Genetic susceptibility to systemic sclerosis from clinical aspect to genetic factor analyses.
ABSTRACT Systemic sclerosis is a rare autoimmune disease mainly characterized by vascular alteration and fibrosis involving skin but also visceral organs such as lungs, digestive tract, and heart. This disease leads to high morbidity and mortality. Its pathogenesis remains unclear, but recent attention has focus on genetic factors.
We first recall the main manifestations associated with systemic sclerosis and leading to its diagnosis and prognosis. Then we propose an overview on human genetics studies, as a number of genetic loci have been identified that appear to be associated with the disease.
Articles concerning association studies with candidate genes encoding for extracellular matrix proteins, cytokines, growth factors, chemokines, and proteins involved in vascular tone and immune regulations are presented and discussed.
Systemic sclerosis is a multigenic complex disorder. Genetic associations are observed in distinct phenotypes such as the diffuse cutaneous form or the limited form, or in association with specific autoantibody pattern. Promising candidate genes are those involved in pathways that lead to the vascular damage and fibrosis. A better knowledge of crucial mediators involved in systemic sclerosis could in the future provide new therapeutic strategies to control the disease.
- SourceAvailable from: Jonathan Kay[Show abstract] [Hide abstract]
ABSTRACT: OBJECTIVE:: To test the hypothesis that the subset of patients with impaired renal function who are exposed to gadolinium-containing contrast agents (GCCAs) and develop nephrogenic systemic fibrosis (NSF) have a genetic predisposition for disease. METHODS:: We examined whether an intronic single-nucleotide polymorphism (SNP) in caveolin-1 (CAV1 rs4730751) and 2 coding SNPs in transforming growth factor-beta 1 (TGFB1 rs1800471, codon 25; and rs1800470, codon 10) were associated with the NSF phenotype. RESULTS:: Forty-one patients with a history of chronic kidney disease and GCCA administration were studied, including NSF cases (n = 17) and control subjects (n = 24) without clinical or histological evidence of NSF. No significant differences in the genotype frequencies at these SNPs in TGFΒ1 and CAV1 were found between patients with NSF and subjects without NSF. CONCLUSIONS:: We conclude that polymorphisms in the genes encoding TGFΒ1 and CAV1 previously associated with the development and progression of fibrosis in several organ systems are not associated with development of NSF in this cohort of patients with renal impairment after GCCA exposure.The American Journal of dermatopathology 10/2012; · 1.30 Impact Factor
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ABSTRACT: Many environmental and genetic factors have been contributed to the development of systemic sclerosis (SSc). To determine whether IL-10 gene polymorphisms are associated with SSc, we conducted a meta-analysis approach. A total of eight studies involving 1,034 SSc cases and 1,815 controls were obtained by electronic database, i.e. Embase, Blackwell, Scopus, China National Knowledge Infrastructure database, Chinese Biomedical database, Google searching. We analyzed three gene polymorphisms, including IL-10 -1082G/A (rs1800896), IL-10 -819C/T (rs1800871), IL-10 -3575T/A (rs1800890). The combined odds ratio (OR) with its 95% confidence interval (95% CI) was calculated using fixed or random effect models. We found that IL-10 819C allele might contribute to SSc susceptibility by fixed effect model and IL-10 3575A allele could be an important risk factor for SSc, especially in European descent. No significant heterogeneity were observed. Under random effect model, there was no evidence of statistically significant association between IL-10 1082G/A polymorphism and SSc. Publication bias was absent in all analyses. However, larger scale primary studies are required to further evaluate the IL-10 polymorphism and SSc.Molecular Biology Reports 02/2012; 39(6):6851-5. · 2.51 Impact Factor
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ABSTRACT: Transforming growth factor-β1 (TGF-β1) plays an important role in the pathogenesis of systemic sclerosis (SSc). To investigate whether TGF-β1 gene promoter polymorphisms were associated with the susceptibility of SSc, we performed a meta-analysis based on all available studies through PubMed, Elsevier Science Direct, Embase, and Chinese Biomedical, China National Knowledge Infrastructure and Google Scholar with the last report up to March 15, 2013. Crude odds ratios with 95 % confidence intervals were used to estimate the strength of the association. A fixed or random effects model was adopted according to heterogeneity test. Heterogeneity among studies was evaluated using I (2) . Meta-regression was used to explore potential sources of between-study heterogeneity. Publication bias was estimated using Begg's and Egger's test. Totally, seven papers with 663 SSc patients and 908 healthy controls were subjected to the final analysis. These studies encompass seven for TGF-β1 codon 10, three for codon 25 and three for -509C/T. We failed to detect any association of these promoter polymorphism with SSc susceptibility. For TGF-β1 codon 10 polymorphism, subgroup analyses by race, genotype testing method and classification of SSc were further performed. Similarly, no association was observed. Significant heterogeneity was detected among the studies in all genetic models of TGF-β1 codon 10 polymorphism. Publication bias was absent. Taken together, our meta-analysis did not provided an evidence of confirming association between TGF-β1 (codon 10, codon 25, -509C/T) gene polymorphism and SSc. Nevertheless, due to smaller sample sizes, larger sample studies including different ethnic groups should be considered in future to confirm our results.Rheumatology International 07/2013; · 2.21 Impact Factor
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Genetic susceptibility to systemic sclerosis☆
From clinical aspect to genetic factor analyses
Brigitte Granela,b,⁎, Fanny Bernarda, Christophe Chevillarda,c
aINSERM, U906, Marseille, F-13385, France
bService de Médecine Interne, Hôpital Nord, Assistance Publique Hôpitaux de Marseille (AP-HM), chemin des Bourrely, 13915 Marseille Cedex 20, France
cLaboratoire de Parasitologie et de Mycologie, Faculté de Médecine, Université de la Méditerranée, Marseille, F-13385, France
Received 25 March 2008; received in revised form 18 June 2008; accepted 7 July 2008
Available online 11 September 2008
Background: Systemic sclerosis is a rare autoimmune disease mainly characterized by vascular alteration and fibrosis involving skin but also
visceral organs such as lungs, digestive tract, and heart. This disease leads to high morbidity and mortality. Its pathogenesis remains unclear, but
recent attention has focus on genetic factors.
