Lupus-associated causal mutation in neutrophil
cytosolic factor 2 (NCF2) brings unique insights to
the structure and function of NADPH oxidase
Chaim O. Jacoba,1, Miriam Eisensteinb, Mary C. Dinauerc, Wenyu Mingd, Qiang Liuc, Sutha Johnd, Francesco P. Quismorio , Jr.a,
Andreas Reiffa,e, Barry L. Myonesf, Kenneth M. Kaufmang, Deborah McCurdyh, John B. Harleyi, Earl Silvermanj,
Robert P. Kimberlyk, Timothy J. Vysel, Patrick M. Gaffneyg, Kathy L. Moserg, Marisa Klein-Gitelmanm,
Linda Wagner-Weinern, Carl D. Langefeldo, Don L. Armstronga,p, and Raphael Zidovetzkia,p,1
aThe Lupus Genetic Group, Department of Medicine, University of Southern California, Los Angeles, CA 90089;bDepartment of Chemical Research Support,
Weizmann Institute of Science, Rehovot 76100, Israel;cWashington University School of Medicine, St. Louis Children’s Hospital, St. Louis, MO 63110;dWells
Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202;eChildren’s Hospital of Los Angeles,
Los Angeles, CA 90027;fTexas Children’s Hospital, Baylor College of Medicine, Houston, TX 77030;gOklahoma Medical Research Foundation, Oklahoma
City, OK 73104;hDepartment of Pediatrics, University of California, Los Angeles, CA 90095;iCincinnati Children’s Hospital Medical Center and the US
Department of Veterans Affairs Medical Center, Cincinnati, OH 45229;jHospital for Sick Children, Toronto, ON, Canada M5G 1X8;kDepartment of Medicine,
University of Alabama at Birmingham, Birmingham, AL 35294;lImperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom;
mChildren’s Memorial Hospital and Northwestern University, Chicago, IL 60614;nLaRabida Hospital and University of Chicago, Chicago, IL 60649;oWake Forest
University Health Sciences, Winston-Salem, NC 27157; andpDepartment of Cell Biology and Neuroscience, University of California, Riverside, CA 92521
Edited* by Stuart H. Orkin, Children’s Hospital and the Dana Farber Cancer Institute, Harvard Medical School and Howard Hughes Medical Institute, Boston,
MA, and approved November 2, 2011 (received for review August 15, 2011)
Systemic lupus erythematosus (SLE), the prototypic systemic auto-
immune disease, is a debilitating multisystem autoimmune disor-
der characterized by chronic inflammation and extensive immune
dysregulation in multiple organ systems, resulting in significant
morbidity and mortality. Here, we present a multidisciplinary ap-
proach resulting in the identification of neutrophil cytosolic factor
2 (NCF2) as an important risk factor for SLE and the detailed char-
acterization of its causal variant. We show that NCF2 is strongly
associated with increased SLE risk in two independent populations:
childhood-onset SLE and adult-onset SLE. The association between
NCF2 and SLE can be attributed to a single nonsynonymous coding
mutation in exon 12, the effect of which is the substitution of
histidine-389 with glutamine (H389Q) in the PB1 domain of the
NCF2 protein, with glutamine being the risk allele. Computational
modeling suggests that the NCF2 H389Q mutation reduces the
binding efficiency of NCF2 with the guanine nucleotide exchange
factor Vav1. The model predicts that NCF2/H389 residue interacts
with Vav1 residues E509, N510, E556, and G559 in the ZF domain
of Vav1. Furthermore, replacing H389 with Q results in 1.5 kcal/
mol weaker binding. To examine the effect of the NCF2 H389Q
mutation on NADPH oxidase function, site-specific mutations at
the 389 position in NCF2 were tested. Results show that an H389Q
mutation causes a twofold decrease in reactive oxygen species
production induced by the activation of the Vav-dependent Fcγ
receptor-elicited NADPH oxidase activity. Our study completes
the chain of evidence from genetic association to specific molec-
type-phenotype correlations is emerging as a difficult hurdle
in the implementation and interpretation of genetic association
studies. Candidate gene studies and, more recently, genome-wide
association studies (GWAS), have begun to elucidate the com-
plex genetic profile of systemic lupus erythematosus (SLE) with
identification of ∼30 risk loci (1–3). However, for almost all these
identified loci, the causal polymorphism that leads to lupus sus-
ceptibility has not been discovered. GWAS have been praised for
representing an “agnostic” approach that is unbiased by prior
assumptions regarding genetic association with the disease.
However, such an approach typically ignores all valuable prior
information collected over decades about the pathogenesis and
genetic basis of diseases that have been previously studied. This
ine localization of the polymorphisms responsible for geno-
inevitably leads to the inclusion of regions (and additional SNPs)
that have little to no possibility of being associated with a disease,
increasing the number of tests. More tests mean a more stringent
multiple testing correction and a reduction of power or a greater
number of subjects to overcome the reduction of power. To avoid
this reduction of power, we have developed a two-step bioin-
formatics-driven design that increases the power of gene associ-
ation studies using a partial Bayesian approach. The first step
uses a family-based study to identify “noteworthy” genes (having
a multitest-corrected probability of being associated with SLE of
less than 0.5) from a larger panel of genes selected on the basis of
increased prior likelihood of association because of their known
function or genomic location (4, 5). The second step follows up
these noteworthy genes in a targeted investigation.
