Genetic Markers Associated with Clinical Outcomes in Patients with Inflammatory Bowel Disease.

BASIC SCIENCE REVIEW ARTICLE

Genetic Markers Associated with Clinical Outcomes in Patients with Inflammatory Bowel Disease Jesús K. Yamamoto-Furusho, MD, MSc, PhD and Gabriela Fonseca-Camarillo, PhD

Abstract: Genetic factors play a significant role in determining inflammatory bowel disease (IBD) susceptibility. Epidemiologic data support genetic contribution to the pathogenesis of IBD, which include familial aggregation, twin studies, and racial and ethnic differences in disease prevalence. Recently, several new genes have been identified to be involved in the genetic susceptibility to IBD. The characterization of novel genes potentially will lead to the identification of therapeutic agents and clinical assessment of phenotype and prognosis in patients with IBD. The development of genetic markers associated with clinical outcomes in patients with IBD will be very important in the future. The progress of molecular biology tools (microarrays, proteomics, and epigenetics) have progressed the field of the genetic markers discovery. The advances in bioinformatics coupled with cross-disciplinary collaborations have greatly enhanced our ability to retrieve, characterize, and analyze large amounts of data generated by the technological advances. The techniques available for markers development are genomics (single nucleotide polymorphism genotyping, pharmacogenetics, and gene expression analyses) and proteomics. This could be a potential great benefit in predicting the course of disease in individual patients and in guiding appropriate medical therapy. (Inflamm Bowel Dis 2015;21:2683–2695) Key Words: inflammatory bowel disease, genetic marker, ulcerative colitis, Crohn’s disease, clinical outcome

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nflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by intestinal chronic inflammation. Although the exact origin of IBD remains unknown, new evidence suggests that the interplay among aberrant immune response, environmental factors, and multiple genes have been associated with susceptibility to IBD.1 Clinical and epidemiological data have shown strong molecular evidence suggesting that CD and UC are polygenic disorders as shown in Figure 1. In recent years, most progress has been made in understanding the genetic contribution to disease pathogenesis. The currently known IBD risk loci showed an almost 75% overlap with genetic risk loci for other immunemediated diseases.2 Current genomic, transcriptomic, epigenetic, and microRNA (miRNA) studies have focused on the identification of new genetic risk factors for specific disease and its application to clinical practice such as prognostic factors involved in the clinical course and response to medical therapy.3 Further genetic studies could provide valuable information and may help to Received for publication April 1, 2015; Accepted May 7, 2015. From the Inflammatory Bowel Disease Clinic, Department of Gastroenterology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México City, México. The authors have no conflicts of interest to disclose. Reprints: Jesús K. Yamamoto-Furusho, MD, MSc, PhD, Department of Gastroenterology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15, Col. Belisario Domínguez Sección XVI, Tlalpan, CP 14080, México City, México (e-mail: [email protected]). Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000500 Published online 4 August 2015.

Inflamm Bowel Dis Volume 21, Number 11, November 2015

distinguish the degree of influence of genetic and environmental pathogenic factors. In this review, we report the current status of the genetic markers associated with clinical outcomes in patients with IBD to have future targets for genomic and epigenetic analysis.

GENETIC MODEL AND MOLECULAR GENETICS IN IBD Genetic Model There is strong evidence that suggests a genetic basis for IBD, including familial clustering and racial and ethnic differences in risk for IBD.1 Ten to twenty percent of affected individuals have family history of IBD, with the highest risk among first-degree relatives. A positive family history is the principal risk factor for IBD, with relatives of affected individuals having at least a 10-fold increased risk for IBD. Increased rates of IBD between identical twins compared with fraternal twins, and siblings compared with spouses of affected individuals, suggest that genetic rather than environmental factors are primarily responsible for the observed familial aggregation for IBD. Racial differences in IBD incidence exist with the highest rates among whites. In addition, there is evidence for ethnic aggregation of IBD with higher rates of IBD among those of Jewish descent. Furthermore, within Jewish populations, rates are higher in Ashkenazi than Sephardic Jews.2,3 These differences occur across different periods and geographic areas, suggesting a genetic basis as the most likely explanation for these findings. www.ibdjournal.org |

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Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. Unauthorized reproduction of this article is prohibited.

Yamamoto-Furusho and Fonseca-Camarillo


FIGURE 1. Evolution of genetic markers involved in patients with IBD.

The study of new susceptibility genes will be of great importance to understand better the underlying pathways that lead to IBD. This could be a potential great benefit in predicting the course of disease in individual patients and in guiding appropriate medical therapy. Such information also could potentially allow for identification of family members at risk for developing IBD.

These findings support the notion that CD and UC are polygenic disorders that share some, but not all, susceptibility loci5–10 as shown in Figure 2.

Genomic Regions Associated with IBD IBD1 Locus on Chromosome 16q12

Molecular Genetics In IBD, the genetic contribution is poorly understood and seems to arise from the small contribution of dozens of genes3 as shown in Table 1. Recently, various candidate genes for IBD have been discovered through genome-wide association studies (GWAS). In genetic epidemiology, a GWAS is an examination of many common genetic variants in different individuals to see whether any variant is associated with a trait. GWAS typically focus on associations between single nucleotide polymorphisms (SNPs) and traits like major diseases. The studies with GWAS have revealed a great number of genetic variants predisposing to different complex diseases, but only 3 genetic polymorphisms related to NOD2, IL23/17, and autophagy have been well established for a direct role in IBD.4 Many susceptibility loci have been associated in IBD, and the current knowledge includes 9 loci, termed IBD 1 to 9.5 Although some loci seem specific to CD (IBD1 on 16q-OMIM 266600) or UC (IBD2 on 12q-OMIM 601458), others seem to confer susceptibility to IBD overall (IBD3 6p-OMIM 604519).

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Hugot et al11 performed a GWAS and identified a putative CD locus on chromosome 16 (P , 0.01) centered near loci D16S409 and D16S419 by using multipoint sib-pair analysis. Important examples of CD candidate genes located to the pericentromeric region of chromosome 16 such as CD19 (107265), involved in B-lymphocyte function; sialophorin (182160), involved in leukocyte adhesion; the CD11 integrin cluster (153370), involved in mycobacterial cell adhesion; and the interleukin 4 receptor (IL-4R; 147781) due to IL4-mediated regulation of mononuclear phagocyte effector functions is altered in IBD. There are several genetic factors involved in CD which may also contribute to UC susceptibility.11 The evidence for linkage in this region is now known to be largely accounted by the association of 3 major, relatively uncommon, amino acid polymorphisms within the NOD2/CARD15 gene.

