Gastroenterology 2014;147:680–689
Variants in Nicotinamide Adenine Dinucleotide Phosphate Oxidase
Complex Components Determine Susceptibility to Very Early Onset
Inflammatory Bowel Disease
Sandeep S. Dhillon,1,2 Ramzi Fattouh,1 Abdul Elkadri,1,2,3 Wei Xu,4 Ryan Murchie,1
Thomas Walters,1,3 Conghui Guo,1 David Mack,5 Hien Q. Huynh,6 Shairaz Baksh,6
Mark S. Silverberg,2,7 Anne M. Griffiths,1,3 Scott B. Snapper,8 John H. Brumell,1,2,9 and
Aleixo M. Muise1,2,3
1
SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, The Hospital for Sick Children,
Toronto, Ontario, Canada; 2Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; 3Division of
Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Toronto, The Hospital for Sick Children,
Toronto, Ontario, Canada; 4Princess Margaret Hospital and Dalla Lana School of Public Health, University of Toronto, Toronto,
Ontario, Canada; 5Division of Pediatric Gastroenterology, Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada;
6
Division of Pediatric Gastroenterology, Stollery Children’s Hospital, Edmonton, Alberta, Canada; 7Mount Sinai Hospital
Inflammatory Bowel Disease Group, University of Toronto Group, Zane Cohen Center for Digestive Diseases, Toronto, Ontario,
Canada; 8Division 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; and 9Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
BASIC AND
TRANSLATIONAL AT
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.
Keywords: VEOIBD; CGD; Genetics; Phagocytes.
C
hronic granulomatous disease (CGD) is a severe immunodeficiency caused by genetic defects in components of the NOX2 nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase complex (Figure 1). The mutations identified in the NADPH oxidase gene in CGD patients
mostly result in the complete loss of phagocytes’ ability to
mount the sufficient respiratory burst required to kill invading pathogens, leading to a severe immunodeficiency with
Abbreviations used in this paper: CGD, chronic granulomatous disease;
CYBA, cytochrome b light chain and encodes p22phox protein; CYBB,
cytochrome b-245 or NADPH oxidase 2 and encodes Nox2 or gp91phox;
GFP, green fluorescent protein; IBD, inflammatory bowel disease; MSMD,
Mendelian susceptibility to mycobacterial disease; NADPH, nicotinamide
adenine dinucleotide phosphate; NBT, nitroblue tetrazolium; NCF1,
neutrophil cytosol factor 1 and encodes p47 phox protein; NCF2, neutrophil
cytosol factor 2 and encodes p67 phox protein; NCF4, neutrophil cytosol
factor 4 and encodes p40 phox protein; PID, primary immunodeficiency;
RAC1 and 2, Ras-related C3 botulinum toxin substrate 1 and 2; ROS,
reactive oxygen species; SNP, single nucleotide polymorphism; VEOIBD,
very early onset inflammatory bowel disease; VEOUC, very early onset
ulcerative colitis.
© 2014 by the AGA Institute
0016-5085/$36.00
http://dx.doi.org/10.1053/j.gastro.2014.06.005
September 2014
VEOIBD and the NOX2 NADPH Oxidase Genes
681
recurrent infections.1,2 Interestingly, up to 40% of CGD patients develop intestinal inflammation that is very similar to
the colitis observed in inflammatory bowel disease (IBD)
patients.3–5 Very early onset inflammatory bowel disease
(VEOIBD) is defined as disease onset before 6 years of age6,7
and has increased in incidence dramatically during the past
decade.8 It primarily affects the colon,1,2 and the disease
extent and location and endoscopic and histologic
BASIC AND
TRANSLATIONAL AT
Figure 1. Functional variants in the NADPH oxidase
genes in VEOIBD and CGD.
(A) Direct-exon sequencing
of VEOIBD yielded a number of novel and rare variants with changes in
function and resulting altered NADPH oxidase
complex activity. A damaging G364R variant of
gp91phox was found near
the FAD-binding domain. In
addition, a R90H variant in
the PI(3,4)P2-binding domain of p47phox, which was
previously shown to result
in reduced ROS production, was found in a subset
of patients. p67phox was
shown to have many functional coding variants, most
notably in protein–protein
binding; these were R38Q,
H389Q, N419I, and G501R,
decreasing binding to
Rac2, Vav1, p40phox, and
p47phox, respectively. R30
8Q, a rare variant in the PB1
domain of p40phox, was
shown to reduce binding to
p67phox. Lastly, 2 noncoding variants were found
to reduce protein expression of Rac2 and p22phox.
(B)
Representation
of
NADPH oxidase genes
variants found in VEOIBD
(purple star) and mutations
in CGD (red circle).
appearance of the colonic inflammation often resembles the
colonic disease observed in CGD.
Recent large-scale genetic studies have identified >160
genetic loci that affect risk of developing IBD.9–11 However,
the causal genes in the loci, the mechanisms by which these
variants contribute to IBD pathogenesis, and the influence
on specific IBD phenotypes, are mostly unclear. Common
single nucleotide polymorphisms (SNPs) in the NADPH
682
Dhillon et al
oxidase complex are associated with adult-onset IBD,
including neutrophil cytosol factor 4 and encodes p40 phox
protein (NCF4)12–14 and Ras-related C3 botulinum toxin
substrate 1 and 2 (RAC1 and 2).12,15 Additionally, in a recent
study,12 a novel variant of p67phox (encoded by neutrophil
cytosol factor 2 and encodes p67 phox protein [NCF2]) that
reduced binding to RAC2 was uniquely associated with
VEOIBD.12 Therefore, we investigated the role of these
NADPH oxidase genes in VEOIBD through Tag SNP genotyping and targeted exome sequencing and identified functional variants that reduced oxidative burst, altered NADPH
oxidase complex protein–protein interaction, or decreased
gene expression. Overall, our study revealed that functional
variants in the NOX2 NADPH oxidase complex genes that do
not cause an overt immunodeficiency are associated with
VEOIBD susceptibility.
