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RESEARCH ARTICLE
Increase in Dickkopf-1 Serum Level in Recent
Spondyloarthritis. Data from the DESIR
Cohort
Gaetane Nocturne
1
, Stephan Pavy
2
, Saida Boudaoud
1
, Raphaèle Seror
2
,
Philippe Goupille
3
, Philippe Chanson
4
, Désirée van der Heijde
5
, Floris van Gaalen
6
,
Francis Berenbaum
7
, Xavier Mariette
1,2
, Karine Briot
8
, Antoine Feydy
9
,
Pascal Claudepierre
10
, Philippe Dieudé
11
, Joanne Nithitham
12
, Kimberly E. Taylor
12
,
Lindsey A. Criswell
12
, Maxime Dougados
8
, Christian Roux
8
, Corinne Miceli-Richard
1,2
*
1Institut Pour la Santé et la Recherche Médicale (INSERM) U1184, Université Paris-Sud 11, Le kremlin
Bicêtre, France, 2Service de rhumatologie, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital
Bicêtre, Le Kremlin Bicêtre, France, 3Service de rhumatologie, CHU, Tours, France; UMR CNRS 7292,
Université François Rabelais, Tours, France; CIC-INSERM 1415, Tours, France, 4Service d’endocrinologie,
Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Bicêtre, Le Kremlin Bicêtre, France, 5Leiden
University Medical Center, Leiden, The Netherlands, 6Department of Rheumatology and Internal Medicine,
LUMC, Leiden, The Netherlands, 7Sorbonne Universités, UPMC University Paris 6, AP-HP, Hôpital Saint-
Antoine, Rheumatology Department, Paris, France, 8Service de Rhumatologie B, Assistance Publique-
Hôpitaux de Paris (AP-HP); Université Paris-Descartes, Paris, France, 9Service de radiologie, Hôpital
Cochin, Assistance Publique-Hôpitaux de Paris (APHP), Paris, France, 10 Service de Rhumatologie, Hôpital
Henri-Mondor, Assistance Publique-Hôpitaux de Paris (APHP), Créteil, France, 11 Service de
Rhumatologie, Hôpital Bichat, AP-HP, Paris, France, 12 Rosalind Russell / Ephraim P Engleman
Rheumatology Research Center, Department of Medicine, University of California San Francisco, San
Francisco, United States of America
*corinne.miceli@bct.aphp.fr
Abstract
Objectives
To investigate DKK-1 and SOST serum levels among patients with recent inflammatory
back pain (IBP) fulfilling ASAS criteria for SpA and associated factors.
Methods
The DESIR cohort is a prospective, multicenter French cohort of 708 patients with early IBP
(duration >3 months and <3 years) suggestive of AxSpA. DKK-1 and SOST serum levels
were assessed at baseline and were compared between the subgroup of patients fulfilling
ASAS criteria for SpA (n = 486; 68.6%) and 80 healthy controls.
Results
Mean SOST serum levels were lower in ASAS+ patients than healthy controls (49.21 ±25.9
vs. 87.8 ±26 pmol/L; p<0.0001). In multivariate analysis, age (p = 5.4 10
−9
), CRP level
(p<0.0001) and serum DKK-1 level (p = 0.001) were associated with SOST level. Mean
DKK-1 serum levels were higher in axial SpA patients than controls (30.03 ±15.5 vs. 11.6 ±
4.2 pmol/L; p<0.0001). In multivariate analysis, DKK-1 serum levels were associated with
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 1/16
OPEN ACCESS
Citation: Nocturne G, Pavy S, Boudaoud S, Seror R,
Goupille P, Chanson P, et al. (2015) Increase in
Dickkopf-1 Serum Level in Recent Spondyloarthritis.
Data from the DESIR Cohort. PLoS ONE 10(8):
e0134974. doi:10.1371/journal.pone.0134974
Editor: Shervin Assassi, University of Texas Health
Science Center at Houston, UNITED STATES
Received: February 18, 2015
Accepted: July 15, 2015
Published: August 27, 2015
Copyright: © 2015 Nocturne et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information file.
Funding: An unrestricted grant from Wyeth
Pharmaceuticals was allocated for the first 5 years of
the follow-up of the recruited patients. The DESIR-
cohort is financially supported by unrestricted grants
from both the French Society of Rheumatology, and
Pfizer Ltd, France. A research grant from Pfizer
“Passerelle”was obtained for DKK-1 and SOST
quantification for the entire cohort and for genetic
analysis of the DKK-1 locus. The Variété cohort was
supported by a grant from the Programme Hospitalier
de Recherche Clinique, French Ministry of Health (no.
male gender (p = 0.03), CRP level (p = 0.006), SOST serum level (p = 0.002) and presence
of sacroiliitis on radiography (p = 0.05). Genetic association testing of 10 SNPs encompass-
ing the DKK-1 locus failed to demonstrate a significant contribution of genetics to control of
DKK-1 serum levels.
Conclusions
DKK-1 serum levels were increased and SOST levels were decreased among a large
cohort of patients with early axial SpA compared to healthy controls. DKK-1 serum levels
were mostly associated with biological inflammation and SOST serum levels.
