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Keywords:

  • pediatrics;
  • Crohn's disease;
  • genetics

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Background: The IL-23 receptor (IL-23R) has been found to be associated with small bowel Crohn's disease (CD) in a whole genome association study. Specifically, the rare allele of the R381Q single nucleotide polymorphism (SNP) conferred protection against CD. It is unknown whether IL-23R is associated with IBD in children. The aim was to examine the association of IL-23R with susceptibility to IBD in pediatric patients.

Methods: DNA was collected from 609 subjects (151 CD and 52 ulcerative colitis [UC] trios). Trios were genotyped for the R381Q SNP of the IL-23R gene and SNP8, SNP12, SNP13, of the CARD15 gene using Taqman. The transmission disequilibrium test (TDT) was used for association to disease using GENEHUNTER 2.0.

Results: The rare allele of R381Q SNP was present in 2.7% of CD and 2.9% UC probands. The CARD15 frequency was 31.5% (CD) and 18% (UC). The IL-23R allele was negatively associated with inflammatory bowel disease (IBD): the R381Q SNP was undertransmitted in children with IBD (8 transmitted [T] versus 27 untransmitted [UT]; P = 0.001). This association was significant for all CD patients (6 T versus 19 UT; P = 0.009), especially for non-Jewish CD patients (2 T versus 17 UT; P = 0.0006). TDT showed a borderline association for UC (2 T versus 8 UT; P = 0.06). As expected, CARD15 was associated with CD in children by the TDT (58 T versus 22 UT P = 0.00006), but not with UC.

Conclusions: The protective IL-23R R381Q variant was particularly associated with CD in non-Jewish children. Thus, the initial whole genome association study based on ileal CD in adults has been extended to the pediatric population and beyond small bowel CD.

(Inflamm Bowel Dis 2007)

The currently accepted etiopathogenic hypothesis suggests that chronic intestinal inflammation and related systemic manifestations characteristic of inflammatory bowel disease (IBD) are due to an overly aggressive or pathologic immune response to resident luminal bacterial constituents. Predisposing factors are genetic dysregulation of mucosal immune responses and/or barrier function, with onset triggered by environmental stimuli. The increased level of concordance between identical twins, increased rates of IBD among the Ashkenazi Jewish population, and the familial risk of IBD provides strong evidence that genetic factors play an important role in the pathogenesis of IBD.1

Genetic advances have lead to the discovery that variants of CARD15 located at 16q12 and the 5q31 (IBD5) haplotype confer susceptibility to Crohn's disease (CD). However, the risks associated with these 2 genes account for only a portion of the overall genetic risk. This has led to additional gene findings efforts, the most recent being the recent genome-wide association study that identified the association of the interleukin 23 receptor gene (IL-23R) with small bowel CD. In that study the rare glutamine allele of Arg381Gln (R381Q single nucleotide polymorphism, SNP) conferred protection against CD.2 Distortion of allele transmission was also observed for non-Jewish ulcerative colitis (UC)-affected offspring. This study was conducted in an adult IBD cohort and it remains unknown whether IL-23R is associated with IBD in children.

The IL-23R, consisting of an IL-12β1 and an IL-23R chain, is highly expressed on memory T cells.3 IL-23 is a novel cytokine formed via the binding of IL-12p40 to a p19 protein.4 After binding to the IL-23 receptor, IL-23 preferentially activates memory T cells. IL-23 does exhibit some similar biological activities to IL-12, however, IL-12 is more involved in the differentiation of naïve T cells into TH1 lymphocytes and subsequent IFNγ production. IL-23, on the other hand, mediates proinflammatory activities in part by the production of IL-17 through activation of TH17 lymphocytes.5 Other research has shown that IL-23 increases IL-6 production that may account for the tissue injury characteristic of IBD.6 It has also been shown that IL-23, not IL-12, is an important promoter of chronic joint inflammation.5 Becker et al7 suggested that there is a predisposition of IL-23-induced chronic inflammation in the terminal ileum, which strengthens the evidence for the recent association between IL-23R in small bowel CD patients.2 The elevation of IL-17 levels in the colonic mucosa of both CD and UC patients, combined with the recent association of IL-23R with UC,2 suggests that IL-23 may also play an important role in UC.8