The hypothesis that neutrophil cytosolic factor 2 (NCF2) is a
candidate gene for SLE was derived from testing our bioinfor-
matics-driven approach in a moderately sized family-based link-
age study using the family-based transmission disequilibrium
test (TDT). In that study, SNP rs2274065 showed likely associ-
ation with SLE [χ2= 15.7, P = 7.28 × 10−5, false discovery rate
(FDR) = 0.15]. In the present study, we demonstrate highly
significant association of NCF2 with SLE by genotyping addi-
tional SNPs in two independent case–control populations, iden-
tify a causal mutation, and characterize the consequences of the
causal mutation on the function of the NADPH oxidase complex.
Results and Discussion
Childhood-onset SLE presents a unique subgroup of patients for
genetic studies because of the likelihood of a higher genetic load
or higher degree of penetrance driving an earlier disease onset,
more severe disease course, greater frequency of family history of
Author contributions: C.O.J., D.L.A., and R.Z. designed research; C.O.J., M.E., M.C.D.,
W.M., Q.L., S.J., K.M.K., D.L.A., and R.Z. performed research; M.E., F.P.Q., A.R., B.L.M.,
D.M., J.B.H., E.S., R.P.K., T.J.V., P.M.G., K.L.M., M.K.-G., L.W.-W., D.L.A., and R.Z. contrib-
uted new reagents/analytic tools; C.O.J., M.C.D., K.M.K., P.M.G., C.D.L., D.L.A., and R.Z.
analyzed data; and C.O.J., M.E., M.C.D., D.L.A., and R.Z. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence may be addressed. E-mail: email@example.com or raphael.zidovetzki@
See Author Summary on page 359.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 10, 2012
| vol. 109
| no. 2
SLE, and lesser effect of sex hormones in disease development
(6, 7). Therefore, in stage 1, we genotyped an independent cohort
of 769 subjects with childhood-onset SLE. In stage 2, we geno-
typed an additional population of 5,139 North American subjects
with adult-onset SLE. Because children can develop SLE later
in life, to ensure that our controls are SLE-free, we used healthy
adults as controls for both stages. A total of 5,163 controls were
genotyped. A number of subjects with SLE and controls were
excluded as a result of principal component analysis (PCA), re-
latedness, or genotyping failure. After applying these quality
control and adjustment measures, 4,578 subjects with adult-onset
SLE, 663 subjects with childhood-onset SLE, and independent
cohorts of 879 controls for the childhood-onset group and 3,910
controls for the adult-onset group were used for analyses. The
ethnic background of patients with SLE and controls are detailed
in Table S1, and clinical characteristics of the patients with SLE
are given in Table S2.
A total of 25 SNPs were genotyped; SNPs were chosen to
provide uniform mapping of the gene with the minor allele fre-
quency (MAF) in at least one previously genotyped population
exceeding 5%. When appropriate, preference was given to
nonsynonymous SNPs. An additional 256 imputed SNPs were
included in the analysis. Imputation was performed using IM-
PUTE2 (8) and the 2010/08/04 release of the 1000 Genomes
Project (9) European cohort. As shown in Fig. 1 and Table S3,
the typed SNP with the highest association, rs17849502, showed
an unparalleled association with P values for European Ameri-
cans (EA; subjects of European ancestry) of 2.95 × 10−22in
adult-onset SLE and 7.13 × 10−13in childhood-onset SLE, cor-
responding to multitest-corrected FDRs of 4.62 × 10−20and 5.97 ×
10−10, respectively. Fig. 2 shows a forest plot depicting the con-
tribution of different ethnicities to the overall odds ratio (OR)
and significance of rs17849502. Significant association of
rs17849502 was observed only for the EA subgroup. The lack of
association in the case of Asian Americans (AsA) and African
Americans (AA) is attributable to the extremely low MAF, 0.00,
for all cohorts in the case of AsA and an MAF varying from 0.00
to 0.02 in the case of AA. Thus, we have no evidence of the
association of rs17849502 with SLE in the AsA and AA pop-
ulations. The association of this SNP in Hispanic Americans
(HA) is inconclusive because the number of subjects was too low
−log10 of p values
EA Adult Conditioned on rs17849502
12345678 10 11 1415
−log10 of p values
EA Childhood Conditioned on rs17849502
1025 100 99 100 10059 100 100
91 100 100 100 100 100 100 100
10034 71 100 100 100 100 100 100
100 1671 100 100 100 100
10 100 100 1009299 100 100 100
100 100 100
100 100 100 100
100 100 100 100
65 10100 100 100
60 91 100 100 100 100
20 79 100 100
100 100 100
100 100 100 100
1009 59 92 1009278
10059 37 100 100 100
79 100 100
81100 100 100 100 100 100
84 100 89
100 100 100 5030
100 100 5050
79 51 50 43
24 1005144 44
20 10044 45
38100 100 100
rs12753665rs10797888 rs10911363 rs35295769
rs2274065 rs34391407 rs35089271rs35438109
0 0.2 0.4 0.6 0.8
and childhood-onset (B) SLE cases in the EA subgroup with or without conditioning on rs17849502. The positions of exons (green rectangles) and introns
(connecting lines) are indicated in the middle plot. The pale green rectangles at the two ends correspond to the UTRs. The dashed horizontal red lines
correspond to P = 0.05. (C) LD heat map shows D′ between each of the typed SNPs in the region plotted, with D′ × 100 reported in each diamond. Chro-
mosomal positions are according to National Center for Biotechnology Information build 37.1.
