ARTICLE
Auteur(s) : Sacide Pehlivan1, Ferda
Ozkinay2, Sibel Alper3, Huseyin Onay2, Eda
Yuksel3, Mustafa Pehlivan4, Cihangir
Ozkinay2
1Gaziantep University Faculty of Medicine,
Department of Medical Biology, 27060 Gaziantep, Turkey
2Ege University Faculty of Medicine, Department
of Medical Genetics, Izmir, Turkey
3Ege University Faculty of Medicine, Department
of Dermatology, Izmir, Turkey
4Gaziantep University Faculty of Medicine,
Department of Haematology, Gaziantep, Turkey
accepté le 8 Decembre 2008
Vitiligo is a skin disorder with progressive depigmentation of
the skin. It affects approximately 1% of the world’s population,
without preference for a specific skin tone or gender [1]. In our
population, vitiligo occurs with a prevalence of 1.4% [2]. Although
the exact etiology of vitiligo has not yet been established, the
abnormal immune responses frequently observed in vitiligo patients
have led to the suggestion that, in some cases, the condition has
an autoimmune component [2]. Autoimmune destruction of melonocytes
has been proposed depending on the fact that the condition often
occurs together with other autoimmune diseases and the presence of
anti-melanocyte and organ specific antibodies [3, 4]. An increasing
amount of genetic research being performed on dermatological
disorders is helping to elucidate the pathogenetic mechanisms of
these diseases [5]. Recently, polymorphisms of a number of genes
which are involved in the immune system have been found to play a
role in the susceptibility to vitiligo disease [6-12].
The human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)
gene which consists of three exons, was mapped to 2q33. Certain of
these polymorphic markers have also been associated with vitiligo,
suggesting that the disorder might occur in some individuals
because of a genetic predisposition to the development of
autoimmunity [13, 14].
The IL-4 gene was mapped to chromosome 5q31. −590 C/T
polymorphism (rs number: 2243250), in the IL-4 gene promoter
region, was selected because this specific SNP has been shown to be
associated with increased IL-4 production [15].
Angiotensin converting enzyme (ACE) is an important regulator of
the rennin-angiotensin system and its main function is to control
blood pressure. An insertion/deletion (I/D) polymorphism of a
287-base pair (bp) sequence in intron 16 of the ACE gene accounts
for the most of the variability of serum ACE activity, with DD
genotypes having the highest and II genotypes having the lowest ACE
activity. The D allele appears to confer susceptibility to vitiligo
[6].
A 32-basepair deletion [32] in the gene encoding CCR5, a
chemokine-receptor, results in a non-functional receptor.
Relationship between vitiligo and CCR5 (Δ32) polymorphism has not
been investigated before but a negative association between CCR5
(Δ32) and rheumatoid arthritis has been described [16].
A protective effect of CCR5 (Δ32) polymorphism was shown for
multiple sclerosis [17]. Because of this paradoxical affect on
autoimmune diseases, the role of CCR5 (Δ32) polymorphism on
vitiligo was investigated in this study.
Variation in the penta-allelic 86 base pair tandem repeat (VNTR)
in intron 2 of IL1-RN, specifically the uncommon allele (IL1-RN
allele 2), has been inconsistently linked to SLE susceptibility
[18, 19]. But a strong association has been detected between
IL1-RN-VNTR polymorphism and rheumatoid arthritis [20].
The aim of this study was to investigate IL4 (–590), ACE
(I)/(D), CCR5 (Δ32), CTLA4(+49) and Interleukin IL1-RN-VNTR
polymorphisms in Turkish vitiligo patients.
