Follow-up study identifies two novel susceptibility
loci PRKCB and 8p11.21 for systemic lupus
Yu-Jun Sheng1,2,*, Jin-Ping Gao1,2,*, Jian Li1,2,*, Jian-Wen Han1,2, Qiang Xu1,2,
Wen-Long Hu1,2, Ting-Meng Pan1,2, Yi-Lin Cheng1,2, Ze-Ying Yu1,2,
Cheng Ni1,2, Sha Yao1,2, Cai-Feng He1,2, Yang-Sheng Liu1,2, Yun Li1,2,
Hong-Mei Ge1,2, Feng-Li Xiao1,2, Liang-Dan Sun1,2, Sen Yang1,2and
Objective. We have performed a large-scale replication study based on our previous genome-wide
association study (GWAS) of SLE in the Chinese Han population to further explore additional genetic
variants affecting susceptibility to SLE.
Methods. Thirty-eight single nucleotide polymorphisms from our GWAS were genotyped in two additional
Chinese Han cohorts (total 3152 cases and 7050 controls) using the Sequenom Massarray system.
Association analyses were performed using logistic regression with gender or sample cohorts as a covariate.
Results. Association evidence for rs16972959 (PRKCB at 16p11.2) and rs12676482 (8p11.21) with SLE
was replicated independently in both replication cohorts (P<0.05), showing high significance for SLE in
combined all 4199 cases and 8255 controls of Chinese Han [rs16972959: odds ratio (OR)=0.81; 95% CI
0.76, 0.87; Pcombined=1.35?10?9; rs12676482: OR=1.26; 95% CI 1.15, 1.38; Pcombined=6.68?10?7).
PRKCB is related to the established SLE immune-related pathway (NF-kB) and 8p11.21 contains
important candidate genes such as IKBKB and DKK4. IKBKB is a critical component of NF-kB and
DKK4 is an inhibitor of canonical Wnt signalling pathway. Interestingly, PRKCB is required for recruiting
IKBKB into lipid rafts, up-regulating NF-kB-dependent survival signal.
Conclusions. Our findings provided novel insights into the genetic architecture of SLE and emphasized
the contribution of multiple variants of modest effect. Further study focused on PRKCB, 8p11.21, should
advance our understanding on the pathogenesis of SLE.
Key words: Systemic lupus erythematosus, Genetics, Association, Single-nucleotide polymorphism.
SLE (OMIM #152700) is a common systemic autoimmune
inflammatory disease with complex aetiology but strong
clustering in families. It is characterized by dysregulated
immune responses mediated by T and B cells, leading to
against several self-antigens. Those autoantibodies are
associated with clinical manifestations in various organs
SLE affects predominantly women (prevalence ratio of
men to women is 1:9), especially during child-bearing
years. Genetic components have been demonstrated to
play important roles in SLE . The disease exhibits famil-
ial clustering, with 10?12% of SLE patients having an
affected first-degree relative; in addition, the available
data reveal a concordance rate for SLE of between 24
1Institute of Dermatology and Department of Dermatology at No. 1
Hospital, Anhui Medical University and2State Key Laboratory
Incubation Base of Dermatology, Ministry of National Science and
Technology, Hefei, Anhui, China.
Correspondence to: Sen Yang, Institute of Dermatology, Anhui
Medical University, 81 Meishan Road, Hefei, Anhui 230032, China.
*Yu-Jun Sheng, Jin-Ping Gao and Jian Li contributed equally to this
Submitted 17 March 2010; revised version accepted 23 August 2010.
! The Author 2010. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: email@example.com
Advance Access publication 6 December 2010
and 69% for monozygotic (MZ) twins, compared with
2?9% concordance for dizygotic (DZ) twins [4, 5].
