# Genetic Variations of Cytokines and Cytokine Receptors in Psoriasis Patients from China

Authors: Xiao-Lan Li; Chun-Feng Wu; Gui-Sheng Wu
Journal: International Journal of Genomics
 (2014)

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## Abstract

Psoriasis is a chronic inflammatory and hyperproliferative skin disease affected by both genetic and environmental factors. The aim of the present study was to investigate polymorphisms in a candidate gene family of interleukin (IL) in unrelated Chinese patients with psoriasis and control subjects without psoriasis. In this case-control study, 200 unrelated Chinese psoriasis patients and 298 age- and sex-matched control subjects were enrolled. Genomic DNA was prepared from peripheral blood obtained from all psoriasis patients and control subjects. We genotyped seven single-nucleotide polymorphisms (SNPs) in candidate genes of six ILs: IL4, IL10, IL12B, IL13, IL15, and IL23R, which have been shown in the literature to be associated with psoriasis in other ethnic groups. Among the seven SNPs in the six IL genes studied, only the rs3212227 in the IL12B gene was found to be associated with psoriasis at genotypic level in the studied population. The C/C genotype in the IL12B gene is a protective factor of psoriasis (P=0.0218; OR = 0.51; 95% CI: 0.27–0.96) in Chinese. Furthermore, the studied Chinese population has extremely low minor allele frequency for IL23R. Together, the data reveal unique genetic patterns in Chinese that may be in part responsible for the lower risk for psoriasis in this population.


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## Body

## 1. Introduction

Psoriasis is an immunologically mediated chronic inflammatory and hyperproliferative skin disease affected by both genetic and environmental factors. The prevalence of psoriasis varied among populations with different genetic backgrounds and habitats, from 3% in Northern Europe and 2% in North America and the UK to 0.1–0.3% in American Indians and East Asia [1, 2]. Psoriasis is proposed to be associated with other immune diseases, such as arthritis and Crohn’s disease [3]. Initial causative research has identified strong association between psoriasis and the interleukin genes (IL4, IL10, IL12B, IL13, and IL23R) in the northern European from US and UK [4–7]. Furthermore, the SNP rs56245420 in the IL15 gene has been found to be associated with psoriasis in the Chinese Han population but not in any of the UK, German, or US Caucasian populations investigated [8–10], since the minor allele frequency for this SNP and others across IL15 differs quite strikingly between the populations, suggesting heterogeneity in the genetic susceptibility to psoriasis.In this study, we aimed to determine if psoriasis is associated with six IL genes that have been strongly associated with psoriasis in Europeans but not well studied in Chinese. The seven included SNPs are rs2243250 in the IL4 gene, rs1800872 in the IL10 gene, rs3212227 in the IL12B gene, rs1800925 and rs20541 in the IL13 gene, rs56245420 in the IL15 gene, and rs11209026 in the IL23R gene.


## 2. Materials and Methods

### 2.1. Study Population

A total of 200 psoriasis patients and 298 healthy controls were recruited in this study (see Table2). All participants did not suffer from any other diseases and belonged to Han nationality in Yunnan Province, China. The study was performed according to the Helsinki Declaration with approval of the institutional review boards of the Affiliated Yan’an Hospital of Kunming Medical College and the Kunming Institute of Botany. Informed consent was obtained from each participant before inclusion in this study.