Methods
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TRANSLATIONAL AT
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 a cohort of VEOIBD patients and
validated in a replication cohort. The second approach was to
analyze a subset of VEOIBD patients previously genotyped for
Tag SNPs covering the NADPH oxidase genes and to replicate
associated variants in an independent VEOIBD cohort.
Study Design
Targeted Exome Sequencing. One hundred and
twenty-two VEOIBD patients were sequenced using targeted
Gastroenterology Vol. 147, No. 3
enrichment of the exon-coding for the NADPH oxidase genes
(CYBA, CYBB, NCF1, NCF2, NCF4, RAC1, RAC2) using Agilent
SureSelect target enrichment and sequenced on the Illumina
HiSeq 2000/2500 with exon primer and sequencing protocols
designed by the Beckman Coulter Genomics (http://www.
beckmangenomics.com/) as described previously17 (Figure 2).
Sanger sequencing and Taqman sequencing were used to verify
all genetic variants and SNPs identified in the NADPH oxidase
genes at The Centre for Applied Genomic (TCAG; http://www.
tcag.ca). A validation cohort of 480 healthy controls and the
replication cohort consisting of 164 VEOIBD patients and 980
healthy control individuals genotyped using Taqman at the
Centre for Applied Genomics, The Hospital for Sick Children, as
described previously.12,15
Tagged
Single
Nucleotide
Polymorphisms
Genotyping. In order to examine intronic variants not
covered in the targeted exome sequencing approach (Figure 2),
a VEOIBD subset of patients identified in a previous NADPH
oxidase gene Tag SNP study were examined for association
with VEOIBD. Briefly, 72 Tag SNPs in the NADPH oxidase genes
(4 in CYBA, 6 in CYBB, 14 in NCF2, 20 in NCF4, 10 in RAC1, and
18 in RAC2) were selected from the International HapMap
Project (www.hapmap.org) phase II, data release 23a (with a
minor allele frequency >1%) as part of an Illumina Goldengate
Custom Chip, as described previously.12,15 From that study, 159
VEOIBD patients were identified and compared with 913
healthy controls. SNPs associated with VEOIBD were replicated
in a cohort consisting of 633 subjects (153 VEOIBD patients
and 480 healthy controls) using Taqman at the Centre for
Applied Genomics, The Hospital for Sick Children.
Analysis. Single nucleotide and insertion/deletion variants identified by either targeted exome sequencing or Tag SNP
genotyping were validated by Sanger sequencing. Function and
minor allelic frequency were searched for using National Heart,
Lung and Blood Institute Exome Sequencing Project Exome
Variant Server (http://evs.gs.washington.edu/EVS/),18 National
Center for Biotechnology Information dbSNP (http://www.
Figure 2. Flow chart of
genetic studies.
September 2014
683
Variables
Constructs and Antibodies. Myc-tagged human
p67phox complementary DNA was cloned into the pCDNA3
vector (Invitrogen, Carlsbad, CA), as described previously12;
green fluorescent protein (GFP)–tagged human p40phox complementary DNA was obtained from the laboratory of Dr John
Brumell and GFP-tagged human p47phox complementary DNA
was obtained from OriGene (pCMV6-AC-GFP, RG201233). Mutations in N419I and G501R of p67phox and in R308Q of p40phox
were generated by site-directed mutagenesis using the
QuikChange II Site-Directed Mutagenesis Kit according to
manufacturer’s instructions. All construct sequences were
verified using Sanger sequencing. For Western blot analysis,
primary antibodies used were mouse monoclonal anti-Myc
(Millipore, Billerica, MA), mouse monoclonal anti-GFP (Medimabs, Mont Royal, Canada), and polyclonal p47phox (R360,
rabbit; from the laboratory of Dr John Brumell). Secondary
antibodies used were goat anti-mouse horseradish peroxidase.
Enhanced chemoluminescence (Pierce, Rockford, IL) was used
to visualize protein bands.
Transient Transfection. HEK293T cells were maintained in Dulbecco’s modified Eagle medium containing 10%
heat-inactivated fetal bovine serum and antibiotic-antimycotic
(Sigma, St Louis, MO) at 37 C in a 5% CO2 humidified incubator. Approximately 1.5 106 cells were plated per 100-mm
tissue culture dish and grown overnight to achieve 60%–70%
confluence. The following day, cells were cotransfected with
plasmids containing p67phox, p47phox, or p40phox complementary DNA using the calcium phosphate transient transfection method according to manufacturer’s instructions. Six
hours post transfection, the media was replaced with normal
tissue culture media. Twenty-four hours post transfection, cells
were lysed using standard protein lysis.
Coimmunoprecipitation Studies
Cells lysates were incubated with 2 mg anti-Myc (Millipore)
or anti-GFP (Medimabs) antibody for 1 hour at 4 C. Forty microliters of Protein G-agarose (50% mixture) (Sigma) was
added to this antibody-lysate solution and incubated for an
additional hour at 4 C. The beads were isolated by centrifugation and washed 4 times with lysis buffer. For Western blot
analysis, beads were boiled at 95 C for 5 minutes in 5 sodium
dodecyl sulfate sample buffer, centrifuged, and then loaded into
the gel. All densitometry measurements to indicate protein
expression were measured using ImageJ software. Electronic
images of Western blots were analyzed; band intensity levels of
binding partners, as measured by ImageJ, were normalized to
that of the immunoprecipitated protein.
