NOX1 loss-of-function genetic variants in patients with inflammatory bowel disease

Genetic defects that affect intestinal epithelial barrier function can present with very early onset inflammatory bowel disease (VEOIBD). Using whole genome sequencing, a novel hemizygous defect in NOX1 encoding NAPDH oxidase 1 was identified in a patient with ulcerative colitis-like VEOIBD. Exome screening of 1,878 paediatric patients identified further seven male IBD patients with rare NOX1 mutations. Loss-of-function was validated in p.N122H and p.T497A, and to a lesser degree in p.Y470H, p.R287Q, p.I67M, p.Q293R as well as the previously described p.P330S and the common NOX1 SNP p.D360N (rs34688635) variant. The missense mutation p.N122H abrogated reactive oxygen species (ROS) production in cell lines, ex-vivo colonic explants and patient-derived colonic organoid cultures. Within colonic crypts, NOX1 constitutively generates a high level of ROS in the crypt lumen. Analysis of 9,513 controls and 11,140 IBD patients of non-Jewish European ancestry did not reveal an association between p.D360N and IBD. Our data suggest that loss-of-function variants in NOX1 do not cause a Mendelian disorder of high penetrance but are a context specific modifier. Our results implicate that variants in NOX1 change brush border ROS within colonic crypts at the interface between the epithelium and luminal microbes.


Introduction
In the gastrointestinal tract, the epithelium is the primary barrier separating the high density of microbes in the intestinal lumen from the immune cells in the lamina propria. Defects in epithelial barrier function predispose to inflammatory bowel disease (IBD) in humans and cause intestinal inflammation in animal models. 1,2 Genetic risk variants for IBD in loci linked to genes relevant for epithelial integrity have been identified by genome wide association studies (GWAS) studies (e.g. GNA12, HNF4A, MUC19 or XPB1). 3,4 In addition, there is an expanding group of monogenic defects associated with significant functional impact on intestinal epithelial barrier function. 5,6 Defects in intestinal epithelial guanylcyclase gene GUCY2C or the solute carrier family 9 member 3 (SLC9A3) encoding the sodium hydrogen exchanger NHE3 cause congenital diarrhea and Crohn's disease-like intestinal inflammation in approximately 30% of cases. 7,8 Likewise, defects in IKBKG causing impaired NF-κB signaling result in epithelial apoptosis and intestinal inflammation. 9 In patients with TTC7A-deficiency, defects in epithelial PI4K and Rho signaling cause disturbed epithelial polarization leading to multiple intestinal atresia or very early onset intestinal inflammation. [10][11][12] Recently, we reported a common NOX1 missense mutation associated with ulcerative colitis (UC) in males of Ashkenazi Jewish ancestry, in addition to a rare NOX1 mutation in a patient with early onset of intestinal inflammation. 13 NOX1 is the catalytic subunit of superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex 1, and is comprised of NOX1, p22 phox , NOXA1, NOXO1 and Rac1-GTP. [14][15][16] NOX1 is a close structural homologue of NOX2, the catalytic subunit of the multimeric NADPH oxidase complex 2 (NOX2, p22 phox , p67 phox , p47 phox , p40 phox and Rac2-GTP), the enzyme responsible for the microbicidal 'respiratory burst' in phagocytes. However, in contrast to NOX2, NOX1 is located in the membrane of the intestinal epithelial cells [17][18][19][20][21] and, may have additional functions in endothelial cells and in non-gastrointestinal epithelium. 22 Here, we investigate several loss-of-function missense variants in NOX1 that were identified by whole genome and whole exome sequencing in patients with VEOIBD. We identified eight males with six hemizygous non-synonymous NOX1 variants, with variable functional impact upon reactive oxygen species (ROS) production as compared to the previously described p.P330S and the common p.D360N (rs34688635) variants. We identified two variants with nearly complete loss-of-function and a further four variants with reduced activity. Primary patient-derived intestinal colonic organoids of the loss-of-function variant p.N122H were used to validate those findings.