Objective: We first recall the main manifestations associated with systemic sclerosis and leading to its diagnosis and prognosis. Then we propose
an overview on human genetics studies, as a number of genetic loci have been identified that appear to be associated with the disease.
Methods: Articles concerning association studies with candidate genes encoding for extracellular matrix proteins, cytokines, growth factors,
chemokines, and proteins involved in vascular tone and immune regulations are presented and discussed.
Results/conclusion: Systemic sclerosis is a multigenic complex disorder. Genetic associations are observed in distinct phenotypes such as the
diffuse cutaneous form or the limited form, or in association with specific autoantibody pattern. Promising candidate genes are those involved in
pathways that lead to the vascular damage and fibrosis. A better knowledge of crucial mediators involved in systemic sclerosis could in the future
provide new therapeutic strategies to control the disease.
© 2008 European Federation of Internal Medicine. Published by Elsevier B.V.
Keywords: Genetic association; Polymorphisms; Systemic sclerosis
Systemic sclerosis (SSc; scleroderma) is a connective tissue
disordercharacterized bymicrovascular alteration,autoimmunity
and fibrosis affecting skin and deep organs. Almost all patients
suffering from SSc have Raynaud's phenomenon (which is the
earlier sign of vasculopathy), have positive serum antinuclear
antibodies (autoimmunity markers) and will develop sclerodac-
tyly (skin thickness secondary to the fibrotic process). The
sclerotic process gave the name to the disease.
SSc remains a complex, polymorphic and heterogeneous
systemic disorder. Still nowadays, diagnosis of the disease is
based on clinical presentation, type of organs involvement, and
serum autoantibodies pattern. It is important to note that no
diagnosis of SSc can sometimes take years after the onset of the
first manifestation that is usually the Raynaud's phenomenon.
Molecular approach of the disease is being developed to obtain a
better understanding of the pathogenesis and mechanisms
involved in severity of the disease. The disease is supposed to
be the consequence of external trigger acting upon a genetically
susceptible host. Our purpose concerns genetic susceptibility of
scleroderma, and is mainly based on genetic association studies.
Up to date, various candidate genes with biological relevance to
disease pathogenesis have been analysed. Promising candidate
genes are those involved in pathways that lead to the vascular
damage and fibrosis. Genetic approach of scleroderma provides
European Journal of Internal Medicine 20 (2009) 242–252
☆Please see related article in this issue: Localized scleroderma: A series of 52
patients, Toledano C. et al.
⁎Corresponding author. INSERM, U906 et Laboratoire de Parasitologie et de
Mycologie, Marseille, F-13385, France. Tel.: +33 4 91 32 45 23; fax: +33 4 91
79 60 63.
E-mail address: firstname.lastname@example.org (B. Granel).
0953-6205/$ - see front matter © 2008 European Federation of Internal Medicine. Published by Elsevier B.V.
Author's personal copy
new insights into its pathogenesis and mechanisms leading to
specific disease phenotypes. However, genetic analyses are not
yet used by physicians to evaluate prognosis and severity of the
disease. A better knowledge of crucial mediators involved in
disease could in the future offer new therapeutic strategies to
control the progression of the disease.
2. Main clinical manifestations and classification of systemic
This review begins by recalling the main manifestations
of the disease. It is important to know the heterogeneous
phenotype which reflects different molecular mechanisms and
genetic susceptibility. Some manifestations are typical of SSc
and can differentiate this disease from other connective tissue
disorders. SSc has a high specific mortality due to organ-based
complications including pulmonary arterial hypertension, lung
fibrosis, renal failure and involvement of the gastrointestinal
2.1. Skin fibrosis
The most apparent feature of SSc is a progressive thickening
and sclerosis of the skin. The affected skin is indurate and firmly
over the hands, fingers (sclerodactyly) and face. The sclerotic
the dorsumof hands and face. Progression of the skin disease can
take between 5 and 15 years. SSc is sub-divided into two forms
and diffuse cutaneous SSc (dcSSc) (see classification below).
Inlargeseries,the typeofassociatedautoantibodycanpredict the
risk of skin evolution , but at the level of patient, it is not
I) autoantibodies are usually associated with a dcSSc form of
disease . However, nearly one third of anti-topo I positive
patients develop lcSS form of disease instead of the expected
diffuse form . Presence of anti-centromeres autoantibodies
(ACA) usually correlates with a limited form of the disease.
Although mortality was found highest among patients with the
worst skin involvement, no simple relationship between visceral
complications of disease and change in skin thickness score was
observed in large single-center cohort study .
2.2. Vascular involvement
Injury to vascular wall and extensive damage of the
microvessels are characteristic of SSc. From a clinical point
of view, vascular alterations may have various presentations,
from Raynaud's phenomenon (present in more than 85% of
patients) to severe digital ischemia leading to ulceration and
peripheral necrosis. Raynaud's phenomenon usually appears
simultaneously with other manifestations but may antedate
them by several years. Digital ulcers are extremely painful and
they lead to substantial functional disability . The injury of
the vascular wall is characterized by presence of giant
capillaries (mega capillaries) and avascular areas, easily seen
at naifold capillaroscopy (NC). The digitized video-capillaro-
scopy (NVC) is developing as a powerful new method to assess
individual capillaries over time. Different morphological
aspects are observed: enlarged and giant capillaries, hemor-
rhages, loss of capillaries, ramified capillaries and vascular
architectural disorganization. By NVC, three patterns of
vascular alteration have been described: early, active and late
. More recently, the magnetic resonance angiography (MRA)
has been proposed to assess hand vascular involvement in SSc
. This new non-invasive device allows the visualisation of the
microcirculation and also small-caliber vessels, which are both
involved in SSc . However, further investigations are
warranted to evaluate the interest in clinical practise of these
SSc involves many vascular beds and contributes to pulmonary,
renal, cardiac and gastrointestinal complications .
