries additional susceptibility gene to Type 1 Diabetes (T1D) on the Major Histocompatibility Complex (MHC). We analyzed 10 DR3/TNFa1b5 homozygous subjects in order to establish the conservation of the AH18.2 and then compared this conserved region with other DR3 haplotype, the AH8.1. The Illumina's HumanHap550 Bead chip was used to perform an extensive genotyping of the MHC region. The AH18.2 was highly conserved between DDR1 and HLA-DQA1 genes; therefore most probably the second susceptibility gene is located within this region. We can exclude the region centromeric to HLA-DRA gene and telomeric to DDR1 gene. A comparison between the AH18.2 and AH8.1 haplotypes showed that 233 SNPs were different in the aforementioned conserved region. These data suggest that the 1.65 Mb MHC region between DDR1 and HLA-DRA genes is likely to carry additional susceptibility alleles for T1D on the AH18.2 haplotype.

Novel association of the interleukin 2-interleukin 21 region with inflammatory bowel disease.

OBJECTIVES: Genome-wide association studies have reported the role of the interleukin (IL) 2-IL21 chromosomal region at 4q27 in several autoimmune conditions. Mice deficient in IL-2 develop a disease with clinical and histological similarity to ulcerative colitis (UC) in humans. Modest evidence of linkage with UC was tentatively proposed for the IL2 gene more than a decade ago. Therefore, we decide to investigate the association of polymorphisms in the IL-2 axis (IL2, IL2RA, and IL2RB genes) with inflammatory bowel diseases (IBDs). METHODS: Seven hundred and twenty-eight white Spanish unrelated IBD patients (356 Crohn's disease (CD) and 372 UC) and 549 ethnically matched controls were included in a case-control study. In addition, a Spanish replication cohort with 562 CD and 430 UC patients and 1,310 controls were analyzed. Eight single-nucleotide polymorphisms previously associated with different autoimmune diseases were analyzed using TaqMan chemistry. RESULTS: The IL2-rs6822844 polymorphism modified CD predisposition (P=0.002; odds ratio, OR (95% confidence interval, CI)=0.61 (0.44-0.84)); this was replicated in the other Spanish cohort, resulting in a strong protective effect of the minor allele in the merged samples (P=0.0002; OR (95% CI)=0.70 (0.58-0.85)). A similar effect of rs6822844 was detected for UC. Another marker, rs11938795, also showed evidence of an association with CD (P=0.006; OR (95% CI)=0.73 (0.58-0.92)). CONCLUSIONS: Polymorphisms within the IL2-IL21 linkage disequilibrium (LD) block show a novel association with IBD, this is concordant with suggestive previous results of whole genome analyses in CD and type 1 diabetes. Our data agree with the effect previously observed for other conditions and delineate a shared underlying mechanism.

6q23 polymorphisms in rheumatoid arthritis Spanish patients.

OBJECTIVE: The aim of this work is to replicate the role of two recently described RA genetic markers (rs10499194 and rs6920220) situated at 6q23 in the autoantibody-positive phenotype. METHODS: A case-control study (630 RA patients and 664 healthy blood donors, all white Spaniards) was performed with two single nucleotide polymorphisms (rs6920220 and rs10499194) situated at 6q23. Genotyping was performed by TaqMan technology; autoantibody-stratified analyses in RA patients were also undertaken to replicate the previously reported effect of these polymorphisms. RESULTS: No association was observed for rs10499194 even after autoantibody stratification. The minor allele frequency of rs6920220 was higher in anti-CCP or RF-positive patients than in controls (P = 0.014 and P = 0.015 respectively), thus replicating previous findings. CONCLUSIONS: Our data replicate the association of rs6920220 with autoantibody-positive RA disease, although not for rs10499194.

NO role of NOS2A susceptibility polymorphisms in rheumatoid arthritis.

