0. PROPOSAL SUMMARY
Inflammatory Bowel disease (IBD) is a chronic inflammation of the gastrointestinal tract of which there are two major types: ulcerative colitis (UC) and Crohn’s disease (CD). Epidemiological studies have confirmed an increased risk of IBD in first-degree relatives, with a significantly higher risk in monozygotic twins compared to dizygotic twins. Genome-wide screening has confirmed multiple genetic loci which confer an increased risk for developing IBD. However, the role for genetic data in predicting disease susceptibility, activity, development and progression of IBD is neither very informative nor sufficient. The development and progression of IBD is multifactorial and the prevailing hypothesis is that IBD development results from an aberrant reciprocal interplay between the intestinal microbiota, the gut luminal contents and the intestinal mucosal immune systems in genetically predisposed individuals. The underlying mechanism of inflammation is driven by immunological responses, including macrophage activation, leucocyte trafficking and production of inflammatory cytokines. Moreover, there is a considerable clinical and experimental evidence that shows that the oral and gut microbiota composition in patients with IBD are altered, but whether this alteration is a cause or a result of the inflammation in IBD is not well described. IBD is usually diagnosed by a combination of clinical, endoscopic, radiological and histological findings. Mucosal biopsies obtained by invasive methods often show a characteristic appearance in keeping with UC or CD. Despite encouraging advances in understanding the underlying pathophysiology and genetic susceptibility, IBD remains a substantial burden on the healthcare system, and its diagnosis and treatment remain unsatisfactory.
In the past few years, studies have described the role that the host immune system plays in shaping the microbial metagenome, and in turn, how the microbiome composition regulates gene expression in the immune system. This bidirectional relationship is required for homeostatic health, whereas alterations in this tightly controlled regulatory network that links the host immunogenome to the microbial metagenome delineate susceptibility to common human diseases including IBD. Combining metagenomics and immunogenomics will make it possible to characterise individual susceptibility and precisely delineate the severity and extent of IBD.
The phenotypic spectrum of children with IBD is highly variable, and studies describing IBD in children living in Qatar are still missing. While the incidence of IBD in kids appear to be on the rise, it becomes vital to establish a national pediatric IBD registry in order to provide a comprehensive care of pediatric IBD patients in Qatar. It is also necessary to employ the latest technologies to dissect various pathophysiological profiles associated with IBD in the least invasive way and to offer patients with IBD the best treatment in a personalized fashion.
In this project, we propose to investigate IBD in children living in Qatar and attending the SIDRA IBD clinic. We aim to create a database where the physicians will register all the pediatric IBD cases attending the clinics in SIDRA and input all the phenotypic, clinical, laboratory data for those patients using Oracle Translational Research Centre (TRC) database solution available at Sidra. We will collect blood, stool and saliva samples from all patients at various timelines as described in the study plan. We will assess the salivary and gut microbiota composition using Next Generation Sequencing technology as well as the immune profile using blood transcriptomics. Integration of the metagenomics and immunogenomics data that will be generated will be used to identify microbial and immunological profile associated with different pathophysiological profiles of pediatric IBD in relation to specific clinical types of disease. These profiles will be compared in different patients with CD or UC before or after treatment and during the various phases of the disease. By combining these profiles, we aim to recognize disease “fingerprints”, which will help characterize and intercept various types of IBD. This can potentially predict particular disease course and likelihood for response to treatment and even identify during this process novel targets for therapies or tailored therapeutic strategies to these “fingerprints”. In the long term, we anticipate that the knowledge about such subclinical markers can be harnessed to better identify and diagnose children with IBD in order to develop personalized strategies to prevent subsequent clinical disease and/or to improve clinical management specifically tailored to these children. We speculate that understanding the interplay between the immunogenome and the metagenome of the gut and oral microbiome, we will not only contribute to clinical decision making but will also help to find individualised therapies for the affected patients but improve the understanding for relevant immune pathways and microbiome-host interactions and that this will be relevant for general understanding of dysregulated pathways in IBD.