Over the past two decades, genetic association studies
have revealed numerous susceptibility genes for SLE,
such as HLA , STAT4 , IRF5 [8, 9], ITGAM  and
PTPN22 . Since 2008, six genome-wide association
studies (GWASs) of SLE have been reported and more
than 30 robust susceptibility genes and loci were identi-
fied [12?17]. To look for additional genetic variants
increasing the risk of SLE, we performed a replication
study in two additional cohorts of Chinese Han based
on our previous GWAS data set (1047 SLE cases and
1205 controls) .
Materials and methods
All SLE cases and controls were obtained from doctors
through collaboration with multiple hospitals in two geo-
graphic regions of China (central and southern China)
[18, 19]. Samples in the GWAS stage (1047 SLE cases
and 1205 controls) were recruited from central China;
samples in the replication studies were recruited from
central (replication 1: 1643 cases and 5930 controls) and
southern (replication 2: 1509 cases and 1120 controls)
China. All cases were diagnosed using the revised criteria
for the classification of SLE from the ACR . Clinical
information was collected from the affected individuals
through a full clinical checkup by physician specialists.
Additional demographic information was collected from
both cases and controls through a structured question-
naire. All controls were clinically assessed to be without
SLE, other autoimmune disorders, systemic disorders or
family history of autoimmune disorders (including first-,
second- and third-degree relatives). All participants pro-
vided written informed consent. The study was approved
by the institutional ethical committee of each hospital
(Anhui Medical University, The Second Hospital of Anhui
Medical University and The First Affiliated Hospital of
Anhui Medical University), and was conducted according
to Declaration of Helsinki principles. The information of all
subjects is summarized in Table 1.
EDTA anti-coagulated venous blood samples were col-
lected from all participants. Genomic DNA was extracted
from peripheral blood lymphocytes by standard proced-
ures using Flexi Gene DNA kits (QIAGEN GmbH, Hilden,
Germany) and was diluted to working concentrations of
50ng/ml for genome-wide genotyping and 15?20ng/ml for
the validation study.
The whole genomic genotyping was conducted using
Illumina Human 610-Quad BeadChips (Illumina, Inc., San
Diego, CA, USA) and the replication study was performed
using the Sequenom Massarray system, which were per-
formed as previously described elsewhere . To evalu-
ate the quality of the genotype data for the validation
analysis, 100 randomly selected samples from the
GWAS stage were re-genotyped in replication samples
by using the Sequenom system (Sequenom, Inc., San
Diego, CA, USA). The concordance rate between the
genotypes from the Illumina and the Sequenom analyses
Single nucleotide polymorphism selection for
After excluding the loci reported in our previous GWASs,
590 single nucleotide polymorphisms (SNPs) were se-
lected for further evaluation from our previous GWAS
data set, which were with high minor allele frequency
(MAF) (>0.05 both in cases and controls), high P-value
of Hardy?Weinberg equilibrium (P50.01 in controls) and
with suggestive association evidence after adjustment by
gender (0.0001<P<0.01). Then, selected SNPs were
further analysed by including another 4023 samples as
controls. These samples were genotyped in a series of
GWASs of various diseases in the Chinese Han popula-
tion, including psoriasis , leprosy , vitiligo  and
atoptic dermatitis Sun et al. (2010, data not published).
The improvement of association evidence was as one of
the criteria for selecting SNPs for validation analysis after
these 4023 samples were used as additional controls.
However, because these additional controls had diseases
other than SLE, these 4023 samples were not included in
the final association analysis. In each locus, we selected
the most significant SNP for replication. Finally, 38 SNPs
(from 30 loci) were selected for the validation analysis.
