# MCP-1−2518 A/G Single Nucleotide Polymorphism in Slovak Patients with Systemic Sclerosis

Authors: Zdenka Navratilova; Jozef  Lukac; Frantisek Mrazek; Eva Kriegova; Maria Bucova; Vladimir Bosak; Martin Petrek
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Recent study in a group of German patients with SSc has implicated the SNP in the MCP-1 gene (−2518 A to G) as a factor of susceptibility to SSc. Reflecting the need for replication of genetic association studies, we investigated if this SNP is associated with SSc in another Caucasian population. MCP-1 −2518 A/G genotypes were determined using PCR-SSP in 46 SSc patients and in 449 healthy subjects, all unrelated and of Slovak (Slavonic) origin. The distribution of MCP-1 −2518 A/G genotypes complied with the Hardy-Weinberg equilibrium both in patient and healthy control groups. There was no difference in MCP-1 −2518∗G allele frequency between SSc patients and healthy subjects (patients: 0.23; controls: 0.24; P>.05). Furthermore, MCP-1 −2518 GG homozygotes were similarly represented among SSc patients and healthy subjects (P>.05). The association of MCP-1 −2518 A/G SNP with SSc observed originally in German population was not replicated in the Slovak population.


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## Body

## 1. Introduction

Systemic
sclerosis (SSc) is a complex autoimmune disease characterised by
fibrotic changes in the connective tissues of skin and internal organs [1]. Aetiology
of SSc is not clear to date, however, polymorphisms in genes for inflammatory mediators,
tissue-matrix proteins, and growth factors have been recently implicated in autoimmune
and fibrotic processes in SSc [1–4].In
particular, chemokine (C–C motif) ligand 2 (CCL2) has been identified in
circulation and in lesions from SSc patients [5–10]. This chemokine, also known
as MCP (monocyte chemoattractant
protein)-1, is chemoattractant for monocytes/macrophages and is
also an inducer of collagen biosynthesis [1, 10, 11]. It has been, therefore,
speculated that MCP-1 upregulation may cause cellular accumulation in SSc
lesions [7, 8] and may be also responsible for massive deposition of
extracellular matrix proteins in SSc skin [1, 10, 11].Recently, a single
nucleotide polymorphism (SNP) in the promoter region of the MCP-1 gene (−2518
A/G), whose G variant may associate with increased MCP-1 gene transcriptional
activity [12], was investigated in German SSc patients [13]. Overrepresentation
of MCP-1 −2518 GG homozygotes among the patients implicated carriage of MCP-1 G allele as a risk factor for
SSc [13]. To date, this observation has not, however, been confirmed in other
cohorts or populations. In agreement with rules of performing association
studies [14], this study aimed at replication of the association between the
MCP-1 −2518 A/G SNP and systemic sclerosis in another patient population of
Caucasian origin, namely in Slavonic population of Slovakia.


## 2. Materials and Methods

### 2.1. Patients and Controls

46 patients with systemic sclerosis (SSc) were
included in the study (Table1). In agreement with fundamental requirements for
replication of genetic association studies [14], SSc patients enrolled in the
present study were classified, characterised, and subgrouped according to the
criteria of American College
of Rheumatology [15];
similar approach was applied also in the study under replication [13]. Patients
were unrelated individuals of Slovak origin and were recruited in one tertiary referral
centre (National Institute of Rheumatic Diseases, Piestany, Slovak Republic). The
control population consisted of 449 healthy unrelated volunteers recruited in
nearby Bratislava
(284 males/165 females, mean age ± SD, 49 ± 10 years), in which the absence of
any autoimmune disease was excluded by health questionnaire and interview.Table 1
Clinical characteristics of 46 patients with systemic sclerosis.
Males/females4 : 42Age (mean, min-max) (y)42 (6–61)Diffuse SSc/limited SSc*9/35Lung involvement (lung fibrosis)33 (1)Gastrointestinal involvement19Kidney involvement4Acral ulcers14Myositis5Acroosteolysis14Sjögren’s syndrome6Anti-DNA-topo I antibodies18Anticentromere antibodies4*In 2 patients the information is not available.Informed
consent for the anonymous usage of their DNA for the purposes of this study was
obtained from all enrolled subjects. The study was performed with the approval of
relevant ethics committees in National
Institute of Rheumatic Diseases, Piestany and Medical Faculties in Bratislava and Olomouc.