Index patient
We studied a male patient of Caucasian origin who presented with bloody stools and failure to thrive at the age of two years (Table 1). He was diagnosed with IBD at the age of five years. His disease progressed from proctitis to pancolitis at the age of eight years without upper gastrointestinal involvement (Figure 1a). Histological examination of colonic specimens showed crypt architectural distortion, cryptitis and crypt abscess formation (Figure 1a). Histologic assessment of epithelial cell proliferation or goblet cell formation did not reveal marked differences compared to inflammatory controls (Supplementary Figure  S1). The patient required oral corticosteroid therapy at diagnosis, followed by thiopurine and then methotrexate therapy. After years of poorly controlled inflammation, mucosal healing was achieved with anti-TNF therapy (adalimumab). Nine years after diagnosis, his disease was complicated by Epstein Barr virus-associated haemophagocytic lymphohistiocytosis (HLH) whilst on azathioprine therapy, requiring intensive care therapy. Mutations in XIAP and SAP were excluded at that time. There was no family history of IBD.
Whole genome sequencing of the patient led to the identification of a novel hemizygous variant in the NADPH oxidase 1 NOX1; NM_007052; c.A364C; p.N122H (Supplementary Figure S2). The mutation was validated with Sanger sequencing and inherited in an X-linked manner from the patient's mother (Figure 1b). The mutation is not present in 1,678 CD, 1,089 UC patients or 1,862 non-IBD controls, or in any public exome database covering an excess of 120,000 human alleles (e.g. 1000 Genomes, Exome Aggregation Consortium ExAC), thus supporting the view that this is an extremely rare genetic variant (Supplementary Table 1). The amino acid N122 is located in the 3 rd transmembrane domain and is evolutionary highly conserved across species (Figure 1c). In silico analysis by several tools including SIFT, PolyPhen-2 and CADD, 23 predicted that the mutation causes a loss-offunction (Supplementary Table 2). Furthermore, the p.N122H variant affects all known isoforms of NOX1 (Figure 1d). Other hemizygous, homozygous or compound heterozygous variants in the index patient were considered unlikely pathogenic (Supplementary Table 3). None of these variants have been described as causative for IBD or EBV-induced hemophagocytosis.

NOX1 expression and epithelial-derived ROS
To understand the relative distribution of ROS-generating NADPH oxidases in the human gastrointestinal tract and to compare non-inflamed homeostasis with inflammatory conditions, we determined the mRNA expression pattern of several NADPH oxidases and their subunits. Consistent with previous literature, 17-20 NOX1 is highly expressed in the colonic epithelium with a gradient from ileum to the distal colon ( Figure 1e). The subunits NOXA1 and NOXO1 are constitutively expressed along the human gastrointestinal tract (Figure 1e). Whereas phagocytic NOX2 can be detected even in the non-inflamed mucosa, we observed no expression of NOX4 and very low expression of NOX5 (Supplementary Figure S3a). In parallel with the NOX1 expression gradient, we found increased ROS generation in ex vivo biopsies obtained from lower compared to the upper gastrointestinal tract suggesting that NOX1 is the primary source of superoxide in the non-inflamed colonic epithelium (Supplementary Figure S3b,c).
We visualized superoxide production of colonic crypts ex vivo using nitroblue tetrazolium chloride (NBT). Reaction of NBT with superoxide results in formation of water-insoluble blue formazan precipitates which can be observed by light microscopy (Figure 1f). Following 60 minutes incubation, we observed the deposition of formazan at the outer margin of colonic crypts indicating the constitutive release of superoxide from the colonic epithelium into the crypt lumen (Figure 1f). Co-incubation with ML171 24 or DPI, both NADPH-oxidase inhibitors, suggested a reduction of superoxide production (Figure 1g). This finding was confirmed by the luminescence assay ( Figure 1h).

Ex-vivo NOX1 biopsy and primary organoids
To assess the functional consequences of p.N122H mutation on colonic epithelial ROS production, we first assessed superoxide release in ex vivo colonic biopsies from the index patient and compared those to biopsies from patients with IBD and non-inflamed controls. Although biopsies from the NOX1 p.N122H variant patient generated low level of superoxide, this was within the range seen in other patients with quiescent IBD and noninflamed controls ( Figure 2a). However, since phagocytes that express NOX2 are present in non-inflamed biopsies (Supplementary Figure S3a) and lamina propria infiltrating phagocytes likely contribute to ROS measurements in IBD patient biopsies, the assay specificity is limited and an epithelial NOX1 defect is potentially masked.