TABLE 1 Summary of samples used in GWASs and replication studies
female Female, %
34.02 (11.53) 29.81 (10.08)
35.36 (12.10) 30.89 (11.21) 136/1507
32.85 (11.16) 28.18 (10.42) 113/1396
34.12 (11.68) 29.65 (10.96) 312/3887
29.64 (11.31) 2729/3201
30.74 (12.18) 3817/4438
aGWAS samples are from central China.
bReplication 1 samples are from central China.
cReplication 2 samples are from
PRKCB and 8p11.21 are associated with SLE
were performed using logistic regression with gender as
a covariate in our GWAS data set. Quality control
for GWASs was the same as that previously reported
. For the replication studies, Hardy?Weinberg equilib-
rium tests in controls were calculated using PLINK 1.06
software (Harvard, Boston, MA, USA) . We further
excluded SNPs with a call rate of <90% in cases or con-
trols and with Hardy?Weinberg equilibrium in the controls
(P40.01). After quality control, 30 SNPs were left for final
analysis, and cluster patterns of the genotyping data
from the Illumina and Sequenom analyses were checked
to confirm their quality. Thirty SNPs that passed quality
control were analysed in each replication study using lo-
gistic regression with gender as a covariate. The joint ana-
lysis of all combined samples was performed using
logistic regression with gender and sample cohorts as
covariates. The P-values adjusted by gender were re-
ported without correction
Recombination plots of each susceptibility locus were
generated using the information from the HapMap
project [Han Chinese in Beijing, China (CHB) and
adapted from the University of California at Santa Cruz
Genome Browser (http://genome.ucsc.edu/).
the GWASs, single-markerassociationanalyses
In the replication stage, 38 SNPs were genotyped in 3152
SLE patients and 7050 controls. Thirty SNPs that passed
quality control were included for further analysis. The
association evidence for two SNPs (rs16972959 and
rs12676482) with SLE was replicated independently in
both the replication populations (P<0.05). When the
genotypic data from the GWAS and the replication stage
were combined, we found that the significance of associ-
threshold [odds ratio (OR)=0.81; 95% CI 0.76, 0.87;
Pcombined=1.35?10?9). The other SNP also showed sig-
nificant association with SLE (rs12676482: OR=1.26;
95% CI 1.15, 1.38; Pcombined=6.68?10?7), although it
did not attain the genome-wide threshold for significance.
The statistical results of 38 SNPs are summarized in
In order to identify susceptibility genes underlying
these newly discovered associations, we investigated
patterns of linkage disequilibrium (LD) around every
risk-associated SNP, including what and where the
located in intron 2 of the PRKCB gene [protein kinase
C-b (PKC-b)] in an ?150-kb LD block at 16q11.2 (Fig.
1a). The PRKCB gene was implicated by the association
at 16q11.2, where a single gene was found within the LD
block harbouring the association. SNP rs12676482 was
mapped to 8p11.21, in which multiple genes were located
In the present study, we selected 38 SNPs that appeared
promising based on the results of the previous GWAS
data set, and genotyped them in other two Chinese
cohorts. Two SNPs were validated in both replication co-
horts and showed highly significant association evidence
in combined analysis, which indicated two novel suscep-
tibility loci for SLE: 16q11.2 (rs16972959) and 8p11.21
At 16q11.2, PRKCB is the only known gene in the as-
sociation interval. PRKCB is a member of the PKC gene
family. PKC enzymes are a family of closely related ser-
ine?threonine protein kinases that are activated by diacyl-
glycerol in the presence of phospholipids. PRKCB has
been reported to be involved in many different cellular
functions, such as B-cell activation, apoptosis induction,
endothelial cell proliferation and intestinal sugar absorp-
tion. It can be activated by oxidative conditions in the cell,
induces phosphorylation of p66 (SHC) and cause alter-
ations of mitochondrial calcium ion responses and
three-dimensional structure, thus causing apoptosis .
It is also specifically required for B-cell receptor (BCR)-
mediated NF-kB activation. Furthermore, inhibition of
PRKCB promoted cell death in B lymphomas character-
ized by exaggerated NF-kB activity . All these further
suggested that PRKCB might play an important role in the
pathogenesis of SLE.
