Clinical and Genomic Correlates of Neutrophil Reactive Oxygen Species Production in Pediatric Patients With Crohn's Disease

Abstract

Background & aims: Individuals with monogenic disorders of phagocyte function develop chronic colitis that resembles Crohn's disease (CD). We tested for associations between mutations in genes encoding reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, neutrophil function, and phenotypes of CD in pediatric patients.

Methods: We performed whole-exome sequence analysis to identify mutations in genes encoding NADPH oxidases (such as CYBA, CYBB, NCF1, NCF2, NCF4, RAC1, and RAC2) using DNA from 543 pediatric patients with inflammatory bowel diseases. Blood samples were collected from an additional 129 pediatric patients with CD and 26 children without IBD (controls); we performed assays for neutrophil activation, reactive oxygen species (ROS) production, and bacteria uptake and killing. Whole-exome sequence analysis was performed using DNA from 46 of the children with CD to examine associations with NADPH gene mutations; RNA sequence analyses were performed using blood cells from 46 children with CD to test for variations in neutrophil gene expression associated with ROS production.

Results: We identified 26 missense mutations in CYBA, CYBB, NCF1, NCF2, and NCF4. Patients with CD who carried mutations in these genes were 3-fold more likely to have perianal disease (P = .0008) and stricturing complications (P = .002) than children with CD without these mutations. Among patients with CD with none of these mutations, 9% had undergone abdominal surgery; among patients with mutations in these NADPH oxidase genes, 31% had undergone abdominal surgery (P = .0004). A higher proportion of neutrophils from children with CD had low ROS production (47%) than from controls (15%) among the 129 patients tested for ROS (P = .002). Minor alleles of the NADPH genes were detected in 7% of children with CD whose neutrophils produced normal levels of ROS vs 38% of children whose neutrophils produced low levels of ROS (P = .009). Neutrophils that produced low levels of ROS had specific alterations in genes that regulate glucose metabolism and antimicrobial responses.

Conclusions: We identified missense mutations in genes that encode NADPH oxidases in children with CD; these were associated with a more aggressive disease course and reduced ROS production by neutrophils from the patients.

Keywords: Genetic Variant; IBD; Neutrophil Oxidative Burst; WES.

Figure 1. Flow Chart for Patient Populations with Whole Exome Sequencing and Neutrophil Function Studies

Whole exome sequencing (WES) was completed for 543 pediatric IBD patients, with a median (range) age at diagnosis of 8(0–18) years. These data defined NADPH oxidase gene mutations in this population, and associations with clinical phenotype. Neutrophil function studies including measures of ROS production and bacterial killing were completed for 129 pediatric CD patients with a median (range) age at diagnosis of 11(1–20) years, and 26 non-IBD controls. 46 CD patients had both WES and neutrophil function testing completed to test for associations between NADPH oxidase gene mutations and neutrophil ROS production and bacterial killing. A sub-group of CD patients (n=40) and non-IBD controls (n=6) with neutrophil function testing completed also had RNASeq performed to define the neutrophil global pattern of gene expression in patients stratified by ROS production.

Figure 2. N-Formyl Peptide Induced Neutrophil ROS Production and Core NADPH Oxidase Gene Mutations in Crohn Disease Patients

Peripheral blood samples were stimulated with N-formyl peptide (FMLP) and neutrophil ROS production was measured by flow cytometry using the dihydrorhodamine 123 (DHR) dye. A) Representative FACS plot for unstimulated (black) and stimulated (red) I) control, II) low ROS CD and III) high ROS CD samples are shown. B) FMLP induced neutrophil ROS production measured by the DHR MFI in healthy controls (n=26), and Crohn Disease patients (CD, n=129) is shown as the median (IQR). Differences between the groups was tested by unpaired t-test of log transformed values. C) Histograms demonstrating the number of subjects with each level of FMLP induced neutrophil ROS production in groups of 100 for the DHR MFI is shown, with the point for a DHR MFI of 360, the 15^th percentile within controls, on the x-axis for each group indicated by the arrows, and those with low ROS production indicated by the blue bar. D) The frequency of subjects with low FMLP induced ROS production defined by a DHR MFI less than 360 is shown. Differences between groups was tested using the chi2 test. E) FMLP induced neutrophil ROS production is shown as the median (IQR) for CD patients lacking any potentially damaging variants in core NADPH oxidase genes (no mutation), or patients carrying the indicated missense mutations. Differences between the no mutation group (n=20) and the groups homozygous for the CYBA Y72H variant (n=16) or carrying one of the other NADPH oxidase gene mutations (NOX2, n=10) were tested by the unpaired t-test. For each of the other missense mutations, only 1–3 patient samples were tested, with the results shown for each. F) The frequency of minor allele carriage is shown for CD patients stratified by a FMLP induced neutrophil ROS MFI of 360, ROS Nl versus ROS Lo. Differences between the ROS Nl versus ROS Lo groups were tested by chi2 test for CYBA Y72H homozygous carriage alone, or for carriage of CYBA V76M, NCF1 S99G, NCF2 R38Q, NCF2 T361S, NCF2 N419I, or NCF2 P454S.

Figure 3. Metabolic and Immune Functions Differentially Expressed in CD Neutrophils with Normal and Low ROS Production

Candidate gene prioritization through functional enrichment analysis, using genes differentially regulated between normal ROS CD neutrophils and controls as the test set and a glycolysis gene list as the training set, ranked test genes in the order of their importance in context of glycolytic functions. Genes with significant glycolytic functional enrichment were submitted to ToppCluster to identify mechanistic enrichments. A) Pathways and mechanisms enriched in genes that were upregulated in low ROS CD neutrophils vs control neutrophils or normal ROS CD neutrophils vs control neutrophils with significant ranking in glycolysis candidate gene analysis (part of the first pipeline). B) Pathways and mechanisms enriched in genes that were downregulated in low ROS CD neutrophils vs control neutrophils or normal ROS CD neutrophils vs control neutrophils with significant ranking in glycolysis candidate gene analysis (part of the first pipeline). C) Pathways and mechanisms enriched in genes that were upregulated in low ROS CD neutrophils vs normal ROS CD neutrophils with significant ranking in glycolysis candidate gene analysis (second pipeline).

Figure 4. Core Genes and Functions which Distinguish CD Neutrophils with Normal and Low ROS Production

A 59-gene ROS panel was generated using an unpaired T-test to compared low ROS CD neutrophils to normal ROS CD neutrophils, assuming unequal variance with significance cutoffs p<0.05 and FC>1.5. A) Hierarchical clustering of the 59-gene panel predictive of Low ROS status in CD neutrophils generated using a Pearson’s Centered distance metric and Average linkage rule to cluster both samples and genes. B) ROC analysis of the discriminant power of the 59-gene panel. C) The 52 genes upregulated in low ROS CD neutrophils were submitted for ontological enrichment analysis through ToppCluster.cchmc.org and Cytoscape, with pathways (bright green), biological processes (bright blue), and previously published microarray gene sets (light green) presented.