Association of NCF2 SNPs with SLE. Data are plotted with their P values (shown as −log10values). Association of NCF2 SNPs with SLE in adult-onset (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1113251108 Jacob et al.
to have enough power, given the low MAF of this SNP in the HA
In the EA subgroup, 9 SNPs exhibited significant association
with SLE in either the childhood-onset or adult-onset group (Fig.
1 and Table S3). Of the 256 imputed SNPs, 16 SNPs showed
strong association with SLE after FDR correction (FDR < 10−3;
Fig. S1 and Table S4). Conditional regression shows that the
significant association is completely explained by rs17849502
(Fig. 1 A and B and Fig. S1). This demonstrates that SNP
rs17849502 is the sole independently significant SNP. Therefore,
the previously reported association of rs10911363 with SLE (10)
was attributable exclusively to linkage disequilibrium (LD) with
rs17849502, with a D′ of 1 (Fig. 1C). SNP rs17849502 is a non-
synonymous coding polymorphism in exon 12 of the NCF2 gene,
the effect of which is a substitution of histidine (His) 389 with
glutamine (Gln) 389 in the PB1 domain of the NCF2 protein. A
comparison of a His/His genotype with a Gln/Gln genotype
shows that the latter is associated with SLE, with an OR of 12.37
[95% confidence interval (CI): 8.34, 18.37] in adult-onset disease
and 33.50 (95% CI: 15.72, 71.39) in childhood-onset disease
(Table 1), indicating that individuals with Gln/Gln are far more
likely to develop SLE than those with His/His, a finding with
significant clinical relevance.
NCF2 is a component of the leukocyte NADPH oxidase com-
plex that produces superoxide (11). The NADPH oxidase complex
also known as NOX2) are localized in cellular membranes, con-
stituting the flavocytochrome b558. On activation, the three cy-
tosolic subunits (NCF1, NCF2, and NCF4) colocalize and trans-
locate to the membrane, where they, along with the membrane
subunits and the small GTPase (Rac1 in monocytes/macrophages
and Rac2 in neutrophils), form the active NOX2 complex (11, 12).
Genetic defects in subunits of NADPH oxidase in humans cause
chronic granulomatous disease (CGD), which is associated with
life-threatening bacterial and fungal infections (13).
To add further complexity, there is evidence for both positive
and negative regulation of the NADPH oxidase complex. In rest-
ing cells, Rac-GDP is present as a complex with Rho-GDP dis-
association inhibitor, a negative regulator ofRho family GTPases,
but this complex rapidly dissociates in stimulated cells, forming
Rac-GTP (12). This process is facilitated by activation of gua-
nine nucleotide exchange factors (GEFs) and is accompanied by
translocation of Rac to the plasma membrane (11, 12). Vav1 is
sucha GEFfound inthecytoplasmofhematopoietic lineagecells.
On phosphorylation, Vav1 has GEF activity for the Rac GTPase,
facilitating Rac’s transition from an inactive GDP-bound state
to an active GTP-bound state (14). Recent work demonstrated
a direct interaction of Vav1 with the C terminus of NCF2 that
enhances the GEF activity of Vav1 and causes a positive feedback
loop for amplifying Rac activation (15).
We next asked whether the H389Q mutation in the PB1 do-
main of NCF2 affects any of its multiple protein-protein inter-
actions. The binding sites on NCF2 to Rac1/2, CYBB, and NCF1
are clearly outside the PB1 domain (11, 12). Although the PB1
domain of NCF2 interacts with the PB1 domain of NCF4, the
structure of this heterodimeric complex (16) shows that residue
H389 makes no contact with NCF4 and is highly exposed in the
complex; hence, the mutation H389Q is unlikely to affect the
NCF2/NCF4 interaction. The structure of the NCF2–Vav1
complex is not resolved. To test if the mutation H389Q is likely
to affect the binding with Vav1, we constructed a model of in-
teraction using a protein modeling method developed by one of
the authors (M.E.), which has been demonstrated to be highly
accurate in predicting other protein-protein interactions (17).
The modeling consisted of several stages, most of which used
only the structures of the molecules and their physical properties,
and did not require that H389 be at the binding interface. First,
His binding sites on the entire surface of Vav1 (His anchoring
spots) were mapped using ANCHORSMAP, a computational
procedure developed by one of the authors (M.E.) (18). The
existence of low ΔG His anchoring spots indicated that an in-
terface can be formed in which an exposed His residue from
a binding partner acts as an anchoring hot spot. To test if NCF2
is the relevant binding partner, we evaluated the physicochemi-
0.5 2.0 4.0 6.0 8.0
rs17849502 Additive OR
overall OR (Mantel–Haenszel). AsA are not shown because the risk allele of rs17849502 was not found in this population. Size of rectangles is proportional to
the weighting of the study in the meta-analysis, the top and bottom of diamond are the overall OR, the left and right whiskers (or left and right diamond
edges) are the ±95% CI of the corresponding OR.
Relative contribution of different ethnicities to the overall OR for SNP rs17849502. Forest plot of additive OR ± 95% CI in indicated subgroups and
Table 1. Comparison of genotypes in position 389 of the NCF2 protein
Adult-onset SLEChildhood-onset SLE OR
1.85 (1.69, 2.03)
12.37 (8.34, 18.37)
6.67 (4.46, 10.99)
2.53 (2.01, 3.2)
33.50 (15.72, 71.39)
13.19 (6.06, 28.73) 8372
Sample counts of genotypes in position 389 are shown in subjects who have SLE (adult/childhood onset) and controls (EA). ORs for
the transition between H/H, H/Q, and Q/Q are shown with 95% CIs in parentheses.