Nitroblue Tetrazolium. Venous blood was collected in
potassium-EDTA tubes where polymorphonuclear cells were isolated and purified using PolymorphPrep (CosmoBio USA, Carlsbad, CA), according to the manufacturer’s instructions. The NBT
colorimetric assay was performed by aliquoting 1.5 106 polymorphonuclear cells per well of a 24-well plate in 500 mL Hank’s
Balanced Salt Solution. Cells were allowed to adhere at 37 C for
30 minutes. Media were replaced with medium with each stimulant: phorbol myristate acetate A (P8139, 1 mM; Sigma), Zymosan
A (Z4250, 20 mg/mL; Sigma), and diphenyleneiodonium chloride
(D2926, 10 mM; Sigma). Three hundred microliters of NBT solution (Sigma; N5514, 10-mg tablet suspended in Hank’s Balanced
BASIC AND
TRANSLATIONAL AT
ncbi.nlm.nih.gov/projects/SNP/),19 National Institute of Environmental Health Sciences FuncPred (http://snpinfo.niehs.nih.
gov/snpinfo/snpfunc.htm),20 Polyphen2 (http://genetics.bwh.
harvard.edu/pph2/),21 SIFT (http://sift.jcvi.org/),22 FastSNP
(http://fastsnp.ibms.sinica.edu.tw/),23 Human Splicing Finder
(http://www.umd.be/HSF/),24 and pfSNP (http://pfs.nus.edu.
sg/).25
Setting. Patients included in the study were recruited
from the Inflammatory Bowel Disease Centre at The Hospital
for Sick Children, University of Toronto, and were diagnosed
with VEOIBD between the years 1994 and 2012, with a
confirmed diagnosis before 6 years of age (definition based on
our recent modification4 to the Paris classification26). All patients were found to have typical IBD without any clinical,
histologic, or biochemical evidence of immunodeficiency.
Patients did not have evidence of chronic granulomatous
disease (CGD). CGD is caused by mutations in any 1 of 5 genes,
including X-linked CGD caused by mutation in the gene
encoding p91-phox (CYBB; 300481); autosomal recessive cytochrome b-negative CGD (233690), caused by mutations in the
CYBA gene (608508); autosomal recessive cytochrome
b–positive CGD type I (233700), caused by mutation in the
NCF1 gene (608512); autosomal recessive cytochrome
b–positive CGD II (608515), caused by mutation in the NCF2
gene (608515); and autosomal recessive cytochrome
b–positive CGD type III (613960), caused by mutation in the
NCF4 gene (601488) (OMIM: http://www.omim.org/entry/
306400). VEOIBD patients did not have CGD as clinically, biochemically, or genetically defined, with no previously described
disease causing CGD mutation for either X-lined or 2 CGD
mutations for autosomal recessive disease, no atypical or
chronic infection, and no diagnostically deficient neutrophil
burst as measured by nitroblue tetrazolium (NBT) or dihydrorhodamine. Therefore, these VEOIBD patients do not have
CGD and would not require prophylactic antibiotics or other
CGD specific treatments.
Participants. This was a cohort study that examined the
genetics of VEOIBD patients. All VEOIBD subjects were
recruited from The Hospital for Sick Children, Toronto, Canada,
and had a confirmed diagnosis of IBD before 6 years of age,
based on our recent modification4 to the Paris classification,26
and were therefore classified as VEOIBD.4 Patients with a
known immunodeficiency, including CGD, were excluded from
the study. Phenotypic information and DNA samples were obtained from study subjects with approval of the Institutional
Review Ethics Board for IBD genetic studies at The Hospital for
Sick Children in Toronto. Replication cohorts had Ethics Board
approval for genetic and phenotypic studies at the individual
institutions. Written informed consent was obtained from all
participants. The healthy controls were obtained from the
Centre for Applied Genomics (Ontario Population Genomics
Platform; plates used: 1–15; a complete description of this
control population can be found at http://www.tcag.ca/cyto_
population_control_DNA.html).
To conduct systematic quality control on the raw genotyping data, we examined SNPs genotyped for the initial cohort
only as reported previously.12,15 No SNP deviated significantly
from Hardy-Weinberg Equilibrium in the controls (P < .001).
The pediatric and healthy control cohorts along with strict
quality-control measures were carried out as described previously for this cohort.12,27
VEOIBD and the NOX2 NADPH Oxidase Genes
5.8 (2.2–15)
4.54 105
7.8 (1.5–40)
CI, confidence interval; MAF, minor allele frequency; OR, odds ratio; VEOCD, very early onset Crohn’s disease.
5.3 (1.6–17.3)
Dominant
VEOCD
<1
2.05 103
4.11 103
1.8 (1.1–2.8)
4.3 (1.4–13)
1.7 102
6.89 103
1.1 (0.5–2.5)
2.8 (0.3–23)
2.7 (1.4–5.2)
5.3 (1.3–21.2)
Dominant
Dominant
VEOUC
VEOUC
<1
2.13 103
9.43 103
7.8 101
3.2 101
7.4 102
0.95 (0.5–1.8)
1.9 (1.2–3.2)
8.7 101
P Value
OR (95% CI)
P value
1.11 102
Dominant
VEOIBD
6.0
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 control individuals and replicated in an independent replication cohort.
We first examined 3 SNPs that were associated with
VEOIBD in the targeted exome sequencing discovery cohort
and in the combined analysis (Table 1 and Figure 3). This
included an intronic RAC1 SNP (rs35761891) conferring a
stronger GATA-1 transcription-factor binding site that was
associated with both VEOIBD and VEOUC. An exonic NCF2
SNP (rs35012521) resulting in p67phox N419I variant was
associated with VEOUC (Pcombined ¼ 6.89 103; odds
ratio ¼ 4.3; 95% confidence interval: 1.4–13). And a promoter SNP in CYBA (rs72550704) causing a loss of an
SP1 transcription factor binding site23 implicated in upregulation of gene expression28 was associated with very
early onset Crohn’s disease (Pcombined ¼ 4.54 105; odds
ratio ¼ 5.8; 95% confidence interval: 2.2–15).