IBD2 Locus on Chromosome 12 The greatest evidence for linkage to IBD has been reported for a region of chromosome 12q14 surrounding the microsatellite

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Genetic Markers Associated with Clinical Outcomes

TABLE 1. Candidate Genes Associated with Susceptibility in IBD CD

UC

LRRK2, ATG16L1, IRGM, NOD2 CPB4

PRDX5, BACH2, ADO, GPX4, GPX1, SLC22A4, LRRK2, NOD2 GCKR FASLG, THADA VAMP3, FGFR1OP, ADAM30, LGALS9 MUC19, ITLN1 STAT3, STAT6 SLC9A4, SLC22A5, SLC22A4 ITLN1, NOD2, ATG16L1 NOD2, ITLN1, VDR, TLR1 CCL11, CCL2, CCL7, CLL8, CCR6 ERAP2, LNPEP, DENND18 NDFIP1, TAGAP, IL2RA, PTPN22, IL-31RA, RASGRP1 IL-5, IKZF1, BACH2, RASGRP1 IL-3, IL-4, IL-5, IL-13, CSF-2 IL-27, SBN02, NOD2

CD/UC

PARK7, DAP SERINC3, AGR2

CUL2 ORDML3, XBP1

ARPC2, LSP1, AAMP, ITGAL, CALM3 HSPA6, DLD, PARK7

CARD9, UTS2, PEX13

ALDOB

SLC2A4RG

DAP TTLL8, CEP72, TPPP GNA12, HNF4A, CDH1, ECM1, MUC12, MUC20, ERRFI1 ERRFI1, HNF4A, PLA2G2A/E AQP12A/B, SLC9A3, SLC26A3, OCTN1, OCTN2

PUS10, MST1 KIF21B

REL, PTGER4, NKX2-3

XBP1 SLC11A1, FCGR2A/B, TLR1, TLR2, TLR6, CARD9, REL CARD11 IL8RA, IL8RB MST1

IL-2, TNFRSF9, PIM3, IL7R, TNFSF8, INF-G, IL-20 IL-7R, IRF5 IL-10, IL-19, IL-20, IL-24, IL-35, IL-37, TGF-B IDO

marker D12S83, with a logarithm of odds score of 5.47 and a positive transmission–disequilibrium test, which was subsequently named IBD2.12 This region is more associated with UC compared with CD. A number of possible candidate genes have been investigated including signal transducer and activator of transcription 6 (STAT6), interferon gamma, metalloproteinase (MMP-18), Vitamin D receptor, and b7 integrin family that could be associated with the susceptibility to IBD.13

IBD3 on Chromosome 6p21.3 Satsangi et al9 found that D6S276, adjacent to the major histocompatibility complex, provided no evidence for linkage with CD or IBD overall, but a distortion in allele sharing was reported for siblings with UC. In general, these data suggest that CD and UC are closely related, but distinct, polygenic disorders that share some, but not all, susceptibility genes. In a genome-wide linkage scan involving 268 IBD families of northern European descent containing 353 affected sibling pairs, Hampe et al8 found suggestive linkage to the major histocompatibility complex locus on chromosome 6, with a maximum multipoint LOD score of 2.07 obtained in a region defined

TNFSF8, IL-23, PRDM1, ICOSLG, IL-12B IL-3, IL-4, IL-5, IL-10, IL-13, CSF-2, IL-19 CREM, IL-10

IBD-related Process Autophagy Endoplasmic reticulum stress Cell migration Oxidative stress Carbohydrate metabolism Apoptosis Intracellular logistics Epithelial barrier Restitution Solute transport Paneth cells Innate immune response Immune cell recruitment Antigen presentation T-cell regulation B cell regulation Immunoregulatory cytokines Immune tolerance

by markers D6S289 and D6S276. They reported on a 2-stage linkage and association analysis of both a basic population of 353 affected sibling pairs and an extension of this population to 428 white affected sibling pairs of northern European extraction. Genotyping 28 microsatellite markers on chromosome 6, they found a peak multipoint LOD score of 4.2 at D6S461 for the IBD phenotype. A transmission/disequilibrium test result of P ¼ 0.006 was detected for D6S426 in the basic population and was confirmed in the extended cohort (P ¼ 0.004; 97 versus 56 transmissions). The phenotypes of CD, UC, and indeterminate colitis contributed equally to this linkage, suggesting a general role for the chromosome 6 locus in IBD. Analysis of 5 SNPs in the TNFA in the TNFA (TNF; 191160) and LTA (153440) genes, both located at 6p21 revealed no evidence for association of these candidate genes with IBD. Actually, there is significant evidence that specific HLA class II associations contribute to overall disease pathogenesis, especially for UC. A meta-analysis combining results from 29 studies showed significant positive associations in UC to DR2, DR9, and DRB1*0103, whereas a negative association was found for DR4. For CD, a positive association was found with www.ibdjournal.org |

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FIGURE 2. Role of genetic markers in different pathophysiological pathways in IBD.

DR7, DRB3*0301, and DQ4, and a negative association with DR2 and DR3.14,15 Fisher et al16 found that multiple major histocompatibility complex markers showed strong association with UC in the first stage, with a peak (P ¼ 4.7 · 1028) around rs6927022 in a 400-kb haplotype block containing the BTNL2 gene (606000) and the HLA loci HLA-DQA1 (146880), HLA-DRA (142860), HLA-DRB5 (604776), and HLA-DRB1 (142857). A residual association with an SNP within the BTNL2 gene (rs9268480; P ¼ 0.0036) suggested contribution of that gene or another in linkage disequilibrium (LD) with it. Koss et al17 found that women but not men with extensive colitis compared with distal colitis were significantly more likely (31% versus 12%; P ¼ 0.028) to bear the 2308G-A promoter polymorphism of the TNF gene. This association was even stronger (56% versus 21%; P ¼ 0.0007) in women who also had rather than a C allele at position 720 in the LTA gene. These genetic polymorphisms were also associated with significantly higher TNF production in patients with CD, whereas an A allele instead of a G allele at position 2238 in the TNF gene was associated with lower production of TNF in patients with UC.

