ARTICLE
Auteur(s) : AC Biral1, RF
Magalhaes2, IJ Wastowski3, R
Simoes3, EA Donadi3, AL Simoes4,
CT Mendes-Junior4, AM Tanaka2, MHS Kraemer1,*
1Immunogenetic Transplant Laboratory, Clinical
Pathology Department, School of Medical Science, Rua Gustavo
Rodrigues Dória, 255, 13083-060 Campinas (SP) BrazilFax: (+19)
3788-9434
2Department of Dermatology; School of Medical Science,
State University of Campinas, Campinas
3Biology Molecular Laboratory, Medical School, State
University of Campinas, Campinas
4Department of Genetics, University of Sao Paulo,
Ribeirao Preto, Sao Paulo, Brazil
accepté le 1 Mai 2006
Psoriasis vulgaris is a chronic inflammatory skin disorder
characterized by hyperproliferation and recruitment of T
lymphocytes and mononuclear cells in affected skin [1, 2]. It is
characterized by abnormal proliferation and complex alterations in
epidermal differentiation, as well as a high number of biochemical,
immunologic, inflammatory, and vascular abnormalities. Distribution
of psoriasis in the world population varies according to ethnic
groups and geographical locations, with a peak prevalence of
approximately 2% of the population [3]. Among the Brazilian
population, approximately 3 million individuals are affected,
although the prevalence rate of the disease has not been precisely
determined and probably lies between 2% and 3%. The onset of
disease is called type I before the age of 40 and type II after the
age of 40, which leads to the hypothesis that there are in fact two
forms of psoriasis [4]. The cause of psoriasis is unknown, but its
mode of inheritance is currently considered to be multifactorial.
One hypothesis refers to a genetic predisposition, which implies
the effect of diverse genes and includes several triggering
factors, which in turn play an important role in the expression of
the disease [5-7].A high number of studies have demonstrated that
genes, particularly those contained within the major
histocompatibility complex (MHC), on chromosome 6, confer
susceptibility to psoriasis and influence disease development [8].
Several associations have been related to increased frequencies in
HLA class I and II among patients of different ethnic groups
[9-12]. Alternatively, the association of the HLA molecules in
psoriasis may be indirect and caused by linkage disequilibrium with
a possible gene located somewhere else on chromosome 6 [13]. Many
genes also located in the HLA region and whose products also
suggest involvement with the pathogenesis of psoriasis include
either the tumor necrosis factor-alpha (TNF-α) or beta (LT-α or
TNF-β) [14]. Tumor necrosis factor-alpha (TNF-α) is a key
pro-inflammatory cytokine in the development of psoriasis, with
strong biological activity. Its is regulated by the TNF gene
cluster which displays a high level of polymorphism [15, 16] and is
encoded within the class III region, on chromosome 6, with a length
of 250kb centromeric to the HLA class genes. TNF loci are 12kb in
length, contain several polymorphic areas, and include five
microsatellites (Short Tandem Repeats- STR), TNFa, b, c, d and e
[17].The possible associations of HLA class I gene polymorphism
with the five microsatellite markers in the TNF locus have been
examined in different populations and results of different studies
have shown strong linkage disequilibrium between certain alleles of
these microsatellites and both MHC class I and class II loci [18].
Therefore, alleles from physically linked polymorphic loci that
occupy a small chromosomal extension form useful haplotypes to
infer population background, as well as for disease association
studies [19].The goal of the present study was to analyze
involvement of the HLA class I region genes and TNFα
microsatellites, regarding genetic predisposition to psoriasis
vulgaris. Therefore, we evaluated the distribution of HLA-A, -B, -C
alleles and haplotypes as well as TNFa, b, c, d, e microsatellites
in a group of patients with psoriasis vulgaris and a control group
to define the associations involved with genetic risk factors for
psoriasis vulgaris in Brazilian patients.
Patients and controls
Typing of HLA class I was carried out in 92 patients with
psoriasis, diagnosed according to clinical criteria [20]. The 41
males and 51 females aged between 10 and 80 attended the
Dermatology Ambulatory Department of the Teaching Hospital, School
of Medical Sciences – State University of Campinas and were
recruited between March 2002 and March 2003. All patients came from
Campinas (State of São Paulo), a city located in the southeastern
region of Brazil. Seventy patients had type I psoriasis and 22
patients type II psoriasis. Patients were chosen at random
regarding onset of disease. Of the 92 psoriasis patients, 49 type I
and 21 type II (a total of 70 patients) were studied regarding the
five microsatellite markers in the TNF locus.
The control group consisted of 160 individuals aged between 18
and 53, 91 males and 69 females, from the region of Campinas. Blood
samples were collected from unrelated blood donors and volunteers
from the blood bank of the Hematology and Hemotherapy Centre of the
State University of Campinas and typing of HLA class I genes was
carried out. Of those 160 controls, 71 were chosen in order to
study the five microsatellite markers in the TNF locus. The study
was carried out and samples were obtained with the informed consent
of patients after approval by the Research Ethics Committee.
Typing of alleles was performed at the Transplantation
Immunogenetic Laboratory, Teaching Hospital, Division of Clinical
Pathology, School of Medical Science – UNICAMP, which is accredited
to perform clinical HLA typing by Brazilian organizations
Integrated Health Service (Serviço Único de Saúde-SUS) and the
Brazilian Association of Histocompatibility (ABH).
