association study of crp gene in systemic sclerosis in european caucasian population
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SHORT COMMUNICATION
Association study of CRP gene in systemic sclerosis in EuropeanCaucasian population
Julien Wipff • Philippe Dieude • Jerome Avouac • Eric Hachulla •
Jean-Luc Cracowski • Elisabeth Diot • Luc Mouthon • Jean Sibilia •
Kiet Tiev • Olivier Meyer • Andre Kahan • Catherine Boileau • Yannick Allanore
Received: 2 May 2011 / Accepted: 8 January 2013
� Springer-Verlag Berlin Heidelberg 2013
Abstract Opsonization and apoptotic cell elements are
critical in systemic lupus erythematosus (SLE) and could act
through the activation of the innate immunity. C-reactive
protein (CRP) belongs to opsonins, and polymorphisms of
CRP gene have been shown to be associated with SLE sus-
ceptibility. Accumulating evidences show that SLE and
systemic sclerosis (SSc) share some genetic susceptibility
factors. To determine whether polymorphisms of CRP con-
fer susceptibility to SSc, four SNPs (rs1130864, rs1205,
rs1800947 and rs1341665), chosen using Hapmap linkage
disequilibrium data and published data, were genotyped in a
cohort of 651 SSc patients (569 with antinuclear antibodies,
258 with anti-centromere and 153 with anti-topoisomerase I)
and 442 controls. All individuals were of French Caucasian
origin. The four polymorphisms were in Hardy–Weinberg
equilibrium in the control population. Allelic and genotypic
frequencies for these four polymorphisms were found to be
similar in SSc patients and controls. Moreover, subpheno-
type analyses in particular for subgroups having antinuclear
antibodies did not detect any difference between SSc patients
and controls. These results obtained through a large cohort of
European Caucasian SSc patients do not support the impli-
cation of CRP gene in the pathogenesis of SSc.
Keywords Systemic sclerosis � CRP � Autoimmunity �Single nucleotide polymorphism
J. Wipff (&) � J. Avouac � A. Kahan � Y. Allanore
Rhumatologie A, APHP, Hopital Cochin, Universite Paris
Descartes,
27 Rue du faubourg St Jacques, 75014 Paris, France
e-mail: julienwipff@gmail.com
J. Wipff � J. Avouac � C. Boileau � Y. Allanore
INSERM U1016, Hopital Cochin, Universite Paris Descartes,
27 Rue du faubourg St Jacques, 75014 Paris, France
P. Dieude � O. Meyer
Rhumatologie, APHP, Hopital Bichat Claude Bernard,
Universite Paris Diderot, 46 Rue Henri Huchard,
75018 Paris, France
E. Hachulla
Medecine Interne, Universite Lille II, Place de Verdun,
59037 Lille Cedex, France
J.-L. Cracowski
INSERM CIC3, CHU Grenoble, BP 217-38043, Grenoble,
Cedex 09, France
E. Diot
INSERM EMI-U 00-10, Medecine Interne, CHU Bretonneau,
2 Bis bd Tonnelle, 37044 Tours, France
L. Mouthon
Medecine Interne, APHP, Hopital Cochin, Universite Paris
Descartes, 27 Rue du faubourg St Jacques, 75014 Paris, France
J. Sibilia
Rhumatologie, Hopital Hautepierre, Universite Louis Pasteur,
Avenue Moliere, 67200 Strasbourg, France
K. Tiev
Hopital Saint-Antoine, Universite Pierre et Marie Curie,
184, Rue du Faubourg Saint-Antoine, 75012 Paris, France
C. Boileau
Biochimie Genetique, Hopital A. Pare 9, UVSQ, Avenue
Charles-de-Gaulle, 92100 Boulogne-Billancourt, France
123
Rheumatol Int
DOI 10.1007/s00296-013-2673-8
Introduction
Systemic sclerosis (SSc) is a chronic autoimmune disease
with a complex pathogenesis that is driven by combination
of genetic risk factors and environmental events [1].
Accumulating evidences have demonstrated shared auto-
immunity between various autoimmune diseases, in par-
ticular between SSc and systemic lupus erythematosus
(SLE) [2, 3]. The pathogenesis of SLE is typically char-
acterized by autoantibody-mediated tissue damage making
intervene opsonization which is a process that enhances
phagocyte binding and subsequent pathogen clearance
during the immune response [4]. Opsonins, including
immunoglobulins (Ig), complement components mannose-
binding lectin (MBL), ficolin-3 (Hakata antigen), pentraxin
3 [5] and C-reactive protein (CRP), seem to play a key role
in autoimmune diseases through innate immunity with
implication of genetic variants [4] and variation of protein
levels in serum [5]. CRP, also called pentraxin, is encoded
by CRP gene, mapping to the 1q21–q23 region. CRP is an
important innate immune modulator that facilitates the
clearance of cellular debris [6]. Phagocytes express
receptors that recognize and interact with opsonins, of
which genetic variants have been showed to contribute to
SLE through candidate gene studies [4]. Indeed, several
convincing associations between CRP gene polymorphisms
and SLE have been previously published [7, 8].