NOX1 p.N122H reduces ROS production in primary organoids
To validate the functional impact of the genetic defect in epithelial cells, we generated primary epithelial organoids from colonic biopsy specimens of the NOX1 p.N122H patient, a male IBD control patient and a healthy female control without IBD (Figure 2b). Both IBD patients were in clinical remission at the time of the endoscopy. Colonic organoids express NOX1 mRNA, along with its subunits NOXA1 and NOXO1 and this expression did not differ between the index p.N122H patient and the IBD control patient (Figure 2c). We found high DUOX2 mRNA expression but only minimal NOX2 expression in the colonic epithelial organoids reflecting the epithelial origin and the absence of phagocytes (Supplementary Figure S3a).
Colonic epithelial organoid cells carrying NOX1 p.N122H mutation produced significantly less constitutive superoxide than control organoid cells derived from the IBD patient or a patient without inflammatory disease (Figure 2d). Of note, whereas PMA stimulation increased superoxide generation by control organoids, PMA stimulation of NOX1 p.N122H organoids did not. Even after stimulation with PMA, superoxide production by NOX1 p.N122H organoids was significantly less than baseline ROS of IBD control organoids ( Figure 2d).

Functional characterization of multiple NOX1 hemizygous variants in VEOIBD
To find additional NOX1 variants in patients with VEOIBD we screened exomes of 1,878 pediatric IBD patients. We identified 5 additional non-synonymous variants in NOX1 in separate patients in whom no other known or plausible genetic monogenic diagnosis was established ( Table 1). The missense variants were predicted to be damaging by SIFT, PolyPhen-2 or CADD scores and were rare (MAF<1%) genetic variants (Supplementary  Tables 1 and 2). All variants changed conserved amino acids (Supplementary Figure S4) and were found in regions with significant homology between NOX1 and NOX2 (Supplementary Figure S5).
To characterize the functional impact of those variants we transfected the multimeric NADPH oxidase complex 1 in the colonic epithelial cell line HCT116. Alongside the rare NOX1 variants p.N122H, p.I67M, p.R287Q, p.Q293R, p.Y470H and p.T497A that we identified in this study, we tested the previously described p.P330S variant 13 and the common polymorphism p.D360N (allele frequency European-non-Finnish, 2.4%). Three variants in patients P14, P15 and P16 affected potential splice sites which we could not assess functionally (Supplementary Tables 1, 2 and 4). We used HCT116 cells since these cells express only low levels of NOX1 subunits (data not shown) and endogenous ROS generation cannot be detected ( Figure 3a). Co-expression of wild-type NOX1 together with the subunits NOXA1 and NOXO1 resulted in superoxide generation in unstimulated and PMA-stimulated condition (Figure 3a,b). In contrast, co-expression with NOX1 variants variably diminished ROS generation. Whereas NOX1 variants p.N122H (index patient) and p.T497A completely abrogated both spontaneous and PMA-induced ROS production, the NOX1 variants p.I67M, p.R287Q, p.Q293R, p.P330S, p.D360N (common polymorphism) or p.Y470H were observed to either decrease spontaneous ROS and/or diminish PMAstimulated ROS (Figure 3a,b). Expression levels of wild-type and variant NOX1 constructs were comparable (data not shown). This suggests that the rare genetic variants affected the ROS production to a variable degree between complete loss-of-function and moderate reduction comparable to polymorphisms.

Characteristics of patients with loss-of-function NOX1 variants
We analyzed the IBD phenotype in all male patients with confirmed loss-of-function NOX1 variants (Table 1 and Supplementary Table 4). Out of the 10 male patients with hemizygous NOX1 mutations, four presented with CD, three with UC and three with IBDU. In congruency with NOX1 expression, disease location was always colonic, with two patients also having perianal disease. Median age of onset was 7.9 years (range 1.8 to 10.5 years). Medical treatment included corticosteroids, aminosalicylates, azathioprine, methotrexate or anti-TNF (infliximab or adalimumab in 4 of 8 patients) and one patient underwent ileocecal resection for stricture. No infections with intestinal pathogens were recorded. With exception of the index patient, no other severe HLH/EBV-driven pathologies were recorded.