As for 8p11.21, several genes were observed in the
association interval. Of these genes, IKBKB might be a
more plausible candidate gene for SLE, considering its
biological implications for SLE. IKBKB is a critical compo-
nent of NF-kB , and plays an important role in the
NF-kB pathway . Notably, PRKCB was reported to
be required for recruiting the IkB kinase (IKK) complex
(including IKBKA and IKBKB) into lipid rafts, activating
IKK, degrading I?B or up-regulating NF-kB-dependent
PRKCB and IKBKB are essential for BCR-mediated
NF-kB activation. Furthermore, B-cell hyperactivity, both
in vivo and in vitro, is a classic characteristic of SLE, and
is likely a consequence of abnormal signalling events
Another interesting candidate gene, DKK4, also seems
to be interesting because of its potential role in immunity
and lymphocytes. DKK4 encodes a protein that is a
member of the Dickkopf (DKK) family. DKK4 has been
described to be a inhibitor of canonical Wnt signalling
pathway , and a promotion factor for angiogenesis
and invasion . The evolutionarily conserved canonical
Wnt?b-catenin?T-cell factor (TCF)/lymphocyte enhancer
binding factor (LEF) signalling pathway regulates key
checkpoints in the development of various tissues and
have been shown to play a role in both T and B cells
[34, 35]. Taken together, all this evidence supported that
IKBKB and DKK4 might likely be the susceptibility genes
for SLE. Further study should be warranted to clarify the
causal gene for SLE in the novel susceptibility locus
(8p11.21) identified in this study.
Yu-Jun Sheng et al.
TABLE 2 Association evidence for 38 SNPs at 30 loci in GWAS, replication and combined studies
Replication 1 (central)
Replication 2 (southern)
1047 cases, 1205 controls
1643 cases, 5930 controls
1509 cases, 1120 controls
4199 cases, 8255 controls
0.89 (0.81, 0.98)
0.92 (0.87, 0.98)
0.88 (0.83, 0.94)
1.12 (1.04, 1.20)
1.11 (1.04, 1.19)
0.88 (0.80, 0.97)
1.26 (1.15, 1.38)
1.17 (1.07, 1.28)
1.20 (1.08, 1.32)
1.07 (1.01, 1.13)
0.99 (0.94, 1.06)
0.99 (0.93, 1.05)
0.91 (0.84, 0.98)
1.09 (1.02, 1.16)
1.11 (1.04, 1.18)
1.02 (0.95, 1.09)
0.94 (0.89, 1.00)
0.91 (0.86, 0.97)
0.90 (0.84, 0.97)
0.87 (0.81, 0.93)
1.17 (1.09, 1.26)
1.06 (1.00, 1.13)
0.81 (0.76, 0.87)
0.79 (0.71, 0.88)
0.87 (0.82, 0.92)
1.09 (1.03, 1.16)
1.08 (1.02, 1.15)
1.06 (1.00, 1.12)
1.04 (0.98, 1.10)
0.94 (0.87, 1.01)
aAssociation statistic adjusted for gender.bAssociation statistic adjusted for gender and study. Chr: chromosome.
PRKCB and 8p11.21 are associated with SLE
In this study, we performed a large-scale replication
study based on our previous GWAS of SLE in Chinese
Han population. Two susceptibility genes/loci for SLE,
(rs12676482), were identified, which should provide novel
insights into the genetic architecture of SLE, and empha-
sizes the contribution of multiple variants of modest effect.
Rheumatology key messages
. PRKCB and IKBKB might be related with SLE by
being involved in NF-kB pathway.
. DKK4 might be related to SLE by being involved in
the canonical Wnt signalling pathway.
FIG. 1 Regional association plots showing signals in GWAS samples for 16q11.2 and 8p11.21. The P-values of SNPs
(shown as ?log10 values in y-axis, from the genome-wide single-marker association analysis using the logistic regression
and gender as a covariate) were plotted against their map positions (x-axis). The colour of each SNP spot reflects its r2
with the top SNP (large red diamond, ¨) within each locus, changing from red to white. Estimated recombination rates
(based on the combined CHB and JPT samples from the HapMap project) were plotted in light blue. Gene annotations
were adapted from the University of California at Santa Cruz Genome Browser (http://genome.ucsc.edu/). (a) 16q11.2;
Yu-Jun Sheng et al.