Genomic DNA extraction from peripheral blood leukocytes
Ten milliliters of heparinized blood were obtained from both
psoriasis patients and controls. Genomic DNA was isolated from
leukocytes in anticoagulated blood using a salting out procedure
[21], precipitated with isopropyl alcohol and resuspended in
sterile water. The DNA concentration (optical density 260) and
purity (optical density 260/280) were determined using a
spectrophotometer. The DNA obtained had a 1.6-1.8 degree of purity
and was suitable for polymerase chain reaction (PCR) analysis.
PCR/SSP typing
HLA-A, -B, -C alleles were differentiated at a medium resolution
level in both psoriasis patients and controls using microplates
with 96 wells containing the specific sequence primers (PCR/SSP)
(One Lambda Inc., Canoga Park, CA, USA), 21 reactions for HLA-A
specificities, 36 reaction specificities for HLA-B allele and 14
reactions for HLA-C allele, according to the methodology developed
by Olerup and Zetterquist [22]. Amplification was carried out in 30
cycles for the PCR, that included denaturation at 94 °C for
10s, annealing at 59 °C for 60 s and extension at
72 °C for 30 s. PCR reaction was analyzed by
electrophoresis in ethidium bromide-stained 2% agarose gel.
Presence of the specific PCR product was visualized by a (UV)
transilluminator and documented by photographs.
TNF microsatellite
The TNFa, b, c, d and e microsatellite alleles were amplified using
PCR with primers according to Udalova [23] (table 1( Table 1 )). An Applied Biosystems Thermal Cycler
was used for all PCR reactions and a general set of conditions and
reagents was used for amplification of all loci. The total reaction
volume for each sample was 21 μL for all amplifications; for
PCR, we used 2 μL (100 ng/μL) of genomic DNA, 10X PCR
buffer, Mgcl2 (50 mM), 20 mM for each dATP, dCTP, dGTP
and dTTP, 0.2 μM for each 3’ and 5’ primer and 0.10 μL
for Taq Polymerase (5 U/μL). Amplification was carried out in
30 cycles for the PCR of the TNFc and the first PCR of the TNFa and
TNFb loci, as described by Udalova [23], with small modifications
in the amplification programs. Each cycle consisted of denaturation
at 94 °C for 60 s, annealing at 61 °C for 60 s
and extension at 72 °C for 60 s. PCR of the TNFd and TNFe
loci and the second PCR of the TNFa and TNFb loci consisted of 34
cycles. Each cycle consisted of denaturation at 94 °C for
50 s, annealing at 60 °C for 30 s and extension at
72 °C for 40 s. To visualize amplified products, samples
were applied in denaturizing vertical gels of acrylamide (12%),
submitted to specific electrophoretic conditions for each locus and
visualized according to Sanguinetti [24].
Table 1 Primers used for TNFα microsatellite typing
|
Primer
|
5’ – 3’
|
|
TNFa
|
GCC TCT AGA TTT CAT CCA GCC ACA
|
|
CCT CTC TCC CCT GCA ACA CAC A
|
|
TNFb
|
GCA CTC CAG CCT AGG CCA CAG A
|
|
GTG TGT GTT GCA GGG GAG AGA G
|
|
TNFc
|
GGT TTC TCT GAC TGC ATC TTG TCC
|
|
TCA TGG GGA GAA CCT GCA GAG AA
|
|
TNFd
|
AGA TCC TTC CCT GTG AGT TCT GCT
|
|
CAT AGT GGG ACT CTG TCT CCA AAG
|
|
TNFe
|
GTG CCT GGT TCT GGA GCC TCT C
|
|
TGA GAC AGA GGA TAG GAG AGA CAG
|
Statistical analysis
Allele frequencies (AF) were calculated using the following
formula: AF (%) = (n/2N) × 100, where n = sum of a particular
allele and N = total number of individuals.
To determine the constitutions of the two haplotypes of each
individual, two different computer methods were used: the algorithm
EM [25], implemented by the HTR program [26], and the PHASE method
[27]. Intrapopulation haplotype diversity evaluations and exact
tests to differentiate between the sample groups based on haplotype
frequencies were carried out using the arlequin software program
[28]. To verify the presence of alleles and haplotypes in
significantly distinct frequencies among the examined groups, the
frequencies of each allele and haplotype were compared between
group pairs (PsV I vs. Control; PsV II vs. Control; PsV total vs.
Control), using Fisher exact test (GraphPad in Stat, v. 3.0). The
corrected p-value (pc) was obtained by multiplying the P-value by
the number of measured alleles detected at each locus. A level of
pc < 0.05 was accepted as statistically significant.
Results
Analysis of HLA class I alleles (HLA-A, -B, -C) using
PCR/SSP
Frequency distribution of alleles found in our 92 psoriasis
patients and 160 controls are shown in table 2( Table 2 ). Significant differences in frequencies
were found in four alleles: HLA-B*13 (6.5% in psoriasis patients
vs. 0.3% in controls, pc = 0.003, OR = 22.3), HLA-B*57 (8.7% in
psoriasis patients vs. 1.9% in controls, pc = 0.02, OR = 4.9),
HLA-B*44 (3.3% in psoriasis patients vs. 10.3% in controls,
p = 0.005, OR = 0.29), HLA-Cw*06 (21.7% in psoriasis patients
vs. 5.0% in controls, pc =0.001, OR = 5.3), HLA-Cw*12 (12.5%
in psoriasis patients vs. 4.7% in controls, pc = 0.03, OR =
2.9 (table 3( Table 3 )).