IBD4 on Chromosome 14q11-q12 Duerr et al18 performed a genome scan using 751 microsatellite loci in 127 CD-affected relative pairs from 62 families. They found significant linkage to D14S261 at 14q11-q12 (LOD ¼ 3.00; maximum multipoint LOD ¼ 3.60).

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Vermeire et al19 in a genome-wide scan in a 149 Belgian IBD-affected families cohort demonstrated the existence of IBD4 on 14q11 as susceptibility loci. The IL-25 gene is located within this susceptibility region at 14q11.2. The candidate genes within or near locus for IBD4 is TCR a/ϒ complex.

IBD5 on Chromosome 5q31 Rioux et al20 identified 3 regions of linkage (3p, 5q31-q33, and 6p) and one region of significant linkage to 19p13. Higherdensity mapping in the 5q31-q33 region revealed a locus of genome-wide significance (LOD score ¼ 3.9) that contributed to CD susceptibility in families with early-onset disease. In a prospective cohort study using the LD approach, Rioux et al21 studied 256 father–mother–child trios in which the child had CD and at least 1 parent was unaffected. They found strong evidence of LD in the 5q31 region. They found a common haplotype spanning 250 kb. This region contains several immunoregulatory cytokines involved in the pathophysiology of CD such as IL4 (147780), IL5 (147850), and IL13 (147683). Furthermore, individuals with CD have increased expression of IRF1 (147575) and increased enzymatic activity of P4HA2 (600608). In this study, an ultra high-density SNP map was used to identity SNP related to CD. Martinez et al22 found conflicting evidence about the role of the SLC22A4 1672C-T and the SLC22A5-207G-C polymorphisms. These findings suggested that certain haplotypes in defined populations might confer susceptibility or protection to CD.




Silverberg et al23 evaluated 1879 affected offspring and parents ascertained by a North American IBD Genetics Consortium for 6 IBD5 SNPs and confirmed association to the IBD5 region on chromosome 5q31 (P , 0.0005 for rs12521868). They also showed that this association to be exclusive to the non-Jewish population (P ¼ 0.00005), with under transmission of the risk allele in Ashkenazi Jews. Using Phase II HapMap data, the authors identified a set of polymorphisms spanning genes from P4HA2 to IRF1 with equivalent statistical evidence of association to the reported SLC22A4 variant, each of which, by itself, could entirely explain the IBD5 association to CD.

IBD6 on Chromosome 19p13 Van Heel et al24 performed a genome-wide scan of 137 CDaffected relative pairs from 112 families. The authors observed linkage of CD to regions on chromosome 3 (IBD9; 608448; P ¼ 0.0009) and chromosome X (P ¼ 0.001) in this cohort study. Linkage to chromosome 16 (IBD1; 266600) was found in CD pairs not possessing common NOD2/CARD15 (605956) mutations (P ¼ 0.0007). There is evidence for linkage to chromosome 19 (IBD6), which was observed in CD pairs not possessing CARD15 mutations (P ¼ 0.0001) and in pairs possessing 1 or 2 copies of the IBD5 risk haplotype (P ¼ 0.0005), with significant evidence for genetic heterogeneity and epistasis, respectively.

IBD7 on Chromosome 1p36 Cho et al25 analyzed 4 multiply affected American Chaldean families, not known to be related, with IBD. All but 2 of the affected family members had UC; those 2 were diagnosed with CD. The authors observed evidence for linkage and LD in precisely the same region of chromosome band 1p36. Maximal evidence for linkage was observed near D1S1597 by multipoint analysis (maximum LOD score ¼ 3.01). A shared haplotype (D1S507 to D1S1628) was observed over 27 cM between 2 families. There was homozygous sharing of a 5-cM portion of that haplotype in 1 family and over a less than 1-cM region in the second family. Homozygous sharing of this haplotype near D1S2697 and D1S3669 was observed in 1 individual in a third multiply affected family, with heterozygous sharing in a fourth family. The authors concluded that this locus is associated with the pathophysiological development of IBD located in 1p36.

IBD8 on Chromosome 16p Hampe et al26 investigated the possibility that NOD2 is not the only IBD gene located on this chromosome. In a high-density linkage study, a triple-peaked configuration of the linkage curve was observed with peak LOD scores of 2.7, 3.2, and 3.1 on proximal 16p, proximal 16q, and central 16q, respectively. Further studies confirmed the importance of the NOD2 locus in IBD and provided evidence for an IBD-susceptibility locus on proximal 16p. This locus was designated IBD8. Imielinski et al27 performed a GWAS of early-onset IBD in 3426 patients and 11,963 genetically matched controls and found significant association (combined P ¼ 2.41 · 1029 for IBD and


2.87 · 1029 for CD) at rs8049439 on chromosome 16p11 in an LD block that contains multiple genes, including IL27 (608273), SULT1A1 (171150), and SULT1A2 (601292). Colonic expression of IL27 was found to be significantly lower in patients with earlyonset CD than controls (P , 0.05); colonic expression of SULT1A1 and SULT1A2 was significantly lower in both earlyonset CD (P ¼ 0.05 and 0.001, respectively) and in UC (P , 0.0001 for both genes).

IBD9 on Chromosome 3p26 The CC-chemokine receptor 5 (CCR5) gene located on chromosome 3p21 coincides with this IBD susceptibility locus identified by genome-wide scanning.28 The CCR5 is the receptor for regulated and normal T-cell expressed and secreted (RANTES), a natural proinflammatory cytokine. A 32-bp deletion (A32) in the CCR5 gene results in a nonfunctional receptor found with high frequency in whites. They found an association between CCR5 delta32 homozygosity and the presence of anal lesions in patients with CD with statistical significance.29

Candidates Genes for Suceptibility of IBD Genes of Innate Immune Response Candidate gene analysis has been focused on the innate part of the immune system or pathogen-associated molecular patterns.30 The discovery that the gene encoding the caspaseactivating recruitment domain 15 (CARD15)—also known as the nucleotide oligomerization domain 2 (NOD2)—is important in the pathogenesis of CD has focused attention on the innate immune response and the interaction between genetic factors and bacterial products.30 CARD15 variants are found in the majority of white patients with CD and vary between 35% and 45%, with the exception of Scandinavian, Irish, and Scottish patients with CD, where the prevalence is much lower.31–36

