BACKGROUND & AIMS:Tumor necrosis factor (TNF) is a key driver of intestinal epithelial inflammation. The baculoviral inhibitor of apoptosis protein repeat-containing 3 (BIRC3) gene encodes the cellular inhibitor of apoptosis protein 2 (cIAP2), a known regulator of TNF signaling. Although genetic variants in components of the TNF signaling pathway have been reported, no human BIRC3 variants have been previously identified. METHODS:We screened exomes obtained from Crohn's disease (CD) patients from multiple centers for BIRC3 variants. We used cellular, mouse organoids, induced pluripotent stem cells-derived intestinal organoids, knock-in and knockout mouse models, and knockout zebrafish, as well as transcriptome analysis of various samples to determine pathogenicity of BIRC3 variants. RESULTS:Rare and damaging BIRC3 variants were identified in 14 patients from 10 unrelated families with CD diagnosed between infancy and adulthood. Functional studies showed that BIRC3 deficiency caused impaired receptor-interacting protein kinase 1 (RIPK1) ubiquitylation, leading to RIPK1 autophosphorylation resulting in increased epithelial cell death. The p.H312Y cIAP2 variant identified in both our index and in another independent patient was mislocalizaed, and a knock-in mouse model of this BIRC3 variant (cIAP2H312Y/+) had exacerbation of chemically induced colitis, whereas ciap1-/+ zebrafish developed spontaneous colitis. Transcriptome analysis of mice organoids and zebrafish showed that BIRC3 deficiency led to inappropriate sustained activation of TNF-responsiveness genes in the absence of stimuli. Small molecule pharmacologic inhibition of RIPK1 or caspases attenuated intestinal inflammation in BIRC3-deficient intestinal organoids and cIAP2H312Y/+ mice. CONCLUSIONS:We establish BIRC3 deficiency as a cause of monogenic CD in both pediatric- and adult-onset patients and identify RIPK1 as a therapeutic target.

Myeloid-derived suppressor cells (MDSCs) play a crucial role in tumor-associated immune suppression and in impeding immune checkpoint blockade (ICB) therapies; however, effective intervention strategies targeting MDSCs remain limited. In this study, we observed a positive correlation between spleen tyrosine kinase (SYK) activity in immune cells and tumor progression in human cancers. Utilizing a mouse model with a gain-of-function SYKS544Y mutation, we demonstrated that SYK activation promotes tumorigenesis in carcinogen-induced and xenograft models across multiple cancer types, largely through MDSC-dependent mechanisms. Mechanistically, SYK activates the JAK-STAT-CXCR2 signaling axis, enhancing MDSC migration and suppressing antitumor T cell responses. Notably, pharmacological inhibition of SYK not only inhibited tumor growth but also enhanced the therapeutic effect of anti-PD-1 in mouse models and patient-derived tumor organoid models. Our findings highlight the immunoregulatory role of SYK in tumor progression and suggest that targeting SYK represents a promising strategy to remodel the tumor microenvironment and enhance the efficacy of checkpoint blockade immunotherapy.

BACKGROUND:Next-generation sequencing has enabled precision therapeutic approaches that have improved the lives of children with rare diseases. Congenital diarrhea and enteropathies (CODEs) are associated with high morbidity and mortality. Although treatment of these disorders is largely supportive, emerging targeted therapies based on genetic diagnoses include specific diets, pharmacologic treatments, and surgical interventions. METHODS:We analyzed the exomes or genomes of infants with suspected monogenic congenital diarrheal disorders. Using cell and zebrafish models, we tested the effects of variants in newly implicated genes. RESULTS:In our case series of 129 infant probands with suspected monogenic congenital diarrheal disorders, we identified causal variants, including a new founder NEUROG3 variant, in 62 infants (48%). Using cell and zebrafish models, we also uncovered and functionally characterized three novel genes associated with CODEs: GRWD1, MYO1A, and MON1A. CONCLUSIONS:We have characterized the broad genetic architecture of CODE disorders in a large case series of patients and identified three novel genes associated with CODEs. (Funded by the National Institutes of Health and others.).