Introduction
Spondyloarthritis (SpA) is one of the most common inflammatory rheumatic diseases. The
prevalence is estimated to be 0.5% to 3.4% [1,2]. In addition to the disabling rheumatic mani-
festations, some SpA patients develop severe extra-articular manifestations such as inflamma-
tory bowel disease, uveitis or psoriasis. SpA is also characterized by the formation of
syndesmophytes in the severe form of the disease. Treatment options are still limited to non-
steroidal anti-inflammatory drugs (NSAIDs) as first-line therapy and biological treatment
strategies that block specific immune mediators (e.g., tumor necrosis factor (TNF) blockers,
and probably soon antibodies targeting interleukin 17A (IL-17A) or IL-23). Anti-TNF agents
are commonly used in the refractory forms of the disease and have considerably improved the
quality of life in patients by reducing clinical and biological disease activity. They also have sig-
nificant efficacy in reducing subchondral-bone inflammatory lesions observed on axial MRI.
Nevertheless, most previous studies have failed to demonstrate a structural benefit of TNF
blockers in radiolographic disease progression as evaluated by the modified Stoke Ankylosing
Spondylitis Spine Score after 2-year follow-up [3–6]. Conversely, Haroon et al. suggested that
TNF blockers may reduce radiographic progression [7]. NSAIDs have been associated with
reduced radiographic disease progression [8,9]. A better understanding of the pathogenic
mechanisms involved in syndesmophyte formation is needed to develop targeted therapies for
structural benefit and subsequent functional improvement in patients.
Secreted Wnt glycoproteins are among the major families of cell signaling molecules. Ini-
tially, they were shown to be involved in embryogenesis and tumorigenesis [10]. In recent
years, several studies have implicated the Wnt canonical pathway in osteo-immunology and
notably the bone formation process [11]. Wnt binding to its receptor complex, which includes
low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled, initiates a number
of intracellular signaling cascades leading to the accumulation of β-catenin in the cytoplasm
and then to its translocation into the nucleus, where it enhances target gene expression. These
genes are involved in osteoblastogenesis and the control of osteoclastogenesis.
Dickkopf-1 (DKK-1) and sclerostin (SOST) are two inhibitory proteins of the Wnt signal-
ling pathway leading to osteoblastogenesis blockade. Both bind to LRP5/6 and block the Wnt/
β-catenin canonical signalling pathway. Several murine models support their involvement in
bone homeostasis. Osteopenia develops in mice transgenic for Dkk-1 [12] or SOST [13]. Con-
versely, mice with an inactivating mutation of DKK-1 show increased bone mass [14]. In
humans, mutation of SOST leads to van Buchem disease, characterized by hyperosteosis [15].
In SpA, syndesmophyte development is secondary to endochondral formation (i.e., initial
cartilage formation further replaced by bone) [16]. Therefore, DKK-1 and SOST may be
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 2/16
P081216 / IDRCB 2009-A00892-55). No funding
bodies had any role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have the
following interests: This study was partly supported
by an unrestricted grant from Wyeth Pharmaceuticals
which was allocated for the first 5 years of the follow-
up of the recruited patients. The DESIR-cohort is
partly financially supported by an unrestricted grant
from Pfizer Ltd, France. A research grant from Pfizer
“Passerelle”was obtained for DKK-1 and SOST
quantification for the entire cohort and for genetic
analysis of the DKK-1 locus. There are no patents,
products in development or marketed products to
declare. This does not alter the authors' adherence to
all the PLOS ONE policies on sharing data and
materials, as detailed online in the guide for authors.
involved in osteoblastogenesis dysregulation associated with syndesmophyte formation. The
role of DKK-1 in the fusion of sacroiliac joints was revealed in human TNF transgenic mice
[17]; DKK-1 blockade inhibited bone erosion of the sacroiliac joints and enhanced sacroiliac
ankylosis, which strongly supports the potential role of Wnt signaling in the fusion of sacroiliac
joints, the hallmark of SpA.
In addition, in mice, DKK-1 was found to induce SOST expression, which suggests complex
cross-regulation between both proteins in bone homeostasis [18]. Moreover, both proteins
bind the same LRP5/6 receptor and should mutually act as competitors in inhibiting the Wnt
signaling pathway. Thus, additional investigation of both DKK-1 and SOST is needed to better
define their roles in SpA.
Studies assessing serum level of DKK-1 in SpA patients are scarce and have generated con-
flicting results [19,20]. Discrepancies between published studies could be explained by the
small number of patients studied, different methods of DKK-1 quantification, and lack of
knowledge of DKK-1 serum levels in healthy individuals (e.g., the impact of age and gender on
DKK-1 serum level). Robust data regarding DKK-1 serum levels among a large cohort of SpA
patients and healthy controls are still lacking, as is our understanding of DKK-1 function in
SpA.
We aimed to assess DKK-1 and SOST serum levels and associated factors in patients fulfill-
ing the ASAS criteria for axial SpA within a large prospective cohort of patients with recent
inflammatory back pain (IBP) (the cohort Devenir des Spondylarthropathies Indifferenciées
Récentes [DESIR] [Outcome of Recent Undifferentiated Spondylarthropathies]). We also
aimed to compare these levels with those in healthy controls to obtain more insight into the
role of both Wnt inhibitors in SpA.
