Polymorphisms in Interleukin-15 Gene on Chromosome 4q31.2 Are Associated with Psoriasis Vulgaris in Chinese Population

ORIGINAL ARTICLE

See commentary on pg 2495

Polymorphisms in Interleukin-15 Gene on Chromosome 4q31.2 Are Associated with Psoriasis Vulgaris in Chinese Population Xue-Jun Zhang1,2,6, Kai-Lin Yan1,2, Zhi-Min Wang3,4, Sen Yang1,2, Guo-Long Zhang1,2, Xing Fan1,2, Feng-Li Xiao1,2, Min Gao1,2, Yong Cui1,2, Pei-Guang Wang1,2, Liang-dan Sun1,2, Kai-Yue Zhang3, Beilan Wang1,2,3,4,5, Da-Zhi Wang3, Shi-Jie Xu3, Wei Huang3,6 and Jian-Jun Liu1,2,5 Through a series of linkage analyses in a large Chinese family cohort of psoriasis, we previously identified and confirmed a non-HLA psoriasis linkage locus PSORS9 within a small region at 4q31.2–32.1. Within the critical region of the PSORS9 locus, IL-15 has been long recognized as a strong candidate gene for psoriasis. In this study, we investigated the association between IL-15 genetic polymorphisms and psoriasis in a large Chinese sample. Highly significant evidence for association was identified at a single-nucleotide polymorphism (SNP) (g.96516A-T) within the 30 -untranslated region (UTR) of the IL-15 gene (P ¼ 0.00006, after correction for multiple testing). Haplotype analysis using the SNPs within the 30 UTR region also provided strong supporting evidence for association (P ¼ 0.00005), where we identified a haplotype of the 30 UTR region of IL-15 associated with increased risk to psoriasis (odds ratio ¼ 1.65). This association was also supported by the results of our expression activity analyses, where we demonstrated that the identified risk haplotype is associated with an increased activity of IL-15. Therefore, we provided early evidence for the important role of IL-15 genetic variants in the pathogenesis of psoriasis, probably by increasing interleukin production and inflammation in the lesions of psoriasis. Journal of Investigative Dermatology (2007) 127, 2544–2551; doi:10.1038/sj.jid.5700896; published online 31 May 2007

INTRODUCTION Psoriasis (OMIM *177900) is recognized as the most prevalent autoimmune disease caused by inappropriate activation of the cellular immune system that has a prevalence of 2–5% in Caucasian populations (Nevitt and Hutchinson, 1996) and 0.1–0.3% in Far East populations (Simons, 1949; Yui, 1984). It is characterized by scaly skin patches caused by the infiltration of inflammatory cells into dermis and epidermis, with secondary keratinocyte hyperproliferation (Bowcock and Krueger, 1

Institute of Dermatology and Department of Dermatology at No.1 Hospital, Anhui Medical University, Hefei, China; 2The Key Laboratory of Gene Resource Utilization for Severe Diseases, Ministry of Education, Hefei, China; 3 Chinese National Human Genome Center at Shanghai, Shanghai, China; 4 MOE Key Laboratory of Contemporary Anthropology and Center for Evolutionary Biology, School of Life Sciences and Institutes of Biomedical Sciences, Fudan University, Shanghai, China and 5Genome Institute of Singapore, Singapore 6

These two authors contributed equally to this work.

Correspondence: Professor Xue-Jun Zhang, Institute of Dermatology, Anhui Medical University, 69 Meishan Road, Hefei, Anhui 230032, PR China. E-mail: [email protected] or Dr Wei Huang, Chinese National Human Genome Center at Shanghai, 250 Bi Bo Road, Shanghai 201203, PR China. E-mail: [email protected] Abbreviations: LD, linkage disequilibrium; SNP, single-nucleotide polymorphism; UTR, untranslated region Received 17 October 2006; revised 21 January 2007; accepted 31 January 2007; published online 31 May 2007