Defining monogenic drivers of autoinflammatory syndromes elucidates mechanisms of disease in patients with these inborn errors of immunity and can facilitate targeted therapeutic interventions. Here, we describe a cohort of patients with a Behçet's- and inflammatory bowel disease (IBD)-like disorder termed "deficiency in ELF4, X-linked" (DEX) affecting males with loss-of-function variants in the ELF4 transcription factor gene located on the X chromosome. An international cohort of fourteen DEX patients was assessed to identify unifying clinical manifestations and diagnostic criteria as well as collate findings informing therapeutic responses. DEX patients exhibit a heterogeneous clinical phenotype including weight loss, oral and gastrointestinal aphthous ulcers, fevers, skin inflammation, gastrointestinal symptoms, arthritis, arthralgia, and myalgia, with findings of increased inflammatory markers, anemia, neutrophilic leukocytosis, thrombocytosis, intermittently low natural killer and class-switched memory B cells, and increased inflammatory cytokines in the serum. Patients have been predominantly treated with anti-inflammatory agents, with the majority of DEX patients treated with biologics targeting TNFα.

Integrin heterodimers containing an Integrin alpha V subunit are essential for development and play critical roles in cell adhesion and signaling. We identified biallelic variants in the gene coding for Integrin alpha V (ITGAV) in three independent families (two patients and four fetuses) that either caused abnormal mRNA and the loss of functional protein or caused mistargeting of the integrin. This led to eye and brain abnormalities, inflammatory bowel disease, immune dysregulation, and other developmental issues. Mechanistically, the reduction of functional Integrin αV resulted in the dysregulation of several pathways including TGF-β-dependent signaling and αVβ3-regulated immune signaling. These effects were confirmed using immunostaining, RNA sequencing, and functional studies in patient-derived cells. The genetic deletion of itgav in zebrafish recapitulated patient phenotypes including retinal and brain defects and the loss of microglia in early development as well as colitis in juvenile zebrafish with reduced SMAD3 expression and transcriptional regulation. Taken together, the ITGAV variants identified in this report caused a previously unknown human disease characterized by brain and developmental defects in the case of complete loss-of-function and atopy, neurodevelopmental defects, and colitis in cases of incomplete loss-of-function.

BACKGROUND:Patients with inflammatory bowel disease (IBD) exhibit considerable interindividual variability in medication response, highlighting the need for precision medicine approaches to optimize and tailor treatment. Pharmacogenetics (PGx) offers the ability to individualize dosing by examining genetic factors underlying the metabolism of medications such as thiopurines. Pharmacogenetic testing can identify individuals who may be at risk for thiopurine dose-dependent adverse reactions including myelosuppression. We aimed to evaluate PGx variation in genes supported by clinical guidelines that inform dosing of thiopurines and characterize differences in the distribution of actionable PGx variation among diverse ancestral groups. METHODS:Pharmacogenetic variation in TPMT and NUDT15 was captured by genome-wide genotyping of 1083 pediatric IBD patients from a diverse Canadian cohort. Genetic ancestry was inferred using principal component analysis. The proportion of PGx variation and associated metabolizer status phenotypes was compared across 5 genetic ancestral groups within the cohort (Admixed American, African, East Asian, European, and South Asian) and to prior global estimates from corresponding populations. RESULTS:Collectively, 11% of the cohort was categorized as intermediate or poor metabolizers of thiopurines, which would warrant a significant dose reduction or selection of alternate therapy. Clinically actionable variation in TPMT was more prevalent in participants of European and Admixed American/Latino ancestry (8.7% and 7.5%, respectively), whereas variation in NUDT15 was more prevalent in participants of East Asian and Admixed American/Latino ancestry (16% and 15% respectively). CONCLUSIONS:These findings demonstrate the considerable interpopulation variability in PGx variation underlying thiopurine metabolism, which should be factored into testing diverse patient populations.