In our VEOIBD discovery targeted exome sequencing cohort,
we also identified 8 variants (5 SNPs and 3 novel variants)
predicted to result in NADPH oxidase complex dysfunction that
Intronic
Strong GATA binding site23
Reduces ROS
Coding
N419I
Reduces binding to NCF4
Promoter SP1 site loss23
Reduces p22phox expression
Targeted Exome Sequencing
Table 1.Replicated SNPs Identified by Targeted Exome Sequencing
BASIC AND
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Results
RAC1
(RAC1)
rs35761891
NCF2
(p67phox)
rs35012521
CYBA
(p22phox)
rs72550704
Discovery cohort
Association analyses of the discovery and replication cohorts were used to test associations of the NADPH oxidase SNPs
and variants with VEOIBD, very early onset Crohn’s disease,
and very early onset ulcerative colitis (VEOUC) vs healthy
controls. Logistic regression analysis was applied for an additive model and Pearson c2 tests were applied for dominant and
recessive models. This analysis was done using the Goldenhelix
(SVS 7.6.4) program. The combined cohort was analyzed using
the same protocol with pooled population data from both
cohorts. For densitometry measurement statistics, the Bonferroni post-test was used with n ¼ 3, with each full experiment
representing 3 triplicate experiments.
OR (95% CI)
Statistical Methods
P value
Replication cohort
Combined analysis
Salt Solution at 1 mg/mL) was added and the total mixture was
incubated at 37 C in 5% CO2 humidified incubator for 30 minutes.
Cells were washed and fixed in 2.5% paraformaldehyde fixative
and then lysed and solubilized in 240 uL 2M KOH and 280 uL
dimethyl sulfoxide. Absorbance was then measured at 570 nm.
Data Sources Measurement. Phenotypic information
and DNA samples were obtained from study subjects with
approval of the Institutional Review Ethics Board for IBD
genetic studies at The Hospital for Sick Children in Toronto.
Replication cohorts had Ethics Board approval for genetic and
phenotypic studies at the individual institutions. Written
informed consent was obtained from all participants. The
affected subjects were recruited from International Early Onset
Pediatric IBD Cohort Study sites and healthy controls were
obtained from the Centre for Applied Genomics.
1.4 (0.9–2.1)
Gastroenterology Vol. 147, No. 3
OR (95% CI)
Dhillon et al
Gene (protein)
SNP name
Resulting functional change MAF, % Disease type Genetic model
684
September 2014
VEOIBD and the NOX2 NADPH Oxidase Genes
685
Figure 3. Forest plot of
NADPH oxidase SNP
associated with VEOIBD.
Forest plot of the odds
ratios of associated variants with VEOIBD phenotypes. For each variant,
the effect allele (EA), minor
allele frequency (MAF), P
value, and odds ratio with
the 95% confidence interval is shown.
Tagged Single Nucleotide Polymorphism
Genotyping
In order to examine intronic SNPs not identified in the
targeted exome sequencing approach, a subanalysis examining a subset of VEOIBD previously genotyped for Tagged
NADPH oxidase SNPs12 was carried out. We found novel
associations with 2 RAC2 SNPs (rs1476002 and rs1476002),
an NCF4 SNP (rs18813313), and an RAC1 SNP
(rs10951982) (Table 3 and Figure 3). In the combined
analysis, all SNPs remained associated with VEOIBD and
rs1476002 had the strongest association with VEOUC
(Precessive ¼ 7.77 107; odds ratio ¼ 7.0; 95% confidence
interval: 2.9–17), suggesting the importance of the RAC2 in
VEOUC susceptibility.
Functional Studies
Next we determined if the identified coding variants had
abnormal NADPH oxidase functions. First, we examined the
NCF2 variant (rs35012521) that resulted in a p67phox N419I
amino acid change in the PB1 domain. When transfected
into HEK 293T cells, on coimmunoprecipitation (Figure 4A),
the N419I variant located in the PB1 domain showed
Table 2.SNPs and Novel Variants Identified by Targeted Exome Sequencing
Gene (protein)
CYBB (gp91phox)
NCF1 (p47phox)
NCF2 (p67phox)
NCF2 (p67phox)
NCF2 (p67phox)
NCF2 (p67phox)
NCF4 (p40phox)
RAC2 (RAC2)
Amino acid change,
SNP name
Protein domain
G364R,
rs141756032
R90H,
rs13447
R38Q,
rs147415774
H389Q,
rs17849502
G501R,
Novel
Ferric reductase
P454S,
rs55761650
R308Q,
rs141160114
Noncoding
chromosome
22, 37627176, (C/T)
PX domain
TPR1 domain
PB1 domain
Between PPR
and SH3 2 domains
MAFa
0.005% (males)
5.5%30
Novel, no data
5.0%
VEOIBD patients
(n ¼ 122), n (%)
1 (0.8)
Predicted damaging21
12 (9.8)
Reduces ROS production30
1112 (9.0)
Reduces binding to
Rac212
Reduces binding to Vav131
14 (11.5)
Novel, no data
1 (0.8)
Exon 15
0.008%
1 (0.8)
PB1 domain
0.0001%
1 (0.8)
Novel, no data
1 (0.8)
Intron 5
Defect
Predicted damaging21
decrease in p47phox
binding
Exonic splicing enhancer
site lost23
Predicted damaging21
decrease in p67phox binding
hnRNP A1 site gain24
splicing dysregulation
hnRNP, heterogeneous nuclear ribonucleoproteins; MAF, minor allele frequency.
a
Population frequencies refer to data collected by National Heart, Lung and Blood Institute Exome Sequencing Project Exome
Variant Server18 (http://evs.gs.washington.edu/EVS/), limited to European Americans.
BASIC AND
TRANSLATIONAL AT
were not observed in the 1560 genotyped healthy controls
(Table 2 and Figure 3). This included a coding R90H p47phox
variant (encoded by NCF1; rs3447) that was previously shown
by Olsson et al in rheumatoid arthritis patients to significantly
decrease reactive oxygen species (ROS) production.29 In addition, an NCF2 SNP (rs17849502) resulting in a p67phox H389Q
variant previously associated with systemic lupus erythematosus,30 was found in 14 VEOIBD patients. This p67phox H389Q
variant was previously shown to reduce ROS production and
was suggested to reduce p67phox binding to VAV1, an important
process for FcyR-dependent ROS production.30
(0.57–0.89)
(1.8–8.6)
(2.9–17)
(0.6–0.9)
(1.3–27)
0.71
3.9
7.0
0.8
6.0
103
104
107
103
103
2.12
2.16
7.77
5.34
7.65
(0.5–0.99)
(0.67–9.56)
(1.31–24.4)
(0.6–1.1)
0.73
2.5
5.7
0.8
NA
4.14 102
1.6 101
9.0 103
2.06 101
NA
(0.5–0.9)
(1.7–12.6)
(2.6–24.5)
(0.6–0.9)
(1.8–35.6)
rs739041
rs1883113
RAC2 (RAC2)
NCF4 (p40phox)
CI, confidence interval; MAF, minor allele frequency; NA, not applicable; OR, odds ratio.