2544 Journal of Investigative Dermatology (2007), Volume 127

2005). Epidemiological and family studies (Bhalerao and Bowcock, 1998) have clearly demonstrated a strong genetic component to the risk to psoriasis, and a combination of genetic and environmental factors is believed to be causative. Many groups around the world have pursued the search for disease gene using linkage analysis in large family cohorts (Tomfohrde et al., 1994; Matthews et al., 1996; Nair et al., 1997; Trembath et al., 1997; Capon et al., 1999; Samuelsson et al., 1999; Lee et al., 2000; Veal et al., 2001; Zhang et al., 2002; Asumalahti et al., 2003; Karason et al., 2005), and nine loci designated PSORS1–9 as well as additional 11 loci have been identified or been suggested as psoriasis susceptibility loci. Despite a major international effort, no susceptibility genes have been identified with certainty till today. In our previously genome-wide scan (Zhang et al., 2002), we localized a non-HLA psoriasis susceptibility locus to chromosome 4q28–q32 (PSORS9) in 61 multiply affected families. The linkage was supported by a subsequent metaanalysis of five genome-wide linkage data, showing that apart from the major linkage locus within the HLA region (PSORS1) on chromosome 6p, the 4q31 interval is another major linkage locus for psoriasis (Sagoo et al., 2004). Recently, we performed a followed-up study using an extended Chinese family cohort of psoriasis and dense marker coverage, which confirmed and refined the PSORS9 locus to a small region at 4q31.2–32.1 (Yan et al., 2007). & 2007 The Society for Investigative Dermatology

X-J Zhang et al. Interleukin-15 Gene Polymorphism and Psoriasis Vulgaris

IL-15 has been recognized as one of the candidate genes within the critical region of the PSORS9 locus (Bowcock, 2004). IL-15 is a Th1-related cytokine that triggers inflammatory cell recruitment, angiogenesis, and production of other inflammatory cytokines. An upregulated IL-15 expression is consistently reported in psoriatic lesions (Ong et al., 2002; Ruckert et al., 2003). Psoriatic dermal pathology is characterized by local T cell, macrophage and neutrophil infiltration and activation, angiogenesis, and epidermal hyperplasia, all features to which IL-15 might plausibly contribute (McInnes and Gracie, 2004). Villadsen et al. (2003) identified that an IL-15-specific antibody reduced the severity of psoriasis in a human psoriasis xenograft model. In this study, we investigate the possibility of IL-15 being a susceptibility gene within the PSORS9 locus through a series of genetic and functional analyses in a large Chinese sample of psoriasis. RESULTS Sequence analysis of IL-15

We first performed a systematic sequence analysis of IL-15 by analyzing all eight known exons, exon–intron boundaries, approximately 2,000 bp upstream of the 50 flank and downstream of the 30 flank sequences in 48 affected individuals of psoriasis and 48 healthy controls. We identified 19 variants, of which 14 are known variants (rs4956403, rs3806798, rs2857261, rs9282742, rs4956406, rs1021446, rs3775596, rs12508335, rs7349640, rs12510030, rs17007610, rs10519613, rs1057972, and rs10833) and five are novel ones (1216A/G, 82704T/C, 82705T/C, 96516A/T, and 99717G/T) (Table 1). Nucleotide position one ( þ 1) is the first basepair of the exon 1(NM_172174). The novel singlenucleotide polymorphisms (SNPs) are named g.xxxx, where the xxx stands for the position of the SNP on the reference sequence NM_172174. None of the identified variants is in the coding sequences or potential splicing sites of IL-15. Linkage disequilibrium pattern characterization

In order to perform a comprehensive genetic association analysis of IL-15, we characterized the linkage disequilibrium (LD) pattern within IL-15. Aiming to achieve a dense and even SNP coverage across the whole gene, without any gap larger than 5 kb, additional 19 SNPs were selected from the public dbSNP database (nine from database) or identified by additional sequence analysis in the same 48 cases and 48 healthy controls (10 from sequence analysis). In total, we analyzed 38 SNPs in the 48 cases and 48 health controls: five variants in the 50 -flanking region, four in the 30 -untranslated region (UTR), 28 within introns, and one in 30 -flanking region (Table 1). All the 38 variants have an estimated minor allele frequency above 5%. The 38 SNPs spanned a total of 100 kb sequence of the entire IL-15 gene, resulting in dense coverage of one SNP per 3 kb sequence. Based on the genotypes from the 48 cases and 48 healthy controls, none of the SNPs showed a significant deviation (Po0.05) from Hardy–Weinberg equilibrium. LD pattern was characterized by calculating pairwise D0 and r2 values among 38 SNPs and summarized in Figure 1.