Nitric oxide has been described as a trigger for the synthesis of proinflammatory mediators and as a cytotoxic molecule with a pivotal role in apoptosis at the joints of rheumatoid arthritis (RA) patients. Polymorphisms in the NOS2A gene, which codes for the inducible nitric oxide synthase [(i)NOS], have been tested for association with several autoimmune diseases such as Crohn's disease or type 1 diabetes. Moreover, the existence of correlated levels of (i)NOS protein and synovial cell apoptosis in RA patients, pointed to NOS2A as a good candidate gene involved in RA predisposition. The role of NOS2A was studied in 405 Spanish RA patients and in 398 ethnically matched healthy controls, through the analysis of five SNPs: two at the NOS2A promoter (rs2779251 and 2779248), other two exonic markers (Asp(346)Asp (rs1137933) and Ser(608)Leu (rs22518)) and the last one located at intron 7 (rs3729508). We also included other two widely-used promoter polymorphisms: the insertion/deletion (TAAA/-) and the (CCTTT)n microsatellite. No individual association of each single-marker or haplotype was found with RA susceptibility. Our data show the low linkage disequilibrium between these NOS2A SNPs and the alleles of the (CCTTT)n microsatellite, corroborating in a Spanish population the observation previously described in British and Gambian population. The present data do not support a causative role of NOS2A polymorphisms in RA predisposition.

[Complex diseases genetics]. / Genética de las enfermedades complejas.

Common diseases are not inherited in a mendelian way, but have got anyway a genetic component, as shown by their increased frequency in siblings. Differences in the genome of individuals are not bigger than 0.1%, but are the reason why the susceptibility to diseases, their prognosis and the response to treatments differ. There are approximately ten million variations in the human genome, most of them single nucleotide polymorphisms (SNP) but also insertions, duplications, deletions, inversions and translocations. Two big international projects finished at the beginning of this century (The Human Genome Project and the HapMap Project) have paved the way to the study of these variations. The rapid technological advances allow today the study of one million SNPs at one time and it is forecasted that is some years the whole human genome will be sequenced in a few hours. All this is making possible new discoveries of the genetic variations that confer susceptibility to diseases, the location of new therapeutic targets and the beginning of a new medicine: personalized medicine.

Polymorphisms in the selenoprotein S gene: lack of association with autoimmune inflammatory diseases.

BACKGROUND: Selenoprotein S (SelS) protects the functional integrity of the endoplasmic reticulum against the deleterious effects of metabolic stress. SEPS1/SelS polymorphisms have been involved in the increased release of pro-inflammatory cytokines interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and IL-6 in macrophages. We aimed at investigating the role of the SEPS1 variants previously associated with higher plasma levels of these cytokines and of the SEPS1 haplotypes in the susceptibility to develop immune-mediated diseases characterized by an inflammatory component. RESULTS: Six polymorphisms distributed through the SEPS1 gene (rs11327127, rs28665122, rs4965814, rs12917258, rs4965373 and rs2101171) were genotyped in more than two thousand patients suffering from type 1 diabetes, rheumatoid arthritis or inflammatory bowel diseases and 550 healthy controls included in the case-control study. CONCLUSION: Lack of association of SEPS1 polymorphisms or haplotypes precludes a major role of this gene increasing predisposition to these inflammatory diseases.

IFIH1-GCA-KCNH7 locus: influence on multiple sclerosis risk.

A recent genome-wide scan of nonsynonymous SNPs and ulterior validation in case-control and family analyses evidenced a susceptibility locus for type 1 diabetes (T1D) on chromosome 2q24.3. We aimed at testing the effect of this locus in other autoimmune diseases with complex genetic background, such as multiple sclerosis (MS). Four SNPs along the locus, rs13422767, rs2111485, rs1990760 and rs2068330, were genotyped using TaqMan MGB chemistry in 311 T1D and 412 MS patients and 535 ethnically matched healthy controls. The previously reported association of this locus was found for the first time in MS (rs2068330, G vs C: P=0.001; OR (95% CI)=0.73 (0.6-0.88)) and a trend for replication was observed in our Spanish diabetic cohort. Therefore, genes included in this locus - IFIH1 interferon induced helicase, GCA grancalcin or the potassium channel KCNH7 - are potential candidates implicated in the pathogenesis of these autoimmune diseases, although strong linkage disequilibrium in the region hampered further localization of the etiologic gene.