### 2.2. Determination of Genotype

Genomic DNAs were isolated from whole blood using regular phenol/chloroform method. The SNP rs11209026 in the IL23R gene was genotyped by the TaqMan allelic discrimination method (Applied Biosystems). New PCR-RFLP methods were generated to genotype the SNP rs56245420 in the IL15 gene. Primers 5′-TTT CTG TTA TTA ACA AAC ATC ACT CTG-3′ and 5′-CAA CAC TTG TAC ATA TTT TTA TTCAAt AT-3′ (mismatch is shown in bold lower case) were used for rs56245420. Other five SNPs were genotyped by PCR-RFLP methods described previously with slight modification [11–15]. PCR reaction was carried out in a total volume of 20 μL containing 20 ng of genomic DNA, 1 × PCR buffer, 1.5 mM MgCl2, 200 μM of each dNTP, 30 ng of each primer, and 1 unit of Taq DNA polymerase (TakaRa). Samples were denatured at 95°C for 2 min followed by 30 cycles of 94°C for 45 sec, 61°C (rs2395029) or 54°C (rs56245420) for 45 sec, and 72°C for 45 sec and ended with a final extension for 7 min at 72°C. PCR products were digested with 4 U of appropriate restriction endonuclease and electrophoresed on 3% agarose gels and stained with ethidium bromide. The restriction endonucleases, PCR product lengths, and restriction patterns are shown in Table 1.Table 1
Primer sequences, PCR product lengths, restriction endonucleases, and restriction patterns for the PCR-RFLP analyzed SNPs.
SNP
Gene
Position of SNP in genomic sequence
Forward primera
Reverse primer
Anneal temperature (°C)
Product length (bp)
Restriction endonucleases
Fragments of frequent allele genotype (bp)
Fragments of heterozygous genotype (bp)
Fragments of rare allele genotype (bp)
Reference
rs2243250
IL4
132009154:C/T
TAAACTTGGGAGAACATGGT
TGGGGAAAGATAGAGTAATA
49
195
AvaII
195
22 + 173 + 195
22 + 173
[1]
rs1800872
IL10
206946407:A/C
AGGTGATGTAATATCTCTGT
TAAATATCCTCAAAGTTCC
57
303
RsaI
65 + 238
65 + 238 + 303
303
[2]
rs3212227
IL12B
158742950:A/C
TTCTATCTGATTTGCTTTA
TGAAACATTCCATACATCC
51
233
TaqI
233
68 + 165 + 233
68 + 165
[3]
rs1800925
IL13
131992809:C/T
GTCGCCTTTTCCTGCTCTTCCCGC
GGAATCCAGCATGCCTTGTGAGG
65
247
Bsh1236I
23 + 224
23 + 224 + 247
247
[4]
rs20541
IL13
131995964:C/T
TAGGCTGAAGACGGGCAGCA
AAGAAACTTTTTCGCGAGGGCC
63
199
MspI
22 + 177
22 + 177 + 199
199
[5]
rs56245420
IL15
142873720:A/T
TTTCTGTTATTAACAAACATCACTCTG
CAACACTTGTACATATTTTTATTCAATAT
54
274
SspI
27 + 247
27 + 247 + 274
274
This study
aMismatch is shown in bold and underlined font.Table 2
Genotyping of seven studied SNPs in psoriasis patients (n=200) and controls (n=298).
SNP
Gene
Population
Genotype (%)
Minor allele (%)
rs2243250
IL4
T/T
T/C
C/C
C
Controls
189 (63.4)
98 (32.9)
11 (3.7)
120 (20.1)
Psoriasis
127 (63.5)
61 (30.5)
12 (6.0)
85 (21.3)
rs1800872
IL10
A/A
A/C
C/C
C
Controls
138 (46.3)
123 (41.3)
37 (12.4)
197 (33.1)
Psoriasis
93 (46.5)
86 (43.0)
21 (10.5)
128 (32.0)
rs3212227
IL12B
A/A
A/C
C/C
C
Controls
119 (39.9)
128 (43.0)
51 (17.1)
230 (38.6)
Psoriasis
77 (38.5)
104 (52.0)
19 (9.5)*a
142 (35.5)
rs1800925
IL13
C/C
C/T
T/T
T
Controls
222 (74.5)
72 (24.2)
4 (1.3)
80  (13.4)
Psoriasis
140 (70.0)
52 (26.0)
8 (4.0)
68 (17.0)
rs20541
IL13
C/C
C/T
T/T
T
Controls
146 (49.0)
126 (42.3)
26 (8.7)
178 (29.9)
Psoriasis
100 (50.0)
80 (40.0)
20 (10.0)
120 (30.0)
rs56245420
IL15
A/A
A/T
T/T
T
Controls
139 (46.6)
135 (45.3)
24 (8.1)
183 (30.7)
Psoriasis
78 (39.0)
97 (48.5)
25 (12.5)
147 (36.8)
rs11209026
IL23R
G/G
G/A
A/A
A
Controls
298 (100)
0
0
0
Psoriasis
200 (100)
0
0
0
*
a
P
=
0
.
0218.