Strong LD was observed across the IL-15 gene region, and at least one historical recombination seems to have occurred, dividing the region into two strong LD blocks. Such a pattern is very similar to the one of Chinese population from the HapMap project (data not shown). In order to perform an effective and efficient genetic association analysis, tagging SNP-based approach was employed by selecting 12 representative SNPs (g.-1216, rs12508866, rs13106911, rs7671458, rs2322301, g.82704, rs2857261, rs10519613, g.96516, rs1057972, rs10833, and g.99717) using r2 ¼ 0.9 as a cut-off value, that is, the genetic association analysis using the 12 selected SNPs will only suffer a slight lose of power comparing with an analysis using all the 38 SNPs. Case–control association analysis

We performed a case–control association analysis of IL-15 common genetic variations by genotyping the 12 selected SNPs in a sample of 632 unrelated psoriatic patients and 485 healthy controls. The genotypes of the 12 SNPs were all in Hardy–Weinberg equilibrium in both case and control groups (P40.05). For association analysis, we first tested each of the 12 SNPs individually for association with disease phenotype. Of the 12 SNPs tested, four SNPs (rs2857261, rs10519613, g.96516, and rs1057972) showed significant association to psoriasis with a nominal P-value below 0.05 (Table 2). The four SNPs spanned 13.9 kb sequence of IL-15, one within intron 3 and the other three in the 30 UTR region of the gene. After a Bonferroni correction for multiple testing, only two SNPs (rs10519613 and g.96516) from the 30 UTR region still showed significant association with disease phenotype (P ¼ 0.048 and 0.00006, respectively). The most significant association evidence was detected at the SNP g.96516A-T, where the T allele was associated with increased risk to psoriasis (odds ratio (OR) ¼ 1.56) at a highly significant P-value (P ¼ 0.000005) (Table 2). Furthermore, under a dominant model, the evidence for association is even stronger with a higher OR value and a smaller P-value (P ¼ 0.0000004; OR ¼ 1.86) (Table 2). We further investigated the association evidence within the 30 UTR region by analyzing the haplotypes of the four SNPs showing positive association evidence. The haplotypes of the four SNPs were constructed from unphased genotype data using the PHASE program. A total of seven haplotypes was identified, but only four haplotypes are common, with a population frequency above 10% and accounted for 87.4% of all the observed haplotypes (Table 3). All the seven haplotypes were tested for association with disease phenotype, and two haplotypes showed significant association evidence, with a nominal Po0.05 (Table 3). A highly significant evidence for association was obtained for the haplotype 4, with a nominal P ¼ 0.00005, which remained highly significant after a Bonferroni correction for multiple testing. This haplotype (ATAC) has a population frequency of 18.1% in affected individuals but 10.9% in healthy controls (Table 3). The estimated relative risk conferred by this haplotype was 1.65 under an assumption of a multiplicative model. When the haplotypes had been taken into account under a dominant model, we did find the distribution of www.jidonline.org 2545

X-J Zhang et al. Interleukin-15 Gene Polymorphism and Psoriasis Vulgaris

Table 1. Characteristics of the polymorphisms that were detected in the IL-15 gene Number