Materials and methods
The study included 50 healthy controls and 48 patients diagnosed
with vitiligo in the Department of Dermatology, Medical School
Hospital, Ege University, Izmir/Turkey. The study was approved by
the local institutional ethical committee and was performed
according to the Declaration of Helsinki of 1975. All participants
were informed about the nature of the study and all consented to
participate. Genomic DNA was extracted from peripheral blood
leucocytes by a salting-out procedure. Polymorphisms (genotypes)
for the genes ACE insertion(I)/deletion(D), CCR5 (Δ32), IL1-RN
(VNTR in intron 2) were detected by PCR and agarose gel
electrophoresis (2.5%) [5, 9, 10]. IL4 (–590) and CTLA4 (+49) gene
polymorphisms were typed PCR-RFLP and agarose gel electrophoresis
(3.5%) (6,8).
SPSS version 14.0 for Windows (SPSS Inc., Chicago, IL; USA) was
used for statistical analysis. Genotypes and allele frequencies
were compared between vitiligo patients and healthy persons using a
Pearson’s chi-square test or Fisher’s exact test when one or more
variables were < 5. Odds ratios (OR) and 95% confidence interval
(CI) were also calculated. HWE was determined using the test
provided by the Institute of Human genetics, Technical University
Munich, (http://ihg.gsf.de/cgi.bin/hw/hwa1.pl). P-values < 0.05
were considered to be statistically significant.
Results
The allele and genotype frequencies for ACE, CCR5, IL1-RN, IL4,
CTLA4 genes found in healthy controls and patients with vitiligo
are shown in table 1. No significant
differences in either the genotype distribution or allelic
frequencies of IL4, CCR5 and ACE gene polymorphisms were observed
between vitiligo patients and healthy controls. GG genotype in
CTLA4 (16.6%) and CTLA4 G allele frequency (26%) were found to be
significantly higher in vitiligo patients compared to the
frequencies found in control groups (p = 0.002, 0.000,
respectively). CTLA4 – AA and IL1-RN – 1/5 genotypes (64.6%, 0%)
and IL1-RN 5 allele frequency (0%) were found to be significantly
lower in vitiligo patients compared to control groups (p = 0.014,
0.015, 0.016, respectively).
The observed genotype counts deviated significantly from those
expected according to the Hardy Weinberg Equilibrium (p < 0.05),
except for CTLA (+49) polymorphism (p = 0.0003).
Table 1 Comparison of different variant gene
frequencies between patients with vitiligo and healthy persons
|
Genotype
|
Vitiligo patients
|
Healthy persons
|
|
|
|
|
|
na (%)
|
nb (%)
|
OR
|
95% CI
|
p-value
|
|
CTLA4
|
AA
|
31 (64.6)
|
43 (86)
|
0.297
|
0.110-0.802
|
0.014
|
|
AG
|
9 (18.8)
|
7 (14)
|
0.705
|
0.240-2.074
|
0.525
|
|
GG
|
8 (16.6)
|
- (0)
|
0.833
|
0.734-0.946
|
0.002*
|
|
Alleles
|
A/G
|
71/25 (74/26)
|
93/7 (93/7)
|
0.214
|
0.088-0.532
|
0.000
|
|
CCR5
|
NN
|
45 (93.8)
|
46 (92)
|
1.304
|
0.276-6.160
|
0.737
|
|
ΔN/ΔΔ
|
3 (6.2)
|
4/0 (8)
|
1.304
|
0.276-6.160
|
1.000a
|
|
Alleles
|
N/ Δ
|
92/4 (96/4)
|
96/4 (96/4)
|
0.958
|
0.233-3.945
|
0.617*
|
|
ACE
|
DD
|
14 (29.2)
|
17 (34)
|
1.251
|
0.532-2.940
|
0.607
|
|
ID
|
25 (52.1)
|
23 (46)
|
0.784
|
0.354-1.733
|
0.547
|
|
II
|
9 (18.7)
|
10 (20)
|
1.083
|
0.397-2.953
|
0.876
|
|
Alleles
|
D/I
|
53/43 (55/45)
|
57/43 (57/43)
|
0.930
|
0.529-1.635
|
0.801
|
|
IL-4
|
TT
|
29 (60.4)
|
28 (54)
|
0.834
|
0.373-1.863
|
0.658
|
|
CT
|
19 (39.6)
|
19 (38)
|
0.935
|
0.415-2.109
|
0.872
|
|
CC
|
- (0)
|
3 (6)
|
1.064
|
0.992-1.141
|
0.129*
|
|
Alleles
|
T/C
|
77/19 (80/20)