### 2.3. Data Analysis

Statistics analysis was performed by SPSS software for windows (SPSS Inc.). The frequencies of genotypes and alleles for all the six studied loci were determined assuming codominant inheritance. The Hardy-Weinberg equilibrium (HWE) for six loci in psoriasis patients and controls was tested by means of chi-square tests. The statistical significance of the genotype and allele frequency variables between the psoriasis patients and control group was evaluated by chi-square test with Yates correction for small numbers. Relative risk associated with the significant genotype was estimated by the odds ratio (OR). OR with 95% confidence intervals (95% CI) was tested using a chi-square distribution and the null hypothesis being tested is OR = 1.P values <0.05 were considered as statistically significant.


## 2.1. Study Population

A total of 200 psoriasis patients and 298 healthy controls were recruited in this study (see Table2). All participants did not suffer from any other diseases and belonged to Han nationality in Yunnan Province, China. The study was performed according to the Helsinki Declaration with approval of the institutional review boards of the Affiliated Yan’an Hospital of Kunming Medical College and the Kunming Institute of Botany. Informed consent was obtained from each participant before inclusion in this study.


## 2.2. Determination of Genotype

Genomic DNAs were isolated from whole blood using regular phenol/chloroform method. The SNP rs11209026 in the IL23R gene was genotyped by the TaqMan allelic discrimination method (Applied Biosystems). New PCR-RFLP methods were generated to genotype the SNP rs56245420 in the IL15 gene. Primers 5′-TTT CTG TTA TTA ACA AAC ATC ACT CTG-3′ and 5′-CAA CAC TTG TAC ATA TTT TTA TTCAAt AT-3′ (mismatch is shown in bold lower case) were used for rs56245420. Other five SNPs were genotyped by PCR-RFLP methods described previously with slight modification [11–15]. PCR reaction was carried out in a total volume of 20 μL containing 20 ng of genomic DNA, 1 × PCR buffer, 1.5 mM MgCl2, 200 μM of each dNTP, 30 ng of each primer, and 1 unit of Taq DNA polymerase (TakaRa). Samples were denatured at 95°C for 2 min followed by 30 cycles of 94°C for 45 sec, 61°C (rs2395029) or 54°C (rs56245420) for 45 sec, and 72°C for 45 sec and ended with a final extension for 7 min at 72°C. PCR products were digested with 4 U of appropriate restriction endonuclease and electrophoresed on 3% agarose gels and stained with ethidium bromide. The restriction endonucleases, PCR product lengths, and restriction patterns are shown in Table 1.Table 1
Primer sequences, PCR product lengths, restriction endonucleases, and restriction patterns for the PCR-RFLP analyzed SNPs.
SNP
Gene
Position of SNP in genomic sequence
Forward primera
Reverse primer
