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

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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)

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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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