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Purpose: There is a critical need for population-based prospective cohort studies because they follow individuals before the onset of disease, allowing for studies that can identify biomarkers and disease-modifying effects, and thereby contributing to systems epidemiology., Participants: This paper describes the design and baseline characteristics of an intensively examined subpopulation of the LifeLines cohort in the Netherlands. In this unique subcohort, LifeLines DEEP, we included 1539 participants aged 18 years and older., Findings to Date: We collected additional blood (n = 1387), exhaled air (n = 1425) and faecal samples (n = 1248), and elicited responses to gastrointestinal health questionnaires (n = 1176) for analysis of the genome, epigenome, transcriptome, microbiome, metabolome and other biological levels. Here, we provide an overview of the different data layers in LifeLines DEEP and present baseline characteristics of the study population including food intake and quality of life. We also describe how the LifeLines DEEP cohort allows for the detailed investigation of genetic, genomic and metabolic variation for a wide range of phenotypic outcomes. Finally, we examine the determinants of gastrointestinal health, an area of particular interest to us that can be addressed by LifeLines DEEP., Future Plans: We have established a cohort of which multiple data levels allow for the integrative analysis of populations for translation of this information into biomarkers for disease, and which will offer new insights into disease mechanisms and prevention., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Recombinant interferon β (IFNβ) is a first-line therapy for relapsing-remitting multiple sclerosis (MS), with a proven effect on the inflammatory activity. Neutralising antibodies against IFNβ (NAbs) promote a loss of IFNβ bioactivity in a titre-dependent way and their development was associated with certain human leucocyte antigen (HLA) alleles. We investigated the contribution conferred by HLA alleles on the development of NAbs in independent cohorts of Southern Europe., Methods: Serum NAbs from 610 MS patients with HLA-genotype data were evaluated by cytopathic effect assay: negative tests included at least one negative result (NAb titres<20 NU/mL) after 1 year treatment; NAb-titres ≥20 NU/mL were positive tests and NAb titres ≥150 NU/mL in any test were classified as high-titre positives., Results: The combined presence of DRB1*07/DQA1*02 with A*26 or B*14 was found in 20% of patients with NAbs at high titres, but only in 5.4% of NAb-negative patients (p=0.00052, OR (95% CI) 4.34 (1.85 to 10.13)). The DRB1*04:01 allele was also more frequently carried by patients with high titres of NAbs (10% vs 4.5%; p=0.046, OR (95% CI) 2.38 (0.93 to 5.92)). The alleles carried at a significantly lower frequency in patients with high persistent NAbs corresponded to the A*11 allele (3.3% vs 13.8%; p=0.023, OR (95% CI) 0.22 (0.02 to 0.87)), as well as the DRB1*03/DQA1*05/DQB1*02 haplotype (16.3% vs 26.8%; p=0.02, OR (95% CI) 0.53 (0.27 to 1.03)) and the DRB1*13/DQA1*01:03/DQB1*06:03 haplotype (2.5% vs 9.1%; p=0.045, OR (95% CI) 0.25 (0.03 to 1.02))., Conclusions: 50% of the studied MS patients carried some of the five independently associated HLA allele/allele combinations described in this work. This relevant percentage of patients could benefit a therapeutic decision., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: STAT4 and IL23R loci represent common susceptibility genetic factors in autoimmunity. We decided to investigate for the first time the possible role of different STAT4/IL23R autoimmune disease-associated polymorphisms on the susceptibility to develop non-anterior uveitis and its main clinical phenotypes., Methods: Four functional polymorphisms (rs3821236, rs7574865, rs7574070, and rs897200) located within STAT4 gene as well as three independent polymorphisms (rs7517847, rs11209026, and rs1495965) located within IL23R were genotyped using TaqMan® allelic discrimination in a total of 206 patients with non-anterior uveitis and 1553 healthy controls from Spain., Results: No statistically significant differences were found when allele and genotype distributions were compared between non-anterior uveitis patients and controls for any STAT4 (rs3821236: P=0.39, OR=1.12, CI 95%=0.87-1.43; rs7574865: P=0.59 OR=1.07, CI 95%=0.84-1.37; rs7574070: P=0.26, OR=0.89, CI 95%=0.72-1.10; rs897200: P=0.22, OR=0.88, CI 95%=0.71-1.08;) or IL23R polymorphisms (rs7517847: P=0.49, OR=1.08, CI 95%=0.87-1.33; rs11209026: P=0.26, OR=0.78, CI 95%=0.51-1.21; rs1495965: P=0.51, OR=0.93, CI 95%=0.76-1.15)., Conclusion: Our results do not support a relevant role, similar to that described for other autoimmune diseases, of IL23R and STAT4 polymorphisms in the non-anterior uveitis genetic predisposition. Further studies are needed to discard a possible weak effect of the studied variant.