In this study the association of IL-23R in a pediatric IBD population was tested using the robust method of transmission disequilibrium test (TDT) analysis. We specifically tested R381Q SNP, since this was the most significant result in the genome-wide association study.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patients and Families

IBD patients and families were enrolled from participating sites of the Western Regional Research Alliance for Pediatric IBD (WRRAPID). A total of 203 families (609 subjects) were enrolled. In order to be eligible all CD patients must have undergone complete colonoscopy with ileal intubation or complete colonoscopy and small bowel follow-through. A diagnosis of CD for this study required at least 2 of the following: 1) a history of abdominal pain, weight loss, short stature, malaise, rectal bleeding, or diarrhea; 2) characteristic endoscopic findings of discontinuous ulcerations, cobblestoning, fistula, or severe perianal disease; 3) radiologic features of stricture, fistula, or evidence of cobblestoning or ulceration of the mucosa; 4) macroscopic appearance at laparotomy of typical bowel wall induration, mesenteric lymphadenopathy, or serosal involvement showing creeping fat or other inflammatory changes; or 5) histopathology showing transmural inflammatory cell infiltrate or epithelial granulomas and absence of identifiable infectious agents.9 A diagnosis of UC for this study required at least 3 of the following after exclusion of infectious or neoplastic disease10: 1) typical case history with diarrhea and/or blood in stools for more than a week or recurrent episodes; 2) typical sigmoidoscopic or colonoscopic findings of granulated, friable mucosa with or without superficial ulcerations; 3) characteristic histopathologic and/or cytologic findings of inflammation; or 4) radiologic findings of inflammation, with spiculated, granulated inner surface of the colon proximal to the rectum and/or frank ulcerations.

Parents of the IBD probands were also included as part of the TDT analysis. Indeterminate colitis patients were excluded from the study. Informed consent was obtained from all study participants and this study was approved by the Ethics Review Board at each participating site.

Genotyping

Whole blood samples were drawn on all IBD trios, each consisting of 1 child with IBD (CD or UC) and their 2 parents at each of the participating sites, and sent via overnight FedEx to the Genotyping Core Facility of the Medical Genetics Institute General Clinical Research Center at Cedars-Sinai Medical Center (CSMC). Both parents and the affected child were genotyped for the reported protective R381Q SNP (rs11209026) of the IL-23R gene and the well-established predisposing 3 variants of the CARD15 gene (SNP8, SNP12, SNP13). R381Q was specifically examined as it had the greatest significance, exceeding genome-wide level significance, in the prior genome-wide association.2 Genotyping methods were adapted to the TaqMan MGB genotyping platform following the manufacturer's instructions and using PrimerExpress design software (Applied Biosystems, Foster City, CA) as previously described.11, 12

Statistical Analysis

The TDT statistic was used to test the association with disease using GENEHUNTER 2.0.13 TDT tests whether a gene is under- or overtransmitted to the affected child. Since the genotyping and analysis was done within a family, it avoided the possibility of spurious associations due to unrecognized population stratification. CARD15 TDT was used as an expected positive control for the study population.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Study Population

A total of 609 subjects were enrolled. Of the 203 trios, 151 were from CD probands and 52 from UC probands. The median age at diagnosis for all IBD patients was 11 years (range, 0.8–18 years). Of the 151 children with CD, 67% had both small and large bowel involvement, 30% large bowel disease only, and the remaining 13% had isolated small bowel disease. Our study population consisted of 62 Jewish ancestry probands; 37% (53/151) and 17% (9/52) were from CD and UC, respectively. The majority of the families (86%) were of the Caucasian ethnic group.