1. OBJECTIVES AND SIGNIFICANCE
Pathophysiology of Inflammatory Bowel disease (IBD): IBD is a relapsing chronic inflammation of the gastrointestinal tract that occurs as a result of an altered immune response to commensal and/or pathogenic microbes in individuals susceptible to the disease . IBD is characterized by intermittent disease flares and periods of remission (Figure.1), and in the absence of adequate treatment, chronic inflammation may result in an irreversible damage to the intestins . IBD includes a spectrum of diseases but the two primary types are ulcerative colitis (UC) and Crohn’s disease (CD) . UC is a debilitating condition of chronic inflammation of the mucosal surface of the colon that leads to a significant morbidity in both adults and children . It is characterized by a continuous inflammation of the colon associated with a bloody mucous diarrhea . CD is a chronic inflammatory disease that affects different regions of the digestive tract, although it most commonly affects the colon and terminal ileum . CD presents with discontinuous, transmural lesions affecting different regions of both the small and large intestines and can be associated with intestinal granulomas, fistulas, and structures . Accurate differentiation between UC and CD is important to ensure early and appropriate therapeutic intervention. However, only few studies aimed at discovering a non-invasive biomarker that enabled to distinguish UC from CD which aids the treating physicians to proceed with the treatment and disease management properly. Burczynski et al. present blood transcriptional profiles in UC vs. CD patients and identified 12 genes which were representative of UC and CD, respectivley. Similarly, Huebenthal et al. reported a miRNA profile of CD and UC whole blood samples which was able to identify a signature of 16 distinct miRNAs but lacks validation in a larger cohort.
Figure 1: IBD disease phases
Inflammatory bowel disease is a chronic illness that has periods of flares (active disease) and remission (being well) .
Epidemiological studies and IBD registries mostly from North America and Europe, suggest that IBD affects approximately 1.5 million Americans and 2.2 million Europeans and the incidence seems to be increasing worldwide . IBD affects both sexes at any age, although the peak incidence is between 10-40 years and 10% of patients are under 18 years of age . Less than 2% of all IBD patients develop intestinal inflammation before the age of 6 and those are termed as very early-onset IBD (VEO-IBD) . In pediatric IBD, the disease phenotype and subsequent disease course are influenced by the age at first diagnosis . In a large North American cohort of pediatric IBD, kids who had VEO-IBD were more likely to have a mild disease at diagnosis but a more aggressive phenotype over time as compared to kids who had an onset between 6 to 10 years of age . In the last few decades an increase in incidence occurred in industrialized zones which earlier had low incidence of IBD such as Iran, Lebanon and North Africa . There are few published reports describing IBD in the Arabian Gulf region, and the majority of these studies described the disease mainly in adults with few pediatric studies . Kalaoui et al., reported the colonoscopic findings in 159 children in Kuwait over a period of nine years and 21% of those had IBD . Another study which reported IBD epidemiology among children in the region was by El Mouzan et al., who reported 50 IBD children seen in Riyadh, Saudi Arabia during 1993-2002 . Similar registries and/or studies investigating IBD in kids living in Qatar are still missing. Additionally, there is a critical need to develop less invasive tools that can be used for an early diagnosis of IBD in kids.
Determinants for disease susceptibility: IBD is a multifactorial disease and despite numerous studies, there is still a critical knowledge Gap in the IBD field where the actual causes of IBD are still not very well known. Genetic studies concluded that more than 200 susceptibility loci are associated with IBD . Therefore, genetic testing is useful in VEO-IBD to identify rare genetic defects, allowing for genetic counseling for family members as well as treatment options such as stem cell transplantation in IL-10R deficiency . However, the role for genetic data in predicting disease susceptibility, activity, development and progression of IBD is not very informative as patients with no known genetic predisposition can still develop the disease. The development and progression of IBD may be affected by the complex interactions between genetic , environmental including breast feeding, diet, smoking, drugs , and microbial factors , causing continuous inflammation supported by altered mucosal barrier and defects in the immune system . The prevailing hypothesis is that IBD development results from an aberrant interplay between intestinal microbiota and gut luminal contents with the intestinal mucosal immune systems in genetically predisposed hosts (Figure. 2) . Whether other host-commensal interactions play an additional role in contributing to IBD progression, complications and/or response to treatment is not well described and whether similar mechanisms in non-genetically predisposed host occurs is also not well known.