Role of IL-10 promoter polymorphisms in the development of severe aorto-iliac occlusive disease.

Aortic severe occlusive disease (ASO) is a peripheral manifestation of atherosclerosis with an inflammatory component. Interleukin (IL)-10 is an anti-inflammatory cytokine that plays a key role in the development of atherosclerosis, promoting the stability of the atherosclerotic plaque. Several polymorphisms within the 5' region of the IL-10 gene have been related to altered transcriptional activity and protein levels. We aimed at studying two microsatellites, IL-10R and IL-10G, at -4 and -1.2 Kb, and three single nucleotide polymorphisms at positions -1082A/G, -819C/T and -592C/A in a collection of 94 ASO patients and 519 ethnically matched controls. Our results show that the IL-10 proximal promoter haplotype IL-10G*11/ -1082G/ -819C/ -592C is more frequent in ASO patients than in controls (28.7% vs 16% p = 0.003; OR = 2.12). Therefore, our data suggest a role of the IL-10 gene on ASO susceptibility.

Interleukin-6 gene variation in Spanish patients with immunoglobulin-A deficiency.

Selective immunoglobulin-A deficiency (IgAD) is the most common immunodeficiency in Caucasian populations. Genetic factors are important in its etiology; however human leukocyte antigen (HLA) genes, which explain 40% of the genetic risk for IgAD, are the only susceptibility factors commonly agreed upon at this time. Because interleukin-6 (IL-6) plays an important role in B-lymphocyte differentiation from plasma cells, we aimed to address the IL-6 genetic influence on IgAD susceptibility. We performed a case-control study that included 305 Caucasian Spanish IgAD patients and 529 ethnically matched healthy control subjects, as well as a familial study with 128 IgAD trios. We genotyped the functional promoter polymorphism -174G>C and nine additional single nucleotide polymorphisms. For the case-control analyses the chi(2) test or Fisher's exact test were used, and for the family study the transmission disequilibrium test was used. We observed an increased frequency of the -174C allele in IgAD patients (p = 0.005, odds ratio [OR] = 1.51, 95% confidence interval [CI] = 1.12-2.04) and a protective effect of the rs2069849_C allele (p = 0.007, odds ratio = 0.29, 95% CI = 0.09-0.76). In conclusion, we described for the first time an association between IL6 polymorphisms and IgAD. Although it is not clear which genetic variants are causing susceptibility/protection, this intriguing finding is remarkable because of the role of IL-6 in antibody production.

Association of MYO9B haplotype with type 1 diabetes.

MYO9B (myosin IXB) polymorphisms were associated with celiac disease and ulcerative colitis susceptibility, presumably through alteration of the intestinal permeability. Recently this gene was also associated with several diseases with an autoimmune component, such as rheumatoid arthritis and systemic lupus erythematosus. We aimed to test, for the first time, the potential role of MYO9B polymorphisms in type 1 diabetes (T1D), an autoimmune condition preceded by changes in intestinal barrier integrity. Three previously associated MYO9B polymorphisms (rs962917, rs2279003, and rs2305764) were studied in 316 T1D patients and 706 ethnically matched controls. Minor alleles of those polymorphisms were more frequent in diabetic patients than in controls and the haplotype carrying major alleles in those positions, rs962917*G/rs2279003*C/rs2305764*G, significantly reduced the risk of T1D in the Spanish population (p = 0.004; OR [95% confidence interval] = 0.68 [0.52-0.90]). Our data suggest an involvement of this MYO9B chromosomal region in T1D predisposition, indicating extensive influence on autoimmune diseases.

ICAM1 R241 is not associated with celiac disease in the Spanish population.

