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 association studies (GWAS) have identified susceptibility loci in complex diseases and, therefore, helped to ascertain the underlying pathogenic processes involved. Some of the signals found, particularly those with a more robust effect such as CARD15/NOD2 polymorphisms in Crohn's disease (CD) or HLA-DRB1*1501 in multiple sclerosis (MS) risk, had been documented previously. Although most of the associations evidenced in GWAS have only modest effects on susceptibility, they provide clues about new pathways with biological effect on these multifactorial diseases, such as the autophagy genes, ATG16L11 and IRGM,2 in CD. The intrinsic value of these high-throughput analyses is recognized, but the need for replication in independent data sets to validate an association should not be forgotten, as apparently stringent significance thresholds of 10–7 in a GWAS are comparable to the usual P-value of 0.05 in a classical hypothesis-driven study.
Interestingly, a growing number of susceptibility genes are found to be shared between common traits, such as the PTPN22 R620W polymorphism3 or the chromosomal region harboring IL2/IL21 genes.4 Risk variants affecting different autoimmune diseases were checked for association with type I diabetes and new shared susceptibility loci were identified.5 One of these shared genes is STAT3 (signal transducer and activator of transcription 3), which had been previously described as a risk factor for CD.6 The association with CD was not replicated in ulcerative colitis (UC) by either Silverberg et al.7 or Fisher et al.,8 although the latter tested only non-synonymous polymorphisms. We aimed at studying the susceptibility conferred by STAT3 polymorphisms to the two main phenotypes of inflammatory bowel disease (IBD), CD and UC, in an independent population. Four polymorphisms in the STAT3 chromosomal region were selected (Figure 1). Two of them were previously associated with IBD: rs744166 with CD by Barrett et al.6 and rs12948909 with both clinical phenotypes by Anderson et al.9 The other two were analyzed on the basis of their suggestive associations with more than one disease in the publicly available data of the GWAS performed by the Wellcome Trust Case-Control Consortium, although they did not exceed the threshold of statistical significance.10
STAT proteins are latent cytoplasmic transcription factors that become activated by tyrosine phosphorylation. They can then dimerize and translocate to the nucleus, in which they induce transcription. It is well established that STAT3 is necessary for the signaling of proinflammatory cytokines such as interleukin-6, which is involved in the pathogenesis of MS and IBD,11 and also for multiple aspects of the biology of Th17 cells.12 In fact, this novel subset of T cells have shifted the accepted hypothesis of balance between Th1/Th2 cytokines, underscoring the critical importance of Th17 cells as mediators of a number of human inflammatory diseases, with CD and MS among them.13 STAT3 is required for differentiation and expansion of these Th17 cells and it also modulates the expression of adhesion molecules that regulate trafficking of encephalitogenic T cells into the central nervous system.14 Moreover, the persistency of high levels of phosphorylated STAT3 in circulating CD4+ T cells has been reported to favor the early conversion to clinically defined MS after the first clinical event,15 and relapsing MS patients showed high levels of pSTAT3.16 Concordantly, mice with targeted deletion of Stat3 in CD4+ T cells (CD4Stat3−/−) do not develop experimental autoimmune encephalomyelitis, which is an animal model of MS, and do not show inflammatory cell infiltrates in the spinal cord.14 These mounting evidences led us to study the role of the STAT3 gene in an immune-mediated disease with a clear Th17 component, such as MS.
The studied polymorphisms conformed to Hardy–Weinberg expectations. As shown in Table 1a, the minor allele frequency of three of them, rs744166, rs1026916 and rs12948909, evidenced a significantly protective effect in UC or CD patients. No significant differences were observed between MS patients and controls for any of the studied polymorphisms (Table 1a). The inferred haplotypes conformed by them were analyzed (Table 1b shows those with a frequency of over 3%) and the most frequent haplotype AGAC increased susceptibility in both CD and UC patients (differences withstood Bonferroni's correction for multiple testing). No effect in MS could be detected. No differences were observed among clinical subphenotypes (CD: ileal, colonic, ileocolonic, inflammatory, stricturing, perforating; UC: left-sided, extensive; MS: relapsing–remitting, primary progressive) or after stratification for the well-known risk alleles, HLA-DRB1*1501 in MS or NOD2/CARD15 in CD (data not shown).
Recent GWAS have greatly contributed to the discovery of genes involved in CD pathogenesis. A proposal of its etiology considers the disease triggered by commensal bacteria infecting cells of the intestinal mucosa. The failure of innate immunity to clear intracellular bacteria would drive the activation of adaptive immunity and inflammation. Some critical genes found associated with CD by these pangenomic studies, such as IL23R17 or STAT3,6 helped to identify the role of the Th17 pathway in this condition. Mice with tissue-specific disruption of Stat3 in bone marrow cells die within 4–6 weeks after birth with CD-like pathogenesis.18 T cell-specific Stat3 knockout mice enhanced T-cell proliferation.19 Moreover, total STAT3 as well as phosphorylated STAT3 were detected in tissue sections from both CD and UC, and correlated with the extent of inflammation of the tissue sections.20 From a genetic standpoint, this issue of the STAT3 implication in both clinical forms of IBD was not exempt of controversy, as Franke et al.21 reported the effect of one STAT3 polymorphism, rs744166, in UC but not in CD (P=0.13) risk. We also found an effect of similar magnitude for this variant in UC and not in CD, most probably due to limited power. However, the present data support the importance of this gene in both clinical phenotypes, CD and UC, as already reported by Anderson et al.9 for rs12948909. The most frequent haplotype evidenced a similar effect for CD and UC (Table 1b). Further characterization and fine mapping of this genetic region is warranted to pinpoint the polymorphism responsible for the effect observed for the associated haplotype.
