1. Role of the C8orf13-BLK region in biopsy-proven giant cell arteritis
Orlando Torres a
, Rogelio Palomino-Morales a
, Tomas R. Vazquez-Rodriguez b
, Santos CastaÒeda c
,
Inmaculada C. Morado d
, Jose A. Miranda-Filloy b
, Norberto Ortego-Centeno e
,
Benjamin Fernandez-Gutierrez d
, Javier Martin a
, Miguel A. Gonzalez-Gay f,
*
a
Instituto de Parasitologìay
Biomedicina LÔpez Neyra, Consejo Superior de Investigaciones Cientificas (CSIC), Granada, Spain
b
Division of Rheumatology, Hospital Xeral-Calde, Lugo, Spain
c
Department of Rheumatology, Hospital de la Princesa, Universidad Autònoma, Madrid, Spain
d
Rheumatology Service, Hospital Clìnico San, Carlos, Madrid, Spain
e
Hospital Clìnico San, Cecìlio, Granada, Spain
f
Division of Rheumatology, Hospital Universitario Marques de Valdecilla, Santander, Spain
A R T I C L E I N F O
Article history:
Received 16 December 2009
Accepted 18 February 2010
Available online 7 March 2010
Keywords:
Giant cell arteritis
Disease susceptibility
C8orf13-BLK
Genetic studies
Gene polymorphism
A B S T R A C T
Giant cell arteritis (GCA) is a complex polygenic disease in which more than one genetic locus is likely to
contribute to disease susceptibility and clinical expression. In the present study, we have analyzed for first
time the implication of rs13277113 and rs2736340 variants from the C8orf13-BLK gene region in the
susceptibility to GCA. A total of 220 biopsy-proven GCA patients and 486 matched controls were assessed.
DNA from patients and controls was obtained from peripheral blood. Samples were genotyped for the
C8orf13-BLK region rs13277113 and rs2736340 using a predesigned TaqMan allele discrimination assay. No
significant differences in the genotype distribution between GCA patients and controls for the rs13277113
and rs2736340 C8orf13-BLK gene variants were found. GCA patients were also stratified according to the
presence of specific clinical features of the disease. In this regard, the allele A of the rs13277113 variant was
overrepresented in patients with severe ischemic manifestations compared with patients without severe
ischemic manifestations (p ϭ 0.04; OR ϭ1.65; 95% CI ϭ 0.99–2.78). In conclusion, our results do not support
a major implication of the C8orf13-BLK gene region in susceptibility to GCA. However, a potential implication
of the rs13277113 variant in the development of severe ischemic complications may exist.
᭧ 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights
reserved.
1. Introduction
Giant cell arteritis (GCA) is the most frequent systemic vasculitis
in the elderly in individuals of white ethnicity from Western coun-
tries [1]. It is characterized by the granulomatous involvement of
large and medium-sized blood vessels derived from the aorta, with
a predilection for the extracranial branches of the carotid artery
[2,3]. Inflammation of the arterial wall and vessel occlusion
through fast and concentric intimal hyperplasia leads to the severe
ischemic complications observed in patients with this vasculitis
[4]. They may be observed in approximately 50% of patients [5].
Among them, visual ischemic manifestations constitute the most
feared complication of GCA [6]. These occur in approximately 25%–
30% of patients, with permanent loss of vision in 10%–15% [5–7]. Of
GCA patients, 40% to 60% of GCA patients also present clinical
manifestations of polymyalgia rheumatica (PMR) [1,8].
Susceptibility to autoimmune disorders may be the result of the
interaction of multiple genetic factors that regulate the threshold of
autoreactivity. GCA is a complex polygenic disease [9], and a num-
ber of gene polymorphisms have been associated with either dis-
ease susceptibility [9,10] or a higher risk of severe ischemic com-
plications [11,12] in patients with this vasculitis. Many of these
genes are susceptibility factors for other autoimmune diseases,
suggesting that they are shared autoimmune disease susceptibility
genes. This fact may support the paradigm of common dysregulated
pathways across multiple autoimmune diseases, such as systemic
lupus erythematosus (SLE). With respect to this, two genome-wide
association studies in SLE have implicated the C8orf13-BLK (B lym-
phoid tyrosine kinase gene) region of chromosome 8p23.1 as a
susceptibility locus for SLE [13,14]. These findings were further
replicated in a Japanese population [15]. Similarly, this C8orf13-BLK
region has also been associated with rheumatoid arthritis [16]. B
lymphoid kinase (Blk), encoded by the BLK gene, is a member of the
Src family kinases (SFKs), which includes Blk, Lck, Fyn, Lyn, c-Src,
c-Yes, Fgr, and Hck [17]. Blk is the only SFK that is exclusively
expressed in B cells and thymocytes but not in mature T cells
[18–20]. Blk transduces signals downstream of the B-cell receptor
* Corresponding author.