Genetic Analyses

Allele Frequencies

The rare glutamine allele of the R381Q SNP was present in only 2.67% of CD and 2.94% of UC probands (Table 1). CARD15 allele frequency (any variant) was 31.5% (CD) and 18% (UC). Similar frequencies were observed for the parents for both genes.

Table 1. Allele Frequencies for CARD 15 SNPs and IL23R R381Q SNP
SNPAlleleCD FamiliesUC Families
ParentProbandParentProband
  1. SNP, single nucleotide polymorphism; CD, Crohn's disease; UC ulcerative colitis.

IL23R R381Q SNPR95.48%97.33%95.05%97.06%
 Q4.52%2.67%4.95%2.94%
CARD15 SNP 8C93.77%92.33%97.03%98.04%
 T6.23%7.67%2.97%1.96%
CARD15 SNP12G96.59%95.24%98.04%97.06%
 C3.41%4.76%1.96%2.94%
CARD15 SNP1394.97%92.25%97.06%95.1%
 C5.03%7.95%2.94%4.9%
CARD 15 Any SNPAny rare allele27.7%31.51518
Transmission Disequilibrium Test (TDT)

TDT in all IBD trios showed a highly significant association between IBD and undertransmission of the rare glutamine allele of R381Q; 8 transmitted (T) versus 27 untransmitted (UT); P = 0.001 (Table 2). This association was significant for all CD patients (6 T versus 19 UT; P = 0.009), especially for non-Jewish CD patients (2 T versus 17 UT; P = 0.0006) (Table 3). TDT showed a borderline association for UC (2 T versus 8 UT; P = 0.06). As expected, CARD15 was associated with CD in children by the TDT: (58 T versus 22 UT P = 0.00006), but not with UC (Table 4).

Table 2. IL23R TDT for IBD, CD, and UC
IL23R VariantIBDCDUC
TUTP valueTUTP valueTUTP value
  1. TDT, transmission disequilibrium test; IBD, inflammatory bowel disease; CD, Crohn's disease; UC ulcerative colitis; T, transmitted; UT, untransmitted.

Glutamine allele8270.0016190.009280.06
Table 3. IL23R TDT in Jewish and Non-Jewish IBD Families
AncestryIBD SubtypeGlutamine Allele TGlutamine Allele UTP value
  1. TDT, transmission disequilibrium test; IBD, inflammatory bowel disease; CD, Crohn's disease; UC ulcerative colitis; T, transmitted; UT, untransmitted.

Non-JewishCD2170.0006
 UC160.06
JewishCD420.41
 UC120.56
Table 4. CARD 15 TDT for IBD, CD, and UC
CARD 15 Allelic VariantsIBDCDUC
TUTP valueTUTP valueTUTP value
  1. TDT, transmission disequilibrium test; IBD, inflammatory bowel disease; CD, Crohn's disease; UC ulcerative colitis; T, transmitted; UT, untransmitted.

702Trp (SNP 8)25180.2923140.14240.41
908Arg (SNP 12)1760.021450.04310.32
Leu1007fsinsC (SNP13)2860.00012350.0007510.1
Any variant58220.0000650170.00006850.41

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

This is the first study to demonstrate a significant association between IL-23R and IBD in a pediatric cohort. The protective allele coding for glutamine of the IL-23R R381Q (Arg381Gln) variant was associated with CD in non-Jewish children. Thus, the initial whole genome association (GWA) study based on ileal CD has been extended to the pediatric population and beyond small bowel CD.2 In this study the CARD15 association acted as a positive control, and the observed association with CARD15 demonstrated that applying the TDT to this pediatric cohort will be useful in further gene finding efforts for IBD.