Jacob et al.PNAS
| January 10, 2012
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cal surface complementarity between NCF2 and Vav1 utilizing
a multistep approach. Most of the steps used only the structures
of the molecules and their physical properties, and did not re-
quire that H389 be at the binding interface. In the first step,
protein-protein docking between NCF2 and Vav1 was conducted
using the geometric-electrostatic-hydrophobic (GEH) version
of the MolFit program (19). Because experimental data regarding
the interaction interface between NCF2 and Vav1 are lacking,
the docking scans were based only on the physicochemical
properties of the molecules calculated from their coordinates.
Hence, in the docking scans, the molecules were not guided to
interact via NCF2/H389 or to avoid the NCF2/NCF4 interface.
The MolFit program produced an ensemble of 12,256 docking
models evaluated by GEH score. These models underwent
postscan filtering in which additional properties of the interface,
desolvation energy and statistical propensity measures, were
tested and rescored (20). This postscan filtering was passed by
366 docking models. In the next step, a condition was added that
the residue NCF2/H389 should be located at the interface; this
condition was fulfilled only in 12 cases. In the last step, the 12
docking models were matched with the anchoring spots map,
searching for docking solutions in which H389 is positioned near
a His anchor with binding ΔG less than −4 kcal/mol. The dis-
tances between the Cβ and Cε1 atoms of H389 and the corre-
sponding atoms in nearby anchors were calculated and averaged.
In three cases, H389 was located within 3 Å of an anchor; in the
other cases, the distance was significantly larger (5 Å or more) or
there was no nearby anchoring site. Remarkably, the highest
scoring docking model was the one that used an anchoring site
with highest preference of His over Gln, by 1.5 kcal/mol. Two
additional tests were used to validate the model. First, it was
verified that the model allowed simultaneous binding of the PB1
domain of NCF2 to Vav1 and NCF4 without clash. Second, in
agreement with experimental results, the best model of simul-
taneous binding of Vav1 and NCF4 to NCF2 involved the Rac-
binding conformation of Vav1.
The NCF2/Vav1 interaction model thus obtained was sup-
ported by the available information and led to the formation of
the hypothesis that the H389Q mutation will affect the in-
teraction of NCF2 with Vav1, and consequently alter the func-
tion of the NADPH oxidase complex. To test the first part of this
hypothesis, we conducted coimmunoprecipitation (Co-IP) experi-
ments showing that NCF2 binding to Vav1 involves their re-
spective PB1 and ZF domains. A series of Co-IP experiments of
deletion and truncation constructs of NCF2 (Fig. 3) and Vav1
(Fig. 4) provides strong evidence that the PB1 domain of NCF2
is directly involved in binding to the ZF domain (also known as
the C1 domain) of Vav1.
bar) for Vav1 based on these experiments. (B and C) Immunoprecipitation (IP) of full-length or truncated NCF2 mutants with full-length Vav from lysates
prepared from COS7 cells transiently transfected with corresponding expression constructs. Lysates were immunoprecipitated with a rabbit polyclonal an-
tibody against Vav1 (Santa Cruz Biotechnology), and immunoprecipitates and lysates were analyzed by immunoblotting with the indicated antibodies. To
detect NCF2, either a rabbit polyclonal antibody that reacts with the N terminus of NCF2 (Santa Cruz Biotechnology) or a mouse monoclonal antibody di-
rected against the C terminus of NCF2 (BD Transduction Laboratories) was used in B and C, respectively. Samples shown in each panel were run on the same
gel from the same experiment, but irrelevant lanes were excised for simplicity and clarity of presentation. Upon C-terminal truncation of NCF2, the removal of
the PB1 domain led to a failure to coimmunoprecipitate with Vav1 (B, lanes ΔC299 and ΔC210), in contrast to an NCF2 derivative that included only the
C-terminal PB1 and SH3 domains (C, lanes ΔN262 and ΔN350). WB, Western blot; Wt, wild type.
(A) Summary of interaction of truncated NCF2 derivatives with Vav1 as determined by Co-IP experiments and proposed binding region (vertical green
| www.pnas.org/cgi/doi/10.1073/pnas.1113251108 Jacob et al.
The docking model presented in Fig. 5 fits all the available
experimental information. Accordingly, our model suggests that
the NCF2 H389 residue sits in a pocket on the surface of the Vav1
ZF domain, surrounded by Vav1 residues E509, N510, E556, and
G559. In addition, an adjacent loop of NCF2 interacts with the
C-terminal part of domain DH of Vav1 (residues 341–361) and
stabilizes a conformation adequate for Rac binding. Previous
studies have suggested that a R394Q substitution in mouse NCF2
(21) and a R395W substitution in human NCF2 (22) affected
NCF2 interaction with NCF4 and resulted in CGD. Indeed, the
experimental structure of the NCF2/NCF4 complex and our
model support the findings that NCF2 amino acids 394 and 395
are involved in NCF2 binding to NCF4 but not in the binding
Most importantly, the model predicts a significantly reduced
binding of NCF2 to Vav1 as a consequence of an H389Q mu-
tation. Gln binds 1.5 kcal/mol weaker than His at the predicted
position. Considering that the GTP-bound form of Rac is the
physiological mediator of oxidase activation in the NOX2 oxi-
dase (11, 12), decreased binding of Vav1 to NCF2 should de-
crease amplification of Rac activation, which, in turn, should
decrease production of reactive oxygen species (ROS).