Figure 2: Prevailing Hypothesis of Factors contributing to IBD: Those include genetic predisposition, aberrant immune response and environmental factors as well as the main role of the microbiome.
IBD and the microbiome: Recent breakthroughs in high-throughput techniques have revolutionized our understanding of the complex composition and the function of the intestinal bacterial ecosystem- thereafter called the microbiota; and have substantially advanced our knowledge of the crucial role that the microbiome (collection of microbes and their genes) plays in maintaining host physiology and homeostasis . The microbiome is highly variable between individuals and these variations are determined in part by host genetics, diet, antibiotic exposure, mode of delivery and maternal colonization at birth . In the human gut, the most abundant bacteria are Firmicutes and Bacteroidetes; less-abundant species vary greatly among individuals . Most humans share a core set of resident bacteria and, to a greater extent, a core set of microbiome-encoded genes and metabolic functions . A growing evidence indicates that disequilibrium of the gut microbiome composition, called dysbiosis, contributes to the development of IBD. Multiple studies have shown that the composition of the intestinal microbiota from IBD patients is characterized by a decreased prevalence of protective microbiota (such as Bifidobacterium species) and an expansion of detrimental bacteria (like Enterobacteriaceae and Fusobacteria species) . Using the current knowledge of the gut microbiome composition in IBD patients, it became partly possible to differentiate, to some extent, between the two major IBD conditions and the progressive stages of IBD; this is greatly important to ensure an early and appropriate therapeutic intervention. Gevers et al. found that some ileal microbiome signatures were predictive of CD and they observed this signature even in the absence of overt inflammation . Ott et al demonstrated in adult patients that the gut microbiota differed between active and quiescent stages of inflammatory bowel disease . In another study, the authors showed the possible use of the gut microbial composition to test whether a treatment is effective or not. They showed that the fecal microbial diversity in patients who responded to the anti-TNF therapy resembled those of the controls as compared to patients who did not respond to treatment . Furthermore, the gut microbial signature was used to predict the disease course in post-operative recurrence of CD, where a decreased population of Faecalibacterium prausnitzii in the resected ileum correlated with an increased risk of recurrence .
Microbial dysbiosis in IBD patients is not only restricted to the gut. Oral manifestations such as aphthous stomatitis, oral ulcer, dry mouth, and pyostomatitis vegetans are observed in IBD patients , suggesting the association of oral microbiota with such manifestations. However only one study described dysbiosis of salivary microbiota in IBD patients where the authors suggested that the oral microbial dysbiosis is possibly linked to the gut dysbiosis in those patients, however they did not assess for the gut microbiota composition in those patients to confirm their hypothesis . More studies assessing the link between oral and gut microbiome composition in IBD patients are needed especially in children where early accurate diagnosis can predict the outcome of the disease.
IBD, the microbiome and the altered immune response: Advances in genomics have also contributed to a revolution in the study of another factor that shapes the microbiome: the host’s immune system . Basic and clinical immunology have been redefined by developments in next-generation sequencing and genome editing techniques . Our molecular understanding in this field now ranges from transcriptome profiles of innate and adaptive immune cells to epigenetic regulation of cytokine expression and the effect of genetic mutations on immune-mediated diseases.
The host’s microbiome plays a pivotal role in the development of IBD but despite the influence of gut microbial colonization on gene regulatory processes the functional link with transcriptional changes remains elusive. The regulation of gene expression mediated by microbiota is most prominent in the immune system which was shown, e.g. in a study where the strongest associations between the microbiome and transcriptome in the pelvic pouch of patients with IBD were found in pathways of the innate and adaptive immune system . Various components of the host’s immune system are implicated in the pathogenesis of IBD and include intestinal epithelial cells, innate lymphoid cells, cells of the innate (macrophages/monocytes, neutrophils, and dendritic cells) and adaptive (T- and B-lymphocytes) immune system, and subsequently secreted cytokines and chemokines. The aberrant chronic activation of an innate immune response may be mediated by enhanced toll-like receptor activity in the intestinal mucosa which is generally involved in maintaining the integrity of the mucosal as well as the commensal homeostasis. Increased epithelial TLR2 and TLR4 expression is associated with inflammatory bowel disease leading to an excessive immune response and chronic inflammation in IBD patients . Similarly, TLR9 expression is positively correlated with disease severity , a finding which has led to the development of a therapeutic agent which acts through TLR9 signaling. DIMS0150 is a synthetic DNA-based immunomodulatory sequence that showed efficacy in 71% steroid-resistant ulcerative colitis patients which went into remission after 12 weeks of administration of this drug .