Taking into account (1) shared autoimmunity between
SLE and SSc, (2) the critical role of innate immunity in
these both connective tissue disorders and (3) the previous
reports showing association between CRP variants and
SLE, CRP appears to be an attractive candidate gene for
contributing to SSc genetic bases. Therefore, we herein
tested the hypothesis that genetic variants of CRP gene
could confer susceptibility to SSc.
Materials and methods
We performed a large case–control association study in a
large cohort consisting of 646 SSc patients and 442 healthy
unrelated controls from France, which has been previously
described [9]. For all SSc patients, we determined LeRoy’s
cutaneous subtype [10] and carried out a phenotypic
assessment, as recommended [11]. The study was approved
by all the necessary local institutional review boards, and
written informed consent was obtained from all subjects.
For testing autoimmune subgroup in SSc, all patients were
tested for antinuclear antibodies by indirect immunofluo-
rescence. We systematically checked for antibodies spe-
cific to SSc: Anti-centromere antibodies (ACAs) based on
their distinctive IIF pattern and anti-topoisomerase I anti-
bodies by counter immunoelectrophoresis.
Genotyping
Subjects were genotyped for the four SNPs: three tagging
SNPs, rs1130864, rs1205 and rs1800947, and one func-
tional SNP that influences CRP level, rs1341665 [12]. A
competitive allele-specific PCR system (Kaspar Genotyp-
ing, Kbioscience, Hoddeston, UK) was used to genotype
these SNPs, as previously reported [9].
Statistical analysis
Statistical analysis was carried out with R software (ver-
sion 2.9.1). Bonferroni’s correction was applied to all tests
of SNP marker (the p value multiplied by n SNP) and to all
‘‘hypothesis-generating steps’’ when subphenotypes are
queried (10 phenotypic subsets).
Power calculation
Power was assessed by a standard non-central chi-square
approximation, as previously described [9]. As an example,
taking into account the expected frequency of the rare
allele of rs1205, the set has a power of 89 % for detecting
an association between SSc and this CRP variant, with an
OR of 1.5 [8, 9], at the 5 % significance level.
Results
All demographic data and disease characteristics of SSc
patients are detailed in Table 1. All the four polymor-
phisms were in Hardy–Weinberg equilibrium for the con-
trol group. Linkage disequilibrium analyses confirmed that
the four SNPs are not in linkage disequilibrium. Observed
frequencies of the different genotypes were very close to
those expected with regard to database (NCBI). Regarding
Table 1 Characteristics of the European Caucasian cohort of patients
with systemic sclerosis for the different CRP polymorphisms
Patients [n (%)] SSc cohort for SLC6A4tag SNP markers
(n = 651)
Age (years ± SD) 57.4 ± 12.9
Sex (female) 573 (88)
Disease duration (years ± SD) 10.8 ± 9.5
Diffuse cutaneous subtype [n (%)] 208 (32)
Antinuclear abs [n (%)] 569 (87)
Positive anti-topoisomerase I Abs [n (%)] 153 (23)
Positive anti-centromere abs [n (%)] 258 (40)
Pulmonary arterial hypertension [n (%)] 48 (7)
Digital ulcerations [n (%)] 207 (32)
SSc systemic sclerosis, Abs antibodies
Rheumatol Int
123
the 4 SNPs, no difference was found between allelic and
genotypic frequencies of patients and controls (Table 2).
When we considered subphenotypes, especially those
related to the autoimmune involvement, that is, presence of
antinuclear antibodies, anti-centromere and anti-topoiso-
merase I, we observed no allelic or genotypic association
(Table 2). Intra-cohort analyses did not reveal any differ-
ence between SSc patients with or without the autoimmune
trait (antinuclear antibodies, anti-centromere and anti-
topoisomerase I) for any of the 4 variants. Frequencies of
minor allele of the four SNPs were also similar to those of
controls in other SSc subsets (pulmonary fibrosis, digital
ulcerations and pulmonary arterial hypertension).
Discussion
Recently, a growing body of evidence suggests shared
autoimmunity pathways between SSc and SLE. Innate
immunity takes a central place in pathogenesis of auto-
immune connective tissue disorders as highlighted by the
identification of IRF5 [2], STAT4 [3], as genetic suscepti-
bility factors in both SLE and SSc.
