Aleixo M. Muise

Common variants at five new loci associated with early-onset inflammatory bowel disease

The inflammatory bowel diseases (IBD) Crohns disease and ulcerative colitis are common causes of morbidity in children and young adults in the western world. Here we report the results of a genome-wide association study in early-onset IBD involving 3,426 affected individuals and 11,963 genetically matched controls recruited through international collaborations in Europe and North America, thereby extending the results from a previous study of 1,011 individuals with early-onset IBD. We have identified five new regions associated with early-onset IBD susceptibility, including 16p11 near the cytokine gene IL27 (rs8049439, P = 2.41 × 10−9), 22q12 (rs2412973, P = 1.55 × 10−9), 10q22 (rs1250550, P = 5.63 × 10−9), 2q37 (rs4676410, P = 3.64 × 10−8) and 19q13.11 (rs10500264, P = 4.26 × 10−10). Our scan also detected associations at 23 of 32 loci previously implicated in adult-onset Crohns disease and at 8 of 17 loci implicated in adult-onset ulcerative colitis, highlighting the close pathogenetic relationship between early- and adult-onset IBD.

The Diagnostic Approach to Monogenic Very Early Onset Inflammatory Bowel Disease

Patients with a diverse spectrum of rare genetic disorders can present with inflammatory bowel disease (monogenic IBD). Patients with these disorders often develop symptoms during infancy or early childhood, along with endoscopic or histological features of Crohns disease, ulcerative colitis, or IBD unclassified. Defects in interleukin-10 signaling have a Mendelian inheritance pattern with complete penetrance of intestinal inflammation. Several genetic defects that disturb intestinal epithelial barrier function or affect innate and adaptive immune function have incomplete penetrance of the IBD-like phenotype. Several of these monogenic conditions do not respond to conventional therapy and are associated with high morbidity and mortality. Due to the broad spectrum of these extremely rare diseases, a correct diagnosis is frequently a challenge and often delayed. In many cases, these diseases cannot be categorized based on standard histological and immunologic features of IBD. Genetic analysis is required to identify the cause of the disorder and offer the patient appropriate treatment options, which include medical therapy, surgery, or allogeneic hematopoietic stem cell transplantation. In addition, diagnosis based on genetic analysis can lead to genetic counseling for family members of patients. We describe key intestinal, extraintestinal, and laboratory features of 50 genetic variants associated with IBD-like intestinal inflammation. In addition, we provide approaches for identifying patients likely to have these disorders. We also discuss classic approaches to identify these variants in patients, starting with phenotypic and functional assessments that lead to analysis of candidate genes. As a complementary approach, we discuss parallel genetic screening using next-generation sequencing followed by functional confirmation of genetic defects.

Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function

Intact interleukin-10 receptor (IL-10R) signaling on effector and T regulatory (Treg) cells are each independently required to maintain immune tolerance. Here we show that IL-10 sensing by innate immune cells, independent of its effects on T cells, was critical for regulating mucosal homeostasis. Following wild-type (WT) CD4(+) T cell transfer, Rag2(-/-)Il10rb(-/-) mice developed severe colitis in association with profound defects in generation and function of Treg cells. Moreover, loss of IL-10R signaling impaired the generation and function of anti-inflammatory intestinal and bone-marrow-derived macrophages and their ability to secrete IL-10. Importantly, transfer of WT but not Il10rb(-/-) anti-inflammatory macrophages ameliorated colitis induction by WT CD4(+) T cells in Rag2(-/-)Il10rb(-/-) mice. Similar alterations in the generation and function of anti-inflammatory macrophages were observed in IL-10R-deficient patients with very early onset inflammatory bowel disease. Collectively, our studies define innate immune IL-10R signaling as a key factor regulating mucosal immune homeostasis in mice and humans.

IL-10R polymorphisms are associated with very-early-onset ulcerative colitis

Background:Interleukin-10 (IL-10) signaling genes are attractive inflammatory bowel disease (IBD) candidate genes as IL-10 restricts intestinal inflammation, IL-10 polymorphisms have been associated with IBD in genome-wide association studies, and mutations in IL-10 and IL-10 receptor (IL-10R) genes have been reported in immunodeficient children with severe infantile-onset IBD. Our objective was to determine if IL-10R polymorphisms were associated with early-onset IBD (EO-IBD) and very-early-onset IBD (VEO-IBD). Methods:Candidate-gene analysis of IL10RA and IL10RB was performed after initial sequencing of an infantile onset-IBD patient identified a novel homozygous mutation. The discovery cohort included 188 EO-IBD subjects and 188 healthy subjects. Polymorphisms associated with IBD in the discovery cohort were genotyped in an independent validation cohort of 422 EO-IBD subjects and 480 healthy subjects. Results:We identified a homozygous, splice-site point mutation in IL10RA in an infantile-onset IBD patient causing a premature stop codon (P206X) and IL-10 insensitivity. IL10RA and IL10RB sequencing in the discovery cohort identified five IL10RA polymorphisms associated with ulcerative colitis (UC) and two IL10RB polymorphisms associated with Crohns disease (CD). Of these polymorphisms, two IL10RA single nucleotide polymorphisms, rs2228054 and rs2228055, were associated with VEO-UC in the discovery cohort and replicated in an independent validation cohort (odds ratio [OR] 3.08, combined P = 2 x 10−4; and OR 2.93, P = 6 x 10−4, respectively). Conclusions:We identified IL10RA polymorphisms that confer risk for developing VEO-UC. Additionally, we identified the first splice site mutation in IL10RA resulting in infantile-onset IBD. This study expands the phenotype of IL10RA polymorphisms to include both severe arthritis and VEO-UC.