Although the complete etiology of IBD is unclear, both CD and UC are believed to be T-cell-driven processes, with inflammation triggered by inappropriate cytokine production released from subsets of TLR-expressing CD4+ T-helper (Th) cells. While CD is associated with Th1 and Th17 responses , UC is typically associated with Th2 cells . It has been suggested that IBD results from defects in the T-cell-mediated regulatory processes that would normally prevent and/or terminate inflammatory responses . Impaired Treg function is associated with a breakdown in intestinal tolerance and thus leading to aberrant immune function which may also contribute to the pathogenesis of IBD .
State of the art technologies in IBD: Since the disease phenotype and subsequent disease course are influenced by the age at first diagnosis, it is vital to make an accurate diagnosis of IBD at early age and this can be facilitated by combining metagenomics and immunogenomics in kids with IBD. Moreover, away from predicting susceptibility and behavior in IBD, there is an unmet need for sensitive biomarkers to monitor molecular perturbations that precede disease activity and guide response to treatment. Monitoring molecular perturbation may allow the identification of molecular changes that reflect an active disease process but precede onset of clinical symptoms, this can be best performed by using omics approaches and integrating clinical data, genetic predisposition, environmental exposure and diet with microbiome signature, inflammatory profiles and transcriptomics. In this proposal, we will develop a registry for IBD children living in Qatar. We will use metagenomics to assess the gut and oral microbiome composition combined with immunogenomics to assess the immune-profiles in kids with IBD. We will combine this data with the clinical, laboratory and environmental data collected by the SIDRA physician. By combining these profiles, we aim to recognize IBD “fingerprints”, which will help the differential diagnosis between patients and types of IBD disease. We can potentially predict disease course and likelihood for response to treatment and even identify during this process novel targets for therapies or tailored therapeutic strategies to these “fingerprints”. By characterizing this unique cohort of kids with IBD, it is a great opportunity for directly testing experimental therapies. By carefully delineating the activity and impact of disease, it will be possible to seek therapeutic benefit in specific rather than generic terms. Ultimately it will be easier to assess treatment response in specific subgroups of patients with severe disease. This has the potential to change the pattern of the disease by targeted therapeutic intervention.
2.1. SCIENTIFIC OR TECHNICAL OBJECTIVES
Registries and studies on IBD in children living in Qatar are still lacking. Since the disease phenotype and subsequent disease course are influenced by the age at which the first diagnosis was made, it is therefore vital to make an accurate diagnosis of IBD at an early age. The genetic predisposition of the disease explains only partly the etiology of the disease, indicating that other factors including dysbiosis of the microbiome and dysregulation of the immune system may play a pivotal role in the pathology of IBD. The overall goal of this project is to define IBD “fingerprints” specific to kids living in Qatar and to find microbial signatures associated with certain disease conditions (UC or CD), disease course or response to treatment. Furthermore, we will assess the immune profile of IBD patients following in vitro exposure of patients’ whole blood to certain immune stimuli to study whether a specific mechanism or signaling network are associated with the disease etiology or progression.
The project has 3 specific objectives:
Objective 1: Build a database for IBD children living in Qatar and implement serial collection of blood, stool and saliva from IBD patients.
A registry for pediatric patients with IBD attending the SIDRA IBD clinic will be established. After recruiting a research coordinator, high frequency sample collection will be implemented in IBD pediatric patients, thanks to the participation of Dr. Elawad, Chief of Gastroenterology/Hepatology/Nutrition and Section Head for the Inflammatory Bowel Disorders in SIDRA and his team members (Dr. McGrogan, Dr. Akobeng, Dr. Al-Mudahka). Blood, stool and saliva samples will be collected from all patients after consent, along with detailed clinical information including a baseline questionnaire, in addition to the Pediatric Crohn’s Disease Acitivity Index (PCDAI) and the Pediatric Ulcerative Colitis Activity Index (PUCAI). Healthy siblings of the IBD patients will be recruited as controls, with parental consent and/ or child assent based on the age.
