Patients with mutations in Tetratricopeptide Repeat Domain 7A (TTC7A) results in a severe and untreatable form of inflammatory bowel disease with the majority dying before 2 years of age. Pathological features of these patients include intestinal apoptosis and disrupted apicobasal polarity. Since the molecular function of TTC7A is still poorly understood, we recently carried out a screen identifying potential TTC7A binding partners in an attempt to reveal its pathway of action. TTC7A is part of an evolutionarily conserved pathway and acts as a scaffolding protein recruiting phosphatidylinositol 4-kinase, PI4KIIIα, to the plasma membrane where it also binds to EFR3B. This conserved pathway helps PI4KIIIα phosphorylate phosphatidylinositol (PI) to PI-4-phosphate (PI4P) which has been implicated in cell survival and the maintenance of cell polarity. In addition, the E3 ubiquitin ligase, UBR5, was identified as a potential TTC7A binding protein and may play a role in stabilizing TTC7A protein levels. Furthermore, UBR5 is suggested to be a colorectal oncogene because it interacts with β-catenin to upregulate canonical Wnt signaling. UBR5 has also been implicated as a tumour suppressor gene and in regulating apoptosis. To understand the role of TTC7A and UBR5 in cell survival. We hypothesize that UBR5 is involved and required in the PI4KIIIα-TTC7A-EFR3B pathway to maintain cell survival. Vector constructs were created harbouring patient derived TTC7A mutations (E71K, Q526X, A832T) and expressed in HEK 293T cells. TTC7A immunoprecipitates (IP) were analyzed by tandem mass spectrometry identifying various binding proteins. To validate these potential TTC7A interacting proteins, co-IP was performed by co-transfecting cells with appropriate expression plasmids and then lysing the cells after 24hrs. Protein of interest was IP using the target antibody. Whole cell lysate and co-IP samples were subject to Western blot analysis. Preliminary results show an interaction between the UBR5 and TTC7A along with the patient derived TTC7A variants. They also show the presence of PI4KIIIα in the UBR5/TTC7A complex. Furthermore, they suggest that β-catenin only interacts with UBR5 in the presence of TTC7A. The presence of PI4KIIIα in the UBR5/TTC7A complex implies that UBR5 is part of the conserved pathway to maintain cell survival through the downstream PI4P levels. Western blot analysis shows that UBR5 interacts most strongly with the TTC7A Q526X. A possible explanation is that the truncated TTC7A Q526X is ubiquitinated by UBR5 for proteasomal degradation, resulting in decreased PI4P levels. Also, the study suggests that TTC7A recruits β-catenin for ubiquitination by UBR5. Together, the research supports the notion that TTC7A is involved in multiple pathways with UBR5 to maintain cell survival or in causing colorectal cancer. None

Very early onset inflammatory bowel disease (VEOIBD) is a severe disease presenting in children <6 years. Patients with TTC7A mutations present with apoptotic enterocolitis, disrupted intestinal architecture, multiple intestinal atresias, and/or combined immunodeficiency. There are few effective treatment options for these patients, and infant fatality is common. Rare autosomal recessive variants for tetratricopeptide repeat domain 7A (TTC7A) have been uncovered in the most severe forms of VEOIBD. The role of TTC7A in the pathogenesis of VEOIBD is largely unknown. The rising prevalence of IBD in Canada is driven by the rapidly increasing incidence (~60%) in children. Compounded with poor responses to standard IBD therapies, the need for therapies motivates researchers to seek effective treatment options. We hypothesize that TTC7A dysfunction results in aberrant intestinal phenotypes related to VEO-IBD. My research aim is to define TTC7A mutant phenotypes in cell-based and zebrafish models; thus, allowing for phenotypic screening and discovery of drugs for use in clinical settings. The overall research goal is to identify compounds that can rescue aberrant phenotypes induced by the TTC7A defect via high throughput drug screening. Since research on TTC7A’s function is scarce, identifying drugs with known targets may provide some insight into TTC7A’s cellular functions, and in VEO-IBD as a whole. Mutant phenotypes were characterized using CRISPR engineered TTC7A knockout HAP1 cells. Assays revolving around apoptosis and cell adhesion were used in a manner amenable to high throughput screening (HTS). For example, 96-well fluorescent and luminescent assays using caspase 3 /7 luciferases. TTC7A mutant zebrafish with fluorescently stained GI tracts allowed for peristaltic activity analyses. HTS using libraries containing FDA approved drugs identified drugs that could rescue TTC7A-related aberrant phenotypes Aberrations in a range of phenotypes were observed in TTC7A in vitro models including round and small morphologies, altered f-actin organization, poor adhesion, compromised viability, and increased susceptibility to apoptosis. Homozygous TTC7A zebrafish show reduced gut motility, narrow intestinal lumens, and increased apoptotic cells. Drug screening has identified several (hits) drugs capable of rescuing TTC7A phenotypes. These drugs will be validated in orthogonal assays as well as in patient derived intestinal organoids. Defining the TTC7A mutant phenotype has provided targets for identifying drugs for use in clinical settings. Furthermore, these findings could elucidate the functional pathways of this relatively uncharacterized protein. Drugs capable of rescuing TTC7A defects could increase patient prognosis and uncover functional pathways contributing to VEOIBD. CIHR

MIRAGE syndrome is caused by heterozygous mutation in the SAMD9 gene. The syndrome is characteristic of enteropathy, and is often fatal within the first 2 years of life. However, the pathogenesis of enteropathy in the syndrome is unknown. We present a case of MIRAGE syndrome (gestational age 35 weeks, birth weight 1330g) who developed restriction of growth, adrenal hypoplasia, genital anomaly, and enteropathy at the time of birth. Sigmoidoscopy showed longitudinal ulcers in rectum. Aim of this study is (1) to identify whether SAMD9 mutation is involved in early onset inflammatory bowel disease (IBD), and (2) to investigate the involvement of SAMD9 mutation in colitis. (1) Whole exome sequence (WES) results performed for IBD patients and their families in The Hospital for Sick Children were reviewed. Mucosal expression of SAMD9 in patients’ biopsy samples were investigated by immunohistochemistry. (2) We assessed difference in TNF-alpha responsiveness between wild type (WT) or mutated (R1293W) SAMD9 using stably expressing HEK293 cell lines. Difference in apoptosis were measured using western blotting and Caspase assays. (1) Among our WES data, 3 patients from 2 families (2 ulcerative colitis, 1 colonic Crohn’s disease) had mutation in SAMD9 gene. Biopsy samples from IBD patients showed increased SAMD9 signals by immunohistochemistry, whereas those from MIRAGE syndrome patient and these 3 patients showed decreased signals. (2) R1293W expressing cells showed increased expression of cleaved Poly ADP-ribose polymerase (PARP) when compared with WT and control. X-Linked inhibitor of apoptosis (XIAP), a known apoptosis inhibitor involves in early onset IBD, were decreased in R1293W mutations. Caspase assay showed increased caspase 3/7 activity in mutated cell lines. Apoptosis was also observed in pathology of the patient’s intestinal mucosal biopsy specimen. SAMD9 might be a novel gene associated with development of pediatric IBD. Suppression of XIAP might result in increased apoptosis and intestinal inflammation observed in patients with SAMD9 mutation. None