Patients and Methods
Patients and controls
This cross-sectional study quantified DKK-1 and SOST serum levels among all patients
enrolled in the DESIR cohort and for whom data were available at baseline.
The DESIR cohort is a large national multicenter cohort developed to facilitate investiga-
tions of diagnostic and prognostic markers and etiologic, pathogenic and socio-economic fac-
tors among patients with early IBP suggestive of axial SpA. In fact, patients included in this
cohort have IBP classified by the criteria of Calin et al. [21] or the Berlin criteria [22] (consider-
ing 2 of 4 items) of recent onset (>3 months and <3 years), with symptoms suggestive of SpA
according to the local investigator’s assessment (score 5ona0–10 numerical rating scale,
with 0, not suggestive of SpA, and 10, very suggestive). Patients included in DESIR cohort are
planned to be followed up to 10 years. The main characteristics of the patients at baseline have
been reported previously [23]. This cohort included 708 patients (mean age 33.8 ± 8.6 years,
46.2% men, and 57.3% positive for human leukocyte antigen B27 (HLA-B27)). The baseline
characteristics included age, ethnicity, date at onset of IBP and peripheral arthritis, nature of
IBP, presence of SpA features, relevant family history, and medication, including the use of
NSAIDs and disease-modifying anti-rheumatic drugs (DMARDs). The duration of axial symp-
toms was defined as the time between the first axial symptom and the initial interview. As pre-
viously described [23], spinal mobility was measured by the Bath Ankylosing Spondylitis
Metrology Index. Patients were asked to complete the Bath Ankylosing Spondylitis Disease
Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath
Ankylosing Spondylitis Global Index, Health Assessment Questionnaire, Medical Outcomes
Survey Short Form 36, and Ankylosing Spondylitis Quality of Life questionnaire. Blood tests
performed in the regional rheumatology centers tested for C-reactive protein (CRP) level,
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 3/16
erythrocyte sedimentation rate (ESR), and HLA–B27 antigen as well as usual biologic parame-
ters. High-sensitivity CRP (hs-CRP) was assessed as described [24]. The Ankylosing Spondyli-
tis Disease Activity Score (ASDAS) [25] was calculated with CRP level. Radiographs were
evaluated by 2 trained central readers blinded to any other data [26]. Radiographs of the sacro-
iliac joints were graded according to New York criteria. Lateral radiographs of the cervical and
lumbar spine were used to calculate the modified Stoke Ankylosing Spondylitis Spine Score
(mSASSS) [3]; an abnormal axial radiograph was defined with mSASSS 1. Data were
extracted from the M0 DESIR database locked on June 30, 2010.
DKK-1 serum level was additionally assessed in 69 SpA patients from the SpondyloArthitis
Caught Early (SPACE) cohort [27]. The SPACE cohort started in January 2009 and is an ongoing
project. Patients 16 years old with chronic (almost daily) back pain for at least 3 months but
<2 years, with onset before the age of 45 years, who were referred to the rheumatology outpatient
clinic of Leiden University Medical Center (LUMC) were included after signing informed con-
sent. The SPACE study protocol was approved by the LUMC medical ethics committee.
Controls were healthy subjects from the French Variété cohort. Variété is an open, prospec-
tive, French national, multicenter, non-randomized study of healthy volunteers established to
determine normative data for insulin-like growth factor 1 (IGF-I) and other hormones in the
general population (ClinicalTrials.gov Identifier: NCT01831648). The project aimed to estab-
lish normative data based on a large random selection from the general population, including
representation from all age groups (about 100 subjects for each decade age range). Subjects
with medical conditions and receiving medications that may affect IGF-I measurement were
excluded. A total of 974 healthy subjects were recruited in 10 centers in France. Each subject
underwent clinical examination. Personal medical history was recorded and gonadal status
evaluated. Patients underwent biological standard workup, and 80 ml blood was sampled;
serum and plasma was aliquoted and frozen and stored at -80°C before hormone measure-
ments. All patients gave their informed consent to participate in the study, which was approved
by the local ethics committee.
DKK-1 and SOST serum levels were assessed at baseline on the whole cohort, but case–con-
trol analyses and assessment of factors associated with increased DKK-1 serum level were
restricted to the subgroup of patients fulfilling the ASAS criteria. DKK-1 and SOST serum lev-
els at baseline were compared with those of 80 healthy controls from the Variété cohort.
Because of no data in the literature on the impact of gender and age on DKK-1 serum level
among the healthy population, 453 healthy controls from Variété cohort were further assessed
for DKK-1 serum level in a broader age range than those matched for the DESIR cohort (18–
79 years old, 47.5% females).
Ethic statement
This study fulfills the current Good Clinical Practice Guidelines (French version) and received
approval from the appropriate ethics committee. All patients gave their written informed con-
sent. A website containing the detailed description of the centers, the organization of the cohort
and the full detailed protocol and Case Record Form is at http://www.lacohortedesir.fr.