2.3. Gastrointestinal involvement
Gastrointestinal involvement is frequent in SSc, occurring in
75 to 90% of patients with diffuse or limited cutaneous disease
. Although all regions of the gut may be affected, the
esophagus is the most common gastrointestinal localization of
this disease. Motility studies show reduced-amplitude or absent
peristaltic contractions of the distal two-thirds of the esophagus
and decreased lower esophageal sphincter pressure. This leads
to clinical symptoms such as reflux, burn, and dysphagia. In
severe cases, chronic esophagitis can lead to esophagal stricture
and endobrachyesophagus, a pre malignant lesion. Other
manifestations include hemorrhage related to watermelon
stomach (gastric antral vascular ectasia), abdominal distension,
pseudo-obstruction syndrome, bacterial overgrowth with sec-
ondary malabsorption, diarrhoea/constipation and weight loss.
Digestive motor disturbances are well evaluated by oesophageal
manometry and intestinal manometry . Manometric findings
indicate that there are both neuropathic and myopathic
component of upper intestinal tract dysfunction in SSc.
Anorectal involvement is frequent in SSc patients (50–70%)
and causes fecal incontinence and rectal prolapse .
2.4. Pulmonary involvement
It represents one of the most serious visceral complications in
SSc, leading to respiratory insufficiency, and often death.
Scleroderma lung disease encompasses vascular (pulmonary
artery hypertension), interstitial lung disease, or both. Other non
specific manifestations are obstructive disease, pleural disease,
lung cancer, and aspiration . Pulmonary arterial hypertension
(PAH) is a serious complication of SSc and a leading cause of
death in patients. Pulmonary arterial hypertension is found in
approximately 10–15% of patients with SSc . The prognosis
remains poor compared to that of idiopathic PAH. A compre-
hensive work-up is required to delineate the underlying cause of
dyspnoea in a scleroderma patient, and to establish the
contribution of each component to the symptoms. For detection
of PAH, it is advised to perform first of all an echocardiography
243 B. Granel et al. / European Journal of Internal Medicine 20 (2009) 242–252
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but also an electrocardiogram, to measure the diffusing capacity
for carbon monoxide/alveolar volume (DLCO/VA) ratio, and
serum N-terminal pro-brain natriuretic peptide (NT-proBNP)
level. If pulmonary artery hypertension is suspected, a right-heart
catheterisation is necessary to confirm the diagnosis . To
detect interstitial lung disease, it is advised to perform pulmonary
function testing and high-resolution thoracic CT scanning.
Bronchoalveolar lavage is optional. The 6-minute walk test, a
sub maximal exercise test, may serve as a simple clinical tool to
assess functional capacity. It measures the distance ambulated
during a 6-minute walk. Lung disease in SSc contributes
significantly to excess morbidity and early mortality, especially
when diffusion capacity drops below 40% and/or forced vital
capacity below 50% .
2.5. Cardiac involvement
Primary myocardial involvement is common in systemic
sclerosis . The underlying mechanism involves microcircu-
latory impairment and abnormal vasoreactivity. Cardiomyopathy
with left ventricular or biventricular failure may result from focal
ischemic-induced myocardial fibrosis. Pericardial involvement is
frequent but usually asymptomatic. Conduction system abnorm-
alities appear common but not serious, while arrhythmias may be
2.6. Renal disease
It was the most rapidly fatal visceral involvement, named
“scleroderma renal crisis” (SRC) characterized by abrupt onset of
malignant hypertension and rapidly progressive renal insufficiency
and microangiopathic hemolytic anaemia. The diffuse form of SSc
is most of the time concerned.Steroiduse has been considered as a
triggering factor of SRC. In spite of treatment with angiotensin-
converting enzyme inhibitors (ACEI), hemodialysis is required
permanently in around 40% . Poor renal outcome is usually
associated with lower blood pressure at presentation. Mortality is
high (59% survival at 5 years), and is higher in men .
2.7. Classification of SSc
Disease heterogeneity and difficulties in separating SSc from
SSc-like conditions make classification an important issue.
Although not the primary intent of such criteria, they can serve
as guidelines to accurate diagnosis by allowing the discrimination
with other diseases. In 1980, preliminary criteria of American
Rheumatism Association for the classification of SSc  have
been referenced as diagnostic criteria. They included one major
criterion, proximal scleroderma, and three minor criteria, 1) scler-
the distal finger pad, and 3) bilateral basilar pulmonary fibrosis.
These proposed criteria had 97% sensitivity for definitive SSc and
98% specificity. As recently noticed by Johnson and Laxer ,
these preliminary criteria were initially created to be specific rather
than sensitive to minimize false-positive diagnosis. Thus, they
exclude some SSc patients, in particular those with the limited
disease, patients with highly disease-specific serologic markers
(such as anti-topoisomerase I), and patients with rapidly
progressive course . This classification could potentially lead
to exclusion of patients with the limited form of the disease.
The other used classification of SSc patients has been proposed
by LeRoy EC et al.  in 1988 (Table 1) and identifies two SSc
subsets; diffuse cutaneous SSc (dcSSc) and limited cutaneous SSc
(lcSSc). In this classification, lcSSc includes and replaces the
CREST variant of SSs: Calcinosis, Raynaud's phenomenon, Eso-
phagal dysmotility, Sclerodactyly, and Telangiectasia syndrome.
In 2002, analysis of a cohort of 309 French Canadian patients
included a classification in four subsets based on the extent of
involvement (restricted to sclerodactyly), 3) intermediate (sclero-
sis of upper extremities proximal to metacarpophalangeal joints
and face without trunk involvement, and 4) diffuse (sclerosis
including trunk) . In this series, the percentage of deaths per
subsets increased from 8% in the normal skin group to 21%,
26.9% and 31% inthe other subsets,respectively(p=0.039). The
major cause of death in this series was SSc, accounting for 53%
skin subset”, the death was SSc-related in 43.8% in the “limited”,
These data provided evidence in favour of a distinct intermediate
SSc subset. However, thisissue remains unsettled,and for others,
there is no convincing evidence of any advantage for distinguish-
ing the limited, intermediate and diffuse forms of SSc rather than
only the limited and diffuse forms .
3. Evidence for a genetic component to systemic sclerosis
Although SSc is not inherited in a Mendelian fashion, the
contribution of genetic factors in susceptibility to the disease is
supported by the following points (for more details, see ).
Subsets of SSc (LeRoy et al. ).