Objective: Interferon (IFN) signaling plays a crucial role in autoimmunity. Genetic variation in interferon regulatory factor 5 (IRF5), a major regulator of the type I interferon induction, has been associated with risk of developing several autoimmune diseases. In the current study we aimed to evaluate whether three sets of correlated IRF5 genetic variants, independently associated with SLE and with different functional roles, are involved in uveitis susceptibility and its clinical subphenotypes., Methods: Three IRF5 polymorphisms, rs2004640, rs2070197 and rs10954213, representative of each group, were genotyped using TaqMan® allelic discrimination assays in a total of 263 non-anterior uveitis patients and 724 healthy controls of Spanish origin., Results: A clear association between two of the three analyzed genetic variants, rs2004640 and rs10954213, and the absence of macular edema was observed in the case/control analysis (P FDR =5.07E-03, OR=1.48, CI 95%=1.14-1.92 and P FDR =3.37E-03, OR=1.54, CI 95%=1.19-2.01, respectively). Consistently, the subphenotype analysis accordingly with the presence/absence of this clinical condition also reached statistical significance (rs2004640: P=0.037, OR=0.69, CI 95%=0.48-0.98; rs10954213: P=0.030, OR=0.67, CI 95%=0.47-0.96), thus suggesting that both IRF5 genetic variants are specifically associated with the lack of macular edema in uveitis patients., Conclusion: Our results clearly showed for the first time that two functional genetic variants of IRF5 may play a role in the development of macular edema in non-anterior uveitis patients. Identifying genetic markers for macular edema could lead to the possibility of developing novel treatments or preventive therapies.

Objective: Endogenous uveitis is a major cause of visual loss mediated by the immune system. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes a lymphoid-specific phosphatase that plays a key role in T-cell receptor (TCR) signaling. Two independent functional missense single nucleotide polymorphisms (SNPs) located within the PTPN22 gene (R263Q and R620W) have been associated with different autoimmune disorders. We aimed to analyze for the first time the influence of these PTPN22 genetic variants on endogenous non-anterior uveitis susceptibility., Methods: We performed a case-control study of 217 patients with endogenous non-anterior uveitis and 718 healthy controls from a Spanish population. The PTPN22 polymorphisms (rs33996649 and rs2476601) were genotyped using TaqMan allelic discrimination assays. The allele, genotype, carriers, and allelic combination frequencies were compared between cases and controls with χ(2) analysis or Fisher's exact test., Results: Our results showed no influence of the studied SNPs in the global susceptibility analysis (rs33996649: allelic P- value=0.92, odds ratio=0.97, 95% confidence interval=0.54-1.75; rs2476601: allelic P- value=0.86, odds ratio=1.04, 95% confidence interval=0.68-1.59). Similarly, the allelic combination analysis did not provide additional information., Conclusions: Our results suggest that the studied polymorphisms of the PTPN22 gene do not play an important role in the pathophysiology of endogenous non-anterior uveitis.

Background: Multiple sclerosis (MS) is a multifactorial disease with a genetic basis. The strongest associations with the disease lie in the Human Leukocyte Antigen (HLA) region. However, except for the DRB1*15:01 allele, the main risk factor associated to MS so far, no consistent effect has been described for any other variant. One example is HLA-DRB1*03:01, with a heterogeneous effect across populations and studies. We postulate that those discrepancies could be due to differences in the diverse haplotypes bearing that allele. Thus, we aimed at studying the association of DRB1*03:01 with MS susceptibility considering this allele globally and stratified by haplotypes. We also evaluated the association with the presence of oligoclonal IgM bands against myelin lipids (OCMB) in cerebrospinal fluid., Methods: Genotyping of HLA-B, -DRB1 and -DQA1 was performed in 1068 MS patients and 624 ethnically matched healthy controls. One hundred and thirty-nine MS patients were classified according to the presence (M+, 58 patients)/absence (M-, 81 patients) of OCMB. Comparisons between groups (MS patients vs. controls and M+ vs. M-) were performed with the chi-square test or the Fisher exact test., Results: Association of DRB1*03:01 with MS susceptibility was observed but with different haplotypic contribution, being the ancestral haplotype (AH) 18.2 the one causing the highest risk. Comparisons between M+, M- and controls showed that the AH 18.2 was affecting only M+ individuals, conferring a risk similar to that caused by DRB1*15:01., Conclusions: The diverse DRB1*03:01-containing haplotypes contribute with different risk to MS susceptibility. The AH 18.2 causes the highest risk and affects only to individuals showing OCMB.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with presumed autoimmune origin, triggered by genetic and environmental risk factors. A recent genome-wide association study conducted on MS identified new biallelic markers outside the HLA (human leucocyte antigen) region involved in disease susceptibility: rs1109670 (DDEF2); rs1458175 (PDZRN4); rs1529316 and rs2049306 (CSMD1); rs16914086 (TBC1D2); rs1755289 (SH3GL2); rs1841770 (ZIC1); rs651477 (EN1); rs7607490 (TRIB2); rs397020 (C20orf46); rs908821 (SLC25A36); rs7672826 (MGC45800) and rs9523762 (GPC5). We aimed at replicating these top association signals in a Spanish cohort of 2863 MS patients and 2930 sex- and age-matched controls. Only rs9523762 mapping in the GPC5 gene was significantly associated (G allele, P=1.6 × 10(-5); odds ratio (95% confidence interval)=1.23 (1.12-1.36)), supporting a role for this proteoglycan in MS predisposition. The independent replication of association signals to validate data generated by genome-wide association scans is a first step in the effort to improve patient care.