DKK-1 locus genotyping
Single nucleotide polymorphisms (SNPs) encompassing the DKK-1 locus were genotyped to
determine whether genetic variants of DKK-1 are associated with DKK-1 serum levels. Ten
DKK-1 SNPs were chosen in order to cover the 74 Kb including DKK-1 locus with 5’and
3’UTR regions of the gene. Seven out these 10 SNPs were previously studied in rheumatoid
arthritis [28]. The 10 selected SNPs captured 66% of the DKK-1 locus when considering SNPs
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 4/16
with MAF higher than 0.10. SNPs were genotyped using a competitive allele-specific PCR sys-
tem (KASpar genotyping, http://www.lgcgenomics.com). All genotyped SNPs had minor allele
frequency (MAF) >0.01 and were assessed for deviation from Hardy-Weinberg equilibrium.
Of an initial 486 SpA patients fulfilling ASAS criteria, 58 patients were excluded from analysis
based on self-reported non-Caucasian ancestry, and 2 individuals were excluded due to geno-
typing calling rate <20%. Control individuals consisted by 1238 healthy individuals of Cauca-
sian ancestry. Forty four control individuals were dropped from analyses based on individual
genotyping calling rate <20%. Thus, case-control analyses were performed based on compari-
sons of 426 SpA patients to 1,194 controls of Caucasian ancestry.
Serum analyses
In the DESIR cohort, serum was prospectively collected from 2009 to 2010 at inclusion and
stored in aliquots at -80°C in the Biological Resources Center at Bichat Hospital (accreditation
AFNOR #34457). SOST and DKK-1 serum levels were assessed by sandwich ELISA (Biomedica
Medizinprodukte, Vienna, Austria). ELISA tests involved an EVOLIS System (Bio-Rad, Hercu-
les, CA, USA). DKK-1 serum samples were diluted 1:4 as recommended by the manufacturer
for quantification. DKK-1 serum level from the DESIR cohort, the SPACE cohort and 80 age-
and sex-matched healthy controls were assessed by the second-generation ELISA kit from Bio-
medica (Lot F112). DKK-1 serum level from 453 additional patients from the Variété cohort
was assessed with the third-generation ELISA kit from Biomedica (Lot F125).
SOST serum level from the DESIR cohort and 80 age- and sex-matched controls was
assessed with the first-generation ELISA kit from Biomedica (Lot Y113). SOST serum level was
not assessed in a larger sample of healthy controls because data were available in the literature
on impact of gender and age on levels [29,30].
For both DKK-1 and SOST, results are expressed in picomole per liter. For DKK-1, the con-
version to picogram per milliliter is as follows: 1 pmol/L = 28.68 pg/mL.
Various quality controls were performed throughout the study: 2 internal controls were
quantified on each ELISA plate for validation of each experiment. The first internal control
(C1) was provided by the manufacturer and was an expected 3.1 to 5.9 pmol/L. All experiments
were validated with a mean variation between all experiments of 4.38 (+/-0.42). The second
internal control was a serum aliquot from a patient (C2) re-quantified on each used ELISA
plate: C2 quantification varied from 24.06 to 33.06 pmol/L. Serum providing D.O. >3.5 (>50
pmol/L) was diluted 1:2 and re-quantified. We used 80 serum samples tested in duplicate,
which demonstrated no significant variation between both quantifications. Finally, we com-
pared the DDK-1 ELISA (second-generation) test from Biomedica with the ELISA kit from
R&D systems (human Dkk-1 DuoSet ELISA kit) and found a correlation between both tests
(Spearman's rho (r
s
) = 0.72; p<0.0001, S1 Fig).
Statistical analysis
Qualitative data are described as number (%) and quantitative data as mean (±SD) or median
(interquartile range (IQR)) as appropriate. The Mann-Whitney test was used to compare inde-
pendent samples. The correlation between serum levels and biochemical variables was evalu-
ated by Spearman’s correlation coefficient (r
s
). Variables included in univariate analysis were
weight, body mass index (BMI; kg/m
2
), disease duration, erythrocyte sedimentation rate (ESR;
mm/h), C-reactive protein level (CRP; mg/L), BASFI, BASDAI, serum calcium or phosphate
level, and lumbar-spine and total-femur bone mineral density. Variables identified as signifi-
cantly associated with DKK-1 or SOST levels on univariate analysis (at p = 0.10) were entered
into non-parametric linear regression models. DKK-1 serum levels are normally distributed
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 5/16
and were studied as a continuous or a dichotomous variable (patients with high levels of DKK-
1[3
rd
and 4
th
quartiles] (DKK-1>36 pmol/L) compared to patients with low levels [1
st
and 2
nd
quartiles]) (DKK-1 36 pmol/L)) in multivariate analyses (linear regression and logistic
regression, respectively) to account for covariates associated with DKK-1 serum levels such as
CRP, SOST serum level and presence of sacroiliitis on radiography. P<0.05 was considered sta-
tistically significant. Statistical analyses involved use of R 3.1.0 (R Core Team [2014], R Foun-
dation for Statistical Computing, Vienna, Austria. http://www.R-project.org/).
Genetic association analyses were performed to determine whether individual SNPs were
associated with disease/phenotype/DKK-1 serum levels using the STATA program (v.12; Col-
lege station, Texas). The contribution of the 10 SNPs was assessed according to a recessive,
dominant or additive model of transmission in uni- and multivariate analyses. For SNPs that
were in linkage disequilibrium (D’>0.95 and r
2
>0.65), haplotypes were estimated using
PLINK and haplotype association analyses (bivariate) were performed using Haploview.