E-mail address: miguelaggay@hotmail.com (M.A. Gonzalez-Gay).
O. Torres and R. Palomino-Morales contributed equally to this work.
J. Martin and M.A. Gonzales-Gay share senior authorship in this study.
Human Immunology 71 (2010) 525–529
Contents lists available at ScienceDirect
0198-8859/10/$32.00 - see front matter ᭧ 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.humimm.2010.02.016

2. (BCR) and plays a role in BCR signaling and B-cell development
[21,22]. B-cell development is dependent on the activation of the
transcription factor nuclear factor kB (NF-␬B) by SFKs (Blk, Fyn,
Lyn) [21]. The C8orf13 gene is ubiquitously expressed but its exact
function is currently unknown.
Since autoantibodies [23], in particular anticardiolipin antibod-
ies [24,25], have also been proposed to play a role in the clinical
spectrum of manifestations of GCA, in the current study we sought
to investigate the potential association of the C8orf13-BLK region
with susceptibility to biopsy-proven GCA, We also assessed
whether polymorphisms in this region might influence the clinical
spectrum of manifestations of this systemic vasculitis. For this
purpose, we selected the rs13277113 and rs2736340 variants of the
C8orf13-BLK gene region, as they were found to be the C8orf13-BLK
gene variants most strongly associated with susceptibility to SLE
[13], and as these gene variants were associated with alterations in
B-cell receptor signaling and NF-␬B signaling pathways [26].
2. Subjects and methods
A total of 220 patients diagnosed with biopsy-proven GCA be-
tween 1991 and 2007 were included in this study. Most of these
patients (n ϭ 128) had diagnoses made in the Division of Rheuma-
tology of the Hospital Xeral-Calde (Lugo, Northwest Spain). The
remaining patients had diagnoses made in two centers from Ma-
drid (Hospital ClÎnico San Carlos and Hospital de la Princesa; n ϭ
82) and Granada (Hospital ClÎnico San CecÎlio; n ϭ 10). A control
population (n ϭ 486) from the corresponding cities matched by age,
gender, and ethnicity with GCA patients was also studied. Patients
and controls were included in this study after written informed
consent. Ethical committee approval was obtained.
All GCA patients had a positive temporal artery biopsy result
showing disruption of the internal elastic laminae with infiltration
of mononuclear cells into the arterial wall with or without giant
cells [27]. Treatment with corticosteroids generally prevents the
development of ischemic complications of GCA. However, severe
ischemic complications, mainly strokes in the vertebrobasilar ter-
ritory, have been reported to occur in some patients after the onset
of corticosteroid therapy. In this regard, strokes may be observed
within the first month after GCA diagnosis [28], and visual ischemic
events have also been described within the first 48–72 hours after
the onset of corticosteroids [29]. In contrast, severe ischemic com-
plications related to the disease are uncommon in corticosteroid-
treated patients for at least 1 month. Because of this, to encompass
the whole spectrum of clinical manifestations directly attributed to
GCA, we assessed all the clinical manifestations that occurred in the
period from the onset of GCA symptoms to 1 month after the onset
of corticosteroid therapy.
GCA patients were considered to have PMR manifestations if
they had severe bilateral ache and pain involving the neck, the
shoulder, and/or the pelvic girdles, associated with morning stiff-
ness [30]. They were considered to have visual ischemic manifes-
tations in the context of GCA if they experienced at least one of the
following ocular complications: transient visual loss, including
amaurosis fugax, permanent visual loss or diplopia [31]. Accord-
ing to previously reported definitions, severe ischemic manifes-
tations were considered to be present if GCA patients had at least
one of the following complications: visual ischemic complica-
tions, strokes and/or transient ischemic attacks, jaw claudica-
tion, or large-artery stenosis of the extremities that caused signs
of occlusive manifestations [32].
There were no significant differences in the demographic and
clinical features between biopsy-proven GCA patients from Lugo
and those from Madrid or Granada (data not shown).
