Genetics and Therapeutics in Pediatric Ulcerative Colitis: the Past, Present and Future

HLA genes

Because of their roles in antigen presentation and T-cell priming, HLA gene variants have long been considered prime candidates for IBD susceptibility^29,30. Indeed, pre-GWAS era linkage disequilibrium studies first described genetic association of IBD susceptibility to major histocompatibility complex (MHC) regions. Hampered by difficulties inherent to the linkage disequilibrium architecture of the area, susceptibility loci identification was slow³¹. However, all GWAS to date have continued to report a strong association between HLA loci and UC. A recent study performed high-density mapping of the MHC region in over 32,000 patients with IBD³². This study found significant enrichment in MHC class II alleles: HLA DRB1, HLA DQA1, and HLA-DRB1*01:03 in particular were strongly associated with UC. The HLA-DRB1*01:03 allele is of particular interest as it also showed a strong association with CD colitis and other studies have linked it to pancolitic phenotype in UC³³. Goyette also found decreased class II HLA heterozygosity, suggesting that broader colonic antigen recognition is important in protection against UC development.

These associations are especially intriguing for pediatric IBD, since young patients, in particular those less than 8 years of age, are more prone to developing extensive colonic disease³⁴. Two pediatric IBD GWAS studies have replicated findings of HLA polymorphisms with UC susceptibility^17,18.

Barrier function

Immune tolerance to food and the normal intestinal microbiota is crucial for the control of intestinal homeostasis. A critical element in maintaining intestinal immune homeostasis is epithelial integrity. In addition, the observation that MUC2^-/- deficient mice develop spontaneous colitis³⁵ suggests that the intestinal mucus layer contributes to barrier function and colonic immune homeostasis. Epithelial barrier function in UC is impaired³⁶ and active UC is characterized by mucin depletion.

Not surprisingly, polymorphisms associated with UC have been found in genes controlling key aspects of barrier function. These include polymorphisms in ECM1, believed to control epithelial basement membrane integrity^37,38. SNPs in the CDH1 locus, encoding the adherens junction protein E-cadherin required for tight junction formation, HNF4a, responsible for epithelial differentiation, and LAMB1, encoding the β1 subunit of basement membrane laminin, have also been identified¹⁴ and replicated³⁹. Finally, polymorphisms in GNA12, associated with tight junction assembly via interactions with Zo-1 and Src, have recently been described⁴⁰.

Unfolded protein response

The unfolded protein response (UPR) is a stress response of the endoplasmic reticulum (ER) aimed at maintaining cellular homeostasis, especially in highly secretory cells such as Paneth cells and goblet cells. ER stress has been linked to environmental influences and loss or dysregulation of the UPR leads to activation of apoptotic pathways. The intestinal surface is the largest epithelium in contact with the environment, so it is not surprising that genes controlling the UPR pathway have been associated with IBD pathogenesis. Bertolotti et al. reported that mice lacking IRE1β expression, a component of the UPR, develop exacerbated dextran sulfate sodium (DSS)-induced colitis⁴¹. Human UPR-associated polymorphisms have also been described. Kaser et al. reported associations of XBP1 variants, a transcription factor involved in the UPR, with development of UC⁴². Similarly, susceptibility-associated variants in PTPN2, a protein tyrosine phosphatase linked to UPR and autophagosome formation^43,44, have also been described^40,45.

Interleukin-23

The interleukin (IL)-23 pathway plays a key role in expansion and differentiation of T helper (T_h) and innate immune cells. Abnormalities in the IL-23 signaling pathway have been linked to development of UC in several cohorts²³. Other components of the pathway such as JAK2 and TYK2 have also been linked to disease pathogenesis³⁷. The importance of this pathway in regulating pro-inflammatory effects seen in IBD is highlighted by the clinical improvement seen in patients with CD when treated with ustekinumab, a monoclonal antibody directed against the p40 subunit shared by IL-12 and IL-23⁴⁶. The ongoing phase 3 international multicenter study UNIFI aims to evaluate this monoclonal antibody in maintenance and induction of patients with moderate to severe UC⁴⁷.