Clinical outcome in IL-10-and IL-10 receptor-deficient patients with or without hematopoietic stem cell transplantation

BACKGROUND Inherited deficiencies of IL-10 or IL-10 receptor (IL-10R) lead to immune dysregulation with life-threatening early-onset enterocolitis. OBJECTIVES We sought to gather clinical data of IL-10/IL-10R-deficient patients and devise guidelines for diagnosis and management, including hematopoietic stem cell transplantation (HSCT). METHODS We enrolled 40 patients with early-onset enterocolitis and screened for mutations in IL10/IL10R using genetic studies, functional studies, or both of the IL-10 signaling pathway. Medical records of IL-10/IL-10R-deficient patients were reviewed and compiled. RESULTS Of 40 patients, we identified 7 with novel mutations, predominantly in consanguineous families with more than 1 affected member. IL-10/IL-10R-deficient patients had intractable enterocolitis, perianal disease, and fistula formation. HSCT was carried out in 2 patients with IL-10 deficiency and 1 patient with IL-10R α chain deficiency and proved to be an effective therapy, leading to rapid improvement of clinical symptoms and quality of life. CONCLUSION Because the defect in patients with IL-10/IL-10R deficiency resides in hematopoietic lineage cells and their colitis is resistant to standard immunosuppressive therapy, HSCT should be considered early as a potentially curative therapeutic option.

Eating Disorders in Adolescent Boys: A Review of the Adolescent and Young Adult Literature

Although a number of studies have investigated eating disorders in adolescence and the topic has been thoroughly reviewed, these studies have typically focused on females, only to state that the approach and treatment should be similar in males. Recently, there have been a number of studies that have explored gender differences in eating disorders. In this article, we review the literature pertaining to two DSM-IV-defined disorders (anorexia nervosa and bulimia nervosa), a DSM-IV-defined research disorder (binge eating disorder), and two DSM-PC-defined disorders (dieting/body image and purging/binge-eating behaviors), highlighting those findings that pertain to disordered eating in adolescent boys.

NADPH oxidase complex and IBD candidate gene studies: identification of a rare variant in NCF2 that results in reduced binding to RAC2

Objective The NOX2 NADPH oxidase complex produces reactive oxygen species and plays a critical role in the killing of microbes by phagocytes. Genetic mutations in genes encoding components of the complex result in both X-linked and autosomal recessive forms of chronic granulomatous disease (CGD). Patients with CGD often develop intestinal inflammation that is histologically similar to Crohns colitis, suggesting a common aetiology for both diseases. The aim of this study is to determine if polymorphisms in NOX2 NADPH oxidase complex genes that do not cause CGD are associated with the development of inflammatory bowel disease (IBD). Methods Direct sequencing and candidate gene approaches were used to identify susceptibility loci in NADPH oxidase complex genes. Functional studies were carried out on identified variants. Novel findings were replicated in independent cohorts. Results Sequence analysis identified a novel missense variant in the neutrophil cytosolic factor 2 (NCF2) gene that is associated with very early onset IBD (VEO-IBD) and subsequently found in 4% of patients with VEO-IBD compared with 0.2% of controls (p=1.3×10−5, OR 23.8 (95% CI 3.9 to 142.5); Fisher exact test). This variant reduced binding of the NCF2 gene product p67phox to RAC2. This study found a novel genetic association of RAC2 with Crohns disease (CD) and replicated the previously reported association of NCF4 with ileal CD. Conclusion These studies suggest that the rare novel p67phox variant results in partial inhibition of oxidase function and are associated with CD in a subgroup of patients with VEO-IBD; and suggest that components of the NADPH oxidase complex are associated with CD.

Interleukin 10 receptor signaling: master regulator of intestinal mucosal homeostasis in mice and humans.