Assessment of allelic frequencies and significance in type I and
II psoriasis patients showed that HLA-B*13, -B*57, -Cw*06, and
-Cw*12 alleles were significantly increased in type I psoriasis (p
< 0.05) when compared to the control group; in type II
psoriasis, HLA B*13 allele was statistically significant (pc =
0.003, OR = 31.9) (table 3).
Table 2 Distribution of frequencies (%) of HLA-A, -B,
-C alleles, in psoriasis vulgaris patients and in controls
|
Allele HLA-A*
|
Psoriasis
|
Control
|
Allele HLA-B*
|
Psoriasis
|
Control
|
Allele HLA-Cw*
|
Psoriasis
|
Control
|
|
N = 92
|
N = 160
|
N = 92
|
N = 160
|
N = 92
|
N = 160
|
|
n AF (%)
|
n AF (%)
|
n AF (%)
|
n AF (%)
|
n AF (%)
|
n AF (%)
|
|
*01
|
12 (6.5)
|
30 (9.4)
|
*07
|
09 (4.9)
|
26 (8.1)
|
*01
|
07 (3.8)
|
09 (2.8)
|
|
*02
|
42 (22.8)
|
83 (25.9)
|
*08
|
08 (4.4)
|
19 (5.9)
|
*02
|
06 (3.3)
|
12 (3.8)
|
|
*03
|
14 (7.6)
|
25 (7.8)
|
*13
|
12 (6.5)
|
01 (0.3)
|
*03
|
10 (5.4)
|
27 (8.4)
|
|
*11
|
03 (1.6)
|
15 (4.7)
|
*14
|
07 (3.8)
|
18 (5.6)
|
*04
|
24 (13.1)
|
46 (14.4)
|
|
*23
|
04 (2.2)
|
07 (2.2)
|
*15
|
13 (7.1)
|
33 (10.3)
|
*05
|
05 (2.7)
|
16 (5.0)
|
|
*24
|
16 (8.7)
|
31 (9.7)
|
*18
|
08 (4.4)
|
12 (3.8)
|
*06
|
40 (21.7)
|
16 (5.0)
|
|
*25
|
00 (0.0)
|
02 (0.6)
|
*22
|
00 (0.0)
|
00 (0.0)
|
*07
|
26 (14.1)
|
54 (16.9)
|
|
*26
|
07 (3.8)
|
11 (3.4)
|
*27
|
07 (3.8)
|
06 (1.9)
|
*08
|
07 (3.8)
|
27 (8.4)
|
|
*29
|
02 (1.1)
|
09 (2.8)
|
*35
|
16 (8.7)
|
28 (8.8)
|
*12
|
23 (12.5)
|
15 (4.7)
|
|
*30
|
12 (6.5)
|
13 (4.1)
|
*37
|
05 (2.7)
|
02 (0.6)
|
*14
|
03 (1.6)
|
11 (3.4)
|
|
*31
|
02 (1.1)
|
09 (2.8)
|
*38
|
05 (2.7)
|
07 (2.2)
|
*15
|
04 (2.2)
|
17 (5.3)
|
|
*32
|
05 (2.7)
|
12 (3.8)
|
*39
|
12 (6.5)
|
11 (3.4)
|
*16
|
03 (1.6)
|
14 (4.4)
|
|
*33
|
04 (2.2)
|
11 (3.4)
|
*40
|
03 (1.6)
|
08 (2.5)
|
*17
|
03 (1.6)
|
09 (2.8)
|
|
*34
|
00 (0.0)
|
05 (1.6)
|
*41
|
03 (1.6)
|
03 (0.9)
|
*18
|
04 (2.2)
|
01 (0.3)
|
|
*36
|
01 (0.6)
|
03 (0.9)
|
*42
|
01 (0.6)
|
06 (1.9)
|
|
|
|
|
*66
|
02 (1.1)
|
01 (0.3)
|
*44
|
06 (3.3)
|
33 (10.3)
|
|
|
|
|
*68
|
07 (3.8)
|
19 (5.9)
|
*45
|
01 (0.6)
|
06 (1.9)
|
|
|
|
|
*74
|
00 (0.0)
|
03 (0.9)
|
*47
|
02 (1.1)
|
03 (0.9)
|
|
|
|
|
|
|
*48
|
00 (0.0)
|
02 (0.6)
|
|
|
|
|
|
|
*49
|
05 (2.7)
|
07 (2.2)
|
|
|
|
|
|
|
*50
|
06 (3.3)
|
03 (0.9)
|
|
|
|
|
|
|
*51
|
09 (4.9)
|
23 (7.2)
|
|
|
|
|
|
|
*52
|
04 (2.2)
|
07 (2.2)
|
|
|
|
|
|
|
*53
|
04 (2.2)
|
03 (0.9)
|
|
|
|
|
|
|
*55
|
03 (1.6)
|
02 (0.6)
|
|
|
|
|
|
|
*56
|
01 (0.6)
|
00 (0.0)
|
|
|
|
|
|
|
*57
|
16 (8.7)
|
06 (1.9)
|
|
|
|
|
|
|
*58
|
07 (3.8)
|
09 (2.8)
|
|
|
|
|
|
|
*67
|
00 (0.0)
|
01 (0.3)
|
|
|
|
|
|
|
*73
|
01 (0.6)
|
00 (0.0)
|
|
|
|
|
|
|
*78
|
00 (0.0)
|
01 (0.3)
|
|
|
|
|
|
|
*81
|
01 (0.6)
|
01 (0.3)
|
|
|
|
|
|
|
*82
|
00 (0.0)
|
01 (0.3)
|
|
|
|
Table 3 Distribution of frequencies (%) and statistical
significance in HLA-A,-B, -C alleles among the total group of