Candidate Genes of Autophagy for Suceptibility of IBD Autophagy-related protein 16-1 is encoded by the ATG16L1 gene in humans. The G allele of SNP rs2241880 has been shown in multiple association studies to confer strong risk for CD. This SNP is in fact a common coding variant, specifically a threonine-to-alanine substitution at amino acid position 300 of the ATG16L1 protein (T300A) and seems to account for all of the disease risk conferred by this locus.37

IL-23/IL-17 Axis in IBD IL-23 is highly produced by lamina propria macrophages in the inflamed gut of patients with CD, and it is now evident that IL-23 plays a key role in promoting the synthesis of both Th1 and Th17-cytokines in CD.38 A relatively rare allele at SNP rs11209026, in the IL23R gene, seems to provide a strong protective effect against the development of CD in both Jewish and non-Jewish populations. Weersma et al39 confirmed the association of IL23R with CD www.ibdjournal.org |

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susceptibility. They demonstrated that the rs11209026 SNP in IL23R had a protective effect for IBD in the case–control analysis (odds ratio [OR] ¼ 0.19; 95% confidence interval [CI], 0.10–0.37; P ¼ 6.6 · 1029). Both CD (OR ¼ 0.14; 95% CI, 0.06–0.37; P ¼ 3.9 · 1027) and UC (OR ¼ 0.33; 95% CI, 0.15– 0.73; P ¼ 1.4 · 1023) were associated with IL23R. For ATG16L1, the rs2241880 SNP was associated with CD susceptibility (OR ¼ 1.36; 95% CI, 1.12–1.66; P ¼ 0.0017). The population-attributable risk of carrying allele G is 0.24 and is 0.19 for homozygosity for allele G in CD. No association was found between IL23R or ATG16L1 and celiac disease.

ASSOCIATION OF GENOMICS WITH CLINICAL OUTCOMES


penetrating behavior (P ¼ 1.26 · 1022, OR ¼ 1.25), the need for surgery (P ¼ 2.28 · 1025, OR ¼ 1.73), and complicated disease course (P ¼ 6.86 · 1026, OR ¼ 2.96). Immunomodulator (azathioprine/6-mercaptopurine and methotrexate) use within 3 years after diagnosis led to a reduction in bowel structuring disease (P ¼ 1.48 · 1026, OR ¼ 0.35) and surgical rate (P ¼ 1.71 · 1027, OR ¼ 0.34). Association between each outcome and genetic scores, created using significant SNPs in the univariate analysis, revealed large differences in the probability of developing penetrating disease (IL23R, LOC441108, PRDM1, NOD2; P ¼ 9.64 · 1024; hazard ratio ¼ 1.43), need for surgery (IRGM, TNFS F15, C13ORF31, NOD2; P ¼ 7.12 · 1023; hazard ratio ¼ 1.35), and structuring disease (NOD2, JAK2, ATG16L1; P ¼ 3.01 · 1022; hazard ratio ¼ 1.29) among patients with CD with low and high scores.41

Genetic Markers A genetic marker is a gene or a DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation (which may arise due to mutation or alteration in the genomic loci). A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (SNP). The genetic markers could help gastroenterologist to predict disease courses and identify patients in need of intensive treatment. Identification of disease- and course-specific genetic marker profiles can be used to identify biological pathways involved in the disease development and treatment. Knowledge of disease mechanisms in general can lead to improved future development of preventive and treatment strategies. Thus, the clinical use of a panel of genetic markers represents a diagnostic and prognostic tool of potentially great value. However, to date, studies have only addressed the influence of single mutations on IBD, resulting in a poor prediction of clinical course or response to therapy in individual patients.40 Recently, a study reported the IBDchip in a European multicenter study, which aims to develop an easy way to use DNA array. This noninvasive tool will allow the simultaneous analysis of around 100 relevant mutations to predict the clinical evolution, the risk of developing IBD-related complications, and the likelihood of responding to certain drugs of each patient with IBD. The “IBDchip” is a new strategy to predict the clinical course and response to medical therapy in patients with IBD and to confirm the important role of genetic factors such as NOD2 variant allele as a strong predictor of a more complicated disease course, and JAK2 was also found to be a predictor of a more complicated disease behavior.40

Genes of Innate Immune Response Associated with Clinical Course of IBD The NOD2 gene was the most important genetic factor, being an independent predictive factor for ileal location (P ¼ 2.02 · 1026, OR ¼ 1.90), structuring (P ¼ 3.16 · 1026, OR ¼ 1.82) and

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Genes of Innate Immune Response Associated with Medical Treatment Response Use of Immunomodulators The use of immunomodulators within the first 3 years after diagnosis was associated with the presence of TNFSF15 and C13ORF31SNPs. They were also found to be independent predictive factors for surgery. Smoking habit was mild associated (P ¼ 5.29 · 10–2, OR ¼ 1.37; 95% CI, 1.00–1.88). NOD2 variant and the use of immunomodulators within 3 years after diagnosis were still the most important drivers for surgical outcome, but TNFSF15 and C13ORF31 were no longer independently associated.41

Glucocorticoid Resistance When looking at potential mechanisms of glucocorticoid resistance, the multidrug resistance gene 1 (ABCB1/MDR1) is of special interest. Using a genome-wide approach with tagging SNPs of ABCB1, Ho et al confirmed the contribution of the ABCB1 gene to susceptibility to UC (especially with the phenotype of extensive disease) but concluded the association with haplotype rather than depending on a single SNP.40,41 Polymorphisms of the MDR1 gene have been associated with refractory CD and UC.42

Association of the Interleukin Gene Polymorphisms with UC

Interleukin 1 antagonist (IL-1ra) and interleukin 1b (IL-1b) seem to have a central role because of its immunoregulatory and proinflammatory activities. The role of IL-1 was observed that IBD colonic macrophages are able to activate IL-1b–converting enzyme and hence release mature IL-1b in a manner similar to circulating monocytes. Common IL-33 and IL1RL1 polymorphisms contribute to the risk of IBD in an Italian cohort of adult and pediatric patients, with some influence on subphenotypes.43 Latiano et al43 showed that a significant allele and genotype associations with IL-33 rs3939286 were found in patients with