We are most grateful to Hou-Feng Zheng, Yong Cui,
Xian-Bo Zuo and all the other members for their voluntary
participation in this study. Y.-J.S., J.-P.G. and J.L. are
Foundation (30972727). S.Y. is currently funded by the
National Natural Science Foundation (30972727) and the
Anhui Skin Genetic Study Innovative Research Team
Funding: This study was funded by the National Natural
Science Foundation (30972727) and the Anhui Skin
Disclosure statement: The authors have declared no
conflicts of interest.
1 Tsokos GC, Nambiar MP, Tenbrock K, Juang YT. Rewiring
the t-cell: signaling defects and novel prospects for the
treatment of SLE. Trends Immunol 2003;24:259?63.
2 Nagy G, Koncz A, Perl A. T- and B-cell abnormalities in
systemic lupus erythematosus. Crit Rev Immunol 2005;25:
Moser KL, Kelly JA, Lessard CJ, Harley JB. Recent
insights into the genetic basis of systemic lupus
erythematosus. Genes Immun 2009;10:373?9.
4 Deapen D, Escalante A, Weinrib L et al. A revised estimate
of twin concordance in systemic lupus erythematosus.
Arthritis Rheum 1992;35:311?8.
5 Block SR, Winfield JB, Lockshin MD, D’Angelo WA,
Christian CL. Studies of twins with systemic lupus ery-
thematosus. A review of the literature and presentation
of 12 additional sets. Am J Med 1975;59:533?52.
6 Graham RR, Ortmann W, Rodine P et al. Specific
combinations of HLA-DR2 and DR3 class II haplotypes
contribute graded risk for disease susceptibility and
autoantibodies in human SLE. Eur J Hum Genet 2007;15:
7 Remmers EF, Plenge RM, Lee AT et al. Stat4 and the risk
of rheumatoid arthritis and systemic lupus erythematosus.
N Engl J Med 2007;357:977?86.
8 Graham RR, Kozyrev SV, Baechler EC et al. A common
haplotype of interferon regulatory factor 5 (IRF5) regulates
splicing and expression and is associated with increased
risk of systemic lupus erythematosus. Nat Genet 2006;38:
9 Sigurdsson S, Nordmark G, Goring HH et al.
Polymorphisms in the tyrosine kinase 2 and interferon
regulatory factor 5 genes are associated with
systemic lupus erythematosus. Am J Hum Genet 2005;
10 Nath SK, Han S, Kim-Howard X et al. A nonsynonymous
functional variant in integrin-alpha(m) (encoded by ITGAM)
is associated with systemic lupus erythematosus. Nat
11 Kyogoku C, Langefeld CD, Ortmann WA et al. Genetic
association of the r620w polymorphism of protein tyrosine
phosphatase PTPN22 with human SLE. Am J Hum Genet
12 Hom G, Graham RR, Modrek B et al. Association of
systemic lupus erythematosus with c8orf13-blk and
ITGAM-ITGAX. N Engl J Med 2008;358:900?9.
13 Kozyrev SV, Abelson AK, Wojcik J et al. Functional
variants in the B-cell gene BANK1 are associated with
systemic lupus erythematosus. Nat Genet 2008;40:211?6.
14 Harley JB, Alarcon-Riquelme ME, Criswell LA et al.
Genome-wide association scan in women with systemic
lupus erythematosus identifies susceptibility variants in
ITGAM, PXK, KIAA1542 and other loci. Nat Genet 2008;
15 Han JW, Zheng HF, Cui Y et al. Genome-wide association
study in a Chinese Han population identifies nine new
susceptibility loci for systemic lupus erythematosus.
Nat Genet 2009;41:1234?7.
16 Gateva V, Sandling JK, Hom G et al. A large-scale repli-
cation study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1
and IL10 as risk loci for systemic lupus erythematosus.