psoriasis patients, type I and type II psoriasis patients, and
controls
|
PsV Total
|
Control
|
p
|
pc
|
OR
|
|
Alleles HLA
|
N = 92
|
N = 160
|
|
|
|
|
n AF (%)
|
n AF (%)
|
|
|
|
|
HLA-B*13
|
12 (6.5)
|
01 (0.3)
|
< 0.0001
|
0.003
|
22.3
|
|
HLA-B*44
|
06 (3.3)
|
33 (10.3)
|
0.005
|
ns
|
0.29
|
|
HLA-B*57
|
16 (8.7)
|
06 (1.9)
|
0.0005
|
0.02
|
4.9
|
|
HLA-Cw*06
|
40 (21.7)
|
16 (5.0)
|
< 0.0001
|
0.001
|
5.3
|
|
HLA-Cw*12
|
23 (12.5)
|
15 (4.7)
|
0.002
|
0.03
|
2.9
|
|
PsV I
|
Control
|
p
|
pc
|
OR
|
|
Alleles HLA
|
N = 70
|
N = 160
|
|
|
|
|
n AF (%)
|
n AF (%)
|
|
|
|
|
HLA-B*13
|
08 (5.7)
|
01 (0.3)
|
0.0004
|
0.01
|
19.3
|
|
HLA-B*44
|
05 (3.6)
|
33 (10.3)
|
0.016
|
ns
|
0.3
|
|
HLA-B*57
|
15 (10.7)
|
06 (1.9)
|
< 0.0001
|
0.003
|
6.3
|
|
HLA-Cw*06
|
35 (25.0)
|
16 (5.0)
|
< 0.0001
|
0.001
|
6.3
|
|
HLA-Cw*12
|
18 (12.9)
|
15 (4.7)
|
0.003
|
0.04
|
3.0
|
|
PsV II
|
Control
|
p
|
pc
|
OR
|
|
Alleles HLA
|
N = 22
|
N = 160
|
|
|
|
|
n AF (%)
|
n AF (%)
|
|
|
|
|
HLA-B*13
|
04 (9.1)
|
01 (0.3)
|
0.0009
|
0.03
|
31.9
|
HLA Class I haplotypes
Haplotype frequencies were obtained through calculated
probabilistic estimates. HLA-B*57 Cw*06, HLA-B*13 Cw*06 and B*39
Cw*12 haplotypes showed increased frequencies in the total
psoriasis group and type I psoriasis patients when compared to the
control group (table 6( Table 4 )( Table 5 )( Table 6
)). Only the HLA-B*13 Cw*06 haplotype showed increased frequencies
in type II psoriasis patients when compared to the controls (table
6).
Table 4 Distribution of frequencies (%) of TNF
microsatellite alleles in patients (N = 70) and controls (N = 71)
|
TNFa
|
TNFb
|
TNFc
|
TNFd
|
TNFe
|
|
Alleles
|
PsV
|
Control
|
PsV
|
Control
|
PsV
|
Control
|
PsV
|
Control
|
PsV
|
Control
|
|
1
|
03 (0.02)
|
06 (0.04)
|
17 (0.12)
|
33 (0.23)
|
85 (0.61)
|
85 (0.60)
|
08 (0.06)
|
07 (0.05)
|
12 (0.08)
|
31 (0.21)
|
|
2
|
28 (0.20)
|
40 (0.28)
|
00 (0.0)
|
01 (0.007)
|
55 (0.39)
|
57 (0.40)
|
08 (0.06)
|
05 (0.03)
|
08 (0.06)
|
02 (0.01)
|
|
3
|
03 (0.02)
|
02 (0.01)
|
15 (0.10)
|
10 (0.07)
|
|
|
53 (0.38)
|
60 (0.42)
|
120 (0.86)
|
109 (0.77)
|
|
4
|
06 (0.04)
|
17 (0.12)
|
56 (0.40)
|
50 (0.35)
|
|
|
44 (0.31)
|
53 (0.38)
|
|
|
|
5
|
09 (0.06)
|
03 (0.02)
|
45 (0.32)
|
43 (0.30)
|
|
|
22 (0.16)
|
14 (0.10)
|
|
|
|
6
|
20 (0.14)
|
19 (0.13)
|
01 (0.007)
|
04 (0.03)
|
|
|
05 (0.03)
|
02 (0.01)
|
|
|
|
7
|
15 (0.10)
|
12 (0.08)
|
05 (0.03)
|
01 (0.007)
|
|
|
|
|
|
|
|
8
|
01 (0.007)
|
01 (0.007)
|
|
|
|
|
|
|
|
|
|
9
|
06 (0.04)
|
03 (0.02)
|
|
|
|
|
|
|
|
|
|
10
|
27 (0.19)
|
25 (0.17)
|
|
|
|
|
|
|
|
|
|
11
|
11 (0.08)
|
10 (0.07)
|
|
|
|
|
|
|
|
|
|
12
|
01 (0.007)
|
00 (0.0)
|
|
|
|
|
|
|
|
|
|
13
|
09 (0.06)
|
04 (0.03)
|
|
|
|
|
|
|
|
|
|
14
|
01 (0.007)
|
00 (0.0)
|
|
|
|
|
|
|
|
|
Table 5 Distribution of frequencies (%) and statistical
significance in TNF microsatellite alleles among the total group of