Population based characteristics of genetic loss of function NOX1 variants
In addition to those rare variants in NOX1, common polymorphisms might similarly contribute to IBD susceptibility. Of note, the common NOX1 polymorphism p.D360N has recently been associated with UC in Ashkenazi Jewish males. 13 To determine whether this is Ashkenazi Jewish-specific or an association across several populations, we performed a genetic analysis of individuals of non-Jewish European ancestry. Although we observed a trend in the US European ancestry cohort (Table 2 and Supplementary Tables 5 and 6), neither analysis of allele counts in UK cohorts nor analysis of summed allele counts from a total of 9,513 healthy controls and 11,140 IBD patients revealed an association (Table 2). This suggests that the association of NOX1 p.D360N with UC in males is not present across different populations. Variation is also reflected by a variable distribution of p.D360N allele frequency across populations (Figure 4a).
To further understand the importance of NOX1 variation, we compared the frequency of hemizygous NOX1 missense and NOX1 loss-of-function variants (STOP codon, frameshift, predicted splice site), with those in NOX2, the causative gene of most patients with chronic granulomatous disease. The frequency of genetic missense variation in NOX1 is higher as compared to the homologous NOX2 ( Figure 4b). Similarly, the constraint metric based on the ExAC dataset indicates that loss-of-function variants in NOX1 are tolerated as indicated by a probability of being loss-of-function intolerant 25 (pLI) of 0 ( Figure 4c). In contrast NOX2 is extremely intolerant to any loss-of-function variation (pLI 0.998) 25 as are many other genes causing X-linked primary immunodeficiencies (Figure 4c). This suggests that NOX1 underwent less evolutionary pressure as compared to NOX2 or other X-linked immunodeficiency genes.
In summary, our data suggest that NOX1 is the key NADPH oxidase for baseline ROS in human colonic crypt epithelium. Complete loss-of-function variants in NOX1 are rare and not associated with a distinct IBD sub-phenotype. Population statistics suggest that loss-offunction variants in NOX1 are rare but likely tolerated.

