Background & Aims
Very early onset inflammatory bowel diseases (VEOIBD), including infant disorders, are a diverse group of diseases found in children less than 6 years of age. They have been associated with several gene variants. We aimed to identify genes that cause VEOIBD.
Methods
We performed whole-exome sequencing of DNA from 1 infants 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 the mutations in 2 unrelated families, each with 2 affected siblings. TTC7A interacts with EFR3 homolog B (EFR3B) to regulate phosphatidylinositol 4-kinase (PI4KA) 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. PI4KA 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.
Conclusion
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 PI4KA–TTC7A–EFR3B pathway are involved in the pathogenesis of VEOIBD.
Background & Aims
Defects in intestinal innate defense systems predispose patients to
inflammatory bowel disease (IBD). Reactive oxygen species (ROS) generated by
nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases in the mucosal barrier
maintain gut homeostasis and defend against pathogenic attack. We hypothesized that
molecular genetic defects in intestinal NADPH oxidases might be present in children with
IBD.
Methods
After targeted exome sequencing of epithelial NADPH oxidases
NOX1 and DUOX2 on 209 children with very early onset
inflammatory bowel disease (VEOIBD), the identified mutations were validated using
Sanger Sequencing. A structural analysis of NOX1 and
DUOX2 variants was performed by homology in silico modeling. The
functional characterization included ROS generation in model cell lines and in in vivo
transduced murine crypts, protein expression, intracellular localization, and cell-based
infection studies with the enteric pathogens Campylobacter jejuni and
enteropathogenic Escherichia coli.
Results
We identified missense mutations in NOX1 (c.988G>A,
p.Pro330Ser; c.967G>A, p.Asp360Asn) and DUOX2
(c.4474G>A, p.Arg1211Cys; c.3631C>T, p.Arg1492Cys) in 5 of 209 VEOIBD
patients. The NOX1 p.Asp360Asn variant was replicated in a male
Ashkenazi Jewish ulcerative colitis cohort. All NOX1 and
DUOX2 variants showed reduced ROS production compared with wild-type
enzymes. Despite appropriate cellular localization and comparable pathogen-stimulated
translocation of altered oxidases, cells harboring NOX1 or
DUOX2 variants had defective host resistance to infection with
C. jejuni.
Conclusions
This study identifies the first inactivating missense variants in
NOX1 and DUOX2 associated with VEOIBD. Defective ROS
production from intestinal epithelial cells constitutes a risk factor for developing
VEOIBD.
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