psoriasis patients, type I and type II psoriasis patients, and
controls
|
PsV Total
|
Control
|
p
|
OR
|
|
Alleles STR-TNF
|
N = 70
|
N = 71
|
|
|
|
n AF (%)
|
n AF (%)
|
|
|
|
TNFa4
|
06 (4.3)
|
17 (12.0)
|
0.03
|
0.33
|
|
TNFb1
|
17 (12.1)
|
33 (23.2)
|
0.02
|
0.46
|
|
TNFe1
|
12 (8.6)
|
31 (21.8)
|
0.002
|
0.33
|
|
PsV I
|
Control
|
p
|
OR
|
|
Alleles STR-TNF
|
N = 48
|
N = 71
|
|
|
|
n AF (%)
|
n AF (%)
|
|
|
|
TNFa4
|
03 (3.1)
|
17 (12.0)
|
0.02
|
0.24
|
|
TNFb1
|
12 (12.2)
|
33 (23.2)
|
0.04
|
0.47
|
|
TNFe1
|
08 (8.3)
|
31 (21.8)
|
0.007
|
0.32
|
|
PsV II
|
Control
|
p
|
OR
|
|
Alleles STR-TNF
|
N = 22
|
N = 71
|
|
|
|
n AF (%)
|
n AF (%)
|
|
|
|
TNFa4
|
03 (7.1)
|
17 (12.0)
|
ns
|
|
|
TNFb1
|
05 (12.0)
|
33 (23.2)
|
ns
|
|
|
TNFe1
|
04 (9.5)
|
31 (21.8)
|
ns
|
|
Table 6 Absolute and relative frequencies of haplotypes
which showed statistical significance among the total group of
psoriasis patients, type I and type II psoriasis patients, and the
controls
|
Haplotype
|
Frequencies
|
Frequencies
|
p
|
- Susceptibility
- or
- Protection
|
|
Absolute
|
Relative
|
Absolute
|
Relative
|
|
PsV I
|
Control
|
|
|
|
HLA-B*57Cw*06
|
10
|
0.0909
|
2
|
0.0108
|
0.001
|
Susceptibility
|
|
HLA-B*13Cw*06
|
6
|
0.0545
|
0
|
0
|
0.002
|
Susceptibility
|
|
HLA-B*39Cw*12
|
8
|
0.0727
|
2
|
0.0108
|
0.007
|
Susceptibility
|
|
PsV II
|
Control
|
|
|
|
6
|
0.1667
|
7
|
0.0565
|
0.044
|
Susceptibility
|
|
HLA-B*13Cw*06
|
2
|
0.0769
|
0
|
0
|
0.015
|
Susceptibility
|
|
PsV Total
|
Control
|
|
|
|
10
|
0.0847
|
21
|
0.1694
|
0.037
|
Protection
|
|
HLA-B*13Cw*06
|
8
|
0.0588
|
0
|
0
|
0.001
|
Susceptibility
|
|
HLA-B*57Cw*06
|
11
|
0.0809
|
2
|
0.0108
|
0.002
|
Susceptibility
|
|
HLA-B*39Cw*12
|
8
|
0.0588
|
2
|
0.0108
|
0.016
|
Susceptibility
|
TNF microsatellite frequency distribution analyses
Table 4 shows allele frequency of the five microsatellite loci. The
TNFa is the most polymorphic one, containing 14 alleles. We
detected a decrease in distribution of TNFa4, b1, e1 alleles in
psoriasis patients (p = 0.03, OR = 0.33; p = 0.02, OR =
0.46; p = 0.002, OR = 0.33) respectively, when compared with
controls (table 5), which lose their significance after being
corrected by the number of investigated alleles.
Distribution of allelic frequencies and significance in type I
psoriasis patients were found to be decreased in TNFa4, b1, e1
alleles; however, type II psoriasis patients did not show any
statistical significance, when compared to controls (table 5).
TNF microsatellite haplotypes
Frequencies of haplotypes were obtained through calculated
probabilistic estimates. The TNFa2b1c2d4e1 haplotype in the total
group of psoriasis patients showed decreased frequencies when
compared to controls (table 6). However, our results showed
increased frequency and statistical significance of the haplotype
TNFa11b4c1d3e3 in type II psoriasis patients when compared to
controls (table 6).