BACKGROUND AND AIMS:Inflammatory bowel diseases [IBD] have a complex polygenic aetiology. Rare genetic variants can cause monogenic intestinal inflammation. The impact of chromosomal aberrations and large structural abnormalities on IBD susceptibility is not clear. We aimed to comprehensively characterise the phenotype and prevalence of patients with IBD who possess rare numerical and structural chromosomal abnormalities. METHODS:We performed a systematic literature search of databases PubMed and Embase; and analysed gnomAD, Clinvar, the 100 000 Genomes Project, and DECIPHER databases. Further, we analysed international paediatric IBD cohorts to investigate the role of IL2RA duplications in IBD susceptibility. RESULTS:A meta-analysis suggests that monosomy X [Turner syndrome] is associated with increased expressivity of IBD that exceeds the population baseline (1.86%, 95% confidence interval [CI] 1.48 to 2.34%) and causes a younger age of IBD onset. There is little evidence that Klinefelter syndrome, Trisomy 21, Trisomy 18, mosaic Trisomy 9 and 16, or partial trisomies contribute to IBD susceptibility. Copy number analysis studies suggest inconsistent results. Monoallelic loss of X-linked or haploinsufficient genes is associated with IBD by hemizygous or heterozygous deletions, respectively. However, haploinsufficient gene deletions are detected in healthy reference populations, suggesting that the expressivity of IBD might be overestimated. One duplication that has previously been identified as potentially contributing to IBD risk involves the IL2RA/IL15R loci. Here we provide additional evidence that a microduplication of this locus may predispose to very-early-onset IBD by identifying a second case in a distinct kindred. However, the penetrance of intestinal inflammation in this genetic aberration is low [<2.6%]. CONCLUSIONS:Turner syndrome is associated with increased susceptibility to intestinal inflammation. Duplication of the IL2RA/IL15R loci may contribute to disease risk.

ABSTRACTBackground and Aims:The initial description of a heterozygous dominant ACTG2 variant in familial visceral myopathy was followed by the identification of additional variants in other forms of intestinal dysmotility disorders. we aimed to describe the diverse phenotype of this newly reported and rare disease.Methods:Report of 4 new patients, and a systematic review of ACTG2‐related disorders. we analyzed the population frequency and used in silico gene damaging predictions. Genotype‐phenotype correlations were explored.Results:One hundred three patients (52% girls), from 14 publications, were included. Twenty‐eight unique variants were analyzed, all exceedingly rare, and 27 predicted to be highly damaging. The median Combined Annotation Dependent Depletion (CADD) score was 29.2 (Interquartile range 26.3–29.4). Most patients underwent abdominal surgery (66%), about half required intermittent bladder catheterization (48.5%), and more than half were parenteral nutrition (PN)‐dependent (53%). One‐quarter of the patients died (25.7%), and 6 required transplant (5.8%). Girls had a higher rate of microcolon (P = 0.009), PN dependency (P = 0.003), and death/transplant (P = 0.029) compared with boys, and early disease onset (<2 years of age) was associated with megacystis‐microcolon‐intestinal hypoperistalsis syndrome (MMIHS) features. There was no statistical association between disease characteristics and CADD scores.Conclusions:Damaging ACTG2 variants are rare, often associated with MMIHS phenotype, and overall have a wide phenotypic variation. Symptoms usually present in the perinatal period but can also appear at a later age. The course of the disease is marked by frequent need for surgical interventions, PN support, and mortality. Poor outcomes are more common among girls with ACTG2 variants.

Spleen tyrosine kinase (SYK) is a critical immune signaling molecule and therapeutic target. We identified damaging monoallelic SYK variants in six patients with immune deficiency, multi-organ inflammatory disease such as colitis, arthritis and dermatitis, and diffuse large B cell lymphomas. The SYK variants increased phosphorylation and enhanced downstream signaling, indicating gain of function. A knock-in (SYK-Ser544Tyr) mouse model of a patient variant (p.Ser550Tyr) recapitulated aspects of the human disease that could be partially treated with a SYK inhibitor or transplantation of bone marrow from wild-type mice. Our studies demonstrate that SYK gain-of-function variants result in a potentially treatable form of inflammatory disease. Individuals with SYK gain-of-function variants develop immunodeficiency and systemic inflammation, which are recapitulated in a knock-in mouse model. Treatment of these mice with bone marrow transplantation or with a SYK inhibitor ameliorates disease symptoms, highlighting potential therapeutic strategies for patients with SYK mutations.