RefSNP ID

1

Novel

2

rs4956403

Position1

Variation

1216

A/G

50 flank

0.175

F2R2

831

C/T

50 flank

0.047

F3R3

Location

0

Minor allele frequency

Primers

3

rs3806798

475

T/A

5 flank

0.156

F3R3

4

rs12508866

7939

T/C

Intron 1

0.215

F7R7

5

rs11946791

8531

A/G

Intron 1

0.346

F7R7

6

rs1519551

12718

A/G

Intron 1

0.222

F8R8

7

rs9790346

12767

G/T

Intron 1

0.085

F8R8

8

rs957077

22062

T/C

Intron 2

0.111

F10R10

9

rs1808109

22414

C/T

Intron 2

0.096

F10R10

10

Novel

30412

C/A

Intron 2

0.178

F11R11

11

rs932209

30693

G/A

Intron 2

0.205

F11R11

12

Novel

30887

T/C

Intron 2

0.026

F11R11

13

rs2090664

39838

C/T

Intron 2

0.222

F12R12

14

rs13104121

39938

A/C

Intron 2

0.054

F12R12

15

Novel

45025

A/T

Intron 2

0.096

F13R13

16

rs12498901

45376

G/C

Intron 2

0.136

F13R13

17

rs13106911

57991

C/T

Intron 2

0.419

F14R14

18

rs13112582

58252

C/T

Intron 2

0.378

F14R14

19

rs13139573

58440

A/T

Intron 2

0.378

F14R14

20

rs7671458

69707

T/G

Intron 2

0.156

F15R15

21

rs1607595

77894

C/T

Intron 2

0.156

F16R16

22

rs2322301

78559

C/T

Intron 2

0.378

F16R16

23

Novel

82704

T/C

Intron 2

0.173

F17R17

24

Novel

82705

T/C

Intron 2

0.173

F17R17

25

rs2857261

82883

A/G

Intron 3

0.364

F17R17

26

rs9282742

83020

T/C

Intron 3

0.432

F17R17

27

rs4956406

84051

G/A

Intron 4

0.398

F18R18

28

rs1021446

90803

A/G

Intron 5

0.444

F20R20

29

rs3775596

92060

A/C

Intron 6

0.455

F20R20

30

rs12508335

93114

A/G

Intron 6

0.415

F21R21

31

rs7349640

93147

A/G

Intron 6

0.176

F21R21

32

rs12510030

93405

T/C

Intron 7

0.415

F21R21

33

rs17007610

93511

T/C

Intron 7

0.415

F21R21

0

34

rs10519613

96330

C/A

3 UTR

0.419

F22R22

35

Novel

96516

A/T

30 UTR

0.265

F22R22

0

36

rs1057972

96678

A/T

3 UTR

0.487

F22R22

37

rs10833

96793

C/T

30 UTR

0.057

F22R22

0.215

F23R23

38

Novel

99717

0

G/T

3 flank

1

Nucleotide position one (+1) is the first base pair of the exon 1 (NM_172174).

haplotype 3 (P ¼ 0.037; OR ¼ 1.39; 95% confidence interval 1.018–1.894) and haplotype 4 (P ¼ 0.000016; OR ¼ 1.88; 95% confidence interval 1.408–2.507) differed significantly between the psoriatic and control groups in our samples. The haplotypes 3 and 4 both showed a higher 2546 Journal of Investigative Dermatology (2007), Volume 127

frequency in cases than in controls. All the haplotypes that were significantly associated with a P-value less than 0.05 were highly correlated with susceptibility T allele of SNP g.96516A-T and may indicate susceptibility to psoriasis.

X-J Zhang et al. Interleukin-15 Gene Polymorphism and Psoriasis Vulgaris

–1216 – 831 – 475 7939 8531 12718 12767 22062 22414 30412 30693 30885 39838 39938 45025 45376 57991 58252 58440 69707 77894 78559 82704 82705 82883 83020 84051 90803 92060 93114 93114 93405 93511 96330 96516 96678 96793 99717

D′

r2 –1216 – 831 – 475 7939 8531 12718 12767 22062 22414 30412 30693 30885 39838 39938 45025 45376 57991 58252 58440 69707 77894 78559 82704 82705 82883 83020 84051 90803 92060 93114 93114 93405 93511 96330 96516 96678 96793 99717

Figure 1. Pattern of LD within IL-15; the numbers on the x-axis correspond to 38 markers selected to cover the IL-15 gene relatively evenly at an average spacing of 3 kb. D0 values are shown in the bottom left and r2 values are shown in the top right triangle.

