Gastroenterology

Gastroenterology

Volume 147, Issue 3, September 2014, Pages 680-689.e2
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Variants in Nicotinamide Adenine Dinucleotide Phosphate Oxidase Complex Components Determine Susceptibility to Very Early Onset Inflammatory Bowel Disease

https://doi.org/10.1053/j.gastro.2014.06.005Get rights and content

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 p22phox protein; cytochrome b-245 or NADPH oxidase 2, and encodes Nox2 or gp91phox; 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.

Section snippets

Methods

All results are presented according to the Strengthening the Reporting of Genetic Associations guidelines.16 Two complementary approaches were carried out to define the genetic association of the NADPH oxidase genes (cytochrome b light chain and encodes p22phox protein [CYBA], cytochrome b-245 or NADPH oxidase 2 and encodes Nox2 or gp91phox [CYBB], NCF1, NCF2, NCF4, RAC1, RAC2) with VEOIBD (illustrated in Figure 2). The first approach used targeted exome sequencing of the NADPH oxidase gene in

Targeted Exome Sequencing

To capture rare and novel variants, we carried out targeted exome sequencing of the NADPH oxidase genes (CYBA, CYBB, NCF1, NCF2, NCF4, RAC1, RAC2; Figure 1) in 122 VEOIBD patients without any clinical or laboratory evidence of CGD (as described in Methods and reported according to the Strengthening the Reporting of Genetic Associations guidelines16). All identified NADPH oxidase mutations were heterozygous and validated using both Sanger sequencing and Taqman and compared with 480 healthy

Discussion

Overall, through targeted exome sequencing, we identified heterozygous SNPs and novel variants in the NADPH oxidase complex genes in VEOIBD patients with either impaired ROS production, reduced protein binding, or reduced gene expression. CGD is an X-linked or autosomal recessive disease in which the vast majority of mutations are found in NOX2 (Figure 1B), and almost all identified mutations are associated with markedly reduced, or absence of, the protein expression, or rare missense variants

Acknowledgments

The authors thank the all patients and their families described here.

The following authors were part of the International Early Onset Pediatrics IBD Cohort Study (www.NEOPICS.org): Sandeep S. Dhillon, Ramzi Fattouh, Abdul Elkadri, Ryan Murchie, Thomas Walters, Conghui Guo, David Mack, Hien Q. Huynh, Anne M. Griffiths, Scott B. Snapper, John H. Brumell, and Aleixo M. Muise.

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    Conflicts of interest The authors disclose no conflicts.

    Funding This project was funded by operating grants from the Canadian Institutes of Health Research to AMM and JHB (MOP97756 and MOP119457). AMM and SS are supported by Leona M. and Harry B. Helmsley Charitable Trust to study very early onset inflammatory bowel disease. JHB held an Investigator in Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. Infrastructure for the Brumell Laboratory was provided by a New Opportunities grant from the Canadian Foundation for Innovation and the Ontario Innovation Trust. SBS is supported by National Institutes of Health grants HL59561, DK034854, and AI50950 and the Wolpow Family Chair in IBD Treatment and Research.

    Author names in bold designate shared co-first authorship.

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