0.67
4.6
8.0
0.8
7.9
102
103
104
102
103
1.21
2.71
2.85
1.13
7.22
Additive
Recessive
Recessive
Additive
Recessive
VEOIBD
VEOIBD
VEOUC
VEOIBD
VEOIBD
rs10951982
rs1476002
RAC1 (RAC1)
RAC2 (RAC2)
24
13
13
42
7
P value
OR (95% CI)
P value
OR (95% CI)
P value
Model
Association
SNP name
MAF, %
Gastroenterology Vol. 147, No. 3
Gene (protein)
Replication cohort
Discovery cohort
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Table 3.Replicated NADPH Oxidase Genes SNPs Associated With VEOIBD Identified by Tag SNP Genotyping
OR (95% CI)
Dhillon et al
Combined analysis
686
reduced binding to p40phox (encoded by NCF4) compared
with the wild-type p67phox, suggesting the importance of
p40phox mediated regulation of p67phox in the formation of
the NADPH complex (Figure 1).
Similar to NCF2 variants, the R308Q p40phox (encoded by
NCF4) variant (rs147415774) also located in the PB1
domain responsible for p40phoxp67phox binding, showed
similarly reduced binding to p67phox (encoded by NCF2),
further supporting the importance of this interaction in
proper NADPH complex formation (Figure 4A). Finally, the
G501R p67phox (encoded by NCF2) novel variant was
located in the SH3 domain, which is responsible for interaction with p47phox, was found in 1 patient from the targeted exome sequencing discover cohort. We found that
the G501R p67pho variant reduced binding between
p47phoxp67phox (Figure 4B), demonstrating the overall
importance of protein–protein interactions in NADPH complex formation in VEOIBD (Figure 1).
In addition to protein-binding defects, we also identified
variants in the noncoding regulatory and splicing elements of
the NADPH oxidase genes in a number of VEOIBD patients.
The CYBA (encoding p22phox) variant (rs72550704) that was
found to be associated with very early onset Crohn’s disease
resulted in a loss of an SP1 binding site.23 This variant was
found to reduce p22phox protein expression in Patient 1 (a
heterozygous carry of the variant), who also had reduced
ROS production (Supplementary Table 1 and Supplementary
Figure 1A), although it was not directly shown that the
reduction in p22phox protein expression resulted in subsequent ROS reduction. In this particular case, both the patient
and mother carried the functional variant and therefore other
genetic or environmental factors must also play a role in
susceptibility to disease.
In addition, a novel variant in RAC2 (chromosome 22,
37627176 [C/T]) that was predicted to alter splicing regulation of RAC2 through the addition of a heterogeneous
nuclear ribonucleoprotein A1 site was also identified in a
patient who developed steroid-dependent colitis (a heterozygous carry of the variant). We determined that this novel
intronic variant in RAC2 led to the 5 new alternative splicing
forms of the RAC2 messenger RNA (Supplementary
Figure 1B, C), resulting in loss of exons and truncations
that were not seen in control samples lacking this variant.
Overall, these altered splicing forms resulted in decreased
RAC2 protein expression, as seen in Western blot analysis
(Supplementary Figure 1B).
Variants That Result in Reduced Reactive Oxygen
Species Production
To determine if the NADPH oxidase variants resulted in
functional defects, we retrospectively examined clinical
NADPH oxidase ROS production and identified only 5 of the
122 targeted exome sequencing VEOIBD patients who had
previous ROS measured by NBT for clinical purposes. The 5
patients with identified NADPH oxidase variants all had
reduced ROS production, although within normal limits
(Table 3). Four patients had heterozygous NADPH oxidase
variants and 1 patient (Patient 4) had compound
VEOIBD and the NOX2 NADPH Oxidase Genes
687
Figure 4. Functional studies of rare and private mutation in the NADPH oxidase genes associated with VEOIBD. (A) HEK293T
cells were cotransfected with wild-type and variant p67phox-myc and p40phox-GFP constructs (p67phox-WT, p67phox-N419I,
p40phox-WT, and p40phox-R308Q) p40phox-GFP was immunoprecipitated from cell lysates using anti-GFP primary antibody.
Levels of p67phox-myc bound to p40phox-GFP were determined by Western blot using anti-myc antibodies. Densitometry of
immunoprecipitated p67phox-myc was normalized to total levels of immunoprecipitated p40phox-GFP. n ¼ 3 experimental
replicates; ***P < .05 as calculated by the Bonferroni post-test. (B) HEK293T cells were cotransfected with wild-type and
variant p67phox-myc and p47phox-GFP constructs (p67phox-WT, p67phox-G501R and p40phox-WT) p67phox-myc was immunoprecipitated from cell lysates using anti-myc primary antibody. Levels of p47phox-GFP bound to p67phox-myc were determined by Western blot using anti-GFP. Densitometry of immunoprecipitated p47phox-GFP was normalized to total levels of
immunoprecipitated p67phox-myc. n ¼ 3 experimental replicates; ***P < .05 as calculated by the Bonferroni post-test.
heterozygous NCF2 variants (encoding p67phox H389Q and
N419I) and was diagnosed at 2 years of age with VEOIBD. In
this patient, known NADPH oxidase–derived ROS-dependent
functions,31 including colorimetric NBT and interleukin-8–
and N-formyl-methionine-leucine-phenylalanine–induced
chemotaxis, were impaired (data not shown).