Discussion
It is now clear that the genetic basis of psoriasis is complex and
that its role varies among patients and their families (genetic
heterogeneity) [3]. Linkage and association analyses have shown
that the major histocompatibility complex (MHC) is the major
genetic determinant related to psoriasis susceptibility and that in
the MHC, HLA-Cw*06 is the allele that shows the strongest
association with psoriasis [2]. Therefore, the linkage
disequilibrium of the HLA system related to recombination events
could modify the haplotypes which contain the HLA-Cw6. The
association of that allele with the HLA-B13 or the HLA-B57 could
indicate that those haplotypes, too, play a role in the
susceptibility to the disease, or that another gene associated with
psoriasis may be located next to those genes [29].
In the HLA-Cw6, the replacement of the amino acid alanine with
tyrosine in position 73 was detected at the groove where binding
with the antigen of the HLA-C molecule occurs [30]. This finding
implies that the site where the binding antigen fits into the
groove of the HLA-C molecule is important for presentation of the
supposed antigen associated with psoriasis [31]. From the
immunological viewpoint, an abnormality of the class I molecule
HLA-Cw6 may result in its being recognized by foreign antigens,
with restriction of class I molecules for T-CD8 cells.
Alternatively, it may lead to preferential binding of heterologous
antigens and autologous antigens, or both, with subsequent
recognition by T-CD8 cells [32]. When the cells expand, they begin
to respond to autologous peptides, resulting in an autoimmune
disease [33].
Studies carried out with TNF microsatellite markers examined
production of TNF-α and pathogenesis of many different diseases
[34, 35]. Production of TNF-α, a potent proinflammatory cytokine
with marked biological activity, is regulated by the TNF locus, has
many polymorphisms and is involved in inflammatory diseases [15,
16]. The polymorphisms of the TNF genes can be related to
susceptibility or severity of several groups of diseases. Research
on genomic extension established a strong association of psoriasis
with MHC and especially the ancestral haplotype 57.1 [36]. The
exact role of MHC in the pathogenesis of psoriasis is still not
clear. KALUZA [37] demonstrated that the allele of promoter TNF-α,
the TNF238.2, which is part of the haplotype B57.1, is often
increased in psoriasis patients and is associated with a
significant diminution of the transcriptional activity in vitro.
Special attention was given to the G-A mutation for position –308;
however, conflicting results were reported.
Although the STRs are usually considered to be neutral
evolutionary markers, they have recently been associated with
several biological processes related to genic expression. It was
further suggested that the STRs function as points of recombination
[38] and, depending on their location, they can also feature
linkage disequilibrium with other regions of the genome, which
would directly influence the control of the genic expression (e.g.
promoter region). Therefore, the relationship between the
polymorphisms and some diseases may be caused by the direct
influence of the genetic variability on the genic expression or,
further, by linkage disequilibrium of the TNF genes with other MHC
genes.
In the present study, we determined HLA class I genotyping and
the five TNF-α microsatellite markers. Analysis of these
polymorphic markers, which are associated with both forms of the
disease (type I and type II), allowed us to suggest the segment
associated with susceptibility and protection against psoriasis in
Brazilian patients.
HLA-B*13, -B*57, -Cw*06 and -Cw*12 alleles showed increased
significant frequencies in the total group of patients with
psoriasis and type I psoriasis; however, only the HLA-B*13 allele
was significant in type II psoriasis when compared to controls. The
disease occurs with a much smaller frequency after the age of 40,
when compared to onset before the age of 40, and with weak
correlation with HLA antigens. However, we must not forget that
only a small sample (n = 22) was examined. A larger number of
individuals with psoriasis should therefore be analyzed in relation
of HLA class I markers to confirm that finding, which has not been
reported in other studied populations.
The HLA-Cw*06 allele shows an important increase in relative
risk for psoriasis in almost all the tested populations and that
important frequency increase occurs in spite of the differences
between genetic groups. In the present study, the frequency of
patients with the allele HLA-Cw*06 was 43.4% (43.4% of patients
with psoriasis compared to 10.0% of the controls, p < 0.0001). A
strong linkage disequilibrium between the HLA-Cw*06 and B*13 and
B*57 was also established. Haplotypes with those two alleles and
the HLA-Cw*06 are frequently present in psoriasis patients.
Further, HLA-B*13 Cw*06, HLA-B*57 Cw*06 and HLA-B*39 Cw*12
haplotypes showed a strong association with psoriasis, as well as
with type I psoriasis in the present research.
Analysis of the five TNF microsatellite markers in the total
group of patients with psoriasis showed a decrease in frequency of
the TNFa4, b1, e1 alleles as well as of the TNFa2, b1, c2, d4, e1
haplotypes when compared to the controls. The same decreased allele
frequency was found in type I psoriasis patients. Two haplotypes
showed statistical significance for type II psoriasis, TNFa11 b4 c1
d3 e1 and HLA-B*13 Cw*06. These findings were not reported by any
other study on psoriasis type II. However, it should be remembered
that the present study examined a Brazilian population constituted
by different ethnicities.
The high degree of MHC polymorphism, sequence and presence of
genes vary between haplotypes. The results of our study show
association with susceptibility of HLA class I alleles, HLA-B*13,
-B*57, -Cw*06, -Cw*12 and HLA-B*13 Cw*06, HLA-B*57 Cw*06 and
HLA-B*39 Cw*12 haplotypes. However, the allele HLA-B*44 showed a
diminished frequency with statistical significance both for the
total group of patients with psoriasis and for the psoriasis type I
group. This finding has been previously described and it has
already been suggested that it could be a protection allele against
the disease. The data confirmed findings of other studies carried
out by our laboratory that suggest that there are genes of
susceptibility for HLA class I [39].