Effect of 30 UTR of IL-15 gene on luciferase activity

To investigate the impact of the haplotype sequences of 30 UTR region on transcriptional activity, we performed a luciferase activity assay. We transfected the luciferase reporter constructs carrying the different haplotypes of the 30 UTR region, as well as the construct without insert (pGL3Hap1, Hap3, Hap4, and pGL3 promoter) into the Jurkat cells. Among the three transfected haplotypes, the haplotype 1(Hap1) is the most common haplotype observed in Chinese population and not associated with the increased risk to psoriasis, whereas haplotypes 3 and 4 (Hap3 and Hap4) are both associated with psoriasis risk. Luciferase activity analysis indicated that the luciferase activities of the Jurkat cells carrying either the pGL3-Hap4 (2.1270.20) or the pGL3Hap3 construct (1.8670.09) are significantly higher than those of the cells carrying the non-risk haplotype 1 (pGL3Hap1, 1.3970.21) (Po0.01). The luciferase activities of the cells carrying the pGL3-Hap3 and pGL3-Hap4 constructs are 33.9 and 52.9% higher than the activity of the cells carrying pGL3-Hap1 construct, respectively (see Table 4). Of interest, the haplotype 4, which showed a stronger association with psoriasis than the haplotype 3, also showed a stronger induction of luciferase activity than the haplotype 3.

DISCUSSION In this study, we investigated the contribution of IL-15 genetic polymorphisms to the development of psoriasis. First, we sequenced the coding, exon–intron boundary and promoter sequences of IL-15 in 48 psoriatic cases and 48 controls, but did not identify any putative functional polymorphisms that might have a role in disease development. To further evaluate the role of other IL-15 genetic polymorphisms, we subsequently performed an efficient genetic association analysis of IL-15 common genetic variants using a tagging SNPbased approach and identified significant evidence for association within the 30 UTR region of IL-15. Both single SNP- and haplotype-based genetic association analyses provided highly significant evidence that the genotypes and haplotypes of the SNPs within the 30 UTR region of IL-15 are strongly associated with increased risk to psoriasis. Furthermore, the results of our transcriptional activity analysis using both luciferase reporter assay and immunohistological staining analysis suggested that the identified risk haplotypes (Hap3 and Hap4) are associated with higher expression activity of IL-15 than the non-risk haplotype (Hap1), and psoriatic lesion skin shows much higher expression activity of IL-15 than the non-lesion skin of psoriasis, as well as the www.jidonline.org 2547

X-J Zhang et al. Interleukin-15 Gene Polymorphism and Psoriasis Vulgaris

Table 2. Summary of the association of Tag SNPs between cases and controls Genotype Case

Control

Allele 1 versus allele 2

Minor allele Minor allele frequency 1/1 1/2 2/2 frequency

Odds ratio (95% CI)

v2

0.142

1.13 (0.88–1.44)

0.94

16

0.181

1.15 (0.92–1.44)

80

0.411

1.03 (0.87–1.22)

342 131

12

0.160

195 232

58

0.359

SNP ID

1/1

1/2 2/2

g.-1216

487

128

17

0.128

360 112

13

Rs12508866

448

164

20

0.161

325 144

Rs13106911

239

275 118

0.404

166 239

Rs7671458

473

138

21

0.142

Rs2322301

290

268

74

0.329

Genotype 1/1 vs 1/2+2/2 Odds ratio (95% CI)

v2

P-value

0.333

1.17 (0.89–1.54)

1.20

0.273

1.56

0.211

1.20 (0.93–1.55)

1.93

0.164

0.11

0.736

1.17 (0.91–1.50)

1.53

0.216

1.15 (0.91–1.45)

1.30

0.254

1.24 (0.95–1.62)

2.60

0.107

1.14 (0.96–1.36)

2.14

0.143

1.26 (0.99–1.66)

3.60

0.058

P-value

g.82704

534

89

9

0.085

425

55

5

0.067

0.78 (0.56–1.07)

2.40

0.121

0.77 (0.54–1.09)

2.22

0.136

Rs2857261

291

267

74

0.328

201 209

75

0.370

1.20 (1.01–1.43)

4.23

0.040

1.21 (0.95–1.53)

2.36

0.125

Rs10519613

172

296 164

0.494

168 215 102

0.432

1.28 (1.0841.52) 8.40

0.004

1.42 (1.10–1.83)

7.14

0.008

g.96516

311

238

83

0.320

312 121

52

0.232

1.56 (1.29–1.88) 20.85

0.000005

1.86 (1.46–2.37) 25.44 0.0000004

Rs1057972

165

314 153

0.491

115 226 144

0.470

0.85 (0.72–1.01)