Platelet α-granule biogenesis in precursor megakaryocytes is critically dependent on VPS33B and VPS16B, as demonstrated by the platelet α-granule deficiency seen in the rare multisystem disorder arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome associated with biallelic pathogenic variants in VPS33B and VIPAS39 (encoding VPS16B). VPS33B and VPS16B are ubiquitously expressed proteins that are known to interact and play key roles in protein sorting and trafficking between subcellular locations. However, there remain significant gaps in our knowledge of the nature of these interactions in primary cells from patients with ARC syndrome.To use primary cells from patients with ARC syndrome to better understand the interactions and roles of VPS33B and VPS16B in platelets and precursor megakaryocytes.The proband and his male sibling were clinically suspected to have ARC syndrome. Confirmatory genetic testing and platelet phenotyping, including electron microscopy and protein expression analysis, was performed with consent in a research setting.We describe the first case of ARC syndrome identified in Costa Rica, associated with a novel homozygous nonsense VPS33B variant that is linked with loss of expression of both VPS33B and VPS16B in platelets.These results indicate that stable expression of VPS16B in platelets, their precursor megakaryocytes, and other cells is dependent on VPS33B. We suggest that systematic evaluation of primary cells from patients with a range of VPS33B and VIPAS39 variants would help to elucidate the interactions and functions of these proteins.

Abstract Background Spleen Tyrosine Kinase (SYK) is a cytosolic, non-receptor tyrosine kinase with an imperative role in immune and non-immune processes. Recently, we identified six gain-of-function SYK variants in patients that presented multi-organ inflammation and immune dysregulation. The SYK variants displayed constitutive SYK phosphorylation in human embryonic kidney (HEK) 293T, colonic epithelial cells (SW480), and in knock-in heterozygous SYK mice. These observations mark SYK as a therapeutic target for autoimmune diseases. Phenotype drug discovery accelerates this process and can be done successfully with an appropriate phenotype. A possible phenotype displayed by SYK variants is SYK phosphorylation, as high-throughput screening can identify hit compounds that reduce the constitutive activation of phosphorylated SYK (p-SYK). Aims Aim 1: Determine the screening phenotype with wildtype (WT) and SYK S550Y variant in HEK293T cells. Recently, we observed increased phosphorylation in gain-of-function SYK variants We hypothesize that we can use phosphorylated-SYK (p-SYK) levels to identify hit compounds that can decrease the kinase activity in these variants. With stable transfected SYK WT and SYK S550Y HEK293T cell-line, protein analyses will be completed to characterize the appropriate screening phenotype. Aim 2 Establish an assay for high-throughput drug screening. We will utilize homogenous time-resolved fluorescence (HTRF) assay. The signal measured from HTRF is positively proportional to the level of p-SYK; therefore, we expect that S550Y cells will have a higher signal than the WT. Aim 3 Validate hit compounds in HEK293T and zebrafish. We will create a dose-response curve with the hit compounds in in vitro and in vivo models. Methods We will use stable transfection to established overexpressing SYK WT and S550Y HEK293T cells. We will apply homogenous time-resolved fluorescence (HTRF) to quantify p-SYK levels during the drug screening. Results Protein analyses have verified high expression of p-SYK in stable transfected HEK293T cells. No stimulation was required, as the cells showed an increased phosphorylation level at baseline. Downstream signaling partners such as p-ERK and p-JNK of the MAPK pathway displayed an upregulation. This suggests that the sustained activation of p-SYK may consequently affect cellular processes and contribute to the clinical manifestations observed in patients. Conclusions This research study will identify hit compounds that can produce a safe and effective biological response in pediatric patients with gain-of-function SYK variants. Personalizing medicine throughout high-throughput drug screening can accelerate drug repurposing for pediatric patients with multiple systemic diseases and immune dysregulation. Funding Agencies None