Other T_h17-centric polymorphisms have been associated with UC, including those in NFKBIZ, a regulator of T_h17 cell development, and AHR, a transcription factor involved in T_h17 cytokine expansion²⁷. Additional important polymorphisms in blocks containing immune regulatory genes have been described. For a detailed review please refer to the article by Khor et al.⁴⁸.

Next-generation sequencing

Although GWAS have dramatically influenced our understanding of complex diseases, one limitation of such studies is that they neglect contribution from rare variants, those with minor allele frequencies less than 5%. High effect rare risk variants missed by GWAS may be contained within known IBD susceptibility loci. Two studies have sought to identify causal functionally relevant genetic variants contained within IBD susceptibility loci by employing deep next-generation sequencing. Results from these studies pointed to three new mutations associated with UC. While variants in CARD9 and IL23R were protective, a variant in RNF186, a RING ubiquitin ligase, was found to confer risk^55,56. In addition to reporting novel associations with IBD risk, these studies provide new insight into future methodological strategies for gene discovery in IBD.

Figure 1. Timeline of genetic discoveries and recent drug developments in IBD.

Adapted from Expert Rev Gastroenterol Hepatol. 2009 513–34.

Diagnosis

Currently, pediatric UC diagnosis is based on aggregate clinical, endoscopic, imaging, and histopathological findings as suggested by North American and European working groups^57,58. The rapid pace of discovery of UC-associated genetic variants suggests that genetic diagnosis is quickly unfolding. To date, UC-specific diagnostic approaches utilizing genetic expression analysis from colonic biopsies^59,60, composite genetic risk scores⁶¹, and combined serological and genetic markers⁶² have shown promise, yet application of these tools has yet to invade day-to-day clinical practice. The VEOIBD age group is an exception. As monogenic forms of IBD cluster in this age group, exome and targeted gene chips are increasingly being suggested and used in the genetic diagnosis of VEOIBD^63,64.

Genotype-phenotype correlations

The best-described genotype-phenotype associations in IBD are NOD2 variants and ileal CD^65–67. Recent evidence has also pointed to UC-associated polymorphisms and phenotype associations. MHC-associated variants are of notable interest. As mentioned earlier, polymorphisms in the MHC region have been associated with extensive colonic involvement and colectomy^33,68. Recently, the international IBD Genetics Consortium (IIBDGC) reported the results of the largest genotype-phenotype study to date, incorporating data from more than 34,000 IBD patients. Results pointed to three loci associated with disease subphenotype: NOD2, MHC, and 3p21. MHC loci were again associated with extensive colonic involvement⁶⁹.

Prognosis

The ability to predict disease course at diagnosis as well as early identification of patients who will develop severe and medically refractory UC may result in changes in treatment algorithms.

Haritunians et al. developed a risk score utilizing a GWAS approach in a well-characterized cohort of patients with medically refractory UC. A modeled risk score using 46 SNPs explained 48% of the variance for colectomy risk in the population studied, with a sensitivity of 79% and a specificity of 86%. Further, this study confirmed associations of severe UC with SNPs within the MHC region, as well as SNPs in TNFSF15, IL-10, IL-12B, 12q15, ZPF90, KIF1A, and GSDML/ORMDL3⁷⁰. A study using the same strategy incorporated data from 703 UC patients from 40 sites within the IIBDGC. This study replicated an association between rs2403456 (11p15.3) with medically refractory, severe UC⁷¹.

In order to identify genetic predictors of response to biologic therapy, Arijs et al. studied colonic mucosal gene expression signatures from two cohorts of anti-tumor necrosis factor (anti-TNF)-naïve UC patients receiving biologic induction with infliximab. Microarray-based colonic biopsy profiling was used to develop a prediction response probe set. Five genes − osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, IL-13 receptor alpha-2, and IL-11 − were found to be differentially expressed in responders versus non-responders. The genes identified are involved in adaptive immune response signaling and TNF pathways⁷². Of note, osteoprotegerin has been suggested as a stool marker for diagnosis and assessment of treatment response in pediatric UC^73,74.

Therapeutics

The intent of our discussion previously on genetics and UC is seeded in the hope that personalization of therapies based on genetics will change disease outcomes and potentially lead us to a cure of UC in the future. Though corticosteroids and mesalamine remain the mainstays of current therapy, the following sections will outline other options within our armamentarium with a special focus on the resurgence of drugs like methotrexate, the debate around antibiotics, and a brief look at potential newcomers.