Objective 2: To implement Molecular profiling of IBD in kids living in Qatar
We will assess the gut and salivary microbial composition in patients with IBD using 16S rRNA gene sequencing. We will also perform high frequency blood transcriptional profiling in order to characterize immunological changes that take place during the course of the disease.
Objective 3: Data analysis and integration to identify IBD “fingerprints”
We will analyze the gut and salivary microbial profiling of patients with IBD as compared to their controls in order to identify specific gut and salivary microbial signatures associated with children with IBD in Qatar. We will also analyze the immune-signaling in patients with IBD versus controls to uncover possible immune mechanism that may explain the chronic inflammation associated with IBD. By exposure of patients’ whole blood to ligands, such as Toll-like receptor agonists among others, we will be able to elucidate signaling defects in UC vs. CD patients vs. controls, and identify defined biomarkers for the disease.
We will then integrate the metagenomics and immunogenomics data with the clinical and phenotypic data of patients with IBD. By comparing patients to their matched control and/or patients from various IBD conditions (UC versus CD) and different time points for the same patient we aim to identify specific microbial and/or immune signatures that maybe used as fingerprints for IBD.
2.2. ADVANCES ON STATE-OF-THE ART
Despite the overlap in presentation, symptoms, and progression of CD and UC, discriminating IBD subtypes is vital for the selection of the most appropriate therapeutic or surgical intervention and patient prognosis. However, the differentiation between UC and CD still lacks a gold standard, and relies on long term follow up, success or failure of existing treatment, and recurrence of the disease. Existing diagnostic methods for the identification and classification of IBD are all invasive yet inexact and rely largely on trial-and-error. IBD is usually diagnosed by a combination of clinical, endoscopic, radiological and histological findings. It is also known that the disease course is influenced by the age at which the first diagnosis was made. Therefore, accurate differential non-invasive diagnosis of IBD at an early age is critically needed for appropriate medical and surgical care, intervention, and prognosis. The development of new technologies that can improve understanding of IBD and aid objective diagnosis may help meet this need. Here, we propose building the first database of pediatric IBD in Qatar. We aim to implement high throughput molecular profiling as part of the clinical decision making . The blood transcriptomic profiling assay combined with microbiome profiling and functional immune assays that will be employed in this study could serve as a basis for a new generation of practical and cost-effective differential diagnosis of IBD. If yielded successful, this study has the potential of developing a cost-effective and non-invasive clinical assay that allows proper segregation of IBD patients in either of the clinical subtypes, CD and UC.
2.3. PRELIMINARY DATA OR STUDIES
Drs Elawad, Akobeng, and Al-Mudahka are currently involved in an ongoing multicenter, international study titled “Colitis Of Early Onset And Rare Diseases Within IBD” which is investigating the genotype/phenotype characterization of early onset IBD and refractory IBD in adults and children.
3. EXPECTED IMPACT OF THE PROJECT
3.1. OUTPUTS AND OUTCOMES
A registry for pediatric patients with IBD attending the SIDRA IBD clinic will be established within the Cerner Millenium electronic medical records system implemented at Sidra. Patients will be offered the opportunity to allow their data contained within the electronic medical records system to be used for the purposes of this research project. Where patients indicate their willingness to participate in the research and sign the consent form, all the phenotypic, clinical, laboratory data of each patient collected at each time they visit the clinic will be stored in another database using the Oracle TRC solution. Coding of patient identifiers will enable data to be viewed in anonymized form by research personnel, and to be analyzed as detailed in this protocol. Once immunogenomics and metagenomics data are collected and processed, these will be loaded into the Oracle TRC database for each patient. This will provide a rich resource for further analysis within the scope of this proposal as well as future projects. Clinician will be able to decode the identity of patients requiring specific treatment modalities as suggested by this research project and make informed decisions for individual patient management.