Results
Patients with early SpA and controls
In total, 708 patients have been included in the DESIR cohort (46.2% male). The mean age was
33.8 ±8.6 years and the mean duration from the onset of symptoms to referral to the rheuma-
tologist was 18.8±11.6 months, corresponding to patients with early IBP suggestive of SpA.
Overall, 486 patients fulfilled the ASAS criteria for axial SpA (mean age 32.5 ± 8.6 years, 50.2%
men, and 83.7% HLA–B27 positive). Among these patients, 80% were exposed to NSAIDs at
baseline. Characteristics of disease activity and disease severity are in Table 1. The 80 healthy
controls (51% men, mean age 32 ±9.1 years) were age- and gender-matched with axial SpA
Table 1. Baseline demographics and disease characteristics of Assessment of Spondyloarthritis
International Society (ASAS+) patients from the DESIR cohort.
No. with available data ASAS+ patients (n = 486)
Gender (male %) 486 50.2
Age (years) 486 32.5±8.6
Disease duration (months) 479 18.8±11.6
HLA-B27+ (%) 485 83.7
CRP level (mg/dl) 469 9.3±13.9
hs-CRP level (mg/dl) 470 8.1±14.2
ESR (mm) 468 14.8±16.8
BASDAI 475 43±20.4
BASFI 475 29.7±22.4
BASMI 465 2.2±0.9
Radiological sacroiliitis (%) 476 27.3
mSASSS 1 unit (%) 460 13.2
Current use of oral NSAIDs (%) 396 80
DKK-1 level (pmol/L) 479 30.3±15.5
SOST level (pmol/L) 478 49.2±26.1
Data are mean±SD unless indicated
HLA-B27, human leukocyte antigen B27; CRP, C-reactive protein; hs-CRP, high-sensitivity CRP; ESR,
erythrocyte sedimentation rate; BASDI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath
Ankylosing Spondylitis Functional Index; BASMI, Bath Ankylosing Spondylitis Metrology Index; mSASSS,
modified Stoke Ankylosing Spondylitis Spine Score; NSAIDs, nonsteroidal anti-inflammatory drugs; DKK-1,
Dickkopf-1; SOST, sclerostin
doi:10.1371/journal.pone.0134974.t001
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 6/16
patients from the DESIR cohort. The age range of the 453 healthy controls from the Variété
cohort (238 males) was 18 to 79 years.
Decreased SOST serum level among patients with early SpA
SOST serum level was significantly lower in axial SpA patients than in controls from the Variété
cohort (mean 49.21 ± 25.9 vs. 87.8 ± 26 pmol/L; p<0.0001) (Fig 1). SOST serum level was signifi-
cantly correlated with age (r
s
= 0.36; p = 2.2 10
−16
), CRP level (r
s
=-0.18;p=0.0001),hs-CRP
level (r
s
=-0.22;p=10
−6
), and ESR (r
s
=-0.12,p=0.007)(Fig 2A–2D)(Table 2). SOST serum
level in axial SpA patients was lower for those with than without sacroiliitis on radiography
(n = 130 vs. n = 346; mean 42.95 ± 18.4 vs 49.25 ± 28.91 pmol/L; p = 0.023). SOST serum level
did not differ between patients with than without axial structural lesions (mSASSS 1unitvs0).
A correlation between SOST and DKK-1 serum levels was observed (r
s
= 0.15, p = 0.0008)
(Fig 2E) as previously described [31]. Nevertheless, such correlation was weak when assessed
on the whole population of SpA patients. This correlation was higher in the subgroup of
patients with increased levels of DKK-1 (DKK-1/SOST ratio >1) corresponding to a third of
ASAS positive patients (rs = 0.83; p<0.0001).
On multivariate analysis, age (p = 5.4 10
−9
), CRP level (p<0.0001) and DKK-1 serum level
(p = 0.001) were associated with SOST level (Table 2).
Increased serum DKK-1 level in SpA patients
DKK-1 serum level was significantly higher in axial SpA patients than controls (mean
30.03 ± 15.5 vs. 11.6 ± 4.2 pmol/L; p<0.0001) (Fig 3A), with almost no overlap between
Fig 1. Serum sclerotin (SOST) level among patients with axial spondyloarthritis (SpA) and controls at baseline. Each point represents 1 patient. Data
are mean ±SD. ***p<0.0001.
doi:10.1371/journal.pone.0134974.g001
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 7/16
patients and controls (Fig 3B). This finding was confirmed in an independent SpA cohort
(SPACE; Fig 3A). DKK-1 serum level was weakly significantly correlated with systemic inflam-
mation assessed by ESR (r
s
= 0.1, p = 0.03), CRP level (r
s
= 0.17; p = 0.0001), hs-CRP level (r
s
=
0.14; p = 0.003), ASDAS-ESR (r
s
= 0.11; p = 0.02) and ASDAS-CRP level (r
s
= 0.13; p = 0.004).
(Fig 4A–4E)(Table 3) but not disease activity assessed by the BASDAI (r
s
= 0.052; p = 0.26).