Anneal temperature (°C)
Product length (bp)
Restriction endonucleases
Fragments of frequent allele genotype (bp)
Fragments of heterozygous genotype (bp)
Fragments of rare allele genotype (bp)
Reference
rs2243250
IL4
132009154:C/T
TAAACTTGGGAGAACATGGT
TGGGGAAAGATAGAGTAATA
49
195
AvaII
195
22 + 173 + 195
22 + 173
[1]
rs1800872
IL10
206946407:A/C
AGGTGATGTAATATCTCTGT
TAAATATCCTCAAAGTTCC
57
303
RsaI
65 + 238
65 + 238 + 303
303
[2]
rs3212227
IL12B
158742950:A/C
TTCTATCTGATTTGCTTTA
TGAAACATTCCATACATCC
51
233
TaqI
233
68 + 165 + 233
68 + 165
[3]
rs1800925
IL13
131992809:C/T
GTCGCCTTTTCCTGCTCTTCCCGC
GGAATCCAGCATGCCTTGTGAGG
65
247
Bsh1236I
23 + 224
23 + 224 + 247
247
[4]
rs20541
IL13
131995964:C/T
TAGGCTGAAGACGGGCAGCA
AAGAAACTTTTTCGCGAGGGCC
63
199
MspI
22 + 177
22 + 177 + 199
199
[5]
rs56245420
IL15
142873720:A/T
TTTCTGTTATTAACAAACATCACTCTG
CAACACTTGTACATATTTTTATTCAATAT
54
274
SspI
27 + 247
27 + 247 + 274
274
This study
aMismatch is shown in bold and underlined font.Table 2
Genotyping of seven studied SNPs in psoriasis patients (n=200) and controls (n=298).
SNP
Gene
Population
Genotype (%)
Minor allele (%)
rs2243250
IL4
T/T
T/C
C/C
C
Controls
189 (63.4)
98 (32.9)
11 (3.7)
120 (20.1)
Psoriasis
127 (63.5)
61 (30.5)
12 (6.0)
85 (21.3)
rs1800872
IL10
A/A
A/C
C/C
C
Controls
138 (46.3)
123 (41.3)
37 (12.4)
197 (33.1)
Psoriasis
93 (46.5)
86 (43.0)
21 (10.5)
128 (32.0)
rs3212227
IL12B
A/A
A/C
C/C
C
Controls
119 (39.9)
128 (43.0)
51 (17.1)
230 (38.6)
Psoriasis
77 (38.5)
104 (52.0)
19 (9.5)*a
142 (35.5)
rs1800925
IL13
C/C
C/T
T/T
T
Controls
222 (74.5)
72 (24.2)
4 (1.3)
80  (13.4)
Psoriasis
140 (70.0)
52 (26.0)
8 (4.0)
68 (17.0)
rs20541
IL13
C/C
C/T
T/T
T
Controls
146 (49.0)
126 (42.3)
26 (8.7)
178 (29.9)
Psoriasis
100 (50.0)
80 (40.0)
20 (10.0)
120 (30.0)
rs56245420
IL15
A/A
A/T
T/T
T
Controls
139 (46.6)
135 (45.3)
24 (8.1)
183 (30.7)
Psoriasis
78 (39.0)
97 (48.5)
25 (12.5)
147 (36.8)
rs11209026
IL23R
G/G
G/A
A/A
A
Controls
298 (100)
0
0
0
Psoriasis
200 (100)
0
0
0
*
a
P
=
0
.
0218.


## 2.3. Data Analysis

Statistics analysis was performed by SPSS software for windows (SPSS Inc.). The frequencies of genotypes and alleles for all the six studied loci were determined assuming codominant inheritance. The Hardy-Weinberg equilibrium (HWE) for six loci in psoriasis patients and controls was tested by means of chi-square tests. The statistical significance of the genotype and allele frequency variables between the psoriasis patients and control group was evaluated by chi-square test with Yates correction for small numbers. Relative risk associated with the significant genotype was estimated by the odds ratio (OR). OR with 95% confidence intervals (95% CI) was tested using a chi-square distribution and the null hypothesis being tested is OR = 1.P values <0.05 were considered as statistically significant.