Methotrexate, an immunosuppressant developed in 1948, has been widely used initially in leukemia and now in oncologic, rheumatologic, and autoimmune diseases. Methotrexate inhibits lymphocyte proliferation as a folate antagonist⁷⁵. Methotrexate has recently garnered attention as an alternative oral or subcutaneous agent in the treatment of UC, particularly in patients who have failed or become intolerant to 6-mercaptopurine (6-MP) or infliximab therapy. Two Cochrane reviews from 2014 and 2015, assessing induction and remission success, respectively, showed no significant difference between methotrexate and placebo in patients with UC^76,77. Currently there are two large prospective trials evaluating the potential of methotrexate in UC. The Comparison of Methotrexate versus Placebo in Corticosteroid-dependent UC (METEOR) trial has completed enrollment and is closed. The Methotrexate Response In the Treatment of UC (MERIT-UC) trial is currently recruiting as multicenter prospective trials comparing methotrexate to placebo in patients who are steroid dependent or who failed 6-MP or infliximab therapy.

Antibiotics present an interesting clinical discussion in the setting of UC. Dysbiosis is present in patients with IBD, even at diagnosis. Previous work has shown that in patients with acute severe colitis, those with a more diverse microbiome at diagnosis respond better to steroid therapy versus those with a less diverse microbiome needing salvage medical therapy⁷⁸. A recent paper by Turner et al. described the use of an oral broad-spectrum antibiotic cocktail (metronidazole, amoxicillin, doxycycline, and vancomycin [MADoV]) in pediatric UC patients who had failed other traditional therapies (steroids, oral immunosuppressants including azathioprine and tacrolimus, and anti-TNF agents, both infliximab and adalimumab). A total of 7 out of 15 patients achieved complete short-term remission with the antibiotic cocktail. A larger pediatric randomized controlled trial to assess this intervention is underway⁷⁹. Additionally, in the discussion of dysbiosis, fecal microbial transplantation (FMT) has been proposed in the treatment of UC. There are several active recruiting studies ongoing; however, the only published data on the subject by Kunde et al. suggested a promising response in a pilot study of 10 children over 1 month⁸⁰.

The latest treatment for UC is a humanized monoclonal antibody named vedolizumab. An α4β7 integrin blocker, the medication targets inflammation by blocking the recruitment of lymphocytes specifically to the gut without interfering with trafficking to the central nervous system. This gut-specific feature makes it unique to the current group of therapeutics by isolating the affected area of involvement and subsequently decreasing the systemic effects⁸¹.

Novel therapeutic targets continue to be developed and there are multiple drugs in different phases of development (Figure 2).

Figure 2. New therapeutic agents for ulcerative colitis.

New therapeutic agents for ulcerative colitis with drug targets in parenthesis^47,83–90.

Treatment algorithms continue to evolve in UC and are increasingly utilizing predictive models to determine the best potential therapies for an individual patient. An integral part of the predictive model will be our continued advancement of the understanding of genetic data. Additionally, clinical variables have been recognized to impact the determination of initial therapy and as a predictive tool for choosing a more aggressive therapy in patients at risk for a poor outcome. Schechter et al. described the importance of the PUCAI score at baseline and moreover at the 3-month mark. The 3-month PUCAI was most predictive of sustained response and steroid-free remission at 1 year. Several other variables, including elevated C-reactive protein (CRP), hypoalbuminemia, and anemia have also been described in other studies, though not found to be significant in the Schechter model⁸².

Cure

Although a cure for polygenic complex diseases is still a hope, lessons from VEOIBD provide proof of principle that, indeed, certain well-characterized monogenic forms of IBD may be amenable to cure. Glocker et al. described a series of patients with infantile fistulizing enterocolitis resulting from mutations in IL-10RA and IL-10RB. Allogeneic stem cell transplantation in one of the patients resulted in complete resolution of gastrointestinal symptoms⁵⁰. Similarly, Worthey et al. described a patient with onset of fistulizing colonic disease at 15 months of age. Exome sequencing of this patient pointed to hemizygous missense mutation affecting XIAP; cord blood hematopoietic stem cell transplantation led to disease remission⁵².