Fig 2. Correlation of SOST serum level with age. (A) (r
s
= 0.36; p<0.0001), C-reactive protein (CRP) level (B) (r
s
= -0.18; p = 0.0001), high-sensitivity CRP
(hs-CRP) level (C) (r
s
= -0.22; p<0.0001), erythrocyte sedimentation rate (ESR) (D) (r
s
= -0.12, p = 0.007) and Dickkopf-1 (DKK-1) serum levels (E) (r
s
= 0.15,
p = 0.0008); r
s
: Spearman correlation coefficient.
doi:10.1371/journal.pone.0134974.g002
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 8/16
The association of DKK-1 serum level and ASDAS-ESR may be related to systemic inflamma-
tion rather than patient-reported disease activity.
DKK-1 serum level was significantly higher in HLA-B27-negative than-positive patients
(n = 79 vs n = 406; mean 33.97 ± 19.39 vs 29.99 ± 14.56 pmol/L; p = 0.04). DKK-1 serum level
was associated but not significantly with sacroiliitis on radiography (mean 33.02 ± 16.47 vs
29.93 ± 15.28, p = 0.056). None of the other studied variables (age, gender, weight, BASDAI,
NSAIDs, corticosteroids or DMARDs intake) were significantly correlated with DKK-1 serum
level.
DKK-1 serum level was increased but not significantly in patients with compared to without
axial involvement (mSASSS 1 unit vs 0; n = 61 vs. n = 399; mean 33.42 ± 17.11 vs
30.66 ± 15.53 pmol/L; p = 0.21).
Multivariate analysis revealed a significant positive association of DKK-1 serum level and
female gender (p = 0.03), CRP level (p = 0.006), SOST serum level (p = 0.002) and the presence
of sacroiliitis on radiography (p = 0.05) (Table 3).
Study of DKK-1 polymorphisms in relation to structural damage at
baseline and DKK-1 serum levels
Univariate analyses revealed a borderline significant association between rs7083441 and
rs11001445 with the presence of syndesmophytes at baseline (P
trend
= 0.08 and P
trend
= 0.07,
Table 2. Correlation between SOST serum level and characteristics of ASAS+ patients of the DESIR cohort.
Characteristic N Spearman r
s
p-value
a
β-coefficient p-value
b
DKK-1 level 479 0.15 0.0008 0.25 0.001
hs-CRP level 477 -0.22 10
−6
CRP level 462 -0.18 0.0001 -0.29 0.0008
ESR 461 -0.12 0.007
Age 479 0.36 <2.2 10
−16
0.83 <.0001
a
: Univariate analysis
b
: Multivariate analysis with hs-CRP and ESR excluded
doi:10.1371/journal.pone.0134974.t002
Fig 3. (A) Serum DKK-1 level among axial SpA patients and controls at baseline. Each point represents 1 patient. Data are mean ±SD. *** p<0.0001. (B)
Distribution of DKK-1 levels in controls and axial SpA patients.
doi:10.1371/journal.pone.0134974.g003
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 9/16
Fig 4. Correlation of DKK-1 serum level with systemic inflammation assessed by ESR. (A) (r
s
= 0.1, p = 0.03), CRP level (B) (r
s
= 0.17; p = 0.0001),
hs-CRP level (C) (r
s
= 0.14; p = 0.003), Ankylosing Spondylitis Disease ActivityScore (ASDAS)-ESR (D) (r
s
= 0.11; p = 0.02) and ASDAS-CRP level (E)
(r
s
= 0.13; p = 0.004); r
s
: Spearman correlation coefficient.
doi:10.1371/journal.pone.0134974.g004
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 10 / 16
respectively). However, multivariate analyses including variables previously associated with
structural damage at baseline (CRP, gender, smoking) failed to demonstrate an association of
these SNPs (or any of the 8 other genotyped SNPs) with structural damage at baseline (data
not shown). None of the studied polymorphisms contributed significantly to DKK-1 serum
levels, regardless of the genetic model assumed (recessive, dominant, additive), in either uni-
variate or multivariate analyses (data not shown). Haplotype analyses also did not reveal evi-
dence of association with DKK-1 serum levels.
Discussion
On investigating the serum levels of DKK-1 and SOST in a large cohort of patients with recent
axial SpA, we have demonstrated increased total DKK-1 level and decreased SOST level among
patients as compared with controls. Of importance, quantifications were not biased by
DMARDs and or anti-TNF treatments because all patients included in the cohort were naïve of
these drugs at baseline. Decreased SOST level in SpA patients was previously described [32]
and is expected in a disease associated with new bone formation. Conversely, results for DKK-
1 are new.
We found a significant association of low SOST serum level and sacroiliitis seen on radiog-
raphy (structural damage) among SpA patients from the DESIR cohort. Appel et al. also
reported low serum level of SOST in SpA patients significantly associated with the formation
of new syndesmophytes [32], and SOST inhibition (associated with TNF inhibition) led to a
significant regression of cortical bone erosions in TNF transgenic mice [33]. Subchondral
inflammation, bone erosion and exuberant bone formation being a continuous process in SpA,
low level of SOST at baseline could be associated with new bone formation resulting from over-
whelming healing occurring after inflammation and bone erosion.