|
75/25 (75/25)
|
1.351
|
0.687-2.656
|
0.382
|
|
IL1-RN
|
2/2
|
6 (12.5)
|
1 (2)
|
0.143
|
0.017-1.235
|
0.050*
|
|
2/1
|
6 (12.5)
|
14 (28)
|
2.722
|
0.948-7.817
|
0.057
|
|
2/5
|
- (0)
|
1 (2)
|
1.020
|
0.981-1.062
|
0.510*
|
|
1/1
|
36 (75)
|
29 (56)
|
0.460
|
0.194-1.089
|
0.075
|
|
1/5
|
- (0)
|
6 (12)
|
1.136
|
1.026-1.259
|
0.015*
|
|
Alleles
|
2
|
18 (18.8)
|
17 (17)
|
0.888
|
0.427-1.844
|
0.749
|
|
1
|
78 (81.2)
|
77 (77)
|
1.294
|
0.648-2.587
|
0.465
|
|
5
|
- (0)
|
6 (6)
|
1.064
|
1.012-1.118
|
0.016*
|
*Fisher’s exact test was used and P-value was reported.
Discussion
Vitiligo is a common skin disorder characterized by patterned
depigmentation, because of the decrease of melanin pigment
resulting from apparent melanocyte loss. The pathogenesis is still
unknown, but several hypotheses have been advocated. Autoimmune
destruction of melanocytes has been proposed, depending on the fact
that the condition often occurs together with other autoimmune
diseases and the presence of anti-melanocyte and organ specific
antibodies [3, 4]. Recently, polymorphisms of a number of genes
which are involved in the immune system have been found to play a
role in the susceptibility to vitiligo disease [6-12].
We investigated interleukin IL4 (-590), ACE (I)/(D), CCR5 (Δ32),
CTLA4(+49) and IL1-RN-VNTR polymorphisms in 48 patients and 50
healthy controls. Association between IL4(-590) polymorphism and
vitiligo has not been investigated before. No significant
differences in either the genotype distribution or allelic
frequencies of IL4(–590) polymorphism were observed between
vitiligo patients and healthy controls. Our study was also the
first study to investigate the role of CCR5(Δ32) polymorphism in
vitiligo. No significant association was detected between this
polymorphism and vitiligo.
In the literature there are three articles which have
investigated the role of ACE (I)/(D) polymorphism in vitiligo.
First, Jin et al. indicated the roles of the DD genotype and D
allele in developing vitiligo [6]. But this result was not
confirmed by two later studies [21, 22]. In our study we also did
not detect any association between ACE (I)/(D) polymorphism and
vitiligo. The different populations in which these studies were
performed or the number of the patients included in these studies
might explain these paradoxical results.
In this study, CTLA4(+49) –GG and IL1-RN–2/2 genotypes (16.6%,
12.5%) and CTLA4(+49) G allele frequency (26%) were found to be
significantly higher in vitiligo patients compared to the
frequencies found in the control groups (p = 0.044, 0.003, 0.000).
CTLA4(+49) – AA and IL1RN – 1/5 genotypes (64.6%, 0%) and IL1-RN 5
allele frequency (0%) were found to be significantly lower in
vitiligo patients compared to control groups (p = 0.01, 0.013,
0.015). In our earlier studies we showed an association between
CTLA4-VNTR polymorphism and vitiligo. In this study we have also
shown the effect of another polymorphism in the CTLA4 gene [23].
This indicates a strong association between CTLA4 and vitiligo. The
association between the IL1-RN gene and vitiligo was shown for the
first time in literature.
As a conclusion, the CTLA4-GG genotype might be associated with
the development of vitiligo and the CTLA4 allele-A and IL1-RN
allele-5 might have protective roles in vitiligo.
Acknowledgements
Financial support: none. Conflict of interest: none.
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