Celiac disease (CD) is a chronic intestinal inflammatory disease that develops in genetically susceptible individuals after gluten ingestion. The ICAM1 gene, located in the CD linkage region 19p13, encodes an intercellular adhesion molecule (ICAM-1) involved in inflammatory processes. Increased levels of ICAM-1 were observed in intestinal biopsies and in sera of CD patients. In addition, an association between the ICAM1 polymorphism G241R and CD patients has been recently described in a French population. Our aim in this study was to analyze the role of ICAM1 polymorphisms in CD susceptibility in the Spanish population. We performed a case-control study with 608 CD patients and 537 healthy control individuals and a family study including 231 trios. Four ICAM1 single nucleotide polymorphisms (SNPs) were analyzed: three nonsynonymous, R478W (rs5030400), P352L (rs1801714) and G241R (rs1799969); and one intronic, rs281432. Despite having above 98% statistical power to detect the association described in the French population (odds ratio = 1.7), we did not find any differences in genotypic or allelic frequencies of the G241R polymorphism between our CD patients and controls, and no differences were observed when the other SNPs were analyzed. Therefore, in our population our results discard the important previously described role of ICAM1 G241R in celiac disease.

TLR4 haplotypes in multiple sclerosis: a case-control study in the Spanish population.

Our aim in the present study is to evaluate the variation present in the TLR4 gene and its relationship with multiple sclerosis susceptibility in the Spanish population. Three hundred and sixty-two multiple sclerosis patients and 467 healthy controls from the Spanish population were included in the present study. Twelve single nucleotide polymorphisms (SNPs) were selected, and analyzed by the TaqMan technique. No statistically significant differences for any polymorphism or haplotypes were observed when patients were compared with controls. In conclusion, TLR4 does not play a major role in the predisposition to suffer from multiple sclerosis in our population.

FcRL3 and multiple sclerosis pathogenesis: role in autoimmunity?

BACKGROUND AND AIMS: A functional promoter polymorphism in the FcRL3 gene, -169 T/C, has been shown to regulate gene expression and to play a role in several autoimmune diseases. We aimed at testing for the first time whether this gene was involved in multiple sclerosis (MS) pathogenesis. METHODS: Case-control study performed with 400 Spanish MS patients and 508 healthy subjects. Genotyping of -169 T/C and -110 G/A was ascertained by using TaqMan MGB chemistry following manufacturer suggestions (Applied Biosystems, CA, USA). RESULTS: As previously seen for other autoimmune diseases, a significant difference was observed in the distribution of -169 T/C FcRL3 genotypes between MS patients and healthy controls (p = 0.03; chi(2) = 6.99). The -169 T allele, recently associated with increased susceptibility to Addison's disease, showed a parallel effect in MS [(TT+TC) vs. CC: p = 0.013; OR = 1.55 (1.08-2.54)]. CONCLUSIONS: An increased susceptibility associated to the -169 T allele was found when MS patients and controls were compared, supporting the role of the FcRL3 locus in MS predisposition and therefore extending the evidence of its general influence on autoimmunity.

Susceptibility to type 1 diabetes conferred by the PTPN22 C1858T polymorphism in the Spanish population.

BACKGROUND: The protein tyrosine phosphatase N22 gene (PTPN22) encodes a lymphoid-specific phosphatase (LYP) which is an important downregulator of T cell activation. A PTPN22 polymorphism, C1858T, was found associated with type 1 diabetes (T1D) in different Caucasian populations. In this study, we aimed at confirming the role of this variant in T1D predisposition in the Spanish population. METHODS: A case-control was performed with 316 Spanish white T1D patients consecutively recruited and 554 healthy controls, all of them from the Madrid area. The PTPN22 C1858T SNP was genotyped in both patients and controls using a TaqMan Assay in a 7900 HT Fast Real-Time PCR System. RESULTS: We replicated for the first time in a Spanish population the association of the 1858T allele with an increased risk for developing T1D [carriers of allele T vs. CC: OR (95%) = 1.73 (1.17-2.54); p = 0.004]. Furthermore, this allele showed a significant association in female patients with diabetes onset before age 16 years [carriers of allele T vs. CC: OR (95%) = 2.95 (1.45-6.01), female patients vs female controls p = 0.0009]. No other association in specific subgroups stratified for gender, HLA susceptibility or age at onset were observed. CONCLUSION: Our results provide evidence that the PTPN22 1858T allele is a T1D susceptibility factor also in the Spanish population and it might play a different role in susceptibility to T1D according to gender in early-onset T1D patients.