CD (P ¼ 0.004) and UC (P ¼ 0.002). After stratifying the cohort for age at diagnosis, the differences remained significant only in the adult-onset IBD. Significant associations were also obtained in patients with CD with 2 IL1RL1 polymorphisms (rs13015714 and rs2058660, P , 0.015). By combining homozygous and heterozygous carriers of the rs13015714 risk allele, differences were still significant for both CD adult- and pediatric-onset. On genotype– phenotype evaluation, an increased frequency of extensive colitis in adult UC (P ¼ 0.019) and in steroid-responsive pediatric patients (P ¼ 0.024) carrying the IL-33 rs3939286 risk genotype was observed. mRNA expression of IL-33 and IL1RL1 in inflamed IBD biopsy samples was significantly increased.43 Lacruz-Guzmán et al44 al showed the pharmacogenetic role of the rs1143634 polymorphism of IL1B and TNF polymorphisms in infliximab-treated patients with IBD. They found an association between the rs1143634 C allele and higher serum IL1b concentrations and a lower response to infliximab treatment in patients with CD that warrants the interest of future studies in larger and independent series. IL-1RN and IL-1B polymorphisms were associated with the genetic susceptibility to develop UC and might be associated with the presence of steroid dependence in patients with UC. Yamamoto-Furusho et al45 found a significant increased frequencies of IL-1RN6/1 TC (rs315952) and RN6/2 CC (rs315951) and decreased frequency of IL-1B-511 TC (rs16944) genotypes in Mexican patients with UC as compared with healthy controls. In the subgroup analysis, they found a significant association between the RN6/2 GG (rs315951) and IL-1B-511 CC (rs16944) genotypes and the presence of steroid dependence in patients with UC (pC ¼ 0.0001, OR ¼ 15.6 and pC ¼ 0.008, OR ¼ 4.09, respectively). Patients with UC showed increased frequencies of IL-1RN “CTC” and “TCG” haplotypes when compared with healthy controls (P ¼ 0.019, OR ¼ 1.43 and P , 1027, OR ¼ 2.63, respectively). Two haplotypes (TTG and CTG) showed decreased frequency in patients when compared with healthy controls (P ¼ 9 · 1027, OR ¼ 0.11 and P ¼ 8 · 1026, OR ¼ 0.11, respectively). Interleukin 15 (rs2254514). Interleukin 15 is a Th1-related cytokine that triggers inflammatory cell recruitment with implications for pathogenesis in UC. The IL-15 gene is located within a 35 kb region of the q28-31 locus of chromosome 4. YamamotoFurusho et al46 showed the role of IL-15 gene polymorphisms as susceptibility markers in patients with UC. Seven polymorphisms of IL-15 (rs3806798, rs10833, rs4956403, rs2254514, rs2857261, rs10519613, and rs1057972) were studied in a group of 199 Mexican patients with UC and 698 Mexican Mestizo healthy unrelated individuals. In this study, the rs2254514 polymorphism was significantly associated with decreased risk of UC as compared with controls under both dominant and additive models (OR ¼ 0.62, Pdom ¼ 0.014 and OR ¼ 0.65, Padd ¼ 0.02). The rs2254514 CC genotype was associated with young age at diagnosis, 40 years (P ¼ 0.03, OR ¼ 3.67). Five polymorphisms (rs1051613, rs2254514, rs2857261, rs1057972, and rs10833)


were in strong LD and were included in 6 haplotypes: H1 (ACAAC), H2 (CCGTC), H3 (CTAAT), H4 (CCAAT), H5 (CTAAC), and H6 (CCAAC). Patients with UC showed an increased frequency of the H6 haplotype (P ¼ 0.005, OR ¼ 3.2) and a decreased frequency of the H5 haplotype (P ¼ 0.031, OR ¼ 0.40). These results suggest that the IL-15 rs2254514 polymorphism might have an important role in the development of UC in the Mexican population. Interleukin 19 (rs2243188 and rs2243193). Interleukin (IL)19 belongs to the IL-10 family and is a potent immunomodulatory cytokine, with implications for pathogenesis in IBD. YamamotoFurusho et al47 studied the role of IL-19 gene polymorphisms as susceptibility markers for UC. Three polymorphisms of IL-19 gene (rs2243188, rs2243191, and rs2243193) were genotyped in a group of 200 Mexican Mestizo patients with UC and 698 healthy unrelated individuals with no family history of UC. Although genotypes AA (rs2243188) and AA (rs2243193) were significantly decreased in patients with UC as compared with healthy controls (P , 0.018 and P , 0.006, respectively), a genetic additive effect for the alleles was not observed (Cochran–Armitage trend test, not significant). In the subgroup analysis, no differences were found between the IL-19 genotypes and the clinical characteristics of UC. The results suggest that IL-19 polymorphisms (rs2243188 and rs2243193) might have a protective role in the development of UC in Mexican individuals. Interleukin 20 (rs2981573 and rs2232360). Interleukin (IL)20 belongs to the IL-10 family and is a potent immunomodulatory cytokine. The IL-20 gene is located within a 200 kb region of q31-32 locus of chromosome 1. Yamamoto-Furusho et al48 evaluated the IL-20 gene polymorphisms as susceptibility markers for UC. Three polymorphisms of IL-20 gene (rs2981573, rs2232360, and rs1518108) were genotyped in a group of 198 Mexican Mestizo patients with UC and 698 ethnically matched healthy unrelated individuals with no family history of UC. We found significant decreased frequencies of 2 IL-20 genotypes: GG (rs2981573) (10.6% versus 17.6%, P ¼ 0.017, OR ¼ 0.55; 95% CI, 0.33–0.93) and GG (rs2232360) (10.6% versus 17.6%, P ¼ 0.017; OR ¼ 0.55, 95% CI, 0.33–0.93) in patients with UC as compared with healthy controls. No significant differences of gene frequencies were found between patients with UC and healthy controls in the rs1518108 polymorphism. In the subgroup analysis, no differences were found between the IL-20 genotypes and the clinical characteristics of UC. The results suggest that the GG genotypes of the IL-20 polymorphisms (rs2981573 and rs2232360) might have an important role in the development of UC in the Mexican population. Genes of HLA associated with clinical outcomes. Data from the meta-analysis of 7 small studies also demonstrated a nonsignificant increase in the common allele HLA-DRB1*04 in CD (OR ¼ 1.62; 95% CI, 0.73–3.61).49 This association is particularly important in Japanese cohorts, in whom the strongest HLA www.ibdjournal.org |