Diffuse cutaneous SSc (dcSSc)Limited cutaneous SSc (lcSSc)
– Onset of Raynaud's phenomenon
within 1 year of onset of skin
– Truncal and acral skin involvement
– Presence of tendon friction rubs
– Early and significant incidence of
interstitial lung disease, oliguric
renal failure, diffuse
gastrointestinal disease, and
– Absence of anticentromere
– Anti-topo I antibodies (30% of
– Nailfold capillary dilatation and
– Raynaud's phenomenon for years
– Skin involvement limited to hands,
face, feet, and forearms or absent
– A significant late incidence of
pulmonary hypertension, with or
without interstitial lung disease,
trigeminal neuralgia, skin
– A high incidence of ACA
– Dilated nailfold capillary loops,
usually without capillary dropout
244 B. Granel et al. / European Journal of Internal Medicine 20 (2009) 242–252
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families), SSc occurs significantly more frequently in families
with scleroderma (1.6%) than in the general population
(0.026%). The relative risks in first-degree relatives in these
cohorts ranged from 10 to 16 (13 combined), and in siblings
they ranged from 10 to 27 (15 combined). A positive family
history of SSc was the strongest risk factor yet identified for
SSc; however, the absolute risk for each family member
remains quite low (b1%).
– The high frequency of autoimmune disorders and
autoantibodies in family members of patients with SSc .
– The difference in prevalence and clinical manifestations
among different ethnic groups. For example, the Choctaw
tribe in Oklahoma is a population with a high prevalence of
SSc (2 times higher than the expected prevalence) and a
homogeneous clinical and immunological phenotype.
– Existence of association between SSc and certain HLA
(Human Leukocytes Antigens) alleles particularly among
different clinical subsets of the disease, or depending on the
specific antibodies . HLA class II genes (DRB1⁎01,
DRB1⁎17 and DRB1⁎11) have been implicated as strong risk
factors in the pathogenesis of SSc, across several continents
. These alleles are associated with particular subsets and
have been associated with anti-topoisomerase I . Anti-
centromereantibodies have been associated with HLA class II
DRB1⁎01 and DRB1⁎11 [25,26] or DQB1⁎alleles .
– Although concordance of SSc among identical twins is
only 4.2% for clinical disease expression and not signifi-
cantly different from the concordance of the disease in
dizygotic twins (5.9%), concordance for the presence of
antinuclear antibodies is substantially higher (40% in
dizygotic versus 90% in monozygotic twins) . Further-
more, micro array and quantitative real-time PCR studies
of cultures dermal fibroblasts in the same cohort showed a
40–50% concordance rate for gene expression profiles in
dermal fibroblasts from monozygotic twins clinically dis-
cordant for SSc .
– Animal models of SSc such as the tight skin mouse (Tsk)
with spontaneous (Tsk1) or induced (Tsk2) fibrillin-1
mutation also showed the role of a genetic susceptibility.
These mice develop a scleroderma-like disease (skin, tendon
and cardiac fibrosis) and autoantibodies . Several mouse
model studies were performed. However, all these studies
were involving mice with different genetic backgrounds that
may induce discrepancies in the results similarly to genetic
backgrounds and ethnic origins in human studies [31,32].
In SSc, the number of multiplex families is too small to allow
linkage analysis. The disease is rare with a prevalence rate
estimated in the United States of 242 to 286 cases/million
population . Disease prevalence differs geographically, with
European and Japanese rates for SSc less than those observed in
the United States or Australia . Variations in genetic
predisposition and environmental exposures may be one
explanation of this geographic discrepancy.
Genetic approach in SSc is based on candidate genes
analysis in cases versus controls population. Gene polymorph-
isms are variants of coding or non-coding DNA sequences
whose prevalence varies within the general population. Single
nucleotide polymorphisms (SNPs) defining a genetic locus in
which two or more alleles have gene frequencies greater
than 1%, and microsatellites markers (short sequences of
repeated nucleotides in non-coding regions) can be analysed.
Genetic susceptibility to SSc probably involves association of
polymorphisms in multiple genes with single altered gene
making a small contribution to the disease. Genes coding for
extracellular matrix (ECM) proteins, cytokines, growth factors,
Genes coding for cytokines and growth factors found associated with SSc.
GenePolymorphismsOrigin of the
Genes encoding cytokines
Interleukin 1 alpha
1) SNP − 889T1) Slovak
2) SNPs −889C,
+4729T, +4845G and
IL1B-31 C allele and
IL1B-511 T allele
associated with lcSSc
1) GCC haplotype less
frequent in dcSSc
2) IL10-3575T/A and
Interleukin 1 beta
Interleukin 2 (IL2)
Interleukin 10 (IL10)1) English
Interleukin 13 (IL13)
1) TNF-1031C and
2) TNF-238A and
anti-topo I (n′=22)
Genes encoding growth factors
factor beta 1, 2, 3
(TGFB1, 2, 3)
1) TGFB1 at codon 10
2) TGFB2 (D1S419)
and TGFB3 (D14S277)
−945GG associated with
SSc and presence of
anti-topo I (n′=155)
Genes encoding chemokines
Interleukin 8 receptor
CXCR2+785 CC and
n: number of whole SSc patients, n′: number of SSc patients in the subgroup.
245 B. Granel et al. / European Journal of Internal Medicine 20 (2009) 242–252
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chemokines, proteins involved in vascular tone and immune
regulations are considered candidate for investigation. Some
crucial reviews have already been published on the topic and
must be recalled: Johnson et al.  in 2002, Jimenez and Derk
 in 2004, Abraham and Varga  in 2005, the excellent
review of Assassi and Tan  on SNPs and microarrays
analysis in SSc published in 2005, and the more recent review
of Allanore Y et al. . We propose here to discuss genes
affecting regulation of fibrosis. They are listed in Table 2. Other
genes encoding for ECM components, vascular/oxidative
factors, and immune regulation are noted in Table 3, and will
not be developed.
4.1. Interleukin 1 alpha (IL1-α)
It has been shown that SSc-affected fibroblasts constitutively
expressed IL-1α. Endogenous IL-1α potentially acting at the
nucleus could be involved in the establishment of a fibrogenic
phenotype: IL-1α increases procollagen production and fibro-
blasts proliferation, and induces IL-6 production . In a
Japanese population, Kawaguchi Y et al. showed a frequency
and carriage rates of alleles C at −889, Tat +4729, and allele G
at +4845 in gene of IL-1α significantly higher in SSc patients
Other genes associated with SSc.