BACKGROUND & AIMS:Advances in genomic technologies have led to increasing reports of monogenic inflammatory bowel disease (IBD). Here, we systematically review the literature to determine the clinical features, genetic profile, and previously used treatment strategies in monogenic IBD.METHODS:A systematic review of MEDLINE articles published between January 2000 and December 2020 was conducted. A total of 750 individual monogenic IBD cases were identified from 303 eligible articles.RESULTS:The most frequently reported monogenic IBD genes were IL10RA/B, XIAP, CYBB, LRBA, and TTC7A. In total, 63.4% of patients developed IBD before 6 years of age, 17.4% developed IBD between ages 10 and 17.9 years, and 10.9% developed IBD after age 18. There was a substantial difference between these age groups and the underlying monogenic disorders. Only 31.7% had any history of extraintestinal comorbidity (EIC) before IBD onset, but 76.0% developed at least 1 EIC during their clinical course. The most common EICs were atypical infection (44.7%), dermatologic abnormality (38.4%), and autoimmunity (21.9%). Bowel surgery, biologic therapy, and hematopoietic stem cell transplantation were performed in 27.1%, 32.9%, and 23.1% of patients, respectively.CONCLUSIONS:Monogenic IBD cases, although rare, have varied extraintestinal comorbidities and limited treatment options including surgery and transplant. Early identification and improved understanding of the characteristics of the genes and underlying disease processes in monogenic IBD is important for effective management.

BACKGROUND AND AIMS: Diagnosis of monogenic disease is increasingly important for patient care and personalizing therapy. However, the current process is nonstandardized, expensive, and time consuming. There is currently no accepted strategy to help identify disease-causing variants in monogenic inflammatory bowel disease (IBD). The aim of the study is to develop a prioritization strategy for monogenic IBD variant discovery through detailed analysis of a whole-exome sequencing (WES) data set. METHODS: All consenting pediatric patients with IBD presenting to our tertiary care hospital during the study periodwere enrolled and underwent WES (n = 1005). Available family members also underwent WES. Variants were analyzed en masse using the GEMINI framework and were further annotated using data from dbNSFP, Combined Annotation Dependent Depletion, and gnomAD. Known disease-causing variants (n = 36) were used as positive controls. Machine learning algorithms were optimized and then compared to assist with identifying monogenic IBD case characteristics. RESULTS: Initial gene-level analysis identified 11 genes not previously linked to IBD that could potentially harbor IBD-causing variants. Machine learning algorithms identified 4 primary variant characteristics (Combined Annotation Dependent Depletion score, dbNSFP score, relationship with a known immunodeficiency gene, and alternate allele frequency), and optimal threshold values for each were determined to assist with identifying monogenic IBD variants. Based on these characteristics, an automated variant prioritization pipeline was then created that filters and prioritizes variants from>100,000 variants per patient downto amean of 15. This pipeline is available online for all to use. CONCLUSION: Leveraging a large WES data set, we demonstrate a statistically rigorous strategy for prioritization of variants for monogenic IBD diagnosis.

Our understanding of inflammatory bowel disease is changing as we identify genetic variants associated with immune dysregulation. Inflammatory bowel disease undetermined, even when diagnosed in older children and adolescents, in the setting of multiple inflammatory and infectious diseases should raise the suspicion of complex immune dysregulation with a monogenic basis. We report a case of inflammatory bowel disease undetermined triggered by exposure to a nonsteroidal antiinflammatory drug in a 16-year-old girl with a background history of juvenile idiopathic arthritis, cytopenias, recurrent respiratory tract and middle ear infections, and esophageal candidiasis. Immunologic assessment included measurement of immunoglobulin levels, lymphocyte immunophenotyping, B-cell functional tests, and whole-exome sequencing. Laboratory investigation revealed defects of humoral immunity, including mild persistent hypogammaglobulinemia affecting all 3 isotypes and absent isohemagglutinins. Whole exome sequencing revealed a heterozygous TNFRSF13B (Tumor Necrosis Factor Receptor Superfamily Member 13B, or Transmembrane Activator and Calcium-modulating cyclophilin ligand Interactor, TACI) gene variant, which is associated with common variable immunodeficiency and the development of autoimmune diseases. In conclusion, a clinical history of recurrent infections, atypical histologic features of inflammatory bowel disease, additional autoimmune manifestations, and an inadequate response to conventional therapy should prompt the physician to refer to an immunologist with the query of inborn error of immunity. We report how extensive immune evaluation and genetic diagnosis can individualize care and facilitate a multidisciplinary team approach.