Genomic DNA was extracted from peripheral blood according to
the manufacturer’s protocol with the Pure Gene genomic DNA
isolation kit (Gentra Systems). The two rs13277113 and rs2736340
variants of the C8orf13-BLK gene region were genotyped using a
predesigned TaqMan SNP genotyping assay from Applied Biosys-
tems (ABI, Foster City, CA). PCR amplification was performed, and
the genotypes were automatically attributed by measuring the
allele-specific fluorescence in the ABI 7900HT system (ABI). Auto-
mated allele calling was performed by allelic discrimination plots
using SDS 2.3 software from ABI.
Statistical analysis was performed using the ␹2
test for assess-
ment of Hardy–Weinberg equilibrium. Genotype and allele fre-
quencies were also analyzed using ␹2
test. Haplotype estimates
were performed according to the method of Gabriel et al. [33]. Odds
ratios (ORs) and 95% confidence intervals (CIs) were calculated
according to the Woolf method using the Statcalc program (Epi Info
2002, Centers for Disease Control and Prevention, Atlanta, GA,
USA). Values of p Ͻ 0.05 were considered statistically significant.
The power calculation was performed using Quanto v 0.5 software
(Department of Preventive Medicine, University of Southern Cali-
fornia, Los Angeles, CA) [34].
Results
The main clinical characteristic of this series of biopsy-proven
GCA patients are shown in Table 1.
A high genotyping success rate was achieved (218 of the 220
biopsy-proven GCA patients were available for the genetic assess-
ment). The same high genotyping rate (higher than 99%) was
achieved in the control group. Therefore, the genotyping study was
based on 218 biopsy-proven patients. No evidence of departure
from Hardy–Weinberg equilibrium was observed in controls. The
case:control ratio achieved was 1:2.2. The estimated power of this
study for an estimated OR between 1.5 and 2.0 was 70%–99% for a
type I error rate of 0.05, dominant inheritance mode and 0.0001% of
population risk.
No significant differences in the genotype and allele frequencies
of the C8orf13-BLK variants were observed when GCA patients from
Lugo were compared with those from Madrid or Granada. More-
over, the allele and genotype distribution of the C8orf13-BLK vari-
ants was similar in controls from the three different Spanish re-
gions (data not shown).
Table 2 shows the genotype and allele frequencies of the
rs13277113 and rs2736340 variants of C8orf13-BLK gene region
in the whole series of biopsy-proven GCA patients and control
subjects. No significant differences in the genotype distribution
between GCA patients and controls for both C8orf13-BLK gene
variants were observed. In this regard, allelic frequencies for
both gene variants were similar to those reported in previous
studies [13,26].
Table 1
Main epidemiologic and clinical features of 220 patients with biopsy-proven GCA
Gender ratio (female:male) 148:72
Headache 173 (78)
Abnormal temporal artery on physical examination 140 (63)
Polymyalgia rheumatica 103 (46)
Jaw claudication 88 (40)
Visual ischemic manifestationsa
54 (25)
Permanent visual loss 23 (10)
Stroke 11 (5)
Severe ischemic manifestationsb
119 (54)
ESR Ͼ40 mm in first hour 216 (98)
Numbers in parentheses indicate percentage of patients (%) with a particular variable.
Patient median age at diagnosis was 74 years (range 52–93 years)
a
Includes at least one of the following visual ischemic complications: transient
visual loss, including amaurosis fugax, permanent visual loss, or diplopia.
b
Includes at least one of the following features: visual manifestations, cerebrovas-
cular accidents (stroke and/or transient ischemic attacks), jaw claudication, or limb
claudication of recent onset.
O. Torres et al. / Human Immunology 71 (2010) 525–529526

3. In addition to the analyses based on a single variant, we per-
formed haplotypes estimations. Nevertheless, these C8orf13-BLK
gene region variants did not form haplotype blocks. Analysis of
linkage disequilibrium disclosed a strong degree of linkage disequi-
librium between both C8orf13-BLK gene variants in the study pop-
ulation (r2
ϭ 0.86; D=ϭ 0.73), which was similar to that reported in
previous studies [26]. However, we found no statistically signifi-
cant differences between the patients and controls.