Interleukin 10 (IL10) is a key anti-inflammatory cytokine that can inhibit proinflammatory responses of both innate and adaptive immune cells. An association between IL10 and intestinal mucosal homeostasis became clear with the discovery that IL10 and IL10 receptor (IL10R)-deficient mice develop spontaneous intestinal inflammation. Similarly, patients with deleterious mutations in IL10, IL10RA, or IL10RB present with severe enterocolitis within the first months of life. Here, we review recent findings on how IL10- and IL10R-dependent signaling modulates innate and adaptive immune responses in the murine gastrointestinal tract, with implications of their role in the prevention of inflammatory bowel disease (IBD). In addition, we discuss the impact of IL10 and IL10R signaling defects in humans and their relationship to very early-onset IBD (VEO-IBD).

Mutations in tetratricopeptide repeat domain 7A result in a severe form of very early onset inflammatory bowel disease

BACKGROUND & AIMS Very early onset inflammatory bowel diseases (VEOIBD), including infant disorders, are a diverse group of diseases found in children younger than 6 years of age. They have been associated with several gene variants. Our aim was to identify the genes that cause VEOIBD. METHODS We performed whole exome sequencing of DNA from 1 infant with severe enterocolitis and her parents. Candidate gene mutations were validated in 40 pediatric patients and functional studies were carried out using intestinal samples and human intestinal cell lines. RESULTS We identified compound heterozygote mutations in the Tetratricopeptide repeat domain 7 (TTC7A) gene in an infant from non-consanguineous parents with severe exfoliative apoptotic enterocolitis; we also detected TTC7A mutations in 2 unrelated families, each with 2 affected siblings. TTC7A interacts with EFR3 homolog B to regulate phosphatidylinositol 4-kinase at the plasma membrane. Functional studies demonstrated that TTC7A is expressed in human enterocytes. The mutations we identified in TTC7A result in either mislocalization or reduced expression of TTC7A. Phosphatidylinositol 4-kinase was found to co-immunoprecipitate with TTC7A; the identified TTC7A mutations reduced this binding. Knockdown of TTC7A in human intestinal-like cell lines reduced their adhesion, increased apoptosis, and decreased production of phosphatidylinositol 4-phosphate. CONCLUSIONS In a genetic analysis, we identified loss of function mutations in TTC7A in 5 infants with VEOIBD. Functional studies demonstrated that the mutations cause defects in enterocytes and T cells that lead to severe apoptotic enterocolitis. Defects in the phosphatidylinositol 4-kinase-TTC7A-EFR3 homolog B pathway are involved in the pathogenesis of VEOIBD.

Incidence, Outcomes, and Health Services Burden of Very Early Onset Inflammatory Bowel Disease

BACKGROUND & AIMS The Paris pediatric modification of the Montreal classification defines very early onset inflammatory bowel disease (VEO-IBD) as a form of IBD distinct from that of older children. We compared the incidence and outcomes of VEO-IBD with those of IBD in older children. METHODS We performed a population-based retrospective cohort study of all children diagnosed with IBD in Ontario, Canada, from 1994 through 2009. Trends in standardized incidence were calculated using Poisson regression. We compared outpatient and emergency department visits, hospitalizations, and surgeries among children diagnosed with IBD when they were younger than age 6, ages 6-9.9, and older than age 10 years. Multivariable models were adjusted for income and stratified by sex. RESULTS The incidence of IBD increased from 9.4 per 100,000 children (95% confidence interval [CI], 8.2-10.8/100,000 children) in 1994 to 13.2 per 100,000 children (95% CI, 11.9-14.6/100,000 children) in 2009 (P < .0001). The incidence increased by 7.4% per year among children younger than 6 years old and 6-9.9 years old, and by 2.2% per year among children ≥10 years old. IBD-related outpatient visits were less frequent among children <6 years old than ≥10 years old (odds ratio for female patients, 0.67; 95% CI, 0.58-0.78; odds ratio for male patients, 0.86; 95% CI, 0.75-0.98). Hazard ratios [HRs] for hospitalization were lower for children <6 years old (female HR, 0.70; 95% CI, 0.56-0.87; male HR, 1.12; 95% CI, 0.94-1.33) than for older children. HRs for surgery among children <6 years old with Crohns disease were 0.35 for female patients (95% CI, 0.16-0.78) and 0.59 for male patients (95% CI, 0.34-0.99). HRs for children <6 years old with ulcerative colitis were 0.88 for female patients (95% CI, 0.47-1.63) and 0.42 for male patients (95% CI, 0.21-0.85). There was no difference in hospitalization or surgery rates among children 6-9.9 years old vs those ≥10 years old. CONCLUSIONS Based on a retrospective cohort study, the incidence of VEO-IBD increased from 1994 through 2009. Children diagnosed with IBD before they were 6 years old used fewer health services and had lower rates of surgery than children diagnosed when they were 10 years or older.