In recent reports, IRF5 polymorphisms showed significant association with multiple sclerosis (MS) susceptibility in three studied populations and Irf5-deficient mice exhibited an increased susceptibility to viral infection, linked to a significant decrease in the induction of serum type I interferon (IFN). In the present study, we evaluated the association of two IRF5 polymorphisms with MS predisposition and we also addressed whether these polymorphisms were associated with active replication of human herpes virus-6 (HHV-6) observed in a subgroup of MS patients, and/or with response to IFN-β therapy. A total of 1494 MS patients and 1506 ethnically matched controls were genotyped for rs4728142 and rs3807306 with TaqMan pre-designed assays. One hundred and six patients were classified as responders to IFN-β therapy (no relapses/increases in EDSS over the 2-year follow-up) and 112 as non-responders (at least two relapses or an increase in expanded disability status scale (EDSS) of at least one point during the same period). The combined analysis of available datasets yielded an effect size on MS with odds ratio (OR)(Mantel-Haenszel)=1.14 (P<0.002) for the IRF5 polymorphisms rs4728142 and rs3807306. Additionally, trends for association were observed between rs3807306T and infection with HHV-6 [p=0.05, OR (95% CI)=1.56 (1.00-2.44)] and response to IFN-β therapy [P=0.09, OR (95% CI)=1.39 (0.95-2.05)].

Genome-wide association studies (GWAS) have revealed that different diseases share susceptibility variants. Twelve single-nucleotide polymorphisms (SNPs) previously associated with different immune-mediated diseases in GWAS were genotyped in a Caucasian Spanish population of 2864 multiple sclerosis (MS) patients and 2930 controls. Three SNPs were found to be associated with MS: rs1678542 in KIF5A (P=0.001, odds ratio (OR)=1.13, 95% confidence interval (CI)=1.05-1.23); rs3184504 in SH2B3 (P=0.00001, OR=1.19, 95% CI=1.10-1.27) and rs763361 in CD226 (P=0.00007, OR=1.16, 95%CI=1.08-1.25). These variants have previously been associated with rheumatoid arthritis and type 1 diabetes. The SH2B3 polymorphism has additionally been associated with systemic lupus erythematosus. Our results, in addition to validating some of these loci as risk factors for MS, are consistent with shared genetic mechanisms underlying different immune-mediated diseases. These data may help to shape the contribution of each pathway to different disorders.

STAT3 (signal transducer and activator of transcription 3) signaling is a critical component of Th17-dependent autoimmune processes. Genome-wide association studies (GWAS) have revealed the role of the STAT3 gene in inflammatory bowel disease (IBD) susceptibility, although confirmation in clinical subphenotypes is warranted. Mice with targeted deletion of Stat3 in T cells are resistant to experimental autoimmune encephalomyelitis, which is a multiple sclerosis (MS) model. Moreover, increased phosphorylated STAT3 was reported in T cells of patients evolving from clinically isolated syndrome to defined MS and in relapsing patients. These evidences led us to analyze the role of STAT3 in Crohn's disease (CD), ulcerative colitis (UC) and MS risk. Polymorphisms in the STAT3 region (rs3809758/rs744166/rs1026916/rs12948909) were genotyped and the inferred haplotypes were subsequently analyzed in 860 IBD and 1540 MS Spanish patients and 1720 ethnically matched controls. The haplotype conformed by the risk alleles of each polymorphism was significantly associated with both clinical phenotypes of IBD (CD: P=0.005, odds ratio 1.25, 95% confidence interval 1.06-1.46; and UC: P=0.002, odds ratio 1.19, 95% confidence interval 1.02-1.38). No evidence of association was detected for MS. The originally described association of IBD with STAT3 polymorphisms is corroborated for the two clinical phenotypes, CD and UC, in an independent population. A major role of this gene in MS seems unlikely.