Inflammatory bowel disease (IBD) is characterized by inappropriate immune responses to the microbiota in genetically susceptible hosts, but little is known about the pathways that link individual genetic alterations to microbiota-dependent inflammation. Here, we demonstrated that the loss of X-linked inhibitor of apoptosis protein (XIAP), a gene associated with Mendelian IBD, rendered Paneth cells sensitive to microbiota-, tumor necrosis factor (TNF)–, receptor-interacting protein kinase 1 (RIPK1)–, and RIPK3-dependent cell death. This was associated with deficiency in Paneth cell–derived antimicrobial peptides and alterations in the stratification and composition of the microbiota. Loss of XIAP was not sufficient to elicit intestinal inflammation but provided susceptibility to pathobionts able to promote granulomatous ileitis, which could be prevented by administration of a Paneth cell–derived antimicrobial peptide. These data reveal a pathway critical for host-microbial cross-talk, which is required for intestinal homeostasis and the prevention of inflammation and which is amenable to therapeutic targeting.

Abstract Background Advances in genomic technologies have led to an increase in reports of monogenic inflammatory bowel disease (IBD). The majority of the studies on monogenic IBD have focused only on young children aged <6 years. There are no detailed reports containing a comprehensive picture of monogenic IBD with specific numbers on the clinical features, genetic profiles and disease course. Since each gene-specific cause of monogenic IBD is rare, it is difficult to collect cases in a single study even with international cohort studies. Aims To elucidate a comprehensive picture of monogenic IBD, we conducted a systematic review of all reported cases of monogenic IBD. Methods A systematic review of MEDLINE articles published between January 2000 and December 2019 was conducted. 662 monogenic IBD cases were identified from 273 eligible articles. Data on clinical manifestation, genotype, and management were collected. Results The most frequently reported genes causative of monogenic IBD patients were IL10RA, CYBB, IL10RB, and TTC7A (Figure). In total, 64.8% of patients developed IBD before six years old, 15.6% between ages 10 and 17.9 years, and 11.6% at age 18 years or older. Only 32.7% had any history of extra-intestinal manifestation (EIM) before IBD onset. There was substantial difference in the onset age groups and the underlying monogenic disorders. 74.4% developed at least one EIM during their clinical course. The most common EIMs were atypical infection (44.1%), dermatologic abnormality (39.2%), autoimmunity (22.7%) and lymphoid organ abnormality (11.5%). Autosomal recessive (62.8%) was the most common inheritance pattern, and missense-variants (44.3%) were the most identified type of genetic variants. Deletions including CNVs, intronic, synonymous and inversion, which have the possibility to be overlooked by whole exome sequencing were shown in some cases. Bowel surgery, biologics, and hematopoietic stem cell transplantation were performed in 28.3%, 32.8%, and 24.4% of patients, respectively. Conclusions Monogenic IBD diagnosis and management is a challenging clinical problem across age groups; the EIMs are more diverse and the management is evidently more difficult compared to non-monogenic IBD. An improved understanding of the characteristics of the genes and underlying disease processes in monogenic IBD is necessary for effective management. Funding Agencies CAG, CIHRUehara Memorial Foundation Fellowship