Blood, saliva and stool samples from the patients will be collected and stored for testing. Data from immunogenomics and metagenomics analysis will be inserted in the database and correlation studies will be performed. A specific, personalized signature will be created and those findings will be discussed with the medical team involved in the care of the IBD patient. We speculate that data from this project will not only help to find individualized therapies for the affected patients but also improve the understanding for relevant immune pathways and microbiome host interactions and that this will be relevant for general understanding of dysregulated pathways in IBD. By carefully delineating the activity and impact of disease, it will be possible to seek therapeutic benefit in specific rather than generic terms. Ultimately, it will be easier to assess treatment response in specific subgroups of patients with severe disease. This has the potential to change the pattern of the disease by targeted therapeutic intervention.
Findings will be presented in multiple publications and results will be disseminated to the international communities through presentations and publications in high-impact scientific journals. Moreover, with the support of the Research and Technology Transfer office at SIDRA Medical and Research Center, we will engage with private stakeholders such as pharmaceutical companies to explore the potential of commercializing the methods we have established for routine clinical use in the IBD clinics, not only in Qatar, but also in other countries. Any potential diagnostic tool developed will be patented.
3.2. ALIGNMENT TO NPRP10 THEMATIC AREAS
The proposed project aims to study IBD, a genetic disease in part, therefore responding to the priority themes that were identified through a consultation between Qatar Foundation Research and Development and the main stakeholders in Qatar. The project falls under the Biomedical and Health Pillar where studying Genetic disease is indicated as a priority theme. The project will employ genomics tools to assess IBD in children living in Qatar, in order to develop a differential diagnosis using immune and microbial signature associated with the disease. Moreover, the project also aligns well with the technology hotspots not identified in the priority themes such as developing tools for personalized medicine in the diagnosis and treatment of IBD. This differential diagnosis will be implemented as part of the clinical decision making. The project supports tools for clinicians in making personalized patient related decisions in terms of diagnosis or treatment. We will employ an integrative data analysis to merge immunogenomics and metagenomics data to clinical and phenotypic data collected from the same patient. This tool is urgently needed in clinical decision making.
3.3. SOCIAL, HEALTH, ECONOMIC, AND ENVIRONMENTAL IMPACT
Despite advances in therapy, hospitalization rates for IBD, particularly CD, have shown significant increase, incurring a substantial rise in inflation-adjusted economic burden. With the high drug costs and rates of surgery (up to 75% of CD and 25-33% of UC patients), IBD remains one of the costliest conditions. We anticipate that our proposed project will have a significant social, health and economic impact. In this proposal, we aim at targeting IBD in children of Qatari nationality and residents. The main goal of the present proposal is to address key questions of the chronic inflammation of the gut, its discrimination in the two subtypes namely CD and UC and to translate that to clinical management, including diagnosis, prevention and therapeutics. The present project will provide a decisive boost towards a research infrastructure that could establish Qatar as a leading force in the immunogenomics and microbiome fields not only in the Gulf region but also all over the world. This project has the potential to bring significant scientific, technological and educational contributions to Qatar. Specifically, it holds the promise of providing a unified framework to (i) establish a comprehensive clinical database of pediatric IBD in Qatar; (ii) develop new techniques to diagnose UC versus CD at a non-invasive, yet molecular level and to produce therapies based on the disruption or correction of the aberrant function of gene products; (iii) help train and educate the future generations of students and specialists that are necessary for the deployment of the current biotechnological revolution in Qatar; (iv) strengthen the Qatar economy through its alignment to the current advances in biotechnology; and (v) increase the international visibility of Qatar through the organization of seminars, workshops and conferences, and development of high-impact technologies. The proposed study is innovative and multidisciplinary through the integration of cellular assays, genomics profiling with high-throughput analyses, bioinformatics and computational approaches to study the incidence, molecular mechanisms and genomic profiling of IBD. Upon finishing the project, we expect it will significantly impact the research, education, and public awareness of IBD in Qatar and will help implement our proposed methodology for the differential diagnosis of IBD in the clinical setting.