Controls were age- and sex-matched with patients. To our knowledge, our work provides
new data based on a large cohort concerning the variation in DKK-1 level by age and gender in
healthy controls. DKK-1 serum level was not severely affected by these demographic character-
istics. Conversely, age was a significant predictor of SOST serum level in SpA patients. The cor-
relation between age and SOST level has not been reported in SpA but has been reported
among healthy women [30].
DKK-1 serum level was greatly elevated in SpA patients, without almost no overlap between
data for patients and controls. We previously demonstrated increased DKK-1 level in the
Table 3. Correlation between DKK-1 serum level and characteristics of ASAS+ patients of the DESIR cohort.
Characteristic N Spearman r
s
p-value
a
β-coefficient p-value
b
SOST level 475 0.15 0.0008 0.088 0.002
ASDAS-ESR 456 0.11 0.02
ASDAS-CRP 394 0.13 0.004 1.54 0.03
hs-CRP level 477 0.14 0.003
CRP level 462 0.17 0.0001 0.15 0.006
ESR 461 0.1 0.03
Gender (male) 486 NA 0.08 -3.18 0.03
Sacro-iliitis 476 NA 0.05 3.37 0.05
HLA-B27 485 NA 0.04 2.42 0.2
ASDAS, Ankylosing Spondylitis Disease Activity Score
a
: Univariate analysis
b
: Multivariate analysis with hs-CRP, ESR, and ASDAS-ESR excluded
doi:10.1371/journal.pone.0134974.t003
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 11 / 16
French cohort ESPOIR of rheumatoid arthritis and associated with increased risk of radio-
graphic progression [34]. Reconciling both results is difficult. In fact, as a marker of local bone
resorption, increased DKK-1 level is somewhat expected in RA but is unexpected in SpA, with
bone formation the hallmark of the disease. This increase may be linked to erosive lesions.
Unfortunately, we cannot answer this question because patients exclusively presenting erosive
lesions are underrepresented in the DESIR cohort. Prospective follow-up will help differentiate
erosive from sclerosing lesions. Nevertheless, the distribution of DKK-1 serum level among
SpA patients poorly supports this hypothesis because increased DKK-1 level largely repre-
sented SpA patients, more so than patients with exclusive erosive lesions.
Diarra et al. previously reported decreased serum DKK-1 level in SpA patients [20], but
Daoussis et al. reported higher serum DKK-1 level among SpA patients than controls [19].
These results are not contradictory because the ELISA test used in each study differed: in the
study from Diarra et al., DKK-1 serum level was assessed with human LRP6-coated plates (also
named functional quantification of DKK-1), whereas Daoussis et al. quantified circulating
DKK-1 level with a classical sandwich ELISA. Therefore, these latter results agree with our
study assessing free DKK-1 serum level. Nevertheless, the study by Daoussis et al. relied on a
small sample of patients (n = 45) and assessed DKK-1 serum level among patients with overt
ankylosing spondylitis fulfilling the New York diagnostic criteria. The results obtained in
DESIR cohort involving SpA patients with a short disease duration (18.8 ±11.6 months) are
thus complementary, showing that increased serum level of free DKK-1 is not restricted to the
overt severe structural forms of the disease but should be a more long-standing process. Daous-
sis et al. also studied the functional consequence of increased circulating DKK-1 level in SpA
patients. The authors assessed the effect of sera from SpA patients and controls on Wnt path-
way activation. Jurkat T cells were treated with LICL, a known activator of the Wnt signalling
pathway, then incubated with sera from SpA patients or controls and Wnt pathway activation
was assessed by measuring the level of dephosphorylated β-catenin (the active form). Serum
from SpA patients was unable to inhibit Wnt signalling pathway as compared with control
serum, despite increased level of circulating DKK-1.
Therefore, in SpA patients, free DKK-1 level is increased, but functional DKK-1 seems to be
decreased. The missing link between these observations could be abnormal binding of DKK-1
to its receptor among SpA patients. The origin of this dysfunction is unclear. DKK-1 and not
its receptor LRP6 may be dysfunctional because results observed for SOST, which shares the
same receptor, were opposite in our study. Second, based on our results genetic variation
appears to be unlikely to explain the increased DKK-1 serum levels. Further, neither linkage
nor genome-wide association studies have demonstrated a linkage or an association between
the DKK-1 locus on chromosome 10 and SpA [35]. Cortes et al previously assessed the role of
several polymorphisms of DKK-1 on SpA structural severity but failed to demonstrate evidence
of association, although only 3 DKK-1 SNPs were studied [36]. Our study, which assessed 10
SNPs encompassing DKK-1 locus failed to provide evidence of genetic association with DKK-1
serum levels and/or with structural damage at baseline. However, it is possible that rare coding
variants might interfere with DKK-1 function for a small subset of SpA patients. Alternatively,
post-translational modifications such as glycosylation or phosphorylation might lead to abnor-
mal binding of DKK-1 on LRP5/6.
The variables most significantly associated with DKK-1 serum level were SOST serum level
and those linked to biological inflammation, which agrees with the induction of DKK-1 by
TNF [18]. Moreover, TNF induces SOST in mature osteoblasts and is primarily mediated by
DKK-1 [18]. However, unlike RA, SpA is not characterized by high systemic inflammation.
Thus, inflammation should not explain alone the increased serum level of DKK-1.
