Sandeep S Dhillon1, Ramzi Fattouh2, Abdul Elkadri3, Wei Xu4, Ryan Murchie2, Thomas Walters5, Conghui Guo2, David Mack6, Hien Q Huynh7, Shairaz Baksh7, Mark S Silverberg8, Anne M Griffiths5, Scott B Snapper9, John H Brumell10, Aleixo M Muise11. 1. SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada. 2. SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada. 3. SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Toronto, The Hospital for Sick Children, Toronto, Ontario, Canada. 4. Princess Margaret Hospital and Dalla Lana School of Public Health, University of Toronto, Toronto, Ontario, Canada. 5. SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Toronto, The Hospital for Sick Children, Toronto, Ontario, Canada. 6. Division of Pediatric Gastroenterology, Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada. 7. Division of Pediatric Gastroenterology, Stollery Children's Hospital, Edmonton, Alberta, Canada. 8. Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; Mount Sinai Hospital Inflammatory Bowel Disease Group, University of Toronto Group, Zane Cohen Center for Digestive Diseases, Toronto, Ontario, Canada. 9. Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Medicine, Children's Hospital Boston; Division of Gastroenterology and Hepatology, Brigham & Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts. 10. SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. 11. SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Toronto, The Hospital for Sick Children, Toronto, Ontario, Canada. Electronic address: aleixo.muise@utoronto.ca.
Abstract
BACKGROUND & AIMS: The colitis observed in patients with very early onset inflammatory bowel disease (VEOIBD; defined as onset of disease at younger than 6 years of age) often resembles that of chronic granulomatous disease (CGD) in extent and features of colonic inflammation observed by endoscopy and histology. CGD is a severe immunodeficiency caused by defects in the genes that encode components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. We investigated whether variants in genes that encode NADPH oxidase components affect susceptibility to VEOIBD using independent approaches. METHODS: We performed targeted exome sequencing of genes that encode components of NADPH oxidases (cytochrome b light chain and encodes p22(phox) protein; cytochrome b-245 or NADPH oxidase 2, and encodes Nox2 or gp91(phox); neutrophil cytosol factor 1 and encodes p47 (phox) protein; neutrophil cytosol factor 2 and encodes p67 (phox) protein; neutrophil cytosol factor 4 and encodes p40 (phox) protein; and Ras-related C3 botulinum toxin substrate 1 and 2) in 122 patients with VEOIBD diagnosed at The Hospital for Sick Children, University of Toronto, from 1994 through 2012. Gene variants were validated in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients with VEOIBD. In a second approach, we examined Tag single nucleotide polymorphisms in a subset of patients with VEOIBD in which the NOX2 NADPH oxidase genes sequence had been previously analyzed. We then looked for single nucleotide polymorphisms associated with the disease in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients. We analyzed the functional effects of variants associated with VEOIBD. RESULTS: Targeted exome sequencing and Tag single nucleotide polymorphism genotyping identified 11 variants associated with VEOIBD; the majority of patients were heterozygous for these variants. Expression of these variants in cells either reduced oxidative burst or altered interactions among proteins in the NADPH oxidase complex. Variants in the noncoding regulatory and splicing elements resulted in reduced levels of proteins, or expression of altered forms of the proteins, in blood cells from VEOIBD patients. CONCLUSIONS: We found that VEOIBD patients carry heterozygous functional hypomorphic variants in components of the NOX2 NADPH oxidase complex. These do not cause overt immunodeficiency, but instead determine susceptibility to VEOIBD. Specific approaches might be developed to treat individual patients based on their genetic variant.
BACKGROUND & AIMS: The colitis observed in patients with very early onset inflammatory bowel disease (VEOIBD; defined as onset of disease at younger than 6 years of age) often resembles that of chronic granulomatous disease (CGD) in extent and features of colonic inflammation observed by endoscopy and histology. CGD is a severe immunodeficiency caused by defects in the genes that encode components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. We investigated whether variants in genes that encode NADPH oxidase components affect susceptibility to VEOIBD using independent approaches. METHODS: We performed targeted exome sequencing of genes that encode components of NADPH oxidases (cytochrome b light chain and encodes p22(phox) protein; cytochrome b-245 or NADPH oxidase 2, and encodes Nox2 or gp91(phox); neutrophil cytosol factor 1 and encodes p47 (phox) protein; neutrophil cytosol factor 2 and encodes p67 (phox) protein; neutrophil cytosol factor 4 and encodes p40 (phox) protein; and Ras-related C3 botulinum toxin substrate 1 and 2) in 122 patients with VEOIBD diagnosed at The Hospital for Sick Children, University of Toronto, from 1994 through 2012. Gene variants were validated in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients with VEOIBD. In a second approach, we examined Tag single nucleotide polymorphisms in a subset of patients with VEOIBD in which the NOX2NADPH oxidase genes sequence had been previously analyzed. We then looked for single nucleotide polymorphisms associated with the disease in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients. We analyzed the functional effects of variants associated with VEOIBD. RESULTS: Targeted exome sequencing and Tag single nucleotide polymorphism genotyping identified 11 variants associated with VEOIBD; the majority of patients were heterozygous for these variants. Expression of these variants in cells either reduced oxidative burst or altered interactions among proteins in the NADPH oxidase complex. Variants in the noncoding regulatory and splicing elements resulted in reduced levels of proteins, or expression of altered forms of the proteins, in blood cells from VEOIBD patients. CONCLUSIONS: We found that VEOIBD patients carry heterozygous functional hypomorphic variants in components of the NOX2NADPH oxidase complex. These do not cause overt immunodeficiency, but instead determine susceptibility to VEOIBD. Specific approaches might be developed to treat individual patients based on their genetic variant.
Authors: Thomas Magg; Anna Shcherbina; Duran Arslan; Mukesh M Desai; Sarah Wall; Vanessa Mitsialis; Raffaele Conca; Ekrem Unal; Neslihan Karacabey; Anna Mukhina; Yulia Rodina; Prasad D Taur; David Illig; Benjamin Marquardt; Sebastian Hollizeck; Tim Jeske; Florian Gothe; Tilmann Schober; Meino Rohlfs; Sibylle Koletzko; Eberhard Lurz; Aleixo M Muise; Scott B Snapper; Fabian Hauck; Christoph Klein; Daniel Kotlarz Journal: Inflamm Bowel Dis Date: 2019-10-18 Impact factor: 5.325