Defects in NADPH Oxidase Genes NOX1 and DUOX2 in Very Early Onset Inflammatory Bowel Disease

Patti Hayes; Sandeep Dhillon; Kim O'Neill; Cornelia Thoeni; Ken Y Hui; Abdul Elkadri; Conghui H Guo; Lidija Kovacic; Gabriella Aviello; Luis A Alvarez; Anne M Griffiths; Scott B Snapper; Steven R Brant; James H Doroshow; Mark S Silverberg; Inga Peter; Dermot P B McGovern; Judy Cho; John H Brumell; Holm H Uhlig; Billy Bourke; Aleixo A Muise; Ulla G Knaus

doi:10.1016/j.jcmgh.2015.06.005

Defects in NADPH Oxidase Genes NOX1 and DUOX2 in Very Early Onset Inflammatory Bowel Disease

Cell Mol Gastroenterol Hepatol. 2015 Sep 1;1(5):489-502. doi: 10.1016/j.jcmgh.2015.06.005.

Authors

Patti Hayes¹, Sandeep Dhillon², Kim O'Neill¹, Cornelia Thoeni², Ken Y Hui³, Abdul Elkadri², Conghui H Guo², Lidija Kovacic¹, Gabriella Aviello¹, Luis A Alvarez⁴, Anne M Griffiths⁵, Scott B Snapper⁶, Steven R Brant⁷, James H Doroshow⁸, Mark S Silverberg⁹, Inga Peter¹⁰, Dermot P B McGovern¹¹, Judy Cho¹², John H Brumell¹³, Holm H Uhlig¹⁴, Billy Bourke¹⁵, Aleixo A Muise², Ulla G Knaus¹⁵

Affiliations

¹ Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland.
² SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada ; Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Toronto, Hospital for Sick Children, Toronto, Ontario, Canada.
³ Program in Computational Biology and Bioinformatics, Yale University, New Haven, Connecticut.
⁴ National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Dublin, Ireland.
⁵ Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Toronto, Hospital for Sick Children, Toronto, Ontario, Canada.
⁶ 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.
⁷ Harvey M. and Lyn P. Meyerhoff Inflammatory Bowel Disease Center, Department of Medicine, School of Medicine and the Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland.
⁸ National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
⁹ Mount Sinai Hospital Inflammatory Bowel Disease Group, University of Toronto, Zane Cohen Centre for Digestive Diseases, Toronto, Ontario, Canada.
¹⁰ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.
¹¹ F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California.
¹² Section of Gastroenterology, Department of Internal Medicine, Icahn School of Medicine at Mount Sinai, New York, New York.
¹³ SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
¹⁴ Translational Gastroenterology Unit, Nuffield Department of Clinical Medicine and Children's Hospital, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.
¹⁵ Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland ; National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Dublin, Ireland.

Abstract

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.

Keywords: DUOX2; Inflammatory Bowel Disease; NADPH Oxidase; NOX1; Reactive Oxygen Species; VEOIBD.

Abstract

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