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associations are with DRB1*0405, *0410, and the linked DQB1*0401, 0402 alleles.50 Association with DRB1*04 has not been widely observed in European and North American patients. However, in the recent Canadian study, a weak association with DRB1*04 was identified in patients with ileal disease (RR ¼ 1.7; 95% CI, 1.1–2.5).50 It is interesting that this association was stronger in patients possessing one of the 3 common CDassociated CARD15 variants, which may indicate an epistatic interaction between this DRB1 allele and CARD15 in determining susceptibility to ileal CD.50 Other studies in whites have reported an increased frequency of HLA-DRB1*0103 allele in patients with UC with extensive colitis and extraintestinal manifestations9,51–53 and also patients who underwent proctocolectomy,52,53 suggesting that this allele may be a prognostic genetic marker in patients with UC. Other study found that HLA-DRB1*0103 allele was associated with UC and its severe manifestations such as colectomy and pancolitis (P ¼ 0.003, OR ¼ 3.6; 95% CI, 1.46–8.9), whereas HLA-DRB1*15 allele was only associated with pancolitis in Mexican patients with UC (P ¼ 0.001, OR ¼ 8.5). Yamamoto-Furusho et al54,55 reported that HLA-DRB1 alleles were associated with the clinical course of disease and steroid dependence in Mexican patients with UC. These studies showed that significant associations were found between some HLA-DRB1 alleles and the clinical course of disease: initial active and then inactive and the HLA-DRB1*14 allele (P ¼ 0.03, OR ¼ 4.63; 95% CI, 1.08–21.23) and HLA-DRB1*08 allele (P ¼ 0.04, OR ¼ 4.34; 95% CI, 1.9–33.3). However, the HLA-DRB1*07 (P ¼ 0.001, OR ¼ 9.76; 95% CI, 1.55– 65.56) was significantly associated with steroid dependence in Mexican patients with UC. In other study, Yamamoto-Furusho et al55 found that p-ANCA–positive Mexican patients with UC had a significantly increased frequency of HLA-DR7 compared with p-ANCA–negative controls (22% versus 5.1%; pC ¼ 0.02, OR ¼ 5.2; 95% CI, 1.06– 37.82). Subgroup analysis showed a significantly increased frequency of the HLA-DRB1*07 allele in 15 of 20 patients with UC with severe activity of UC and p-ANCA positivity (100% versus 0%; pC ¼ 0.0000001, OR ¼ 35). No significant differences were found between p-ANCA–positive patients, HLA-DR alleles, and other clinical features such as extraintestinal manifestations, proctocolectomy, and extension. Lappalainen et al56 observed that the rare HLA-DRB1*0103 allele was found to be associated with UC (P ¼ 0.008) in the Finnish population. Walker et al57 found that the HLA-DRB1*0103 allele was absent in all Indian patients with UC and controls in contrast to white European patients with UC (9/50: 18%). The HLADRB1*1502 allele was significantly more frequent in the Indian UC cohort (29.2%) than controls (17.6%) (P ¼ 0.04), and the allele was absent in the white European cohort. There were no significant differences in the frequency of the CARD15/NOD2 (rs2066842) variant or IL23R (rs11209026) between the different ethnic groups.

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GENE EXPRESSION PROFILING In the recent years, advances have been performed in the understanding of the genetics in UC. Gene expression profiling is considered a predictive marker in IBD as shown in Table 2.

Transporters Gene Expression Associated with Medical Treatment Response and Long-Term Remission The multidrug resistant gene 1 (MDR1), also known as ABCB1, is the first member of the ATP binding cassette family

TABLE 2. Genetic Markers Associated with Clinical Outcomes in IBD

Genetic Marker IL-15

Gene Expression SPNs Profile ✓

✓

Clinical course of disease and steroid dependence Histological inflammatory activity Independent predictive factor for ileal location, structuring, penetrating behavior, the need for surgery, and complicated disease course Use of immunomodulators within 3 years after diagnosis Predictor of a more complicated disease behavior CD susceptibility Histological inflammatory activity Clinical course of disease Clinical course of disease and steroid dependence Steroid dependence

✓ ✓ ✓

Steroid-responsive pediatric patients Need of steroid use Proctolectomy Pancolitis Clinical course of disease

✓

TLR5, TLR8 y TLR9 NOD2/CARD15

✓

JAK2

✓

IL-23R IL-6

✓ ✓ ✓ ✓

PPARS MDR1 RN6/2 GG (rs315951) IL-1b-511 CC (rs16944) IL-18 HLA-DRB1*0103 HLA-DRB1*15 HLA-DRB1*14 y HLA-DRB1*08 HLA-DRB1*07 CXLC8

Clinical Outcome

✓

✓ ✓

Steroid dependence Presence of extraintestinal manifestations Histological inflammatory activity Duration of disease




(ABC) proteins.58 The relevance of MDR1 gene in IBD is based on data provided from Mdr1a2/2 knockout mice that spontaneously develop colitis around 12 weeks of age.59 Yamamoto-Furusho et al60 showed that MDR1 expression was decreased in patients with active UC compared with patients with UC in remission and the normal control group (P ¼ 0.034 and P ¼ 0.002, respectively). The MDR1 levels were decreased in UC remission compared with the normal control group (P ¼ 0.04). The medical treatment response and long-term remission were associated with high gene expression of MDR1 (P ¼ 0.009 and P ¼ 0.002, respectively).

Gene Expression of Adaptative Immune Response Mediators Associated with the Use of Steroids Elevated levels of interleukin 18 (IL-18) are found in many chronic inflammatory disorders, including IBD, and polymorphisms in the IL18R1-IL18RAP locus are associated with IBD susceptibility. IL-18 is an IL-1 family cytokine that has been proposed to promote barrier function in the intestine, but the effects of IL-18 on intestinal CD4+ T cells are poorly understood. Harrison et al61 demonstrated that IL-18R1 expression is enhanced on both effector and regulatory CD4+ T cells in the intestinal lamina propria, with T-helper type 17 (Th17) cells exhibiting particularly high levels. They further show that, during steady state, intestinal epithelial cells constitutively secrete IL-18 that acts directly on IL-18R1-expressing CD4+ T cells to limit colonic Th17 cell differentiation, in part by antagonizing IL-1R1 signaling. In addition, although IL-18R1 is not required for colonic Foxp3+ regulatory T (Treg) cell differentiation, we found that IL-18R1 signaling was critical for Foxp3+ Treg cell–mediated control of intestinal inflammation, where it promoted the expression of key Treg effector molecules.61 Villeda Ramírez et al62 showed that IL-18 mRNA expression was significantly increased in the mucosa from patients with active and remission UC compared with the healthy control group (P ¼ 0.006 and P ¼ 0.007, respectively). No significant difference was found between active and remission UC groups. The high gene expression of IL-18 was associated with the use of steroids (P ¼ 0.04). These findings confirmed the potential role of the IL-18 gene in the pathogenesis of UC and suggest that IL-18 gene expression from intestinal mucosa could be used as a potential marker to predict the use of steroids in patients with UC.