NCF2 His-389 is conserved across species (Fig. 6), suggesting
that there is selective pressure to maintain this residue. Func-
tional activation of neutrophils and macrophages via integrins or
Fcγ receptors (FcγR) is markedly impaired in mice lacking both
Vav1 and Vav3 (23–25), consistent with the finding that these are
the main Vav isoforms expressed by these cells (23, 24). Vav1
residues E509, N510, E556, and G559 in the ZF domain, pre-
dicted to interact with NCF2 His-389 (Fig. 5), are either identical
in Vav3 or have conservative substitutions capable of mediating
interactions with NCF2 His-389 (Fig. 6).
To test the prediction that NCF2 H389Q would reduce the
function of NADPH oxidase, site-specific mutations at the 389
position in NCF2 were tested experimentally. Experiments were
conducted in human K562 myeloid cells expressing endogenous
CYBA, CYBB, and NCF1 as stable transgenes and transiently
expressing NCF2 and NCF4. We compared WT NCF2 (with His
at position 389) and NCF2 with either Gln or alanine sub-
stitutions at position 389. We also compared WT NCF2 and
H389Q NCF2 derivatives tagged at the C terminus with en-
hanced yellow fluorescent protein (EYFP). NADPH oxidase was
activated either by cross-linking FcγR with IgG-opsonized latex
beads or by phorbol 12-myristate 13-acetate (PMA). ROS pro-
duction was measured by a luminescence-based assay system. As
shown in Fig. 7, IgG bead-elicited ROS responses for either the
Q389 or A389 mutation were reduced by approximately twofold
compared with WT NCF2, whereas there was no effect on PMA-
elicited ROS release. The differential effect of NCF2 H389
substitutions on PMA- vs. IgG bead-elicited ROS release is es-
pecially relevant, because it is consistent with the lack of par-
ticipation of any Vav isoform in PMA-elicited NADPH oxidase
activity, whereas Vav1 and Vav3 are essential for FcγR-elicited
NADPH oxidase activity (25). Furthermore, these results are
consistent with previous results in which an R395W mutation
reduced the PMA-induced (Vav-independent) NADPH oxidase
activity and caused CGD (16), supporting the notion that amino
acid 395 is involved in the binding to NCF4 rather than to Vav1.
bar) for NCF2. (B–D) Immunoprecipitation of full-length or truncated Vav1 mutants with full-length NCF2 from lysates prepared from COS7 cells transiently
transfected with corresponding expression constructs. Lysates were immunoprecipitated with antibodies directed against Vav1 or against epitope tags
present on truncated Vav1 derivatives as indicated, and immunoprecipitates and lysates were analyzed by immunoblotting with the indicated antibodies. A
Vav1 derivative lacking the ZF domain failed to coimmunoprecipitate with NCF2 (B, lane ΔN608), in contrast to full-length Vav1 or other truncated Vav1
proteins that included the ZF domain (B–D, all lanes except ΔN608). IP, immunoprecipitation; WB, Western blot.
(A) Summary of interactions of truncated Vav1 derivatives with NCF2 as determined by Co-IP experiments and proposed binding region (vertical cyan
Jacob et al.PNAS
| January 10, 2012
| vol. 109
| no. 2
It is noteworthy that a variant NCF1 allele identified by po-
sitional cloning based on its association with the severity of
pristane-induced arthritis in a susceptible rat strain results in a
reduction in neutrophil oxidase activity of a similar magnitude
(26). Hence, a twofold reduction in NADPH oxidase activity is
highly likely to be biologically significant.
That a CGD phenotype is not seen in our SLE subjects even in
the presence of a homozygous 389Q genotype indicates that the
ability of phagocytes to generate a respiratory burst sufficient for
antimicrobial activity is not impaired by reduced NCF2 function.
However, in other immune cells (e.g., antigen-presenting cells),
ROS production is much more limited (26–28) and the role of
ROS is not microbicidal. NADPH oxidase activity regulates
phagosomal pH, and derivative ROS can function as signaling
molecules within and between neighboring immune cells; reduced
levels of ROS in these cells may thus influence antigen process-
ing, immunoregulation, control of cell activation, and differenti-
ation (28, 29).
Our results corroborate more recent findings suggesting that
ROS produced by the NOX2 complex are used in fine-tuning
inflammatory responses, depending on when, where, and at what
level the ROS are produced (29, 30). A point mutation in NCF1
results in decreased ROS production (comparable to those seen
in our H389Q experiments), leading to increased susceptibility to
autoimmune arthritis in rodents (31, 32). Furthermore, both
patients who have CGD and carriers have a higher risk for de-
veloping autoimmune diseases (33). Finally, patients who have
CGD and mothers carrying X-linked CYBB mutations have a 5–
10% incidence of cutaneous and/or mucosal lesions character-
istic of those seen in SLE (34–36).