Discussion
In this study we investigated rare X-linked variants in NOX1 in eight male IBD patients.
Functional analysis suggests a variant-dependent loss of NOX1-mediated superoxide generation. The clinical phenotype of affected patients was early onset of IBD with progressive and severe colonic disease, refractory to conventional therapy. The affected cohort demonstrates that NOX1-dependent ROS production is essential for superoxide production in colonic crypts.
Our characterization of six rare variants in NOX1 (p.I67M, p.N122H, p.R287Q, p.Q293R, p.Y470H and p.T497A) expands the recent description of the rare NOX1 variant p.P330S and the common NOX1 polymorphism p.D360N in two male VEOIBD patients. 13 Furthermore, the NOX1 variant p.R287Q, which is functionally characterized in this study, has also been described but not characterized in an independent cohort of children with VEOIBD, adding further weight to the significance of epithelial NOX1. 26 Diverse mechanisms maintain intestinal epithelial integrity, which serves as a critical barrier to luminal bacteria, viruses and fungi. 1, 2 Amongst these, NOX1 is likely to play a relevant role. 27,28 The generation of superoxide by NOX1 at the colonic epithelial border may have an anti-adhesive or anti-invasive effect on bacteria, contributing to the barrier function of the inner mucus layer within colonic crypts. Indeed, our data confirm previous findings that the gene expression of NOX1 follows the density of bacterial colonization in the gastrointestinal tract. [17][18][19][20] Reduced generation of superoxide due to genetic variants in NOX1 may affect crypt homeostasis allowing subsequent crypt colonization by luminal microbiota. NOX1derived superoxide also influences intestinal epithelial differentiation and goblet cell function. [29][30][31] Colonic mucus produced by goblet cells provides an important barrier between the intestinal microbiota and the colonic epithelium, maintaining sterility at the epithelial border. 32 The importance of NADPH oxidases for barrier function is also exemplified by DUOX2, an NADPH oxidase family member, which is expressed throughout the GI tract. Notably, defects in DUOX2 have been recently described as a potential susceptibility factor in patients with VEOIBD. 13,33 In animal models, defects in the Drosophila duox2 homolog or in Zebrafish increased susceptibility against enteropathogens. 34,35 Whereas Duoxa−/− mice on thyroid hormone replacement showed normal growth and no evident phenotype, 36 recent studies suggested that colonization of segmented filamentous bacteria in the ileum, bacterial translocation into mesenteric lymph nodes, and Salmonella typhimurium systemic dissemination, is dependent on DUOX activity. 37 Although DUOX2 expression is inducible and is increased in patients with intestinal inflammation 33, 38 thereby potentially contributing to epithelial stress and intestinal tissue damage, these data indicate that DUOX activity may a functional mechanism that restricts bacterial colonization at the mucosal interface.
Interestingly, NOX1 activity regulates DUOX2 expression in the intestinal epithelium during C. rodentium infection. 39 Although loss of epithelial Cyba protected against C. rodentium and L. monocytogenes infection, this is likely an indirect effect due to the resulting overgrowth of H 2 O 2 -producing lactobacilli. 39 This suggests an unexpected level of functional hierarchy and complex collaboration between NOX1 and DUOX2. 39 Despite the complete loss of brush border ROS that has been observed in two NOX1 variants (p.N122H and p.T497A), we have not observed an increased susceptibility to intestinal infections such as Salmonella or Yersinia. This is in agreement with recent data suggesting that lack of Nox1 does not affect infection susceptibility in a mouse model of Salmonella typhimurium-induced typhlitis. 40 Nonetheless, larger patient numbers with complete loss-offunction NOX1 variants are required to more definitively determine the impact of these mutations on susceptibility to infection. Interestingly, the patient with the p.N122H mutation developed severe HLH in the context of EBV infection whilst on azathioprine therapy. 41 Viral spreading has been shown to be increased in Nox1-deficient mice infected with influenza virus, 42 implicating a role for NOX1 in viral control. Again, larger patient numbers are required to fully understand the penetrance of the NOX1 variant phenotype.
Given the relatively high load of non-synonymous mutations of NOX1 compared to NOX2 and the large number of DUOX2 mutations in humans, it is likely that NOX/DUOX enzymes have partially redundant functions. It is therefore likely that both NOX1 and DUOX2 play a role in susceptibility to intestinal inflammation, and loss-of-function variants alone cause not highly penetrant Mendelian type disorders. In keeping with a second hit hypothesis, Nox1-deficiency in Il10-deficient mice resulted in earlier and more severe intestinal pathology, but loss of Nox1 alone did not cause intestinal inflammation. 30 Similarly, patients with congenital secretory diarrhea due to activating mutations in the guanylate cyclase C (GC-C) 7,43 or inactivating mutations in SLC9A3 encoding for the sodium/proton exchanger NHE3, 8 a downstream target of GC-C, may also present with an IBD phenotype. In these patients the decreased epithelial uptake of sodium results in changes of mucosal ion content and pH, and Nhe3-deficiency in mice leads to an altered composition of the intestinal microbiota. 44,45 This dysbiosis drives intestinal inflammation in a T cell-dependent model of intestinal inflammation. 46 Our studies suggest that loss of ROS at the colonic crypt may have a similar effect on barrier function.
Altogether these data suggest a highly context specific role of epithelial NADPH oxidases in intestinal homeostasis. Whilst in some animal models, a lack of Il10 and Nox1 increases epithelial stress and susceptibility to colitis 30 , in other instances, lack of NADPH oxidase activity is protective. Indeed a protective role of Nox1 and Duox2 is suggested in a mouse model of ileocolitis caused by lack of protective glutathione peroxidase factors. [47][48][49] In this model GPx1-and GPx2-knockout leads to oxidative stress caused by Nox1 and Duox2 with a dominant role of Nox1 for crypt epithelial apoptosis. 48,49 This oxidative stress is dependent on the presence of the intestinal microbiota since germ free mice are protected and colitis develops within one week of bacterial colonization. 50 Given the role of NOX1 as a modifier of intestinal microbiota, we can only speculate whether IBD patients with loss-offunction NOX1 variants would benefit from therapeutic microbiome-altering strategies (e.g. antibiotics, nutrition, probiotics or fecal transplant). Further studies characterizing the human gut microbiota in the setting of NOX1 deficiency are required.
It is possible that the human population genetics reflect this context specific role of NOX1.
Whereas in populations like Ashkenazi Jews, the hemizygous NOX1 p.D360N variant is associated with UC, in European descent populations it is not. Substantially stronger genetic variation of NOX1 compared to NOX2 suggests that the overall impact on disease susceptibility of these genes is significantly different despite the high degree of protein conservation and similarity. It is even possible, that the degree of genetic variation in NOX1 is caused by environmental factors such as infections where reduced ROS is protective.
In conclusion, we report six novel variants in the NOX1 gene in patients with IBD, two of which are associated with complete loss-of-function of the gene product and with loss of ROS production. Our study illustrates the complexity of epithelial barrier function in the pathogenesis of IBD and the need better to understand the functional impact of ROS production at the colonic crypt epithelium as a cause of colonic inflammation.