Gene Polymorphisms Origin of the population Ref.Comments
Components of the extracellular matrix
SPARC Microsatellite markersPopulation Choctaw (n=20)Conflicting: no association with SPARC SNPs in
UK Caucasian patients (n=121) 
No other data
Conflicting: no association with SNP in intron
C and 5 microsatellites markers in European
Caucasian patients including 243 French SSc and
266 Italian SSc 
Comment: only associated with lung fibrosis
No other data
1) Haplotype 2cM on 15q
2) SNP1 (intron C, +49)
Population Choctaw (n=18)
and Japanese (n=53)
FNRFLP SNPsEnglish (n=161)
TIMP-1Microsatellite markersEnglish (only male SSc n=29) 
Vascular and oxidative factors
EDN1, EDNRA, EDNRBEDN1-1370, +138, +85, +23
EDNRA−312, +69, +105
EDNRB+2841, −2547, −2446
UK (n=205)Comment: no significant association between all
these SNPS and the SSc group as a whole.
However, SSc patients with diffuse skin
involvement (n′=69) had a significant
increased frequency of EDNRB−2446A,
−2547A and +2841G
Comment: Association of this polymorphism with
risk of PÄH in European Caucasians SSc (n′=29)
Conflicting: 2 additional studies of G894T SNP in
French Caucasian (n=77)  and African
American (n=28), White (n=76) and Hispanic
(n=53)  showed no association
with this polymorphism
Conflicting: no association with SSc patients and
the ACE D allele or ACE I/D genotype in
population of various ethnic origins (n=157) 
ENGInsertion polymorphism in intron
7 associated with PAH
Caucasian (n=180 but 29
eNOS+894T alleleItalian (n=73)
ACEACE deletion (ACE D) in
CTLA-41) SNPs +49
2) SNP −1722, −1661 and −318
1) African American (n=37)
2) Iranian (n=83)
[88,89]Conflicting: Takeuchi et al. did not find any
significant association between SNPs +49 and
−308 (n=62) , SNPs −1772 and
−1661 (n=61) , and SSc patients in the
No other data
−499T allele in the promoter and
(GT)14allele in the 3′-untranslated
PTPN22T allele associated with
anti-topo I antibodies in white
Japanese (n=134) 
PTPN22 American (n=1120) Conflicting: no association between PTPN22
620W allele and SSc patients in French
Caucasian population (n=121) 
n: number of whole SSc patients, n′: number of SSc patients in the subgroup.
ACE: angiotensin-converting enzyme, CD19: B cell receptor, CTLA4: cytotoxic T-lymphocytes-associated antigen 4, COLA1: type I collagen, ENG: endoglin,
EDN1: endothelin 1, EDNRA: endothelin receptor A, EDNRB: endothelin receptor B, ENOS: endothelial nitric oxide synthase, FBN1: fibrilline-1, FN: fibronectin,
PAH: pulmonary arterial hypertension, PTPN22: protein tyrosine phosphatase non-receptor type 22, RFLP: restriction fragment length polymorphism, SPARC:
secreted protein, acidic and rich in cysteine, TIMP1: tissue inhibitor of metalloprotease-1.
246B. Granel et al. / European Journal of Internal Medicine 20 (2009) 242–252
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(n=60) than in healthy controls (n=70) (pb0.0001) . These
SNPs were inlinkage disequilibrium.The frequency of the CTG
haplotype was significantly higher in SSc patients than in
healthy controls [pb0.0001, (odds ratio) OR 19.2, 95%CI 8.0–
46.6)]. These results indicate that the CTG haplotype increases
the risk of SSc around 20 times. However the association
between C allele at −889 has not been confirmed in two
independent cohorts of patients from other origin. Matuzzi et al.
 found no association with IL1A C-889T SNP in 78 patients
with SSc compared with 692 healthy blood donors from Italian
origin. However, they did not analyse the CTG haplotype. In 46
Slovak patients with SSc, Hutyrova et al.  reported that the
T allele at −889 (and not C) was significantly overrepresented
in SSc (38% compared with 25.7% in the 150 healthy controls,
p=0.02, OR 1.78, 95%CI 1.09–2.91). One explanation of the
contradictory result observed in Japanese and Slovak popula-
tion could be related to the difference of genetic background
with different allele frequencies between these two populations.
This is illustrated by thefact that frequency of the IL-1α −889T/
T genotype which was of 0% in Japanese SSc patients 
whereas it was of 13% in Slovak SSc patients . Moreover,
as these two studies were performed on a limited number of
patients (respectively 60 and 46), they could be not powered
enough for such conclusion. Recently, genetic association
analysis performed in a larger population of patients from Italy
(n=204) did not found any association between the IL1AC-
889T SNP and the disease .
4.2. Interleukin 1 beta (IL-1β) and interleukin 2 (IL-2)
9 SNPs in 7 cytokine (inflammatory related) genes: IL10, IL1B,
IL1A, IL1RN, LTA, IL2 and IL6 . In their work, IL1B-31 C
allele and IL1B-511 T allele showed a significantly different
distribution between cases (n=78) and controls (n=692). Carriers
of at least one copy of the IL1B-31Callele had an increased riskof
SSc (pb0.001, OR 2.8, 95%CI 1.6–5.2). A similar strong
association was also evident for IL1B-511T allele (pb0.001, OR
3.1, 95%CI 1.7–5.7). When analysing SSc subsets, they showed
among patients with lcSSc (80.8%, n=47), compared to patients
with the dcSSc (45.1%, n=31) (OR 5.1, 95% CI 1.8–14.3,
p=0.001) and in subjects positive to anticentromere antibodies
(n=43) (OR 4.2, 95% CI 1.5–11.9, p=0.007). The authors
concluded that IL1B and IL2 gene polymorphisms may be
involved in susceptibility to SSc, with IL2-384G allele possibly
being a marker for the limited phenotype of SSc . However, as
these data relied on a limited number of patients, they should be
on an Italian population and on a larger number of SSc patients
and the disease .