The present study demonstrates the advantages of our study
design, where the utilization of prior information coupled with a
two-step experimental approach allows for more efficient iden-
tification of disease-associated genes. Our findings demonstrate
that a single identified mutation in NCF2 leads to decreased
ROS production and plays a role in predisposition to SLE. This
finding provides further support for the emerging paradigm shift
that ROS are not solely proinflammatory byproducts of cellular
responses to infectious or inflammatory stimuli but may have a
more nuanced function in immunoregulation and inflammation-
limiting processes, with notable consequences for autoimmune
Materials and Methods
Recruitment and Biological Sample Collection. Subjects were enrolled in the
Lupus Genetic Study Groups at the University of Southern California, Okla-
homa Medical Research Foundation, University of Alabama at Birmingham,
and Imperial College London using identical protocols. All patients met the
revised 1997 American College of Rheumatology criteria for the classification
of SLE (37). Controls were collected in parallel with the SLE cases in the same
institutions. Ethnicity was self-reported and verified by parental and
grandparental ethnicity when known. Controls were defined as adults with
self-reported absence of SLE or any other autoimmune disease in the sub-
jects or their first-degree relatives. Blood samples were collected from each
participant, and genomic DNA was isolated and stored using standard
methods. Cases were defined as childhood onset according to the criterion
that the diagnosis of SLE was made before the age of 13 y by at least one
pediatric rheumatologist participating in the study. Previously typed trios
from our published TDT studies (5) were not regenotyped, and they were
not used in the present study. The University of Southern California In-
stitutional Review Board for research on human subjects approved this
study. The study was also approved by human subject institutional review
board at each institution where subjects were recruited. Informed consent
was obtained from all subjects.
ence of NCF4. Starting coordinates of Vav1 and NCF2 were taken from the
PDB. The structure of the NCF2 PB1 domain was extracted from the complex
with the PB1 domain of NCF4 [chain A in PDB entry 1oey (16)], and the
structure of the DH-PH-ZF domains of Vav1 was taken from the complex
with full-length Rac1 [chain B in PDB entry 2vrw (42)]. Coordinates of missing
residues in the Vav1 domain DH were completed by superimposing the
corresponding domain in the autoinhibited structure of Vav1 [PDB entry
3ky9 (43)]. The solvent-accessible surface of Vav1 is shown in yellow; NCF2 is
shown as a ribbon diagram in green with H389 indicated. The nearby His
anchoring spot is shown in magenta. NCF4 is shown in dark green. (Inset)
Interaction site of H389 detected in the anchoring spots mapping is high-
lighted. The surface of Vav1 was made transparent to show the side chains
of residues that are within 3 Å and interact with H389 in the docking model.
Model of the interaction of NCF2 with Vav1 via His389 in the pres-
binding pocket in the ZF domain of Vav. (A) H389 (boxed in yellow) is con-
served from Gallus gallus (chicken) to human. (B) Sequence alignment of
amino acid residues in the binding pocket in the ZF domain of the three Vav
isoforms in the human and mouse are shown. Each human Vav isoform is
identical to its relevant mouse homolog. The target amino acids (509, 510,
556, and 559) in Vav1 (yellow) are identical or have conservative substitution
in Vav3 (yellow). Replacement of E by D in position 509 is considered a
conserved charge; position 510 is less conserved, but the model suggests that
NCF2 H389 binds Y510 similar to N510. The conservation of P508 and the
general similarity of the character of the residues indicate that this fragment
has a similar fold in the three Vav isoforms. The second fragment is even
better conserved. E556 is identical in all three variants, and G559 is con-
served in Vav1 and Vav3.
Conservation of H389 in NCF2 and its target amino acids in the
| www.pnas.org/cgi/doi/10.1073/pnas.1113251108Jacob et al.
Genotyping. Genotyping was performed using Illumina iSelect Infinium II
Assays on the BeadStation 500GX system (Illumina). For analysis, only ge-
notype data from SNPs with a call frequency greater than 90% in the samples
tested and an Illumina GenTrain score greater than 0.7 were used. GenTrain
scores measure the reliability of SNP detection based on the distribution of
genotypic classes. The average SNP call rate for all samples was 97.18%. To
minimize sample misidentification, data from 91 SNPs that had been pre-
viously genotyped on 42.12% of the samples were used to verify sample
identity. In addition, at least one sample previously genotyped was randomly
placed on each Illumina Infinium BeadChip and used to track samples
throughout the genotyping process.
Stratification Analyses. To account for potential confounding substructure or
admixture in these samples, PCAs were performed (38) for all samples using
a large set of SNPs (18,446, which were genotyped on these subjects as part
of a larger effort). Four principal components were identified that explained
a total of ∼60% of the observed genetic variation. These were used to
identify individuals who were genetically distant from other samples in the
same ethnic subset, and thus capable of introducing admixture bias. In-
creasing the number of principal components to 10 did not significantly
change the inflation factor. We then performed genomic control analysis to
calculate the inflation factor λ using the same set of SNPs. This yielded a λ of
1.13 in EA samples, 1.03 in HA samples, 1.08 in AA samples, and 1.04 in AsA
samples. Q/Q plots were performed to verify a lack of departure from unity
and possible substructure (Fig. S2). The Q/Q plots show no meaningful sys-
tematic departure from expectation (i.e., line of unity), suggesting that
population structure is not a biasing influence in these results.
Statistical Analyses. Testing for association in the case–control studies was
completed using the freely available programs SNPGWA (http://www.phs.
wfubmc.edu/web/public_bios/sec_gene/downloads.cfm) and PLINK (39). For
each SNP, missing data proportions for cases and controls, MAF, and exact
test results for departures from Hardy–Weinberg expectations were calcu-
lated. In addition to an allelic test of association, the additive genetic model
was used as the primary hypothesis of statistical inference. The R module
genetics (available from http://cran.rproject.org/web/packages/genetics/index.
html) were used to estimate the LD between markers and haplotype structures
in different ethnicities.
To have separate and independent control groups for both adult-onset
and childhood-onset cases, the control cohort was split into two control
theratio ofchildhood toadultcasesinallsubgroups,excepttheEAsubgroup,
where the ratio was threefold the ratio of childhood to adult cases to
maximize the power of the childhood stage while maintaining the power of
the adult stage.