3.4. COMMUNICATION AND EXPLOITATION OF RESULTS
We plan to publish the results of this research work in top-notch journals and conferences in the areas of microbiome, genomics and immunology in order to ensure high quality and very comprehensive reviews for our work. The feedback of reviewers as well as comparisons with the existing state-of-the-art results will be used to assess the merits and improve the quality of our research work. Seminars and workshops will be organized periodically, and the feedback of other researchers and collaborators will be also sought to ensure the progress of our work. Weekly seminars and meetings will be organized to coordinate and discuss the progress of the proposed research work. We believe that a very effective plan to evaluate the success of this research work is to monitor the number of publications and new results that we will report each year. We plan to maintain a very high publication rate (i.e., to report at least two journal papers and two conference papers in the most respectable international journals, conferences and workshops) and establish strong collaborations with the existing research institutions in Qatar. This project holds the promise of providing a comprehensive analytical framework to improve the performance of the existing state-of-the-art genomic and immunologic approaches and to develop more efficient tools for prevention and treatment of IBD in children. By combining cellular/phenotypic with high-throughput genomic/immunologic data, the proposed study has the potential of uncovering not only the genetic and molecular profiles associated with this health threat in children, but also new aspects of the logic that governs the transcriptional control and interactions between genes, epigenetic modifications and the role of the immune system with regards to the onset and progression of IBD. By addressing questions about the molecular mechanisms underlying the discrimination between ulcerative colitis and Crohn’s disease, this project has the potential to identify genes, epigenetic patterns and immunologic pathways involved in the development of either (or both), which could provide a new approach to the diagnosis, treatment and/or prevention of IBD and its complications.
4. METHODOLOGY AND PROJECT STRUCTURE
In order to address our 3 objectives, our work plan breaks down in the following work packages (WP) and tasks:
Objective 1/ WP 1: Build a database for IBD children living in Qatar and implement serial collection of blood, stool and saliva from IBD patients.
Task 1.1. Establish a registry for IBD kids living in Qatar and create a database containing their phenotypic, clinical and laboratory data collected through various questionnaires.
Task 1.2. Recruit a research coordinator.
Task 1.3. Implement a high frequency sample collection from IBD pediatric patients attending the SIDRA IBD clinic. Establish a biorepository for blood, stool and saliva samples along with detailed clinical information. Siblings will function as healthy controls.
Objective 2/ WP 2: To implement Molecular profiling of IBD in kids living in Qatar.
Task 2.1. Assess the gut microbial composition in patients with IBD and controls using 16S rRNA gene sequencing
Task 2.2. Assess the salivary microbial composition in patients with IBD and controls using 16S rRNA gene sequencing
Task 2.3. Perform high frequency blood transcriptional profiling in order to characterize immunological changes that take place during the course of the disease.
Objective 3/ WP 3: Data analysis and integration to identify IBD “fingerprints”
Task 3.1. Analyze the gut and salivary microbial profiling of patients with IBD as compared to their controls
Task 3.2. Analyze the immune-signaling in patients with IBD versus controls
Task 3.3. Integrate the metagenomics and immunogenomics data with the clinical and phenotypic data of patients with IBD
4.2. RESEARCH PLAN REQUIREMENTS REGARDING THE SECTIONS ON THE PROTECTION OF HUMAN SUBJECTS SECTION; AND/OR ON THE VERTEBRATE ANIMAL CARE AND USE.
Human Subjects Research
The research proposed here falls under the category of non-exempt human subject research. Approval for human research will been obtained from the SIDRA institutional review board (IRB).
Risks to Human Subjects
a. Human Subjects Involvement, Characteristics, and Design
The research proposed here involves children which have been diagnosed with either UC or CD of different health status, who are seen regularly at Sidra Medical and Research Center IBD clinic. In addition, we will be assaying samples from healthy siblings that serve as age-matched controls and for the microbiome related analysis to control for their diet and environmental factors. All children with IBD and living in Qatar will be recruited to the study and no one will be excluded based on ethnicity or gender. We estimate that approximately 250 to 300 study subjects will be involved in this study, including patients with IBD and control subjects. The rationale of including children in our study, which represent a vulnerable population, is that we aim to specifically conduct research for the benefit of kids with IBD attending the SIDRA IBD clinic to provide the best care and the most updated start of art technologies to offer a differential diagnosis of the various IBD conditions. The earliest the IBD is correctly diagnosis, the best the disease outcome will be. A registry for all the IBD kids living in Qatar will be created, to facilitate the follow-up for patients and all data from those patients will be inserted in a database that will merge clinical, phenotypic and molecular profile of patients. This will facilitate personalized treatment for IBD patients in Qatar and may help predict the disease outcomes and progression. For the safeguard of data and identifiable information. A data security plan will be submitted to the SIDRA IRB as part of our IRB protocol. For the clinical and biospecimen collection timeline, patients will be asked to visit the SIDRA IBD clinic every 3 month or regular check-up and at the onset of any unwell episode. Sample and data collection will continue throughout the project timeline.