In a further step, we aimed to determine the potential influence
of C8orf13-BLKgene variants in the clinical spectrum and the sever-
ity of GCA. For this purpose we stratified biopsy-proven GCA
patients according to the presence/absence of PMR, visual isch-
emic manifestations, and severe ischemic complications of this
vasculitis. As shown in Table 3, no significant differences for the
rs13277113 variant between GCA patients with or without PMR or
visual ischemic manifestations were found. However, when this
gene variant was assessed in GCA patients with or without severe
ischemic manifestations, we found that the allele “A” was overrep-
resented in the subgroup of patients with severe ischemic manifes-
tations compared with patients without severe ischemic manifes-
tations (p ϭ 0.04; OR ϭ1.65; 95% CI ϭ 0.99–2.78) (Table 3). This was
due to an increased frequency of heterozygous (A/G) for the
rs13277113 variant genotype in the subgroup of patients with
severe ischemic manifestations (39.3%) compared with patients
without severe ischemic manifestations (26.7%) (p ϭ 0.05; OR:
1.78; 95% CI: 0.96–3.29) (Table 3).
No significant differences were found for the rs2736340 variant
of the C8orf13-BLK gene region when patients were stratified ac-
cording to specific clinical features of the disease.
Discussion
This study constitutes the first attempt to determine the poten-
tial influence of rs13277113 (A/G) and rs2736340 (C/T) polymor-
phisms of the C8orf13-BLK gene region in the susceptibility and
phenotypic expression of a large series of patients with biopsy-
proven GCA.
The rs13277113 and rs2736340 variants are located between
C8orf13 and BLK genes, which have reading frames in the opposite
directions. In SLE patients, the rs13277113 variant showed an in-
creased expression of C8orf13 and decreased expression of BLK
mRNA based on the risk haplotype [13]. BLK expression is highly
restricted to the B-cell lineage [17]. B-cell–receptor signaling is
important for establishing the B-cell repertoire through induction
of anergy, deletion, and receptor editing during B-cell development
[35]. Deficiency in Blk, Fyn, and Lyn (SFK deficient) decrease the
number of pre-B cells and lead to a substantial reduction in the
numbers of peripheral B cells [36]. SFK plays an important role in
Table 2
BLK gene polymorphisms in biopsy-proven GCA patients and healthy controls
BLK
(rs13277113)
GCA patients
n ϭ 218 (%)
Controls
n ϭ 486 (%)
p OR (95% CI)
AA 6 (2.7) 33 (6.7) 0.03 0.39 (0.14–0.99)
AG 73 (33.5) 167 (34.4) 0.82 0.96 (0.68–1.37)
GG 139 (63.8) 286 (58.8) 0.22 1.23 (0.87–1.74)
A 85 (19.5) 233 (24.0) 0.06 0.77 (0.58–1.02)
G 351 (80.5) 739 (76.0) 0.06 1.30 (0.98–1.74)
BLK
(rs2736340)
GCA patients
n ϭ 217 (%)
Controls
n ϭ 486 (%)
p OR (95% CI)
CC 130 (59.9) 283 (58.2) 0.72 1.06 (0.76–1.49)
CT 77 (35.5) 171 (35.2) 0.94 1.01 (0.71–1.44)
TT 10 (4.6) 32 (6.6) 0.30 0.69 (0.31–1.48)
C 337 (77.6) 737 (75.8) 0.46 1.11 (0.84–1.46)
T 97 (22.4) 235 (24.2) 0.46 0.90 (0.68–1.19)
Table 3
Association between BLK genotypes and typical disease features in biopsy-proven GCA patients
BLK
(rs13277113)
With
n (%)
Without
n (%)
p OR (95% CI)
PMR AA 3 (2.9) 3 (2.6) 1.0 1.12 (0.18–7.14)
AG 34 (33) 39 (33.9) 0.9 0.96 (0.53–1.75)
GG 66 (64.1) 73 (63.5) 0.9 1.03 (0.57–1.85)
A 40 (19.4) 45 (19.6) 0.97 0.99 (0.60–1.64)
G 166 (80.6) 185 (80.4) 0.97 1.01 (0.61–1.67)
Visual manifestations AA 2 (3.8) 4 (2.4) 0.63 1.58 (0.19–10.43)
AG 18 (34) 55 (33.3) 0.93 1.03 (0.51–2.07)
GG 33 (62.3) 106 (64.2) 0.79 0.92 (0.46–1.83)
A 22 (20.8) 63 (19.1) 0.71 1.11 (0.62–1.97)
G 84 (79.2) 267 (80.9) 0.71 0.90 (0.51–1.61)
Severe ischemic manifestations AA 4 (3.4) 2 (2.0) 0.68 1.75 (0.27–14.10)
AG 46 (39.3) 27 (26.7) 0.05 1.78 (0.96–3.29)
GG 67 (57.3) 72 (71.3) 0.03 0.54 (0.29–0.99)
A 54 (23.1) 31 (15.3) 0.04 1.65 (0.99–2.78)
G 180 (76.9) 171 (84.7) 0.04 0.60 (0.36–1.01)