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
that result in profound or complete disruption of enzyme
activity. In contrast, only one NOX2 variant was identified in
VEOIBD patients, and the majority of those variants identified in the NADPH oxidase genes occurred in binding
domains of the cytosolic components of the complex
(p40phoxp67phoxp47phox; Figure 1A and B) that are
uncommonly found in CGD (Figure 1B).
It has been previously observed that defective binding
between 2 NADPH oxidase complex proteins can alter other
protein–protein interactions in the complex, as illustrated by a
study demonstrating that defective p67phox binding to RAC2
also reduced binding between p67phoxNOX2.32 Therefore,
we suggest that the NADPH oxidase variants identified in
VEOIBD patients that reduce protein–protein interactions
might also limit or delay complex formation, leading to
reduced ROS production without the complete loss of function
observed in CGD. Overall, these studies suggest that impaired
NADPH oxidase assembly at the membrane can be a critical
step in the development of colitis in these young patients.
Although these VEOIBD patients share similarities with
the colonic inflammation observed in CGD patients, the
VEOIBD patients described here had heterozygous variants
in the autosomal genes of the NADPH oxidase complex and a
small number of patients studied had low-normal ROS
production, with a minimum of 10% of normal ROS
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Dhillon et al
BASIC AND
TRANSLATIONAL AT
production. Therefore, these VEOIBD patients did not have
CGD or suffer from chronic infection or multisystemic disease associated with CGD. Female carriers of X-linked CGD
with pronounced X-linked inactivation (lyonization) who
have only 10% of normal ROS production do not develop
CGD.33 This also suggests that the levels of ROS production
observed in VEOIBD patients with NADPH oxidase variants
are above the threshold of ROS production observed in CGD
and, therefore, result in susceptibility to VEOIBD and not
immunodeficiency. However, the majority of VEOUC patients with identified functional variants in the NADPH oxidase pathway had left-sided disease, which is the
predominant location of the colitis observed in CGD patients,7,8 as opposed to the pancolitis, which is more
commonly observed in VEOIBD34 (Supplementary Table 2).
Therefore, these studies also suggest that hypomorphic
NADPH oxidase functional variants increase the risk for
developing VEOIBD and the colonic inflammation is similar
to that seen in the more severe immunodeficiency CGD.
Growing evidence suggests the importance of defective
innate immunity in IBD pathogenesis,35–37 including defective neutrophil functions, such as reduced superoxide production,38 decreased recruitment,39 and impaired monocyte
functions.35 Primary immunodeficiencies (PIDs), including
CGD, are typically characterized by pediatric onset,35 and this
might explain the higher degree of association of functional
NADPH oxidase genes variants observed in our VEOIBD
cohorts. The role of PID genes in VEOIBD is exemplified in
infants who develop colitis before 1 year of age (infantile
VEOIBD), where mutations in IL10RA/B were found in patients with a distinct phenotype of severe colitis with perianal
disease and folliculitis17,40 and, more recently, mutations in
TTC7A in infantile VEOIBD patients with apoptotic enterocolitis.41 Similar to the results presented here, variants in
IL10RA have been found in higher prevalence in VEOUC
without evidence of PID,17 suggesting that hypomorphic
variants in PID genes that do not result in overt immunodeficiency can result in susceptibility to VEOIBD.
Interestingly, mirroring the binding defects described
here in VEOIBD, changes in the assembly of the NADPH
oxidase complex have been implicated in Mendelian susceptibility to mycobacterial disease (MSMD), resulting in
selectively reduced oxidative burst in macrophages.42
MSMD is a disease in which patients cannot mount resistance to mild strains of mycobacterial microbes and,
although traditionally defective mutations in NOX2 (encoded
by CYBB) cause CGD, in a rare MSMD case, 2 functional
coding variants of NOX2 resulted in a decrease in
macrophage-specific NADPH oxidase complex formation.42
This further suggests the importance of NADPH oxidase
gene variants in diseases less severe than CGD. In addition,
p47phox (encoded by NCF1) has been genetically linked with
rheumatoid arthritis through genetic association as well as
animal model studies.29,43 In addition, the H389Q coding
variant of p67phox (encoded by NCF2), also identified in this
study, was recently associated with systemic lupus erythematosus.30 These studies suggest that mutations in NOX2
results in immunodeficiency (CGD or MSMD), and functional
variants in other components of the NADPH oxidase
Gastroenterology Vol. 147, No. 3
complex contribute to susceptibility to immune-related
diseases, including a significant role in the development of
VEOIBD in a large cohort.
Supplementary Material
Note: To access the supplementary material accompanying
this article, visit the online version of Gastroenterology at
www.gastrojournal.org, and at http://dx.doi.org/10.1053/j.
gastro.2014.06.005.
References
1. Marks DJ, Miyagi K, Rahman FZ, et al. Inflammatory
bowel disease in CGD reproduces the clinicopathological
features of Crohn’s disease. Am J Gastroenterol 2009;
104:117–124.
2. Schappi MG, Smith VV, Goldblatt D, et al. Colitis in
chronic granulomatous disease. Arch Dis Child 2001;
84:147–151.
3. Werlin SL, Chusid MJ, Caya J, et al. Colitis in chronic granulomatous disease. Gastroenterology 1982;82:328–331.
4. Muise AM, Snapper SB, Kugathasan S. The age of gene
discovery in very early onset inflammatory bowel disease. Gastroenterology 2012;143:285–288.
5. Levine A, Griffiths A, Markowitz J, et al. Pediatric modification of the Montreal classification for inflammatory
bowel disease: the Paris classification. Inflamm Bowel
Dis 2011;17:1314–1321.
6. Benchimol EI, Guttmann A, Griffiths AM, et al. Increasing
incidence of paediatric inflammatory bowel disease in
Ontario, Canada: evidence from health administrative
data. Gut 2009;58:1490–1497.
7. Huang JS, Noack D, Rae J, et al. Chronic granulomatous
disease caused by a deficiency in p47(phox) mimicking
Crohn’s disease. Clin Gastroenterol Hepatol 2004;2:690–695.