b. Sources of Materials
Human biological specimens that will be collected for this study include human whole blood for transcriptional and immune profiling, as well as saliva and stool samples for 16S rRNA gene sequencing to study the microbial composition in the oral cavity and the gut respectively (WP2). In addition, the research coordinator will collect phenotypic and environmental related information during the baseline visit (WP1). The clinicians will collect clinical information in order to assess the severity of the disease including PCDAI and PUCAI scores (WP1) which will be analyzed in this study (WP3). This information will include individual identifiable private information. Only the physician assessing the patient, the Lead PI and the research coordinator will have access to the individual identifiable private information. All other research staff will only have access to de-identified private information and research data coded by a study subject number. The rational for collecting identifiable private information is that we will use this information to follow-up patients and call them back in the clinic in case flare detected by the molecular profiling can be prevented or intervened by personalized treatment. Moreover, we predict that the high-throughput data that will be collected from each patient may redefine treatment options available for those patients.
c. Potential Risks
Potential risks in this study are no more than what is considered minimal risk. The study subjects may experience some pain (like a pinch) to their skin from the peripheral venipuncture as well as some feeling of discomfort from being restrained during the blood draw and from the rubber tourniquet that will be placed on the upper arm. The IBD patients will have blood withdrawn as part of their routine exam, and therefore they are used to this procedure. The discomfort explained above may be more associated with the heathy siblings but we will explain in the consent and assent (documents provided to the SIDRA IRB) how the procedure will be performed. The amount of whole blood which will be collected for this study will be kept at a minimal amount and will be in line with international guidelines addressing blood draws for research purposes in children (http://clinicalcenter.nih.gov/recruit/volunteers.html). All other biological samples such as stool and saliva are being collected non-invasively and do not bear any potential risks to the participants.
Additional, potential nonphysical risks, including breach of confidentially, are minimal.
Adequacy of Protection against Risks
a. Recruitment and Informed Consent
We intend to recruit from patients currently under the care of the Gastroenterology Unit at SIDRA, and new patients presenting to the service during the time course of the study. The initial contact with patients will be performed by the physician and research coordinator. We anticipate that a minimum of 80% of patients in the SIDRA IBD clinic will participate in the research cohort project. Patients will be presented with verbal and written information about this project. For all participants, informed consent and assent will be obtained according to the standards of SIDRA IRB and before any biological samples and data are collected.
Study participants will be recruited as follows:
• Inclusion criteria:
– Diagnosis ≤18 years of age
– Consent to data and sample collection by parents (and patients depending on age at time of consent)
– Clinically determined inflammatory bowel disease (Crohn’s disease, Ulcerative colitis, IBD unclassified)
– No suspicion / or exclusion of common / specific enteric pathogens that can mimic the specific clinical picture (Yesinia, Tuberculosis, Rotavirus, Norovirus, HIV…).
To enroll healthy sibling of children already included in the study, we will ask for parental consent and kids assent (depending on their age). The study design and recruitment is described below:
Baseline Questionnaire characteristics: (after consent)
First name, surname, date of birth, gender, home address, residence at birth, contact telephone numbers, IBD diagnosis and date of diagnosis (for IBD patients only), weight, height, sub-type of disease, general medical history, lifestyle exposures including smoking, physical activity and diet, psychological state, and family history including parents and/or siblings with IBD.
For follow-up: All patients enrolled in this study will be followed up at least every 3 months for the duration of the study. Other clinic visits for unwell episodes or other interventions that occur will be recorded in the study database.