4.3. Interleukin 10 (IL-10)
IL-10 is an anti-inflammatory cytokine produced primarily by
monocytes, T cells and B cells. It is encoded by a gene on
chromosome 1q32. IL-10 plays an important role in regulatory T
cells. IL-10 also regulates the expression of several genes
involved in ECM synthesis and remodelling in human dermal
of type I collagen, whereas it greatly enhances collagenase and
stromelysin . The response to IL-10 of dermal fibroblasts in
SSc patients is similar to that observed in normal dermal
fibroblasts . IL-10 also down regulates the TGF-β-induced
increase of mRNA expression of type I collagen and fibronectin
of IL-10 gene in SSc. Hudson et al.  have reported an
association between SNPs located in the promoter of the gene
(IL10-3575T/A and IL10-2763C/A) and SSc (with p=0.0005
and 0.002 respectively) in a Caucasian population (including 105
SScand143controls):theriskofdevelopingSSc was threetimes
In the second study published by Crilly et al. , no association
was found between SSc and three IL10 SNPs located within a
1.3 kb region proximal to the gene: −1082G/A, −819C/T, and
−592C/A. However, after construction of haplotypes, their
analysis found a lower frequency of the GCC haplotype
(−1082G−819C−592C), which is functionally characterized
by high IL-10 production, in patients with dcSSc (n=51)
compared to control (n=94) and lcSSc groups (n=89) (controls
versus dcSSc; 29 versus 4%, p=0.005, lcSSc versus dcSSc; 22
versus 4%, p=0.002). Thus, genetically controlled low Il-10
production could represent a risk factor for SSc and particularly
the diffuse cutaneous form. These two studies (Hudson et al. and
Crillyetal.)are not comparable: 1.Crillyetal. studiedIL-10
SNPs located in the proximal region of the promoter whereas
Hudson et al.  examined distal SNPs, 2. Crilly et al. 
analysed homogeneous Scottish Caucasians whereas Hudson
replication of these results has been published. Beretta et al. did
not find any association (at single SNP level) between the IL10-
1082G/A and SSc (n=204) in an Italian population .
However, this group did not perform haplotype analysis.
4.4. Interleukin 13 and its receptor
Interleukin 13 (IL-13) is an immunoregulatory cytokine
predominantly secreted by activated Th2 cells. IL-13 appears to
be necessary in the effector phase of inflammation and has been
shown to be involved in abnormal fibrosis . Therefore, we
evaluated its possible involvement analysing four IL13 SNPs in
107 unrelated SSc patients (40 dcSSc and 67 lcSSc) and in 170
controls . All subjects were Caucasians. In whole SSc
population and in dcSSc subset, an association was observed
with a functional IL13 rs1800925 SNP (named IL13-1055), and
with IL13 rs2243204 (p=0.03). The IL13 rs2243204T allele
was more common in SSc patients (p=0.01, OR 2.3 95%CI
1.21–4.38) and in dcSSc form (p=0.01, OR 2.95, 95%CI 1.35–
6.49) than in control subjects.
In the same population, IL13RA1 and IL13RA2 genes
coding for the two chains of the IL-13 receptor, were analysed
. As they are located on the X chromosome and SSc is far
more common in women than in men, only women were
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genotyped (n=97). We detected an association between
IL13RA2 rs638376 and SSc (p=0.004, OR 1.85, 95%CI
1.22–2.74), and with dcSSc subgroup of patients (p=0.01, OR
2.22, 95%CI 1.27–3.89). IL13RA2 rs638376G allele frequency
was higher in women with SSc (51.6%) and in dcSS subgroup
(57.6%) than in controls (36.4%) (with respectively p=0.003,
OR 1.86, 95%CI 1.24–2.79 and p=0.003, OR 2.37, 95%CI
1.35–4.15). These data suggest that IL13 and IL13RA2 gene
polymorphisms may be involved in susceptibility to SSc. So far,
no replication of these results has been published.
4.5. Tumor necrosis factor alpha (TNF-α) and tumor necrosis
factor beta (TNF-β)
TNF-α is a proinflammatory cytokine known to be involved
in inflammatory rheumatic disease. TNF-α may have anti-
fibrotic activities: it blocks TGF-β induced genes and signalling
pathways in SSc dermal fibroblasts and decreases production of
type I collagen and tissue inhibitor of metalloproteinases 1 .
In an English population of 214 UK SSc patients and 354
healthy controls, 5 TNFA promoter SNPs have been analysed
. An increase in the frequency of C allele at position −1031
was observed (46.3% in SSc versus 35.3% in controls,
p=0.05). Stratification of patients based on their autoantibodies
profile showed a striking association between anticentromere
antibodies and TNF-1031C allele (p=0.00005), and also with
TNF-863A allele (p=0.00002). One year later, the work of
Tolusso et al.  performed in an Italian population of SSc
(n=114), revealed a weak association between two other SNPs,
with presence of A allele in position −238 and the AG genotype
in position +489 of the TNFA in SSc as a whole when compared
with healthy blood donors (p=0.03 for −238 and p=0.02 for
+489, respectively). The strength of the association was mainly
due to the dcSSc phenotype (p=0.04 for TNFA-238, pb0.01
for TNFA+489). Finally, the contribution of microsatellite
polymorphisms of TNFA to the pathogenesis of SSc was
studied in a Japanese population (54 SSc and 69 controls): the
frequency of TNFA13 was significantly increased in the 22
Japanese SSc with anti-topo I (p=0.028, OR 6.88, 95%CI
1.16–22.60) compared with controls . However TNFA13
was positively in linkage disequilibrium with HLA-
DRB1⁎1502 (LD=0.053, t=2.69). So, these two markers are
travelling together in the study populations, and thus it was
difficult to conclude to a real contribution of TNFA13 .
These results should be replicated in a larger population to be
considered as valuable. Moreover, as mentioned previously,
strong linkage disequilibrium between TNF-α gene and HLA
class II does not allow the identification of causal
One study evaluating the frequency of TNF-β (lymphotoxin
alpha) gene polymorphisms in SSc from Japan showed that
homozygous genotypes of the TNFB+252 locus located at
6p21.3 were significantly associated with SSc (n=50) .