Human subjects
The recruitment of participants was approved by relevant ethics committees or institutional review boards at the individual contributing institution. Written informed consent was obtained from all patients/legal guardians and healthy controls.
Patients with IBD (including the index patient P1) and controls without intestinal inflammation were prospectively recruited as a part of the Oxford IBD cohort study or the Oxford Gastrointestinal Illness Biobank (REC 09/H1204/30 & REC 11/YH/0020).
Replication genotyping of the p.N122H variant was performed in 2,767 IBD patients and 1,862 non-IBD controls. These individuals were enrolled by the UK IBD Genetics Consortium (http://www.ibdresearch.co.uk/). Exomes of 1,878 pediatric onset IBD patients without genetic diagnosis were screened for additional rare NOX1 variants. Samples were previously genotyped as part of the COLORS in IBD study (n = 150; Oxford, UK), the NEOPICS consortium (n = 545; Toronto, Canada, www.neopics.org) or the RISK cohort 51 (n = 1,183; Atlanta, USA). Genetic association testing of NOX1 p.D360N (rs34688635) was conducted on genotype data from IBD patients of non-Jewish European ancestry from the following cohorts: the European ancestry exome chip dataset 52 (n = 10,523), the Oxford IBD cohort study (n = 513) and the COLORS in IBD study (n = 104). Sequence data from healthy individuals were obtained from the European ancestry exome chip dataset 52 (n = 5,726) and the INTERVAL study (n = 4,436; www.intervalstudy.org.uk). Population controls were also obtained from the UK10K project (www.uk10k.org).

Genotyping
Whole genome sequencing and NOX1 variant p.N122H identification-The index mutation in NOX1 was identified by whole genome sequencing as part of the WGS500 project. 53 DNA was analyzed from peripheral blood mononuclear cells. In summary, whole genome sequencing was performed on 3.5-7.5 ng DNA on either the Illumina HiSeq2000 or the HiSeq2500 run in standard mode using v2.5 or v3 sequencing chemistry. The genomic DNA was fragmented, end-paired, A-tailed and adapter-ligated before size selection and amplification for a multiplexed library preparation as described in Taylor et al. 53 The libraries were paired end sequenced and sequenced to an average coverage between 27 and 40 quality reads per base. Resulting 100 base pair reads were mapped to the GRCh37d5/hg19 human reference sequence using Stampy and the SNVs and short InDels were called with Platypus. 54 Variant files were analyzed using QIAGEN's Ingenuity® Variant Analysis™ software (www.qiagen.com/Ingenuity, QIAGEN Redwood City). High quality calls were analyzed. For candidate analysis, we investigated a gene list of known variants associated with monogenic forms of IBD. 5 Filters for variant frequency (<1%; 1000 Genomes Project) and predicted deleterious filters (SIFT and PolyPhen) were applied to prioritize high quality calls for non-synonymous and likely pathogenic variants.
Replication genotyping of the p.N122H variant-Replication genotyping on the Sequenom plex was carried out at the Sanger Institute.
Whole exome sequencing-Regeneron performed whole exome sequencing on a cohort of 1,183 probands with pediatric onset IBD (ages 0-18.5 years), including their affected and unaffected parents and siblings, where available (total samples = 2,704). Sample preparation, whole exome sequencing, and sequence data production were performed as previously described. 55 The exomes of RISK cohort study participants (n = 545) were captured using SureSelect Human All Exon kit v2 (Agilent Technologies), using 3 µg of genomic DNA. Subsequently, captured libraries were sequenced on Illumina HiSeq2000 by 100-base pair paired-end (PE) sequencing. Image processing and base calling was performed using standard Illumina software. Raw sequence reads were mapped relative to the human genome Schwerd