Associations observed between alleles and haplotypes of the HLA
class I and the STR of the TNF suggest a genetic risk for the
disease, but studies should be carried out with a larger number of
samples to achieve total reliability of the results found in
Brazilian patients with psoriasis vulgaris.
Acknowledgements
Our special thanks to the patients and healthy individuals who
consented in participating in our research, to the Clinical
Hospital of the State University of Campinas, as well as to Dr.
Paulo Eduardo Neves Ferreira Velho, from the Dermatology Department
and to Dr Luiz Alberto Magna, from the Medical Genetic Department,
for their cooperation and numerous helpful suggestions. This
research was supported in part by FAEP (Teaching and Research
Support Fund), UNICAMP, and in part by the Biology Molecular
Laboratory of the Medical School of University of São Paulo,
Ribeirao Preto (FMRP-USP), State of São Paulo, Brazil.
References
1 Farber EM, Nall L, Watson W. Natural history of
psoriasis in 61 twin pairs. Arch Dermatol Rev 1974; 109: 207-11.
2 Martínez-Borra J, González S, Juanes-Santos J,
Sánchez Del Rio J, Alonso-Torre JC, Vásquez-López A,
Blanco-Geláz M, López-Larrea C. Psoriasis vulgaris and
psoriatic arthritics share a 100 kb susceptibility region telomeric
to HLA-C. Rheumatol 2003; 42: 1089-92.
3 Barker JN. Genetic aspects of psoriasis. Clin Exp
Dermatol 2001; 26: 321-5.
4 Henseler T, Christophers E. Psoriasis of early and
late onset: characterization of psoriasis vulgaris. J Am Acad
Dermatol 1985; 13: 450-6.
5 Bowcock AM, Barker JN. Genetics of psoriasis: The
potential impact on new therapies. J Am Acad Dermatol 2003; 49:
S51-S56.
6 Koczan D, Guthke R, Thiesen HJ,
Ibrahim SM, Kundt G, Krentz H, et al. Gene
expression profiling of peripheral blood mononuclear leukocytes
from psoriasis patients identifies new immune regulatory molecules.
Eur J Dermatol 2005; 15: 251-7.
7 Borgiani P, Vallo L, D’Apice MR,
Giardina E, Pucci S, Capon F, et al. Exclusion
of CARD15/NOD2 as a candidate susceptibility gene to psoriasis in
the Italian population. Eur J Dermatol 2002; 12: 540-2.
8 Bhalerao J, Bowcok AM. The genetics of psoriasis: a
complex disorder of the skin and immune system. Hum Mol Genet 1998;
7: 1537-45.
9 Elder JT, Nair RP, Guo SW, et al. The
genetics of psoriasis. Arch Dermatol 1994; 130: 216-24.
10 Choonhakarn C, Romphruk A, Puapairoj C,
Jirarattanapochai K, Romphruk A, Leelayuwat C.
Haplotype associations of the major histocompatibility complex with
psoriasis in Northeastern Thais. Int J Dermatol 2002; 41(6):
330-4.
11 Kraemer MHS, Uthida-Tanaka AM, Oliveira VC,
Biral AC, Cardoso CB, Magalhães RF, Magna LA.
Early-onset of Psoriasis in Brazilian patients: Support for
HLA-Class I and Class II Analysis. In: Sirisinha S,
Chaiyaroj SC, Tapchaisri P, eds. 2nd Congress of the
Federation of Immunological Society of Asia-Oceania. Bologna,
Italy: Monduzzi Editore, International Procedures Division, 2000;
(vI: 69-73).
12 Cardoso CB, Uthida-Tanaka AM, Magalhaes RF,
Magna LA, Kraemer MH. Association between psoriasis
vulgaris and MHC-DRB, -DQB genes as a contribution to disease
diagnosis. Eur J Dermatol 2005; 15: 159-63.
13 Trembath RC, Clough RL, Rosbotham JL.
Identification of a major susceptibility gene locus on chromosome
6p and evidence for further disease loci revealed by a two stage
genome-wide search in psoriasis. Hum Mol Genet 1997; 6: 813-20.
14 Balendran N, Clough RL, Arguello JR.
Characterization of the major susceptibility region for psoriasis
at chromosome 6p21.3. J Invest Dermatol 1999; 113: 322-8.
15 Pociot F, Briant L, Jongeneel CV,
Molvig J, Worsaae H, Abbal M, Thomsen M,
Nerup J, Cambon-Thomsen A. Association of tumor necrosis
factor (TNF) and class II major histocompatibility complex alleles
with the secretion of TNF-alpha and TNF-beta by human mononuclear
cells: a possible link to insulin-dependent diabetes mellitus. Eur
J Immunol 1993; 23: 224.
16 Hashigucci K, Niizeki H, Naruse T, Ota M,
Inamoto N, Nishikawa T, Inoko H. A clinical feature
associated with polymorphisms of the TNF region in Japanese
patients with palmoplantar pustulosis. Hum Immunol 2003; 64:
530-7.