Genome-wide studies highlighted the effect in Crohn's disease (CD) and ulcerative colitis (UC) susceptibility of single nucleotide polymorphisms (SNPs) in 3p21, where BSN (bassoon), MST1 (macrophage stimulating-1) and MST1R (MST1 Receptor) genes map. MST1R expression was significantly downregulated in multiple sclerosis (MS) compared with control brains, resembling findings in the MS mouse model. We pursued to replicate the effect of this locus on inflammatory bowel diseases and to evaluate its contribution to MS risk. Polymorphisms rs9858542, rs2131109 and rs1128535 were analysed by TaqMan assays in Spanish patients (370 CD, 405 UC and 415 MS) and 800 ethnically matched controls. Allele frequencies of these SNPs were significantly different in CD patients compared with controls [rs9858542: P=0.001, Odds ratio (OR)=1.35; rs2131109: P=0.0005, OR=1.37; rs1128535: P=0.007, OR=0.78] and, specifically, in the ileal phenotype [rs9858542: P=0.0004, OR=1.47; rs2131109: P=0.00009, OR=1.52; rs1128535: P=0.02, OR=0.69]. No differences were detected between UC or MS patients and control individuals. The effect of this locus on CD predisposition was replicated, but no influence on UC or MS predisposition could be detected. This susceptibility locus seems to affect mainly to the ileal CD subphenotype, although this point awaits further corroboration in independent cohorts.

Objective: The STAT4 gene encodes a transcription factor involved in the signaling pathways of several cytokines, including interleukin-12 (IL-12), the type I interferons, and IL-23. Recently, the association of a STAT4 haplotype marked by rs7574865 with rheumatoid arthritis (RA) and systemic lupus erythematosus was reported. The aim of this study was to investigate the role of this STAT4 tagging polymorphism in other immune-mediated diseases., Methods: The study group comprised 2,776 consecutively recruited Spanish individuals: 575 with RA, 440 with multiple sclerosis, 700 with inflammatory bowel disease, 311 with type 1 diabetes, and 723 ethnically matched healthy control subjects. The STAT4 polymorphism rs7574865 was genotyped using a predesigned TaqMan assay. Allele and genotype frequencies in patients and control subjects were compared by chi-square test., Results: The association of STAT4 polymorphism rs7574865 with RA was validated in patients of Spanish origin (for T versus G, P = 1.2 x 10(-6), odds ratio [OR] 1.59, 95% confidence interval [95% CI] 1.31-1.92), and the association was described for the first time in both clinical forms of inflammatory bowel disease, Crohn's disease and ulcerative colitis (for T versus G, P = 0.006, OR 1.29, 95% CI 1.07-1.55), and in type 1 diabetes mellitus (for T versus G, P = 0.008, OR 1.36, 95% CI 1.07-1.71). In contrast, the genotypic distribution of this polymorphism showed no difference between patients with multiple sclerosis and healthy control subjects (for T versus G, P = 0.83, OR 1.02, 95% CI 0.82-1.28)., Conclusion: The STAT4 gene is emerging as a novel common risk factor for diverse complex diseases.

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.

Recent studies have shown association of the IL23R gene with inflammatory bowel disease, psoriasis and ankylosing spondylitis. We aimed at studying the involvement of IL23R in celiac disease (CD) and multiple sclerosis (MS). We performed a case-control study including 598 patients with CD, 414 with MS and 546 healthy controls, all of them white Spaniards. All samples were genotyped for two single nucleotide polymorphisms: rs7517847 and rs11209026 (Arg381Gln). Statistical analyses were performed using chi(2-)tests or the Fisher's exact test. The minor allele (Gln) of the coding variant Arg381Gln was significantly increased in CD and MS patients when compared to controls (8% in CD vs 6% in controls, P=0.02; 9% in MS, P=0.006). In MS, a stronger effect was observed in patients showing primary-progressive disease (16%, P=0.004). Moreover, heterozygotes for rs7517847 were significantly increased in this group of MS patients (81% in MS vs 48% in controls, P=0.0002). In conclusion, contrary to what has been described previously, the less frequent allele of the functional polymorphism Arg381Gln (rs11209026) seems to be increasing susceptibility to CD and MS, although in this last group of patients a stronger effect is observed in patients affected of a primary-progressive form.