4.89

0.027

1.14 (0.86–1.50)

0.84

0.360

Rs10833

538

88

6

0.079

417

64

4

0.074

0.93 (0.68–1.28)

0.18

0.67

0.93 (0.66–1.31)

0.16

0.688

g.99717

417

195

20

0.186

329 145

11

0.172

0.91 (0.73–1.13)

0.70

0.402

0.92 (0.72–1.18)

0.43

0.514

CI, confidence interval; SNP, single-nucleotide polymorphism. For each SNP, we named ‘‘1/1’’ the homozygous wild-type genotype, ‘‘2/2’’ the homozygous mutated genotype, and ‘‘1/2’’ the heterozygous genotype. In each test, a Po0.05 was considered statistically significant.

Table 3. Haplotype structure of 30 UTR and their frequencies SNP ID

Haplotype frequency

rs10519613

g.96516

rs1057972

rs10833

Case (n=632)

Control (n=485)

v2

Haplotype 1

C

A

T

C

0.293

0.323

1.314

Haplotype 2

A

A

A

C

0.249

0.243

0.047

0.866

1.021 (0.846–1.233)

Haplotype 3

C

T

T

C

0.151

0.118

3.919

0.048

1.281 (1.002–1.638)

Haplotype 4

A

T

A

C

0.181

0.109

16.292

0.00005

1.651 (1.292–2.109)

Haplotype 5

C

A

A

T

0.044

0.054

0.936

0.333

0.826 (0.561–1.217)

Haplotype 6

A

A

T

C

0.045

0.064

3.458

0.063

0.706 (0.488–1.021)

Haplotype 7

C

A

A

C

0.037

0.054

3.193

0.074

0.694 (0.463–1.038)

Haplotype ID

P-value

Odds ratio (95% CI)

0.252

0.904 (0.761–1.074)

CI, confidence interval; UTR, untranslated region.

Table 4. The mean difference with relative luciferase activity one-way ANOVA VAR00002 pGL3-Hap1

pGL3-Hap3

VAR00002

Mean difference

SE

Sig. 0.0048

pGL3-Hap3

0.47

0.13

pGL3-Hap4

0.73

0.13

0.00026

pGL3-Hap4

0.26

0.13

0.068

ANOVA, analysis of variance.

normal skin. Interestingly, the strength of the association between the haplotypes of the 30 UTR region and disease phenotype seems to be correlated with the strength of the luciferase activity induction of the haplotypes. Therefore, our initial genetic association and subsequent functional analyses 2548 Journal of Investigative Dermatology (2007), Volume 127

provided first evidence for association between the genetic variants of IL-15 and risk to psoriasis. The biological functions of IL-15 support its role as a predisposition gene to psoriasis. Recent evidence indicates that the epidermal changes in psoriasis occur in response to cellular immune infiltrates in the skin, leading to the current general consensus that psoriasis is a T-cell-mediated inflammatory disease. T cells and dendritic cells, together with the proinflammatory cytokines and chemokines that they secrete, collaborate to create a proinflammatory environment in the skin that in turn stimulates the proliferation of resident keratinocytes and endothelial cells, producing a pattern of tissue growth that is recognized as psoriasis (Bowcock and Krueger, 2005). The proinflammatory action of IL-15 includes upregulating the cytokines tumor necrosis factor-a, interferon-g, granulocyte colony-stimulating factor, macrophage