Compared to controls, the frequency of the TNF-1 genotype
was decreased (6% versus 23%, p=0.016), whereas that of
TNF-2 was increased in SSc patients (56% versus 32%,
p=0.012), and particularly in diffuse patients (n=19) compared
to controls (68% versus 32%, p=0.0068). So far, no other
genetic association studies on TNF-β gene have been
4.6. Transforming growth factor beta 1, 2, 3 (TGF-β1, -2, -3)
Growth factors of the TGF-β family (including isoforms 1, 2
and 3) have been implicated in the pathogenesis of SSc because
of their ability to stimulate ECM proteins production and to
inhibit ECM degradation . TGF-β mRNA for all isoforms
were detected in inflammatory skin areas (early stage of SSc)
but not in sclerotic or healthy skin . Crilly et al. ,
analysed the distribution of TGF-β1 genotypes at codon 10
(+869 polymorphism) and codon 25 (+915 polymorphism) in
patients with SSc from UK (152 SSc patients, 63 dcSSc and 89
lcSSc). The authors had previously calculated that at 90%
power to show a 25% difference, they would require 61 subjects
in each study group. They observed significantly more patients
with SSc than controls carrying allele C at codon 10 (48%
versus 38%, p=0.004, OR 1.95, 95%CI 1.16–3.27). The
difference remained significant when patients with SSc were
split into those with dcSSc and lcSSc forms (controls versus
dcSSc, p=0.02 and controls versus lcSSc, p=0.013). Posses-
sion of allele C at codon 10 gave an OR 4.8, 95%CI 2.8–8.4
. No difference in allele frequency was seen between
patients with SSc and controls at codon 25. As presence of allele
C at codon 10 was associated with higher TGF-β1 mRNA and
protein levels, these results suggest that patients with SSc are
genetically predisposed to high TGF-β1 production . The
associated polymorphism does not, however, explain the
difference in the clinical phenotypes of limited and diffuse
SSc . However, and in marked contrast with Crilly et al.,
two other studies have not confirmed these results [57,58]. As
for other cases of failure of replication, difference in the ethnic
background could be an explanation (Choctaw population
living in Oklahoma in the study of Zhou et al. and Japanese
patients in the study of Sugiura et al., compared with unrelated
Scottish subjects in the investigation of Crilly et al.). Difference
in linkage disequilibrium between the populations may also
contribute to these restricted TGF-β1 genotype associations. As
noted by Pandey and LeRoy , genotype frequencies for the
data presented by Crilly et al. were not in Hardy–Weinberg
equilibrium, preventing any conclusion. Finally, in addition to
the host genetic factors, exposure to various occupational and
environmental chemicals have been involved in SSc pathogen-
esis. Thus, population differences in exposure to these
environmental risk factors may also contribute to the differences
in disease susceptibility .
Moreover, Susol et al.  investigated whether six
microsatellite markers (MM) known to map closely to genes
involved in fibrosis (including TGFB1, TGFB2, TGFB3,
PDGFB, TIMP1 and COL5A2) were associated with SSc in a
Caucasian population from UK (191 SSc and 196 controls).
Associations were found between SSc and markers for TGFB3
(p=0.02), TGFB2 (p=0.02) and TIMP1 (only male SSc, n=29,
p=0.02) . Between lcSSc (n=151) and dcSSc (n=40)
patients, the allele frequency distribution differed only for the
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TGFB3 marker (p=0.01) . These data suggest a role for
TGFB3, TGFB2 and TIMP1 in genetic susceptibility to SSc and
for TGFB3 in determining the degree of cutaneous fibrosis .
No replication study has been published.
4.7. Connective tissue growth factor (CTGF)
CTGF (CCN2), discovered more than a decade ago as a
protein secreted by human endothelial cells, is induced by TGF-
β and is considered a downstream mediator of TGF-β on
fibroblasts [61,62]. Expression of the gene encoding CTGF was
found greatly up-regulated in skin biopsy specimens and in
fibroblasts cultured from skin and lung tissues from patients
with SSc . Fonseca et al.  genotyped a polymorphism
(G-945C) in the promoter of CTGF gene in 1000 subjects (500
SSc) from the UK including two groups: group 1 of 200 patients
and 188 control subjects; and group 2 of 300 patients and 312
control subjects. They showed that GG genotype was
significantly more common in patients with SSc than in control
in both groups, with an OR for the combined group of 2.2 (95%
CI 1.5–3.2, pb0.001) . Analysis of the combined group of
patients with SSc showed a significant association between
homozygous for the G allele and the presence of anti-topo I
antibodies (n=155) (OR 3.3, 95%CI 2.0–5.6, pb0.001) and
fibrosing alveolitis (n=207) (OR 3.1, 95%CI 1.9–5.0,
pb0.001) . With functional tests, they showed that the
G-945C substitution repressed CTGF transcription, with the
G allele strongly linked to transcriptional activation of CTGF.
Thus G allele was associated in their series to the risk of SSc,
particularly in patients with anti-topo I antibodies and pulmonary
fibrosis . However, recent replication of this association in a
large and independent US cohort of patients failed . It
included 2315 subjects (white North American, African Amer-
ican, Hispanic-American) divided in two independent cohorts of
patients with SSc and controls matched for race or ethnic group.
No association between CTGF (G-945C) and SSc or its
autoantibodies was observed in both of the cohorts in all three
ethnic groups. Once again, difference in genetic association
studies between different populations (North Americans of
could explain these discrepancies.