8. Matute JD, Arias AA, Wright NA, et al. A new genetic
subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective
defects in neutrophil NADPH oxidase activity. Blood
2009;114:3309–3315.
9. Jostins L, Ripke S, Weersma RK, et al. Host-microbe
interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 2012;491:119–124.
10. Franke A, McGovern DP, Barrett JC, et al. Genomewide meta-analysis increases to 71 the number of
confirmed Crohn’s disease susceptibility loci. Nat Genet
2010;42:1118–1125.
11. Anderson CA, Boucher G, Lees CW, et al. Meta-analysis
identifies 29 additional ulcerative colitis risk loci,
increasing the number of confirmed associations to 47.
Nat Genet 2011;43:246–252.
12. Muise AM, Xu W, Guo CH, et al. NADPH oxidase complex and IBD candidate gene studies: identification of a
rare variant in NCF2 that results in reduced binding to
RAC2. Gut 2012;61:1028–1035.
13. Rioux JD, Xavier RJ, Taylor KD, et al. Genome-wide association study identifies new susceptibility loci for
Crohn disease and implicates autophagy in disease
pathogenesis. Nat Genet 2007;39:596–604.
14. Roberts RL, Hollis-Moffatt JE, Gearry RB, et al. Confirmation of association of IRGM and NCF4 with ileal
Crohn’s disease in a population-based cohort. Genes
Immun 2008;9:561–565.
15. Muise AM, Walters T, Xu W, et al. Single nucleotide
polymorphisms that increase expression of the guanosine triphosphatase RAC1 are associated with ulcerative
colitis. Gastroenterology 2011;141:633–641.
16. Thoeni C, Amir A, Guo C, et al. A novel nonsense mutation
in the EpCAM gene in a patient with congenital tufting
enteropathy. J Pediatr Gastroenterol Nutr 2014;58:18–21.
17. Moran CJ, Walters TD, Guo CH, et al. IL-10R polymorphisms are associated with very-early-onset ulcerative colitis. Inflamm Bowel Dis 2013;19:115–123.
18. Exome Sequencing Project. NGESP. Exome Variant
Server. Seattle, WA: University of Washington, 2013.
19. dbSNP Short Genetic Variants. Volume 2013. Bethesda,
MD: National Center for Biotechnology Information, 2013.
20. SNP Function Prediction (FuncPred). Volume 2013. Triangle, NC: National Institute of Environmental Health
Sciences, 2013.
21. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and
server for predicting damaging missense mutations. Nat
Methods 2010;7:248–249.
22. Kumar P, Henikoff S, Ng PC. Predicting the effects of
coding non-synonymous variants on protein function
using the SIFT algorithm. Nat Protoc 2009;4:1073–1081.
23. Yuan HY, Chiou JJ, Tseng WH, et al. FASTSNP: an always up-to-date and extendable service for SNP function analysis and prioritization. Nucleic Acids Res 2006;
34:W635–W641.
24. Desmet FO, Hamroun D, Lalande M, et al. Human
Splicing Finder: an online bioinformatics tool to predict
splicing signals. Nucleic Acids Res 2009;37:e67.
25. Wang J, Ronaghi M, Chong SS, et al. pfSNP: an integrated potentially functional SNP resource that facilitates
hypotheses generation through knowledge syntheses.
Hum Mutat 2011;32:19–24.
26. Levine A, Kugathasan S, Annese V, et al. Pediatric onset
Crohn’s colitis is characterized by genotype-dependent
age-related susceptibility. Inflamm Bowel Dis 2007;
13:1509–1515.
27. Tyler AD, Milgrom R, Stempak JM, et al. The NOD2insC
polymorphism is associated with worse outcome following
ileal pouch-anal anastomosis for ulcerative colitis. Gut
2013;62:1433–1439.
28. Li L, He S, Sun JM, et al. Gene regulation by Sp1 and
Sp3. Biochem Cell Biol 2004;82:460–471.
29. Olsson LM, Nerstedt A, Lindqvist A-K, et al. Copy number variation of the gene NCF1 is associated with rheumatoid arthritis. Antioxid Redox Signal 2012;16:71–78.
30. Jacob CO, Eisenstein M, Dinauer MC, et al. Lupusassociated causal mutation in neutrophil cytosolic factor
2 (NCF2) brings unique insights to the structure and
function of NADPH oxidase. Proc Natl Acad Sci U S A
2012;109:E59–E67.
31. Hattori H, Subramanian KK, Sakai J, et al. Smallmolecule screen identifies reactive oxygen species as
key regulators of neutrophil chemotaxis. Proc Natl Acad
Sci U S A 2010;107:3456–3551.
VEOIBD and the NOX2 NADPH Oxidase Genes
689
32. Diebold BA, Bokoch GM. Molecular basis for Rac2
regulation of phagocyte NADPH oxidase. Nat Immunol
2001;2:211–215.
33. Lekstrom-Himes JA, Gallin JI. Immunodeficiency diseases caused by defects in phagocytes. N Engl J Med
2000;343:1703–1714.
34. Griffiths AM. Specificities of inflammatory bowel disease
in childhood. Best Pract Res Clin Gastroenterol 2004;
18:509–523.
35. Vinh DC, Behr MA. Crohn’s as an immune deficiency:
from apparent paradox to evolving paradigm. Expert Rev
Clin Immunol 2012;9:17–30.
36. Khor B, Gardet A, Xavier RJ. Genetics and pathogenesis
of inflammatory bowel disease. Nature 2011;474:307–317.
37. Rakoff-Nahoum S, Bousvaros A. Innate and adaptive
immune connections in inflammatory bowel diseases.
Curr Opin Gastroenterol 2010;26:572–577.
38. Hayee B, Rahman FZ, Tempero J, et al. The neutrophil
respiratory burst and bacterial digestion in Crohn’s disease. Dig Dis Sci 2011;56:1482–1488.
39. Marks DJ, Harbord MW, MacAllister R, et al. Defective
acute inflammation in Crohn’s disease: a clinical investigation. Lancet 2006;367:668–678.