Gene Expression of Immune Response Associated with Histological Inflammatory Activity Cytokines have validated roles in the immunopathogenesis of IBD. Matsuda et al63 showed the expressions of tumor necrosis factor (TNF)-a, interleukin (IL)-6, IL-8, and IL-10 mRNAs in the colonic mucosa of patients with UC during active and quiescent UC, and they found that during active UC, all 4 cytokine mRNA levels were high; only IL-6 and IL-8 mRNAs decreased to normal levels during remission. IL-8 mRNA was high even at sites of


endoscopically quiescent UC during active disease. Steroid-naive patients respond better to granulocytapheresis.

Interleukin 6 The gene expression of IL-6 was associated with histological activity in Mexican patients with UC.64 The expression of IL-6 increases in patients with active UC compared with controls (P ¼ 0.004) and patients with UC in remission (P ¼ 0.014). No significant difference was found between patients with active UC and controls (P ¼ 0.446). Gene expression of TNF-a was higher in biopsies from patients with UC activity compared with control subjects (P ¼ 0.004) and those in remission (P ¼ 0.001). The expression of IL-6 correlated significantly (P ¼ 0.02) with histological activity. Interleukin 6 is the best marker of intestinal inflammation and that its expression correlates with histological activity.

Toll-like Receptors Dysregulation of innate immune response by Toll-like receptors (TLRs) is a key feature in IBD. Sanchez-Munoz et al65 evaluated the mRNA profiles of TLR1 to 9 in colonic mucosa of patients with UC, according to disease activity. Toll-like receptors TLR8, TLR9, and IL6 mRNA levels were significantly higher in the colonic mucosa from patients with UC (both quiescent and active) as compared with healthy individuals (P , 0.04). In the UC patients group, the TLR2, TLR4, TLR8, and TLR9 mRNA levels were found to be significantly lower in patients with quiescent disease as compared with those with active disease (P , 0.05), whereas TLR5 showed a trend (P ¼ 0.06). IL6 and TNF mRNA levels were significantly higher in the presence of active disease and help to discriminate between quiescent and active disease (P , 0.05). In addition, IL6 and TNF mRNA positively correlate with TLRs mRNA with the exception for TLR3, with stronger correlations for TLR5, TLR8, and TLR9 (P , 0.0001). TLR9 protein expression was mainly in the lamina propria infiltrate. This study demonstrates that TLR2, TLR4, TLR8, and TLR9 expression increases in active patients with UC and that the mRNA levels positively correlate with the severity of intestinal inflammation and with inflammatory cytokines.64–66

Genes of Adaptative Immune Response Associated with Duration of Disease and Presence the Extraintestinal Manifestations CXCL8 (IL-8) is a proinflammatory CXC chemokine associated with the promotion of neutrophil chemotaxis and degranulation. CXCL8 protein is secreted by a variety of cell types, including intestinal epithelial cells.67 CXCL8 has also been shown to play an important but nonspecific role in the pathogenesis of IBD, and its mRNA has been shown to be restricted to areas with histological signs of inflammatory activity and mucosal destruction.68 CXCL8 mRNA expression was increased from colonic mucosa in patients with active UC as compared with healthy control group (P ¼ 0.003) and UC remission (P ¼ 0.010), and UC in remission was upregulated when compared with healthy control www.ibdjournal.org |

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group (P ¼ 0.04). The extraintestinal manifestations and duration of disease were associated with high gene expression of CXCL8 (P ¼ 0.049 and P ¼ 0.016, respectively) suggesting a role of CXCL8 in patients with UC due to its inflammatory activity. No significant differences were found in relation to CXCL8 gene expression and other demographic and clinical characteristics such as age at diagnosis, gender, extent of disease, medical treatment, and clinical course of disease. These findings confirmed the potential role of CXCL8 gene in the pathogenesis of UC and suggest that CXCL8 gene expression from rectum biopsies could be used as an inflammatory marker.69


REGULATORS OF GENE EXPRESSION (miRNAs) IN IBD

Wu et al75 identified 10 intestine region-specific miRNAs. Three miRNAs were increased and 1 miRNA was decreased in the terminal ileum as compared with the colon. Six other miRNAs expressed varying levels of expression among the colon regions. Five miRNAs were found to be differentially expressed in tissues of patients with active colonic CD, with 3 increased and 2 decreased as compared with normal healthy controls. Similarly, 4 miRNAs were found to be significantly increased in tissues of patients with active ileal CD. The expression differences between ileal CD, colonic CD, and previously identified UC-associated miRNAs support the likelihood that miRNAs influence differing inflammation-related gene expression in each IBD subtype and may form the basis for future diagnostic tests and therapeutic targets for IBD. Lin et al76 demonstrated that miR-31, miR-206, miR-424, and miR-146a are novel specific biomarkers of IBD. Furthermore, miR-31 is universally expressed in both UC and CD not only in fresh-frozen but also in formalin-fixed paraffin-embedded tissues. In this sense, other studies have demonstrated the role of miRNA in IBD; Fasseu et al showed that the expression of mature miRNAs in inactive colonic mucosa of patients with IBD was identified in quiescent colonic mucosa, 8 being commonly dysregulated in noninflamed UC and CD (mir-26a, -29a, -29b, -30c, -126*, -127-3p, -196a, and -324-3p). Several miRNA genes with dysregulated expression colocalize with acknowledged IBD-susceptibility loci, whereas others (e.g., clustered on 14q32.31) map on chromosomal regions not previously recognized as IBD-susceptibility loci. In addition, in silico clustering analysis identified 5 miRNAs (mir-26a, -29b, -126*, -127-3p, and -324-3p) that share coordinated dysregulation of expression both in quiescent and in inflamed colonic mucosa of patients with IBD. Six miRNAs displayed significantly distinct alteration of expression in noninflamed colonic biopsies of patients with UC and CD (mir-196b, -199a-3p, -199b-5p,-320a, -150, and -223).77 Finally, all research conclude that miRNAs as crucial players in the onset and/or relapse of inflammation from quiescent mucosal tissues in patients with IBD. It allows speculating a role for miRNAs as contributors to IBD susceptibility and suggests that some of the miRNAs with altered expression in the quiescent mucosa of patients with IBD may define miRNA signatures for UC and CD and help to develop new diagnostic biomarkers.77 Additional studies about miRNAs in the gut mucosal inflammatory process associated with clinical outcomes in patients with IBD can provide a new therapy targeting on the functions of these key genes that might provide novel perspective for IBD treatment.