Nat Genet 2009;41:1228?33.
17 Graham RR, Cotsapas C, Davies L et al. Genetic variants
near TNFAIP3 on 6q23 are associated with systemic lupus
erythematosus. Nat Genet 2008;40:1059?61.
18 Chen J, Zheng H, Bei JX et al. Genetic structure of the
Han Chinese population revealed by genome-wide SNP
variation. Am J Hum Genet 2009;85:775?85.
19 Xu S, Yin X, Li S et al. Genomic dissection of
population substructure of Han Chinese and its
implication in association studies. Am J Hum Genet 2009;
20 Hochberg MC. Updating the American College of
Rheumatology revised criteria for the classification of
systemic lupus erythematosus. Arthritis Rheum 1997;40:
21 Zhang XJ, Huang W, Yang S et al. Psoriasis genome-wide
association study identifies susceptibility variants within
LCE gene cluster at 1q21. Nat Genet 2009;41:205?10.
22 Zhang FR, Huang W, Chen SM et al. Genomewide asso-
ciation study of leprosy. N Engl J Med 2009;361:2609?18.
23 Quan C, Ren YQ, Xiang LH et al. Genome-wide associa-
tion study for vitiligo identifies susceptibility loci at 6q27
and the MHC. Nat Genet 42:614?8.
24 Purcell S, Neale B, Todd-Brown K et al. PLINK: a tool set
for whole-genome association and population-based
linkage analyses. Am J Hum Genet 2007;81:559?75.
25 Pinton P, Rimessi A, Marchi S et al. Protein kinase c beta
and prolyl isomerase 1 regulate mitochondrial effects of
the life-span determinant p66shc. Science 2007;315:
26 Su TT, Guo B, Kawakami Y et al. PKC-beta controls I
Kappa B kinase lipid raft recruitment and activation in
response to BCR signaling. Nat Immunol 2002;3:780?6.
27 Lam LT, Davis RE, Ngo VN et al. Compensatory IKKalpha
activation of classical NF-kappaB signaling during
IKKbeta inhibition identified by an rna interference
sensitization screen. Proc Natl Acad Sci USA 2008;105:
28 Shinohara H, Maeda S, Watarai H, Kurosaki T. IkappaB
kinase beta-induced phosphorylation of carma1 contrib-
utes to CARMA1 bcl10 MALT1 complex formation in B
cells. J Exp Med 2007;204:3285?93.
PRKCB and 8p11.21 are associated with SLE
29 Fauci AS, Moutsopoulos HM. Polyclonally triggered b cells
in the peripheral blood and bone marrow of normal indi-
viduals and in patients with systemic lupus erythematosus
and primary Sjogren’s syndrome. Arthritis Rheum 1981;
30 Suzuki N, Sakane T. Induction of excessive B cell prolif-
eration and differentiation by an in vitro stimulus in culture
in human systemic lupus erythematosus. J Clin Invest
31 Liossis SN, Kovacs B, Dennis G, Kammer GM,
Tsokos GC. B cells from patients with systemic lupus
erythematosus display abnormal antigen receptor-
mediated early signal transduction events. J Clin Invest
32 Niehrs C. Function and biological roles of the
Dickkopf family of Wnt modulators. Oncogene 2006;25:
33 Pendas-Franco N, Garcia JM, Pena C et al. Dickkopf-4 is
induced by TCF/beta-catenin and upregulated in human
colon cancer, promotes tumour cell invasion and angio-
genesis and is repressed by 1alpha,25-dihydroxyvitamin
d3. Oncogene 2008;27:4467?77.
34 Verbeek S, Izon D, Hofhuis F et al. An hmg-box-containing
t-cell factor required for thymocyte differentiation. Nature
35 Reya T, O’Riordan M, Okamura R et al. Wnt signaling
regulates B lymphocyte proliferation through a lef-1
dependent mechanism. Immunity 2000;13:15?24.
Yu-Jun Sheng et al.