Histology
Historic routine haematoxylin and eosin histology sections from the patient identified to harbor the NOX1 p.N122H mutation and IBD controls were analyzed. Staining of goblet cells or the proliferation marker Ki67 was performed on archived paraffin-embedded colonic epithelial biopsies. Specimens were stained using an Alcian blue and periodic acid-Schiff's reagent staining kit (Clin-Tech Limited, UK) or rabbit monoclonal antibody to Ki67 (clone SP66, AbCam, Cambridge, USA), respectively. Nuclear fast red and haematoxylin (Sigma-Aldrich, Gillingham, UK) were used to counterstain cell nuclei.

Protein sequence alignment
Multiple sequences were aligned using ClustalW2. 56

Expression analysis
Colonic biopsies were stored in RNAlater (Qiagen) prior to analysis. RNA was extracted using RNAeasy kit (Qiagen), transcribed in cDNA with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and expression data were obtained using TaqMan primers (Life Technologies) for NOX 1-5, NOXO1, NOXA1, DUOX1 and DUOX2. Results were normalized to GAPDH expression. Relative gene expression was calculated using the ΔCt method.

ROS assays
ROS generation by colonic epithelial biopsies, organoid cells and transiently transfected HCT116 cells was measured using L-012 enhanced chemiluminescence (Wako laboratories).
For standardization, all intestinal epithelial biopsies were collected using Olympus Endojaw needle forceps with a cup-opening size of 7.2 mm and analyzed within 90 minutes of collection with minimal handling. Organoids were trypsinised into single cell suspension using TrypLETM express (Gibco), counted and equal cell numbers dispensed into white 96well plates.
For microscopy of ROS-producing epithelial cells, ex vivo colonic biopsies were incubated with 100 µg/mL nitroblue tetrazolium chloride (NBT, Sigma) in PBS-glucose (1 g/L) with or without NADPH-oxidase inhibitors. After 60 minutes incubation, formation of formazan was analyzed by light microscopy (Carl Zeiss Axioskop2, 200× magnification) on glass slides without cover slips. Images were photographed using Q-Imaging software. Microscopy images were cropped and analyzed using ImageJ software. 59 Disease activity amongst IBD patients was characterized as active or quiescent on the basis of clinical symptoms (diarrhea, rectal bleeding, and abdominal pain), endoscopic features (mucosal vascular pattern, bleeding, or ulceration), histological features (cryptitis and crypt abscesses, lamina propria polymorphomuclear cell infiltrate), and serum biomarkers (Creactive protein).

Data and statistical analysis
All the data analyses were performed using GraphPad Prism version 5.02 (GraphPad Software, San Diego, California, USA) or R (The Foundation for Statistical Computing, R version 3.2.2). Unpaired data were compared using Mann-Whitney U test and paired data were compared with Wilcoxon signed-rank test. Significance of differences between multiple groups was performed with ANOVA and post-test correction with Dunnet. Genetic association testing in cases and controls was conducted with the ALLELIC test. 60 Pooled analyses were performed on the summed allele counts of different cohorts. P-values of less than 0.05 after correction for multiple testing were considered significant.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
We thank all our patients and their families for participation in this study. We like to thank all contributors of the  . Results are derived from 17 patients with colonic Crohn's disease (active disease n = 10), 11 patients with ulcerative colitis (active disease n = 6), 16 non-inflamed controls and the patient with NOX1 p.N122H mutation. One symbol represents the mean of 1-3 pan-colonic biopsy specimens (both right and left sided) per patient. Active versus quiescent disease dichotomised according to composite assessment including endoscopic appearance (mucosal ulceration, loss of vascular pattern, bleeding), biomarkers (CRP), and