In the host, clearance of nuclear contents of apoptotic
cells by phagocytic cells via CRP opsonization may pre-
vent the development of specific autoimmune responses [6,
12, 13]. Some authors suggested that CRP level may be
influenced by CRP antibodies [12, 13] or by CRP gene
Table 2 Genotypic and allelic
frequencies of the CRP gene
SNPs
SSc systemic sclerosis, ANAantinuclear antibodies
OR1 SSc versus controls
OR2 SSc with ANA versus
controls
n (%) SSc
(n = 651)
ANA ? SSc
patients
(n = 569)
Controls
(n = 442)
Minor allele
frequency for
Hapmap CEU
p and OR values for allelic
comparisons 1 SSc versus controls 2
SSc ANA ? versus controls
rs1130864
Allele
T 406 (31) 346 (31) 275 (31) 0.309 p1 = 0.87, OR1 = 0.88 CI [0.84–1.22]
C 886 (69) 756 (69) 609 (69) p2 = 0.89, OR2 = 1.01 CI [0.84–1.23]
Genotypes
T/T 62 (10) 54 (10) 49 (11)
C/T 282 (44) 238 (43) 177 (40)
C/C 302 (46) 259 (47) 216 (49)
rs1205
Allele
T 441 (34) 384 (35) 295 (33) 0.333 p1 = 0.41, OR1 = 1.08 CI [0.90–1.29]
C 845 (66) 712 (65) 609 (67) p2 = 0.26, OR2 = 1.11 CI [0.92–1.34]
Genotypes
T/T 82 (9) 72 (13) 41 (9)
C/T 277 (47) 240 (44) 213 (47)
C/C 284 (43) 236 (43) 198 (43)
rs1341665
Allele
A 440 (34) 385 (35) 230 (33) 0.333 p1 = 0.66, OR1 = 1.05 CI [0.86–1.27]
G 838 (66) 709 (65) 458 (67) p2 = 0.45, OR2 = 1.08 CI [0.88–1.32]
Genotypes
A/ A 79 (12) 71 (13) 32 (9)
G/A 282 (44) 243 (44) 166 (48)
G/G 278 (44) 233 (43) 146 (43)
rs1800947
Allele
C 140 (5) 126 (6) 53 (6) 0.067 p1 = 0.63, OR1 = 0.91 CI [0.63–1.32]
G 1,162 (95) 984 (94) 853 (94) p2 = 0.87, OR2 = 0.97 CI [0.66–1.41]
Genotypes
C/C 0 (0) 0 (0) 1 (1)
C/G 70 (11) 63 (11) 51 (11)
G/G 581 (89) 492 (89) 401 (88)
Rheumatol Int
123
variants [14]. Noteworthy, associations between SLE and
CRP gene polymorphisms have already been convincingly
replicated [8, 9].
Our results show that the included CRP polymorphisms
do not contribute to susceptibility to SSc and its subphe-
notypes, in particular subgroups having specific autoanti-
bodies (Table 2).
Methodological limitations of genetic studies must
always be considered. Appropriate sample sizes for case
and control cohorts are critical to provide sufficient sta-
tistical power. In this study, the large sample size of the
cohort allowed us providing a strong power (89 %).
Moreover, the genetic background of the studied popula-
tion should be as homogeneous as possible, thereby limit-
ing bias by population stratification. To avoid this bias, we
have focused on European Caucasian individuals. Finally,
allelic and genotypic frequencies in our controls were
found in agreement with previous frequencies reported in
the European Caucasian populations (NCBI; Table 2). Our
results raise discrepancies between SLE and SSc with
regard to their respective pathogenesis. It is noteworthy
that, in comparison with SLE, SSc seems not to be a cir-
culating immune complex-driven disease and nuclear
antibodies are not correlated with disease activity [15].
Thus, clearance of cell fragments by opsonization may
be less important in SSc compared to SLE, but this will
need further investigations.
In conclusion, the CRP gene is not contributing to the
SSc genetic bases in the French European Caucasian
population. Further genetic investigations are needed to
determine the potential role of opsonization in the patho-
genesis of SSc.
Acknowledgments This work was funded by Association des
Sclerodermiques de France, INSERM, and it was supported by
Groupe Francais de Recherche sur la Sclerodermie and Agence
Nationale pour la Recherche (grant R07094KS). For DNA sample of
control population, EFS, Dr Joelle Benessiano (CRB Bichat Claude
Bernard), Dr Nadem Soufir and Pr Bernard Grandchamp are
acknowledged. For DNA sample of Lille Scleroderma population, Dr
Isabelle Fajardy is acknowledged, Molecular biology and biochem-
istry Centre, Lille CHRU.
Conflict of interest None.
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