### 2.2. Assessment of MCP-1 −2518 Single Nucleotide Polymorphism

Genomic DNA was extracted from peripheral blood leukocytes by standard salting-out method. SNP MCP-1 −2518
(rs1024611) was
genotyped by polymerase chain reaction with sequence specific primers (PCR-SSP)
as described elsewhere [16]. Briefly, standard and mutant alleles were
amplified in two separate reactions. The constant reverse primer (5′ TGA
GTG TTC ACA TAG GCT TC 3′) was used for amplification with the forward
specific primers either for the standard allele A (5′ GTG
GGA GGC AGA CAG CTA 3′) or for the mutant allele G (5′ GTG
GGA GGC AGA CAG CTG 3′). In both cases, the amplicon size was 175 bp. Reaction conditions and usage of internal
controls, adopted from phototyping methodology, are also described elsewhere [16].


### 2.3. Statistics

Hardy-Weinberg equilibrium was tested using aχ2
test of observed and expected genotype frequencies. Differences between allelic,
genotype, and phenotype (carriage) frequencies in patient and control groups
were assessed by χ2
test using Woolf-Haldane correction in cases of small numbers. P value
<.05 was considered to be significant. Statistical
power of the present study to replicate main result from the initial German
study [13] (the difference between the proportion of MCP-1 −2518 GG homozygotes
in SSc patients and control subjects) was determined according to the protocol
described elsewhere [17].


## 2.1. Patients and Controls

46 patients with systemic sclerosis (SSc) were
included in the study (Table1). In agreement with fundamental requirements for
replication of genetic association studies [14], SSc patients enrolled in the
present study were classified, characterised, and subgrouped according to the
criteria of American College
of Rheumatology [15];
similar approach was applied also in the study under replication [13]. Patients
were unrelated individuals of Slovak origin and were recruited in one tertiary referral
centre (National Institute of Rheumatic Diseases, Piestany, Slovak Republic). The
control population consisted of 449 healthy unrelated volunteers recruited in
nearby Bratislava
(284 males/165 females, mean age ± SD, 49 ± 10 years), in which the absence of
any autoimmune disease was excluded by health questionnaire and interview.Table 1
Clinical characteristics of 46 patients with systemic sclerosis.
Males/females4 : 42Age (mean, min-max) (y)42 (6–61)Diffuse SSc/limited SSc*9/35Lung involvement (lung fibrosis)33 (1)Gastrointestinal involvement19Kidney involvement4Acral ulcers14Myositis5Acroosteolysis14Sjögren’s syndrome6Anti-DNA-topo I antibodies18Anticentromere antibodies4*In 2 patients the information is not available.Informed
consent for the anonymous usage of their DNA for the purposes of this study was
obtained from all enrolled subjects. The study was performed with the approval of
relevant ethics committees in National
Institute of Rheumatic Diseases, Piestany and Medical Faculties in Bratislava and Olomouc.


## 2.2. Assessment of MCP-1 −2518 Single Nucleotide Polymorphism

Genomic DNA was extracted from peripheral blood leukocytes by standard salting-out method. SNP MCP-1 −2518
(rs1024611) was
genotyped by polymerase chain reaction with sequence specific primers (PCR-SSP)
as described elsewhere [16]. Briefly, standard and mutant alleles were
amplified in two separate reactions. The constant reverse primer (5′ TGA
GTG TTC ACA TAG GCT TC 3′) was used for amplification with the forward
specific primers either for the standard allele A (5′ GTG
GGA GGC AGA CAG CTA 3′) or for the mutant allele G (5′ GTG
GGA GGC AGA CAG CTG 3′). In both cases, the amplicon size was 175 bp. Reaction conditions and usage of internal
controls, adopted from phototyping methodology, are also described elsewhere [16].


## 2.3. Statistics

Hardy-Weinberg equilibrium was tested using aχ2
test of observed and expected genotype frequencies. Differences between allelic,
genotype, and phenotype (carriage) frequencies in patient and control groups
were assessed by χ2
test using Woolf-Haldane correction in cases of small numbers. P value
<.05 was considered to be significant. Statistical
power of the present study to replicate main result from the initial German
study [13] (the difference between the proportion of MCP-1 −2518 GG homozygotes
in SSc patients and control subjects) was determined according to the protocol
described elsewhere [17].