40. Glocker EO, Kotlarz D, Boztug K, et al. Inflammatory
bowel disease and mutations affecting the interleukin-10
receptor. N Engl J Med 2009;361:2033–2045.
41. Avitzur Y, Guo C, Mastropaolo LA, et al. Mutations in
tetratricopeptide repeat domain 7A result in a severe
form of very early onset inflammatory bowel disease.
Gastroenterology 2014;146:1028–1039.
42. Bustamante J, Arias AA, Vogt G, et al. Germline CYBB
mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous
mycobacterial disease. Nat Immunol 2011;12:213–221.
43. Olofsson P, Holmberg J, Tordsson J, et al. Positional
identification of Ncf1 as a gene that regulates arthritis
severity in rats. Nat Genet 2003;33:25–32.
Author names in bold designate shared co-first authorship.
Received January 2, 2014. Accepted June 3, 2014.
Reprint requests
Address requests for reprints to: Aleixo M. Muise, MD, PhD, The Hospital for
Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8.
e-mail: [email protected]; fax: 416-813-6531.
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.
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.
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Supplementary Figure 1. Functional variants conferring decreased NADPH oxidase gene expression. (A) Densitometry of
Western blot of p22phox variant (encoded by CYBA; SNP rs72550704), which was associated with Crohn’s disease diagnosis
at younger than the age of 6 years and resulted in lower p22phox expression. Both the patient and her mother were heterozygous for this variant. (B) Densitometry of Western blot of Rac2 for a novel variant gaining an intronic heterogeneous nuclear
ribonucleoprotein (hnRNP) A1 (noncoding chromosome 22, 37627176 [C/T]) disrupting splicing regulation, which decreased
overall expression levels. (C) Splicing variants of the RAC2 gene for the novel variant found in a single patient conferring a gain
of an intronic hnRNP A1 site, disrupting splicing regulation. To assess for RAC2 splicing changes, patient and healthy control
messenger RNA (approximately 1 ug) were reverse transcribed (random decamers [5 uM], 1 RT buffer, deoxynucleoside
triphosphate solution [0.5 mM], RNase inhibitor [10 U], and reverse transcriptase [100 U]) by incubating the solution at 44 C for
1 hour and then inactivated at 92 C for 10 minutes. Thereafter, complementary DNA was amplified by polymerase chain
reaction (PCR) using primers specific to RAC2 and then visualized on a 1% agarose gel. PCR products were cloned into
plasmids using the CloneJET PCR Cloning Kit, as per manufacturer’s instructions, before sending to sequencing. (C) RAC2
splice variants complementary DNA: variant 1: deleted parts of exon 3–7 (244–294, 1146–1439); variant 2: deleted part of exon
7 (244–294, 1146–1439); variant 3: deleted parts of exons 3–7 (244–300, 1050–1439); variant 4: deleted part of Exon 7
(445–809, 1275–1439); and variant 5: deleted parts of exons 5 and 6 (244–534, 1052–1439).
September 2014
VEOIBD and the NOX2 NADPH Oxidase Genes 689.e2
Supplementary Table 1.NADPH Oxidase Variants Found in Patients With Low-Normal ROS Production
Patient no./NOBI
SNP
1/14.7 and 19.1 on
2 occasions
2/34
rs72550704
3/40 and 53 on
2 occasions
4/53
rs13447
rs147415774
rs17849502
rs35012521
5/48
rs35761891
Gene (protein)
CYBA
(p22phox)
NCF1
(p47phox)
NCF2
(p67phox)
NCF2
(p67phox)
NCF2
(p67phox)
RAC1
(RAC1)
Heterozygous variant/
functional defect
Promoter variant
reduces p22phox expression
R90H
PX domain
reduces ROS production in
transfected COS cells30
R38Q
reduces binding to Rac212
H389Q
reduces binding to Vav131
N419I
reduces binding to NCF4
GATA binding site lost
Clinical features
Dx at age 3 years with pancolitis VEOIBD
Dx at age 19 months with pancolitis VEOIBD
Dx at age 9 months with pancolitis VEOIBD
Dx at age 2 years with pancolitis VEOIBD
Loop ileostomy at age 6 years and end
ileostomy at age 10
Dx at age 6 years with pancolitis VEOIBD
Dx, diagnosis; NOBI, neutrophil oxidative burst index (NOBI was carried out at The Hospital for Sick Children diagnostic
laboratory. Normal value defined as 32 to 300); COS, CV-1 (simian) in Origin.
Supplementary Table 2.Association of Presence of Functional Variants Found in the Targeted Exome Sequencing Project
With Crohn’s Disease and Ulcerative Colitis Disease Locations
Paris classification category
L1: isolated ileal disease
L2: colonic disease
L3: ileocolonic disease
L4a: proximal to ligament of Treitz
L4b: distal to ligament of Treitz
E1: ulcerative proctitis
E2: left-sided UC
E3: extensive disease
E4: pancolitis
Phenotype
present or absent
Absent
Present
Absent
Present
Absent
Present
Absent
Present
Absent
Present
Absent
Present
Absent
Present
Absent
Present
Absent
Present
Without functional
variant, n
With functional
variant, n
52
8
15
46
57
4
54
7
52
9
65
2
57
10
60
7
19
48
25
5
12
18
27
3
25
5
25
6
28
0
16
12
25
3
15
13
OR (95% CI)
P value
1.30 (0.39–4.38)
.75
0.49 (0.19–1.25)
.14
1.03 (0.25–4.30)
.68
1.54 (0.45–5.34)
.52
1.39 (0.44–4.326)
.76
NA
.58
4.28 (1.56–11.69)
.0047
NA
0.34 (0.138–0.85)
1.00
.033
CI, confidence interval; NA, not applicable; OR, odds ratio; UC, ulcerative colitis.
All disease locations are based on the Paris Classification systems with L1–L4b designating Crohn’s disease locations and
E1–E4 designating locations for ulcerative colitis. Association was tested based on absence compared with presence of
functional and rare variants listed in Tables 1 and 2 using a c2 association test.