miRNAs represent a recently discovered class of small noncoding RNAs, and the miRNAs have revealed an important role of gene expression regulation and described as an important player in the regulation of immune response.74 MicroRNAs (miRNAs) are small noncoding RNAs that act as potent negative regulators of gene expression and are differentially expressed in chronic inflammatory diseases, including UC.75

Epigenetic factors can mediate interactions between the environment and the genome; their study could provide new insight into the pathogenesis of IBD. The contribution of epigenetic alterations to disease pathogenesis is emerging as a research priority.

Transcription Factors Associated with Response to Treatment and Mild Clinical Course Peroxisome proliferator-activated receptor gamma (PPARg) is a member of a family of nuclear receptors, which interacts with nuclear proteins acting as coactivators and corepressors. Colon is a major tissue which expresses PPARg in epithelial cells and, to a lesser degree, in macrophages and lymphocytes and plays a role in the regulation of intestinal inflammation.70 PPARg ligands have beneficial effects in different models of experimental colitis with possible implication in the therapy of IBD. Functional, biological, pharmacological, and chemical evidence has shown that aminosalicylates are a new functional synthetic ligand for PPARg in colonic epithelial cells.71 PPARg is indeed the key receptor mediating the 5-ASA activity, by inhibiting several key target genes such as NFkB, signal transducers, and activators of transcription. PPAR-a gene expression was significantly decreased in patients with active UC compared with remission UC group (P ¼ 0.001) and controls (P ¼ 0.001). They found that low gene expression of PPAR-a in mucosa confers a higher risk of UC activity (P # 0.0001, OR ¼ 22.6). They observed an increase of PPAR-a expression in patients with UC who were treated with 5-aminosalicylates compared with those who received any other combined therapy (P ¼ 0.03, OR ¼ 0.08). PPARg gene expression was decreased in the active UC group compared with UC in remission (P ¼ 0.001) and control group (P ¼ 0.001). An increased expression of PPARg gene was associated with mild clinical course of the disease (P # 0.001, OR ¼ 0.05).72 Previously showed decreased gene expression of PPARg in colonic biopsies from patients with active UC and its expression was negatively correlated with the severity of endoscopic disease activity.73

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EPIGENETICS AND IBD




Karatzas et al78 evaluated the DNA methylation changes in peripheral blood and tissue biopsies from patients with IBD. In UC, 5 genes (CXCL14, CXCL5, GATA3, IL17C, and IL4R) were hypermethylated compared with healthy controls. Some of the examined genes showed different methylation patterns between CD and UC. Concerning tissue samples, they found that all hypermethylated genes appear the same methylation pattern and confirmed a moderate–strong correlation between methylation levels in colon biopsies and peripheral blood (Pearson’s coefficients r ¼ 0.089–0.779 and r ¼ 0.023–0.353, respectively). The epigenetic changes observed in this study indicate that CD and UC exhibit specific DNA methylation signatures with potential clinical applications in IBD noninvasive diagnosis and prognosis. A number of potential clinical applications of epigenetics in diagnostics and therapeutics are receiving attention. The diagnostic applications of epigenetics include the use of biomarkers to confirm diagnosis, stratify disease course and response to chemotherapy, and predict development of cancer. Particularly pertinent for IBD, methylation changes in SFRP2, measured in fecal DNA samples, have been used to identify patients with colorectal cancer with approximately 75% sensitivity and specificity.79,80 Biomarkers have been found in a range of body fluids, including sputum, urine, lung, bladder, and head and neck cancers.81–83 DNA methylation is a quantitative trait and therefore an attractive biomarker. A panel of relevant hypomethylated or hypermethylated CpGs might someday be used to distinguish between UC and CD, enable disease stratification, and predict treatment response.84 The tissue-specific nature of epigenetic changes becomes especially relevant when considering their use as biomarkers.

CONCLUSIONS The techniques available for the development of markers are genomics (SNP genotyping, pharmacogenetics, and gene expression analyses) and proteomics, and epigenetics could provide biomarkers for use in diagnosis of IBD, along with predicting disease progression and response to therapy. The use of biomarker profiles could deliver clinically useful results in the next decade to help in personalizing care in patients with IBD. This could be a potential great benefit in predicting the course of disease in individual patients and in guiding appropriate medical therapy and stratifying the patient risk profile.

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New serological markers in pediatric patients with inflammatory bowel disease.

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Prevalence of and outcomes associated with corticosteroid prescription in inflammatory bowel disease.

Genetic association of apolipoprotein E polymorphisms with inflammatory bowel disease.

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National trends and inpatient outcomes of inflammatory bowel disease patients with concomitant chronic liver disease.

Factors Associated with Anxiety and Depression in Korean Patients with Inactive Inflammatory Bowel Disease.

Factors associated with readmission to the hospital within 30 days in patients with inflammatory bowel disease.

Factors associated with the use of probiotics in patients with inflammatory bowel disease.

Early infliximab trough levels are associated with persistent remission in pediatric patients with inflammatory bowel disease.

Inflammatory and Lipid-Associated Markers of Cardiovascular Diseases in Children with First Exacerbation of Inflammatory Bowel Disease.

Evaluation of factors associated with response to hepatitis B vaccination in patients with inflammatory bowel disease.

Fatigue severity and factors associated with high fatigue levels in Korean patients with inflammatory bowel disease.

Patients' information-seeking activity is associated with treatment compliance in inflammatory bowel disease patients.

Association between Genetic Polymorphisms and Response to Anti-TNFs in Patients with Inflammatory Bowel Disease.

Noninvasive Markers of Disease Activity in Inflammatory Bowel Disease.

Serum inflammatory markers and risk of colorectal cancer in patients with inflammatory bowel diseases.

Th17 plasticity and its changes associated with inflammatory bowel disease.