## 3. Results

Distribution of alleles, genotypes, and phenotypes
of MCP-1 −2518 A/G SNP in patients with SSc and control population is presented
in Table2. The distribution of MCP-1 −2518
genotypes was in compliance with Hardy-Weinberg equilibrium in both patient and
control groups (P>.05). Statistical
power of present study to replicate observations originally reported for the German
population [13] reached 99%.Table 2
Genotype, allele, and phenotype (carriage rates)
frequencies of the MCP-1 −2518 SNP in Slovak patients with systemic sclerosis
and healthy controls compared with the data from German population according to
the study by Karrer et al. [13].
FrequencyGenotypeAllelePhenotypeAAAGGGAGAGSlovak patients
(n=46)63.0
(29)28.3
(13)8.7*
(4)77.2
(71)22.8+
(21)91.3
(42)37.0#
(17)Slovak controls (n=449)57.5 (258)36.7 (165)5.8 (26)75.8 (681)24.2† (217)94.2 (423)42.5 (191)German patients (n=18)44.4
(8)27.8
(5)27.8§
(5)58.3
(21)41.7
(15)72.2
(13)55.6
(10)German controls
(n=139)51.1
(71)42.4
(59)6.5
(9)72.3
(201)27.7
(77)93.5
(130)48.9
(68)Data are given as proportions (%) of particular
genotype/allele/phenotype with their absolute number in parentheses.P values for comparison of allele, genotype, and
phenotype frequencies between the Slovak SSc patients and controls: P+=.81;P*=.67;P#=.51. P value for comparison of GG genotype frequencies
between the German SSc patients and controls [13]: P§=.02. P value for
comparison of G allele frequencies between the Slovak and German control
subjects [13]: P†=.23.No significant differences in
distribution of MCP-1 −2518*G allele were observed between patients with SSc and healthy controls (P>.05).
Furthermore, MCP-1 −2518 GG homozygotes and MCP-1  −2518*G allele carriers were
similarly represented among SSc patients and healthy
control subjects (P>.05).Because different
phenotypes of SSc have been recently thought not to necessarily share a common
genetic background [4], we were further interested if MCP-1 −2518 A/G variants
may be related to internal organ involvement or limited/diffuse form of SSc in
our patients. The GG genotype was associated with GI affliction (P=.007), when
the distribution of MCP-1 −2518 A/G SNP was compared between the patients with
GI involvement and healthy controls. The allele and phenotype frequencies in
patients with the GI affliction or the diffuse form of SSc did not differ from
those in healthy controls (P>.05). When the patients with GI
involvement were compared with patients without the affliction, we found no
difference in the distribution of MCP-1 −2518 SNP (P>.05, Table 3).
Furthermore, the allele, genotype, and phenotype frequencies in patients with
limited SSc were similar to those observed in diffuse form of SSc (Table 3). Similarly,
MCP-1 −2518 SNP was not related to the pulmonary (P>.05, Table 3) or renal
involvement in our SSc patients (data not shown).Table 3
Genotype, allele, and phenotype (carriage rates)
frequencies of the MCP-1 −2518 SNP in the subgroups of patients with systemic
sclerosis (SSc) according to the particular clinical manifestation of the
disease (diffuse versus limited
form, presence versus absence
of lung or gastrointestinal (GI) involvements).
FrequencyGenotypeAllelePhenotypeAAAGGGAGAGDiffuse
SSc (n=9)66.7 (6)11.1 (1)22.2 (2)72.2 (13)27.8 (5)77.8 (7)33.3 (3)Limited
SSc (n=35)62.9 (22)31.4 (11)5.7 (2)78.6 (55)21.4 (15)94.3 (33)37.1 (13)Lung
involvement (n=33)57.6 (19)30.3 (10)12.1 (4)72.7 (48)27.3 (18)87.9 (29)42.4 (14)Without
lung involvement (n=13)76.9 (10)23.1 (3)0.0 (0)88.5 (23)11.5 (3)1.0 (13)23.1 (3)GI  involvement (n=19)68.4
(13)10.5
(2)21.1
(4)73.7
(28)26.3
(10)78.9
(15)31.6
(6)Without
GI  involvement (n=27)59.3 (16)40.7 (11)0.0 (0)79.6 (43)20.4 (11)1.0 (27)40.7 (11)Data are given as proportions
(%) of particular genotype/allele/phenotype with their absolute number in
parentheses.P≥.05 for diffuse versus limited
form of SSc and presence versus absence
of lung or gastrointestinal (GI) involvements.