4.8. Monocyte chemotactic protein (MCP-1)
MCP-1 (CCL2) is a predominant monocyte chemoattractant
and activator of mononuclear cells. In addition to its
chemoattractant activities, MCP-1 has profibrotic activities by
stimulating type I collagen gene expression in fibroblasts. This
effect is mediated in part by stimulating fibroblasts production
of TGF-β then acting in an autocrine manner to stimulate ECM
formation . Another important profibrotic mechanism of
MCP-1 is to limit prostanglandin E2 production in alveolar
epithelial cells after injury, thus promoting fibrosis . In SSc,
abnormal regulation of MCP-1 has been observed and this
chemokine could play an active role in initiation of inflamma-
tion and fibroblast activation [66,67]. Only one study concerns
MCP-1 gene analysis in SSc : the frequency of the
functionally relevant −2518G MCP-1 promoter polymorphism
was investigated in 18 patients with SSc and 139 healthy
controls. Despite the very small sample size, genotyping for the
−2518 A/G MCP-1 promotor polymorphism showed that GG
homozygotes were significantly more frequent in patients than
in controls (28% versus 6%, p=0.023). In addition, character-
ization of MCP-1 expression by immunohistochemistry
revealed that MCP-1 was expressed in keratinocytes, infiltrating
inflammatory cells, fibroblasts, and endothelial cells in
scleroderma skin, whereas normal control skin showed no
MCP-1 expression . Finally, basal as well as TNF-induced
MCP-1 expression of fibroblasts isolated from a GG-homo-
zygous SSc patient was significantly higher than from
heterozygous or AA-homozygous donors . Although this
represents a small study, the functional data strongly suggest
that the −2518G polymorphism of the MCP-1 gene may
predispose patients to SSc . However, confirmation by
replicate analysis should be performed.
4.9. Interleukin 8 receptor family (CXCR2)
Interleukin 8 (IL-8) is a chemokine with a potent
chemoattractant activity for neutrophils. High concentration of
IL-8 was observed in bronchoalveolar lavage fluid from patients
with SSc-related fibrosing alveolitis . Two SNPs (CXCR2+
785 and CXCR2+1208) in the 3′ untranslated region of the
gene coding for CXCR2, a member of the IL-8 receptor family,
were found more common among patients with SSc (with and
without fibrosing alveolitis, n=128) compared with controls
(for CXCR2+785 CC, 37% versus 22%, p=0.01 and for
CXCR2+1208 TT, 33% versus 17%, p=0.003) . This
result has not been replicated yet. To date, no association have
been reported with IL8 gene.
4.10. Other analysed genes
(see comments in the last column of the table). Moreover, lack of
associations have been reported in SSc with p22phoxNADPH
oxidase , vascular endothelial growth factor gene (VEGF)
, arylamine N-acetyltransferase 2 (NAT2), COL15 gene .
and Bone Morphogenetic Protein type 2 receptor (BMPR2), a
member of the TGF-β receptor superfamily .
5. Discussion on genetic association studies
Some points must be highlighted concerning genetic
analyses in SSc:
1) The genetic background plays a role in addition to
environmental factors in the induction of autoimmunity. Each
gene associated with SSc disease is expected to have a small
contribution and relative risk for disease . Instead of having
a single selected candidate gene, combination of common
genetic variants issued from several genes must probably be
associated to confer genetic susceptibility to disease.
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2) One must interpret single positive reports of association with
disease with great caution . Methodological limitations of
genetic studies must always be considered since many have
independent groups. In general, association studies must be
confirmed in an independent cohort to be considered as “true”:
as mentioned in Table 3 of this review, most genes analysis
remain in controversy. The overall genetic background of the
study group should be as homogeneous as possible, as gene
allelic frequency and linkage disequilibrium blocks may be
different between populations of different origin.
three possibility must be considered: a) the association is real
and the SNP influences pathogenesis of the disease, b) the
associated SNP is in linkage disequilibrium with other poly-
morphismsthatare inassociationand these last polymorphisms
are those implicated in the pathogenesis, c) the association de-
tected is not real and this can be due to inadequate statistical
analysis (such as Hardy–Weinberg disequilibrium leading to a
confounding factors (genetic background variability, clinical
heterogeneity ….) .
3) Selectionof genetic markers represents a great challenge due
to the elevated numbers of SNPs in the human genome. Only
some are likely to have influence on the disease phenotype.
They are several approach to choose a candidate genes: a) the
been identified in other autoimmune diseases, c) the third is to
choose genes that play a keyrole in the abnormal pathway for a
scleroderma. Inside the candidate gene, the “candidate”
polymorphisms must be chosen. In these genes, several
polymorphisms have been described. All these SNPs are not
independent. Some of them are in linkage disequilibrium and
some others are in strong correlation. SNPs that are in strong
correlation usually give the same result in association studies.
So, association studies need to test only a limited number of
markers in each of these groups that will consider as Tag SNPs
4) Contribution of an associated SNP is rarely obtained in
genetic association studies (biological relevance to disease
pathogenesis). In the recent paper of Fonseca et al. , the
authors not only showed that CTGF-945G allele was
significantly associated with susceptibility to SSc, but also
showed the functional role of this polymorphism on
transcription of this gene. Thus, in SSc patients, presence
of G allele could explain the over expression of CTGF and
persistent wound-healing response .
5) Recent developmentof DNAmicroarraywill provide a new
tool for exploring simultaneous and multiplicative gene ex-
pression in pathologic tissues. Expression microarrays are de-
vices containing tens of thousands of short DNA probes of
specified sequences arrayed in an orderly fashion and tethered
to a flat surface. This new approach will determine SSc gene
expression profiling and will give a more dynamic molecular
in SSc fibroblasts using complementary DNA microarray have
reported meaningful results: increased expression of Secreted
Protein, Acidic and Rich in Cysteine (SPARC) and other ex-
fibroblasts from SSc patients (unaffected skin) compared with
normal controls [76,77]. Finally, it could be very informative to
perform microarray analysis on endothelial cells or immune
cells such as Tand B lymphocytes from patients. To our know-
ledge, this has never been performed.
The diagnosis of SSc still remains on its clinical manifesta-
tions and specific serum autoantibodies production. Candidate
genes or pathways identified through microarrays could be
soon explored as potential biomarkers, and used for molecular
phenotyping of this disease. However, the complexity of this
disorder, requiring the interplay of multiple genes, each con-
tributing a modest effect along environmental exposure or other
still unknown events limits a unique molecular characterization.
A better understanding of the pathogenesis of this incurable
disorder will help to determinate targeted therapies in the future.
This study was supported by the National Public Research
Institute (INSERM), the Assistance publique hôpitaux de
Marseille (AP-HM) (Appel d'Offre de Recherche Clinique
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taking care of patients suffering from Systemic Sclerosis. Fanny Bernard is an
a scientific searcher particularly involved in genetic control of fibrotic diseases.
252B. Granel et al. / European Journal of Internal Medicine 20 (2009) 242–252