## 3. Results

Only one of the fourteen Hardy-Weinberg tests (seven polymorphic loci each in the psoriasis patient and control groups) hadP values smaller than 0.05 (P=0.01 at rs3212227 in controls). All nine remaining genotype frequencies fit Hardy-Weinberg expectations according to chi-square tests in psoriasis patients and controls (P>0.05). Therefore, there is no meaningful deviation from WHE, and our population is derived from random mating.Polymorphism (minor allele frequency > 1%) has been found for all studied SNPs except for rs11209026 in the IL23R gene (Table2). Table 2 shows that rs3212227 in the IL12B gene (P=0.0218) was associated with psoriasis at genotypic level in the studied population. Other SNPs examined were not associated with psoriasis considered from single locus. As shown in Table 3, while the A/C genotype (OR = 1.48; 95% CI: 0.95–2.30) and the alleles (OR = 0.84; 95% CI: 0.63–1.13) at rs3212227 in the IL12B were not a risk factor of psoriasis, the C/C genotype was a protective factor of psoriasis (OR = 0.51; 95% CI: 0.27–0.96).Table 3
Correlation of psoriasis symptoms with SNPs in the IL genes.
SNP
Genotype
Psoriasis
Controls
OR (95% CI)
Allele
Psoriasis
Controls
OR (95% CI)
rs2243250
TT
127
189
T
315
476
TC
61
98
0.93 (0.63–1.37)
C
85
120
1.07 (0.78–1.46)
CC
12
11
1.62 (0.69–3.78)
TC-CC
73
109
1.00 (0.69–1.45)
rs1800872
AA
93
138
A
272
399
AC
86
123
1.04 (0.71–1.52)
C
128
197
0.95 (0.72–1.25)
CC
21
37
0.84 (0.46–1.53)
AC-CC
107
160
0.99 (0.69–1.42)
rs3212227
CC
19
51
C
142
230
AC
104
128
2.18 (1.21–3.92)
A
258
366
1.14 (0.88–1.48)
AA
77
119
1.71 (0.96–3.17)
AC-AA
181
247
1.97 (1.12–3.45)
rs1800925
CC
140
222
C
332
516
CT
52
72
1.15 (0.76–1.74)
T
68
80
1.32 (0.93–1.86)
TT
8
4
3.17 (0.94–10.72)
CT-CC
60
76
1.25 (0.84–1.86)
rs20541
CC
100
146
C
280
418
CT
80
126
0.93 (0.64–1.36)
T
120
178
1.01 (0.77–1.33)
TT
20
26
1.12 (0.59–2.12)
CT-TT
100
152
0.96 (0.67–1.37)
rs56245420
AA
78
139
A
253
413
AT
97
135
1.28 (0.87–1.87)
T
147
183
1.28 (1.00–1.71)
TT
25
24
1.85 (0.99–3.46)
AT-TT
122
159
1.36 (0.94–1.96)


## 4. Discussion

The etiology of psoriasis is a complex interaction of environmental and biological factors. Genetic factors may play a significant role in the risk of psoriasis in Chinese [10, 16]. Recent genetic studies indicate that the location of these genes varies considerably among populations and families. We are interested to know if psoriasis is associated with the genes that have been strongly associated with psoriasis in Europeans but not well studied in Chinese.Our results showed that the IL12B gene was associated with psoriasis in Chinese at genotypic level (P<0.05), which is in line with the findings from European studies [17]. The C/C genotype for rs3212227 in IL12B is a protective factor (OR = 0.51) from psoriasis. Similar result from studying SNP rs6887695 in IL12B showed that the minor allele C was a protective factor from psoriasis [5].The nonsynonymous SNP in IL23R, rs11209026, widely thought to be the primary psoriasis-associated SNP in IL23R in Europeans, was found not to be polymorphic in Chinese, which is in agreement with the findings of others [18]. The low frequencies of variant in IL23R are accordingly of low risk for psoriasis in Chinese. With single SNP analysis, no association is found between the psoriasis and the IL4, IL10, IL13, and IL15 genes.



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*Source: 870597-2014-05-25.xml*