FDR estimates using q values were calculated for different ethnicities using
the q value package (available from http://cran.r-project.org), which imple-
ments the q value extension of FDR (40). The FD for combined results were
estimated using the Benjamini and Hochberg procedure (41), because the
proportion of correctly rejected null hypotheses was possibly overestimated
when using the q value extension and the Benjamini and Hochberg pro-
cedure provides a more conservative estimation of FDR (with less power).
The FDR corresponds to the proportion of false-positive results among the
total results. Thus, an estimate of FDR less than 0.05 signifies that less than
5% of the results accepted as true are false-positive results and is taken as
a measure of significance.
Molecular Modeling of the NCF2/NCF4/Vav1/Rac1 Complex. Coordinates of the
molecules.Startingcoordinates ofVav1andNCF2were taken fromtheProtein
Data Bank (PDB). The structure of the NCF2 PB1 domain was extracted from
the complex with the PB1 domain of NCF4 [chain A in PDB entry 1oey (16)],
and the structure of Vav1 was taken from the complex with Rac [chain B in
PDB entry 2vrw (42)]. Coordinates of missing residues in Vav1 domain DH
were completed by superposing the corresponding domain in the auto-
inhibited structure of Vav1 [PDB entry 3ky9 (43)].
Computational anchoring spots mapping. ANCHORSMAP (developed by M.E.)
identifies preferred binding sites of amino acid side chains (anchors) on the
surface of a protein, taking into consideration that this amino acid is part of
a hypothetical protein (18). The procedure detects small cavities and sub-
cavities on the surface of a protein, which are adequate for binding single
amino acid side chains. Next, thousands of amino acid probes are scattered
near the cavities, and optimal probe positions are determined through it-
plasmids for expression of NCF4 and NCF2 WT, NCF2 H389A mutant, or NCF2 H389Q mutant with the Amaxa Nucleofector kit V, under the T-16 program.
Derivatives of NCF2 tagged at the C terminus with EYFP were also tested. According to our model, a Q in position 389 binds 1.5 kcal/mol weaker than H,
whereas an A binds 2.0 kcal/mol weaker than H in this position. (A) ROS production in K562 cells in response to hIgG beads in the presence of luminol and HRP
(n = 4; mean ± SEM; *P < 0.05). (B) ROS production in K562 cells in response to PMA in the presence of isoluminol and HRP (n = 4; mean ± SEM; In A and B, the
total relative light unit (RLU) values over 60 min, measured at 1-min intervals, are expressed as the percentage of the RLU output of NCF2 WT-transfected cells
in each experiment. Data were normalized to correct for interexperiment variability and log-transformed to stabilize variance; they were tested for sig-
nificance using the Student t test. Representative kinetic curves are taken from one of four independent experiments for ROS production in K562 cells in
response to hIgG beads (C) or PMA (D).
NCF2 H389Q mutation leads to decreased ROS release in K562 cells. K562 cells stably transfected with CYBB and NCF1 were cotransfected with
Jacob et al.PNAS
| January 10, 2012
| vol. 109
| no. 2
erative energy minimization and spatial clustering. The binding ΔGs of the
optimally posed probes are calculated by means of a scoring function that
includes van der Waals, electrostatic, and solvation energy terms. The elec-
trostatic energy is corrected for the dielectric shielding exerted by the
approaching protein. Finally, the anchoring spots are clustered to produce
an ensemble of mean anchoring spots, which were found to be very useful
for detecting interacting surfaces. The standard parameters described by
Ben-Shimon and Eisenstein (18) were used in the current study. Only an-
choring spots with ΔG less than −4 kcal/mol were considered in subsequent
analyses, because such low-energy anchoring spots often correspond to
experimentally detected hot spots (18).
Rigid body docking with MolFit. Protein-protein docking was executed using the
docking program MolFit4. MolFit performs a comprehensive stepwise rota-
tion/translation docking scan. It uses grid representations of the molecules
and fast Fourier transformations to evaluate the surface complementarity for
each pose (44). In this study, we used the GEH version of MolFit, which tests
the shape complementarity of the contacting surfaces, together with the
electrostatic (45) and hydrophobic (19) complementarity. Standard trans-
lation and rotation grid intervals, 1.05 Å and 12°, respectively, were used.
The resulting lists of docking models were intersected to produce a list of
models evaluated by a GEH score (19). Higher scores indicate better shape,
electrostatic, and hydrophobic complementarities of the interacting surfaces.
Propensity and solvation postscan filter. The comprehensive docking scan was
followed by an efficient postscan filtering and rescoring procedure that tests
additional descriptors of the interfaces (20). The procedure determines the
interface core (46) for each docking model and calculates several interface
core residue and residue-residue propensities and a measure of the spatial
clustering of the interface core atoms. These descriptors, together with the
whole interface solvation energy, are used in class-specific filters that retain
models for which any M of the N descriptors exceed their calibrated thresh-
olds. These models are reevaluated by class-specific scoring functions that
combine the same interface descriptors with the GEH score.
Normal modes analysis. The analysis was performed using the ElNemo Web
server, which computes the low-frequency normal modes of a protein (http://
igs-server.cnrs-mrs.fr/elnemo) (47). The low-frequency normal modes (ex-
cluding the 6 translational and rotational modes) represent gross defor-
mations of the molecule, such as domain movements.