## 4. Discussion

Despite an adequate methodical approach, the present study did not reveal
an association between MCP-1 −2518 A/G single nucleotide polymorphism and systemic
sclerosis in Slovak population. Therefore, our data do not confirm the findings
from German population [13] and do not support hypothesis that MCP-1 −2518 A/G
SNP is directly involved in the genetic susceptibility to SSc.To explore
the association of MCP-1 −2518 A/G SNP with susceptibility for SSc, our
case-control study used the same methodological strategy as the original report
[13], but importantly the size of the case group in our study was 2.5 fold bigger
than the original one (46 versus 18). Moreover, the statistical power of the current study to replicate the initial
observation reaches 99%. This value is sufficiently high in order to detect the
genetic association described in original study, because it exceeds 80%
threshold [17].One may argue that discrepant observations in
North-European and Eastern-European populations may be due to different ethnic
background. However, there were no differences in frequencies of the MCP-1 −2518 G allele and also of
GG homozygote between German and Slovak healthy populations and further, these
were similar to those reported in other Caucasian populations [13, 18]. In this
regard, Slavonic and German populations also did not differ in the distribution
of a spectrum of twenty-two cytokine SNPs located across 1, 2, 4, 5, 6, 7, 12,
16, and 19 chromosomes [19, 20].Since patients in both centres were enrolled according
to unified international criteria, it is improbable that discrepancies between
these two studies were caused by heterogeneous disease phenotypes. In our
opinion, the most straightforward explanation of the positive genetic
association reported in the original study may be the very small sample size of its patient group (n=18) that may
not reflect real distribution of MCP-1 −2518 A/G SNP in German patients [13]. In support of
this explanation, also a recent study investigating a spectrum of chemokine
polymorphisms in non-Caucasian (Korean) population [2] included more SSc
patients than the original German study [13] and similar to us, no relationship
between the MCP-1 A/G SNP and disease in Koreans was observed [2].Despite the
number of SSc patients in our study was much higher than in original report [13],
in one particular situation it may be considered as “suboptimal:” our study may
not be powerful enough to detect possible “true association” in case that it
would be much weaker than observed in initial German study [13]. This situation
could occur if this MCP-1 polymorphism confers a risk to only particular
phenotype of SSc that is overpresented in German patients [13], but minor or totally
absent in Slovak patients group. In this regard, German study recruited SSc patients
presenting with skin involvement while affection of other internal organs was
not reported here [13] by contrast to our Slovak patients. Taking this into
account, we can speculate that MCP-1 −2518 A/G SNP may be related to a particular
phenotype of SSc.Finally, from a geneticist’s point of view, the contradiction between
the present study and the initial observations [13] can be explained by the
presence of linkage disequilibrium (LD). Although MCP-1 −2518 A/G has been the
most commonly investigated MCP-1 SNP, there are other SNPs located in the MCP-1
gene [12]. Moreover, the data on the functional significance of MCP-1 −2518 A/G
SNP are not consistent [13, 16], and the investigated SNP is not the only factor involved in regulation
of MCP-1 gene transcriptional activity [12]. It may be, therefore, speculated
that in setting of systemic sclerosis, the MCP-1 −2518*G allele represents a
marker in linkage disequilibrium (LD) with supposed “casual” allele located
nearby. In this context, the investigations of the role for MCP-1 gene
polymorphisms should be directed at wider range of SNPs and take into account
haplotype combinations [12].In conclusion, this case-control study performed in Slovak
population does not provide further evidence for recently formulated concept
that MCP-1 −2518 A/G SNP is involved in the genetic predisposition to systemic
sclerosis as a whole. However, it remains to be elucidated whether MCP-1 −2518 A/G
SNP may be in linkage disequilibrium with a causative variant conferring
susceptibility to SSc, which may be located within the polymorphic CC chemokine
genetic cluster on the chromosome 17.



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