17 Höhler T, Grossmann B,
Stradmann-Bellinghausen B, Kaluza W, Reuss E, Vlam
de K, Veys E, Märker-Hermann E. Differential
association of polymorphisms in the TNFα region with psoriatic
arthritis but not psoriasis. Ann Rheum Dis 2002; 61: 213-8.
18 Hajeer AH, Hutchinson IV. Influence of TNFalpha
gene polymorphisms on TNFalpha production and disease. Hum Immunol
2001; 62: 1191-9.
19 Foissac A, Salhi M, Cambon-Thomsen A.
Microsatellites in the HLA region: 1999 update. Tissue Antigens
2000; 55: 477-509.
20 Sampaio SAP, Rivitti EA. Erupções
eritemato-escamosas. In: Dermatologia, 2a ed. São Paulo: Artes
Médicas, 2000: 183-97.
21 Miller AS, Dykes DD, Polesky HF. A Simple
Salting out Procedure for Extracting DNA for Human Nucleoted Cells.
Nuclei Acids Res 1988; 16: 1215.
22 Olerup O, Zetterquist H. HLA-DR typing by PCR
amplification with sequence-specific primers (PCR-SSP) in 2 hours:
an alternative to serological DR typing in clinical practice
including donor-recipient matching in cadaveric transplantation.
Tissue Antigens 1992; 39: 225-35.
23 Udalova IA, Nedospasov A, Webb GC,
Chaplin DD. Turetskaya Rl. Highly Informative Typing of the
Human TNF Locus Using Six Adjacent Polymorphic Markers. Genomics
1993; 16: 180-6.
24 Sanguinetti CJ, Neto ED, Simpson AJG. Rapid
silver staining and recovery of PCR products separated on
polyacrylamide gels. Biotechniques 1994; 17: 915-9.
25 Excoffier L, Slatkin M. Maximum-likelihood
estimation of molecular haplotype frequencies in a diploid
population. Mol Biol Evol 1995; 12: 921-7.
26 Zaykin DV, Westfall PH, Young SS,
Karnoub MA, Wagner MJ. Ehm M.G. Testing association of
statistically inferred haplotypes with discrete and continuous
traits in samples of unrelated individuals. Hum Hered 2002; 53:
79-91.
27 Stephens M, Smith NJ, Donnelly P. A New
Statistical Method for Haplotype Reconstruction from Population
Data. Am J Hum Genet 2001; 68: 978-89.
28 Schneider S, Roessli D, Excoffier L. Arlequin
ver. 2000: A software for population genetics data analysis.
Switzerland: Genetics and Biometry Laboratory, University of
Geneva, 2000.
29 Roitberg-Tambur A, Friedmann A, Tzfoni EE,
Batta S, Ben HR, Safirman C, et al. Do specific
pockets of HLA C molecular predispose Jewish patients to psoriasis
vulgaris? J Am Acad Dermatol 1994; 31: 964-8.
30 Henseler T, Christophers E. Disease concomitance in
psoriasis. J Am Acad Dermatol 1995; 32: 982-6.
31 Ikäheimo I, Silvennoinen-Kassinen S,
Karvonen J, Tiilikainen A. Alanine at position 73 of
HLA-C is association with psoriasis vulgaris in Finland. Br J
Dermatol 1994; 131: 257-9.
32 Griffiths TW, Griffiths CEM, Voorhees JJ.
Immunopathogenesis and immunotherapy of psoriasis. Dermatol Clin
1995; 13: 739-49.
33 Nickoloff BJ, Turka LA, Mitra RS. Direct and
indirect control of T-cell activation by keratinocytes. J Invest
Dermatol 1995; 105: 25-9.
34 Niizeki H, Naruse T, Hecker KH,
Taylor JR, Kurimoto I, Shimizu T, Yamasaki Y,
Inoko H, Streilein JW. Polymorphisms in the TNF genes are
associated with susceptibility to effects of ultraviolet-B
radiation on induction of contact hypersensitivity. Tissue Antigens
2001; 58: 369.
35 Weissensteiner TH, Lanchbury JS. TNFB polymorphisms
characterize three lineages of TNF region microsatellite
haplotypes. Immunogenetics 1997; 47: 6.
36 Jenisch S, Westphal E, Nair RP, Stuart P,
Voorhees JJ, Christophers E, et al. Linkage
disequilibrium analysis of familial psoriasis: identification of
multiple disease-associated MHC haplotypes. Tissue Antigens 1999;
53: 135-46.
37 Kaluza W, Grossmann S, Hug R, Schopf ER,
Galle PR, Maerker-Hermann E, Höehle T. Different
Transcriptional activity and in vitro TNFα production in psoriasis
patients carrying the TNF-α 238 A promoter polymorphism. J Invest
Dermatol 2000; 114: 1180-3.
38 Biet E, Sun J, Dutreix M. Conserved sequence
preference in DNA binding among recombination proteins: an effect
of ssDNA secondary structure. Nucleic Acids Res 1999; 27:
596-600.
39 Biral AC, Cardoso CB, Magalhaes RF,
Magna LA, Tanaka AM, Kraemer MH. Association of
psoriasis with specific alleles of the HLA-B*57 CW*06; B*13 CW*06
haplotypes in Brazilian patients: in search of genetic
susceptibility. Hum Immunol 2003; 64: S137.
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