BLK
(rs2736340)
With
n (%)
Without
n (%)
p OR (95% CI)
PMR CC 66 (64.1) 64 (56.1) 0.34 1.31 (0.73–2.36)
CT 34 (33.0) 43 (37.7) 0.47 0.81 (0.45–1.48)
TT 3 (2.9) 7 (6.1) 0.34 0.46 (0.09–2.04)
C 166 (80.6) 171 (75.0) 0.16 1.38 (0.85–2.24)
T 40 (19.4) 57 (25.0) 0.16 0.72 (0.45–1.17)
Visual manifestations CC 32 (61.5) 98 (59.4) 0.78 1.09 (0.55–2.18)
CT 17 (32.7) 60 (36.4) 0.63 0.85 (0.42–1.73)
TT 3 (5.8) 7 (4.2) 0.71 1.38 (0.27–6.25)
C 81 (77.9) 256 (77.6) 0.95 1.02 (0.58–1.79)
T 23 (22.1) 74 (22.4) 0.95 0.98 (0.56–1.72)
Severe ischemic manifestations CC 67 (57.8) 63 (62.4) 0.49 0.82 (0.46–1.48)
CT 43 (37.1) 34 (33.7) 0.60 1.16 (0.64–2.11)
TT 6 (5.2) 4 (4.0) 0.75 1.32 (0.32–5.77)
C 177 (76.3) 160 (79.2) 0.47 0.84 (0.52–1.36)
T 55 (23.7) 42 (20.8) 0.47 1.18 (0.73–1.91)
O. Torres et al. / Human Immunology 71 (2010) 525–529 527

4. the pre-BCR mediated NF-␬B activation and B cell development
[20]. In B cells, the NF-␬B family of transcription factors is an
important regulator of immune and inflammatory responses [37].
Altered protein levels of BLK may influence tolerance mechanisms
in B cells, predisposing to autoimmunity.
There are no GCA-specific autoantibodies. However, a broad
range of human autoantigens have been found in patients with this
vasculitis [23]. In this regard, anticardiolipin antibodies have been
reported, particularly in GCA patients with biopsy-proven vasculi-
tis [24]. Furthermore, antiendothelial antibodies have been dem-
onstrated in up to 50% of GCA patients [38]. Since an association
between BLK and primary antiphospholipid syndrome was previ-
ously described [39], we aimed to determine whether a potential
association of the C8orf13-BLK gene region with GCA might exist.
However, in the present study, we could not confirm an association
of GCA with rs13277113 and rs2736340 variants located within the
C8orf13-BLK region. Nonetheless, GCA is a relatively uncommon
disease, and although our study encompassed the largest series of
biopsy-proven patients assessed for genetic studies, our cohort is
probably underpowered for the detection of an association. There-
fore, we cannot completely rule out the possibility that these gene
variants might exert a weak effect that might require a higher
number of patients to be disclosed.
Interestingly, a potential effect of the C8orf13-BLK rs13277113
(A/G) gene variant was observed in the subgroup of biopsy-proven
GCA patients with severe ischemic manifestations. This result is in
keeping with a recent study reported by Hom et al., which found
that the rs13277113 (A/G) variant was associated with SLE suscep-
tibility [13]. In this regard, SLE patients showed an increased ex-
pression of the C8orf13 variant and decreased expression of BLK
mRNA [13]. With respect to this, SFK plays an important role in
pre-BCR mediated NF-␬B activation and B cell development [20].
Also, in B cells, the NF-␬B family of transcription factors is an
important regulator of immune and inflammatory responses [37].
However, further replication studies are required to confirm
whether this dysregulation in BCR and NF-␬B signaling may play a
role in the development of severe ischemic complications in pa-
tients with GCA.
In conclusion, the present study does not support an association
of polymorphisms within the C8orf13-BLK gene region with the
susceptibility to biopsy-proven GCA. However, further studies in
biopsy-proven GCA patients from different genetic backgrounds
are required to fully exclude an association between the C8orf13-
BLK gene region and this vasculitis.
Acknowledgments
This study was supported by a grant from Fondo de Investiga-
ciones Sanitarias PI06-0024 (Spain) and in part by RETICS Program,
RD08/0075 (RIER) from Instituto de Salud Carlos III (ISCIII), within
the VI PN de IϩDϩI 2008-2011.
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