CARMIL2 is required for CD28-mediated co-stimulation of NF-κB signaling in T cells and its deficiency has been associated with primary immunodeficiency and, recently, very early onset inflammatory bowel disease (IBD). Here we describe the identification of novel biallelic CARMIL2 variants in three patients presenting with pediatric-onset IBD and in one with autoimmune polyendocrine syndrome (APS). None manifested overt clinical signs of immunodeficiency before their diagnosis. The first patient presented with very early onset IBD. His brother was found homozygous for the same CARMIL2 null variant and diagnosed with APS. Two other IBD patients were found homozygous for a nonsense and a missense CARMIL2 variant, respectively, and they both experienced a complicated postoperative course marked by severe infections. Immunostaining of bowel biopsies showed reduced CARMIL2 expression in all the three patients with IBD. Western blot and immunofluorescence of transfected cells revealed an altered expression pattern of the missense variant. Our work expands the genotypic and phenotypic spectrum of CARMIL2 deficiency, which can present with either IBD or APS, aside from classic immunodeficiency manifestations. CARMIL2 should be included in the diagnostic work-up of patients with suspected monogenic IBD.

Background and Aims: Very early onset inflammatory bowel disease [VEOIBD] is characterized by intestinal inflammation affecting infants and children less than 6 years of age. To date, over 60 monogenic aetiologies of VEOIBD have been identified, many characterized by highly penetrant recessive or dominant variants in underlying immune and/or epithelial pathways. We sought to identify the genetic cause of VEOIBD in a subset of patients with a unique clinical presentation. Methods: Whole exome sequencing was performed on five families with ten patients who presented with a similar constellation of symptoms including medically refractory infantile-onset IBD, bilateral sensorineural hearing loss and, in the majority, recurrent infections. Genetic aetiologies of VEOIBD were assessed and Sanger sequencing was performed to confirm novel genetic findings. Western analysis on peripheral blood mononuclear cells and functional studies with epithelial cell lines were employed. Results: In each of the ten patients, we identified damaging heterozygous or biallelic variants in the Syntaxin-Binding Protein 3 gene [STXBP3], a protein known to regulate intracellular vesicular trafficking in the syntaxin-binding protein family of molecules, but not associated to date with either VEOIBD or sensorineural hearing loss. These mutations interfere with either intron splicing or protein stability and lead to reduced STXBP3 protein expression. Knock-down of STXBP3 in CaCo2 cells resulted in defects in cell polarity. Conclusion: Overall, we describe a novel genetic syndrome and identify a critical role for STXBP3 in VEOIBD, sensorineural hearing loss and immune dysregulation.

Inflammatory bowel disease (IBD), clinically defined as Crohn’s disease (CD), ulcerative colitis (UC), or IBD-unclassified, results in chronic inflammation of the gastrointestinal tract in genetically susceptible hosts. Pediatric onset IBD represents ≥ 25% of all IBD diagnoses and often presents with intestinal stricturing, perianal disease, and failed response to conventional treatments. NOD2 was the first and is the most replicated locus associated with adult IBD, to date. However, its role in pediatric onset IBD is not well understood. We performed whole-exome sequencing on a cohort of 1,183 patients with pediatric onset IBD (ages 0–18.5 years). We identified 92 probands with biallelic rare and low frequency NOD2 variants accounting for approximately 8% of our cohort, suggesting a Mendelian inheritance pattern of disease. Additionally, we investigated the contribution of recessive inheritance of NOD2 alleles in adult IBD patients from a large clinical population cohort. We found that recessive inheritance of NOD2 variants explains ~ 7% of cases in this adult IBD cohort, including ~ 10% of CD cases, confirming the observations from our pediatric IBD cohort. Exploration of EHR data showed that several of these adult IBD patients obtained their initial IBD diagnosis before 18 years of age, consistent with early onset disease. While it has been previously reported that carriers of more than one NOD2 risk alleles have increased susceptibility to Crohn’s Disease (CD), our data formally demonstrate that recessive inheritance of NOD2 alleles is a mechanistic driver of early onset IBD, specifically CD, likely due to loss of NOD2 protein function. Collectively, our findings show that recessive inheritance of rare and low frequency deleterious NOD2 variants account for 7–10% of CD cases and implicate NOD2 as a Mendelian disease gene for early onset Crohn’s Disease.