Plasmid Constructs. Constructs for mouse Vav1 and human NCF2 cDNAs were
subcloned into pcDNA3.1 (Invitrogen), NCF2 in pEYFP-N1 (BD Biosciences
Clontech), and human NCF4 in pRK5 (BD Biosciences) as previously described
(15, 48). Site-directed mutagenesis was performed in NCF2 in pcDNA3.1 using
the QuikChange site-directed mutagenesis kit (Stratagene) to generate NCF2
H389A. An NCF2 H389Q cDNA was synthesized by Genscript and cloned into
pcDNA3.1 and pEYFP-N1. The constructs were confirmed by sequencing.
Truncated derivatives of human NCF2 were generated using internal re-
strictionsites orPCR,exceptforthefragmentsN1-210and N1-299, which were
gifts from D. Lambeth (Emory University, Atlanta, GA) and cloned into
were also cloned into pcDNA3.1 or pCMV-Tag3c (Myc tagged; Stratagene).
Some derivatives were based on a truncated derivative of Vav1 tagged at the
N-terminus with green fluorescence protein using pEGFP-C3 (BD Biosciences
Clontech), or a full-length Vav1 tagged at the N-terminus with a Flag epitope
tag (gift of D. Billadeau, Mayo Clinic, Rochester MN). Details of constructs are
available on request.
Analysis of Interactions Between NCF2 and Vav1. Immunoprecipitation ex-
periments to analyze interactions between NCF2 and Vav1 derivatives
expressed in COS7 cells were performed as previously described (15). COS7
cells were transiently transfected with expression constructs for either full-
length Vav1 in combination with full-length or truncated NCF2 derivatives
or full-length human NCF2 and truncated Vav1 derivatives. Lysates were
immunoprecipitated with antibodies against either Vav1, NCF2, Myc (9E10;
Upstate Biotechnology), or Flag (F-3165; Sigma–Aldrich) as appropriate, and
lysates and immunoprecipitates were analyzed by immunoblotting (15).
Preparation of Transfected Lines for NADPH Oxidase Activity Studies. K562
myeloid cells (American Type Culture Collection) were grown in RPMI 1640
with 10% (vol/vol) FCS and 1% penicillin/streptomycin at 37 °C in 5% CO2as
previously described (48). To generate K562 cells stably expressing human
CYBB and NCF1, K562 cells were transected with the CYBB cDNA in pEF-
PGKpac and NCF2 cDNA in pEF-PGKhygro (49) using an Amaxa Nucleofector
(Lonza) and Amaxa program T-16. Cells were selected in the presence of 250
μg/mL hygromycin B and 2 μg/mL puromycin. For analysis of NADPH oxidase
activity, the Amaxa Nucleofector kit V (Lonza) was used to transfect 2 × 106
CYBB/NCF1-expressing K562 cells transiently with 2 μg of pRK5-pNCF4 and
2 μg of pcDNA3-pNCF2 WT or mutant (H389Q or H389A). After 24 h,
transfected K562 cells were collected for NADPH oxidase assays and for
immunoblot analysis. NCF1, NCF2, and NCF4 expression was evaluated by
immunoblotting as described (48) using anti-NCF1 monoclonal antibody (BD
Biosciences Clontech), anti-NCF2 monoclonal antibody (BD Biosciences
Clontech), and anti-NCF4 polyclonal antibody (Millipore), respectively. Rep-
resentative blots are shown in Fig. S3.
NADPH Oxidase Activation in K562 Cells. NADPH oxidase activity was assayed
using chemiluminescence enhanced by luminol or isoluminol, which is
membrane-impermeable as described (48); both compounds detect super-
oxide in a peroxidase-dependent reaction (48). In our hands, the chem-
iluminescence signal for luminol is greater than for isoluminol; thus, we used
the former to assay human IgG (hIgG) latex bead-elicited ROS release more
sensitively, which is less than that elicited by PMA. Note that although the
K562 cells used in these studies bind hIgG beads, they are not ingested. For
measurement of ROS release, PMA (300 ng/mL) or hIgG-latex beads (6.25 ×
105hIgG-latex particles) were used to activate 2 × 105K562-CYBB/NCF1 cells,
which were cotransfected the previous day with plasmids for expression of
NCF4 and WT or mutant NCF2, in the presence of 20 μM isoluminol (for PMA
stimulation) or 20 μM luminol (for hIgG-latex bead stimulation) and 20 units/
mL HRP, without or with superoxide dismutase (SOD; final concentration of
75 μg/mL). A Spectromax L two-channel microplate luminometer (Molecular
Devices) was used to record luminescence every 1 min at 37 °C for a total of
60 readings. Chemiluminescence was not detected in the presence of SOD.
Activity was normalized to NCF2 expression determined by densitometry.
ACKNOWLEDGMENTS. We thank Y.X. Wu for technical assistance, C. Marchal
and M. Stefanovic for assistance with generating some of the NCF2 ex-
pression constructs, J. Matute and X. Li for generating the transgenic K562
cell line, and Dr. XingPing Cui for fruitful discussions. Support for this study
was provided by National Institutes of Health Grants AR043815 (to C.O.J.);
HL45635 (to M.C.D.); AR62277 (to K.L.M.); AI08394, AI024717, AR042460 (to
J.B.H.); AR049084 and AR33062 (to R.P.K.); and RR020143 and AI063274 (to
P.M.G.). Additional support was provided by the Alliance for Lupus Research
(C.O.J., J.B.H., and R.Z.), Riley Children’s Foundation (M.C.D.), Children’s Dis-
covery Institute (M.C.D.), and US Department of Veterans Affairs Medical
Center, Cincinnati (J.B.H.).
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