The IL-23R protein is comprised of an extracellular domain, a single transmembrane domain, and a 252 amino acid cytoplasmic domain.3 The Arg381Gln variant identified in the genome-wide association encodes for a subunit of the IL-23R located on chromosome 1p31. The glutamine allele is the rare allele and the arginine is the common allele. The frequency of the rare allele in our IBD probands was 2.67% for CD and 2.94% for UC. In the prior case-control association study, the glutamine allele was shown to protect against CD in both the Jewish (odds ratio [OR]: 0.26) and non-Jewish populations (OR: 0.45). TDT analysis herein demonstrated that the glutamine allele was undertransmitted in all CD-affected offspring. Our study confirms the negative association of R381Q with CD using TDT, most significantly observed herein in non-Jewish families. Further studies need to be done to confirm an association between IL-23R and UC, as some susceptibility was observed in the prior GWA and a trend was seen in our pediatric patients using TDT.

Further research is certainly needed to examine other variants on the IL-23R gene and its adjacent region. Of interest is the proximity of the IL-23R gene to the IL-12RB1 gene, which encodes the second subunit of the IL-23R. Research suggests that both IL-23 and IL-12 promote a Th1 proinflammatory response. Based on more recent gene targeted animal model studies it appears that IL-23 may be a more significant driver of inflammation. Murphy et al5 demonstrated that the targeted absence of IL-23 was protective, whereas loss of IL-12 exacerbated collagen-induced arthritis. In these studies the IL-23 gene knockout mice did not develop clinical signs of disease or suffer any joint and bone pathology. In contrast, IL-12 deficiency appeared to exacerbate the signs of inflammation. A resistance to joint inflammation and bone pathology was found to be correlated with an absence of IL-17–producing CD4+T cells. This is in contrast to the IL-12–deficient mice who paradoxically developed more Il-17–producing CD4+ lymphocytes with increased expression of the proinflammatory cytokines; tumor necrosis factor, IL-1, IL-6, and IL-17. Such gene-targeted models are lacking in models of gut inflammation but recent evidence suggests that IL-23 does play an important role in the inflammatory cascade characteristic of IBD.6, 7, 14 Becker et al7 demonstrated that the small bowel, more specifically the terminal ileum, is predisposed to develop chronic inflammation through IL-23. The blockade of the proinflammatory cytokine TNFα has set the stage for an era of monoclonal antibody blockage of key cytokines involved in autoimmune diseases.15 Anti-TNFα therapies serve as a model for development of other therapies that will block the formation of or signaling pathway for proinflammatory cytokines. The IL-23 pathway has also been a target of antibody blockade. A small pilot study demonstrated that the administration of anti-IL-12p40 resulted in the downregulation of both IL-23 and IL-12, as well as their downstream proinflammatory mediators, given that both cytokines share an identical p40 chain that is bound to a p19 chain in the case of IL-23 and a p35 chain in IL12.6 Further research is needed to understand how variants of the IL-23R gene contribute to disease susceptibility in IBD. This will also help guide the future direction of therapeutic interventions.

The results of our study demonstrate that the allele coding for glutamine at the IL-23R R381Q (Arg381Gln) variable site protects against pediatric CD. Studies are planned to examine the association between IL-23R and phenotypic expression in IBD patients. It is hoped that the identification of other susceptibility variants and an understanding of the contribution of the IL-23R pathway to the susceptibility of chronic intestinal inflammation will lead to improved therapeutic interventions and outcomes for patients with IBD.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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    ABI: Assays on demand SNP genotyping products. Publication 127PB04–01, 2002. Available from www.appliedbiosystems.com
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    Spielman RS, McGinnis RE, Ewens WJ. Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet. 1993; 52: 506516
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    Schmidt C, Giese T, Ludwig B, et al. Expression of interleukin-12-related cytokine transcripts in inflammatory bowel disease: elevated interleukin-23p19 and interleukin-27p28 in Crohn's disease but not in ulcerative colitis. Inflamm Bowel Dis. 2005; 11: 1623.
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    Targan SR, Hanauer SB, van Deventer SJ, et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group. N Engl J Med. 1997; 337: 10291035.