In contrast with the present results in IBD, no influence of these STAT3 polymorphisms on MS risk was observed in our cohorts, although the statistical power to detect the effect originally described in CD (rs744166, OR=1.18) was 90% and it ranged between 80 and 90% depending on the specific polymorphism. Furthermore, a recent meta-analysis of two GWAS in MS,22, 23 which included 2624 patients and 7220 controls,24 has shown association of rs2293152 with the disease, but not at genome-wide significance level (P=1.13 × 10−5) and this association could not be validated in the same study on a set of 2215 cases and 2116 controls (P=0.972). This single-nucleotide polymorphism maps in the same linkage disequilibrium block of the STAT3 polymorphisms analyzed in this study. Therefore, evidence argues against the implication of this gene in MS risk.
In summary, although previous studies in CD25 and MS26 found suggestive linkage at chromosome 17q21, in which the STAT3 gene is located, association with this gene has only been corroborated for the IBD. Despite the already mentioned data in the MS mouse model and in MS patients pointing to an involvement of the STAT3 pathway in MS etiology (see Introduction), our results with more than 1500 patients seem to rule out the association with the STAT3 gene. Paradoxical as it could seem, this scenario is reminiscent of the situation found with osteopontin, one of the most frequently transcribed genes in MS lesions and the fifth most common transcript that was unique to the brains of patients with MS.27 Furthermore, patients with relapsing–remitting MS have higher serum levels of osteopontin, specifically during relapses.28, 29 However, in terms of the genetic association with MS, most studies in independent populations failed to find a role of the osteopontin polymorphisms in disease susceptibility,30, 31, 32 including the more recent GWA studies.22, 23 One possible explanation for these results might involve the synergistic interaction with some other gene(s), in such a way that the effect of one of them is masked when analyzed separately and awaits the discovery of epistatic companions.33 Coordinated impairment of some other component of the STAT3 pathway seems a plausible hypothesis and further studies are warranted to ensue the correct ascertainment of the genetic determinants in MS. Strategies that include epistatic interactions are beginning to yield interesting leads in MS and in other complex diseases.34, 35
Genetic studies stand out as approaches to define pathogenic pathways and ultimately, the integration of genetic together with functional data will promote a clearer understanding of the clinical forms of autoimmunity.
Materials and methods
Spanish patients (860 IBD and 1540 MS) and 1720 ethnically matched controls, mostly blood donors and staff, were consecutively recruited from Hospital Clínico (Madrid), Hospital Virgen de las Nieves (Granada), Hospital Clínico San Cecilio (Granada), Hospital Virgen Macarena (Sevilla), Hospital Carlos Haya (Málaga) and Blood Bank (Granada) and included in the study after informed consent. The ethics committees of these hospitals approved the study.
Diagnosis of IBD patients was based on standard clinical, radiological, endoscopical and histological criteria.36 The mean age at onset for the 452 UC patients was 38 years; 41% of them suffered pancolitis, 47% extraintestinal manifestations and 13% colectomy. The 408 CD patients were classified according to the location of the lesions in ileal (L1, 48%), colonic (L2, 16%), ileocolonic (L3, 32%) and upper gastrointestinal tract (L4, 3%) and according to the disease behavior in inflammatory (B1, 43%), stricturing (B2, 15%) and perforating (B3, 42%). Only 20% of the CD patients debuted after the age of 40 years.
MS patients were diagnosed based on the Poser criteria37 and 36% of patients carried the HLA DRB1*1501 allele. Most patients were relapsing–remitting (80%) and their mean age at onset was 29±9 years.
Genotyping of the samples was carried out with pre-designed TaqMan Assays (rs3809758, rs744166, rs1026916 and rs12948909) from Applied Biosystems, Inc. (Foster City, CA, USA), using 384-well plates in a 7900HT Fast Real-Time PCR system, under conditions recommended by the manufacturer. Genotyping call-rate success was over 95% for the four single-nucleotide polymorphisms and all patient groups and controls.
The statistical analysis to compare allelic and genotypic distributions was performed using chi-square test or Fisher's exact test (when expected values were below 5) included in a standard statistical package (Epi Info v. 6.02; World Health Organization, Geneva, Switzerland). Odds ratios were calculated, and their 95% confidence intervals were estimated using the Cornfield method. Haplotypic frequencies were inferred with the expectation-maximization algorithm implemented in the Arlequin v2.000 software. Linkage disequilibrium was measured by calculating two parameters, r2 and D’ (Figure 1).
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We thank Carmen Martínez and Angel García for their skillful technical assistance. Elena Urcelay works for the Fundación para la Investigación Biomédica-Hospital Clínico San Carlos. This work was supported by grants from Fundación Alfonso Martín Escudero, Fundación Ramón Areces, Fondo de Investigaciones Sanitarias PI07/0353, PI08/1676, PI08/1636 and RETICS 2007 (REEM), PN-SAF2006-02023, Junta de Andalucía Tin2007-67418-C3-03, SAS PI0168/2007.
The authors declare no conflict of interest.
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Cénit, M., Alcina, A., Márquez, A. et al. STAT3 locus in inflammatory bowel disease and multiple sclerosis susceptibility. Genes Immun 11, 264–268 (2010). https://doi.org/10.1038/gene.2010.10
- Crohn's disease
- ulcerative colitis
- multiple sclerosis
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