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 12 / 16
Univariate analyses revealed high DKK-1 level (P
trend
= 0.056) and low SOST level
(P = 0.02) among patients with sacroiliitis on radiography. As well, DKK-1 serum level was sig-
nificantly reduced among HLA-B27–positive patients. In fact, these patients are expected to
have fewer structural or inflammatory lesions on radiography, thus fulfilling the “clinical arm”
of the ASAS criteria. Thus, DKK-1 level is increased when SOST level is decreased among
patients with structural lesions seen on radiography. DKK-1 might be unable to bind LRP5/6
correctly among some SpA patients, as discussed previously. DKK-1 and SOST may compete
for binding at LRP5/6, assuming that a higher affinity of SOST for its receptor would lead to
increased levels of free DKK-1. Nevertheless, the positive and significant correlation between
DKK-1 and SOST does not support this latter hypothesis, at least among one third of the SpA
patients corresponding to those with a DKK-1/SOST ratio >1.
In conclusion, we demonstrate higher total serum DKK-1 levels but lower serum levels of
SOST in SpA patients compared to controls. We also demonstrate an association between
DKK-1 and SOST levels and systemic inflammation and between SOST levels and age among
SpA patients. Our results suggest that increased DKK-1 serum levels among SpA patients is
unlikely to be explained by genetic variation at that locus. Prospective follow-up will help
improve our knowledge of the role of Wnt/DKK-1/SOST pathways in SpA. First it will help
clarify the interaction between treatment (NSAIDs, TNF-blockers) and DKK-1 or SOST levels;
Second, it will help better delineate the role of DKK-1 and SOST in structural disease progres-
sion (i.e., syndesmophyte formation) and/or in systemic bone loss in SpA. Finally, these results
raise the question of a potential dysfunction of DKK-1 linked with post-transcriptional modifi-
cations. Further studies are needed to unravel this puzzle to open up new therapeutic
perspectives.
Supporting Information
S1 Fig. Correlation of DKK-1 serum level assessment between 2 different ELISA kits (R&D
and Biomedica).
(TIF)
Acknowledgments
The DESIR cohort is conducted under the control of Assistance Publique-Hopitaux de Paris
via the Clinical Research Unit Paris-Centre and under the umbrella of the French Society of
Rheumatology and INSERM (Institut National de la Santé et de la Recherche Médicale). The
database management is performed within the department of epidemiology and biostatistics
(Professor Jean-Pierre Daurès, D.I.M., Nîmes, France). We also wish to thank the different
regional participating centres: Pr Maxime Dougados (Paris—Cochin B), Pr André Kahan
(Paris—Cochin A), Pr Olivier Meyer (Paris—Bichat), Pr Pierre Bourgeois (Paris—La Pitié-Sal-
petrière), Pr Francis Berenbaum (Paris—Saint Antoine), Pr Pascal Claudepierre (Créteil), Pr
Maxime Breban (Boulogne Billancourt), Dr Bernadette Saint-Marcoux (Aulnay-sous-Bois), Pr
Philippe Goupille (Tours), Pr Jean-Francis Maillefert (Dijon), Dr Xavier Puéchal (Le Mans), Pr
Daniel Wendling (Besançon), Pr Bernard Combe (Montpellier), Pr Liana Euller-Ziegler (Nice),
Pr Philippe Orcel (Paris—Lariboisière), Pr Pierre Lafforgue (Marseille), Dr Patrick Boumier
(Amiens), Pr Jean-Michel Ristori (Clermont-Ferrand),
Dr Nadia Mehsen (Bordeaux), Pr Damien Loeuille (Nancy), Pr René-Marc Flipo (Lille),
Pr Alain Saraux (Brest), Pr Corinne Miceli (Le Kremlin Bicêtre), Pr Alain Cantagrel
(Toulouse), Pr Olivier Vittecoq (Rouen). Furthermore, we want to thank all radiology
departments involved in the DESIR cohort.
DKK-1 and SOST in Spondyloarthritis
PLOS ONE | DOI:10.1371/journal.pone.0134974 August 27, 2015 13 / 16
We thank Dr Yassine Taoufik, Dr Pascale Chrétien, Bruno Oualid and Emilie Rouyer for
technical assistance and unrestricted access to the microplate system (Department of Immu-
nology, Hôpitaux Universitaires Paris Sud, France). We thank Annie Chou and Kevin Chen
for their assistance in genetic data analyses on STATA software.
Variété cohort was supported by the Programme Hospitalier de Recherche Clinique, French
Ministry of Health (no. P081216 / IDRCB 2009-A00892-55).
Author Contributions
Conceived and designed the experiments: CMR. Performed the experiments: GN SB PG P.
Chanson DvDH FvG FB XM KB AF P. Claudepierre MD CR CMR. Analyzed the data: GN SP
SB RS PG P. Chanson DvDH FvG FB XM KB AF P. Claudepierre PD JN KET LAC MD CR
CMR. Contributed reagents/materials/analysis tools: GN SP SB RS PG P. Chanson DvDH FvG
FB XM KB AF P. Claudepierre PD KET JN LAC MD CR CMR. Wrote the paper: GN SP SB RS
PG P. Chanson DvDH FvG FB XM KB AF P. Claudepierre PD KET JN LAC MD CR CMR.
Had full access to all of the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analyses: CMR.
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