X-J Zhang et al. Interleukin-15 Gene Polymorphism and Psoriasis Vulgaris

inflammatory protein-1a, and macrophage inflammatory protein-1b, IL-1b, IL-10 (Fehniger and Caligiuri, 2001). IL-15 also activates human neutrophils, with effects including morphologic changes, increased phagocytosis, increased de novo RNA and protein synthesis, and delayed apoptosis (Girard et al., 1996). Multiple positive disease associations have recently been described, with increased expression of IL-15 protein appearing to be critical to disease pathogenesis (D’Auria et al., 1999; Asadullah et al., 2000; Muro et al., 2001; Gonzalez-Alvaro et al., 2003; Shi et al., 2004). Specifically, with respect to psoriasis, the use of monoclonal antibodies (146B7) to block the actions of IL-15 results in an inhibition of the onset in a human psoriasis xenograft model (Villadsen et al., 2003). In addition, Yano et al. (2003) have also provided further insight into the mechanisms by which IL-15-mediated rescue from apoptosis could operate to promote epidermal proliferation in psoriasis. Complementary to these biological and pharmacological findings, our results provide the first genetic evidence for the association between IL-15 and the development of psoriasis. Our results show that genetic variants within the 30 UTR region of IL-15 are associated with greater risk to psoriasis by regulating IL-15 expression activity. This observation was supported by the results of our in vitro transfection experiments using reporter constructs containing IL-15 30 UTR variants, as well as by the results from our immunohistological staining analysis (data no shown), which is similar to a previous report (Ruckert et al., 2000). The 30 UTR region of genes has been recognized as important for controlling gene expression activity at different levels such as nuclear export, polyadenylation, translation efficiency, and mRNA degradation (Conne et al., 2000; Guhaniyogi and Brewer, 2001; Macdonald, 2001). It has also shown that from fungus to human, the 30 UTR sequences of genes evolve more rapidly and thus become more complex than the 50 UTR sequences (Mazumder et al., 2003), suggesting that the 30 UTR region probably carries more important genetic information than previously thought. Some human diseases, for example, myotonic dystrophy, have been shown due to mutations in the 30 UTR of genes (Conne et al., 2000). Interestingly, using multilocus haplotype analysis, Kurz et al. (2004) identified a strong association between a haplotype covering the 30 UTR of IL-15 and the susceptibility to asthma in German population. Although indirect, these results provided supporting evidence for a functional role of the 30 UTR region in regulating IL-15 activity. To investigate whether any of the risk-associated SNPs within the 30 UTR region is functional by interrupting or creating any transcription factor binding sites, we submitted the sequence containing three SNPs of the 30 UTR region of IL-15 to the ‘‘Genomatix’’ online program (http://www. genomatix.de/online_help/help_matinspector). The analysis did not identify any transcription factor-binding sites that overlap with the SNPs (data not shown). Although we cannot totally rule out the possibility of the risk-associated SNPs being functional ones by interrupting unknown transcription factor binding sites, we believe that the identified riskassociated SNPs are more likely to be polymorphisms that are

in strong LD with the causal variant for disease. Although the pathogenic variant within IL-15 is still unknown, our results suggest that the pathogenic variant will increase the risk to disease by increasing interleukin production and inflammation in the lesions of psoriasis. In conclusion, our studies suggest that IL-15 is a potentially important gene in psoriasis susceptibility in the Chinese populations, and that IL-15 polymorphism may contribute to the development of psoriasis by increasing the expression level of IL-15. Further studies will be needed to replicate this associations in additional samples of both Chinese and nonChinese populations. Our study provides further support for a pharmacological inhibition of IL-15 as a mean to treat and prevent the development of psoriasis. MATERIALS AND METHODS Study subjects We recruited a total of 632 (297 females and 341 males) individuals affected with psoriasis vulgaris and 485 population-based (225 females and 274 males) unaffected controls for collection of genomic DNA from the Dermatology Departments of the Anhui Medical University using an ascertainment procedure described previously (Zhang et al., 2002). The mean age of the 638 case individuals with psoriasis was 45.3 years (range, 3–75 years). Control subjects comprised 485 individuals from the general population with mean age 39.4 years (range, 13–69 years). All the participating individuals provided informed written consent. The study was approved by the Ethics Committee of the Anhui Medical University and was conducted according to Declaration of Helsinki Principles.

SNP identification and genotyping Genomic DNA was extracted from peripheral blood leukocytes using a standard procedure (Miller et al., 1988). Primers for sequencing and SNP genotyping analysis were designed using Primer 3.0 software (http://www:genome.wi.mit.edu/cgi-bin/primer/ primer3/www.cgi) and the genomic sequence NM_172174 from the GenBank (http://www.ncbi.nlm.nih.gov). All the primer sequences are listed in the Tables S1 and S2. Sequence analysis was performed as previously described (Zhang et al., 2004). We identified the SNPs by sequencing the exons, exon–intron boundaries and approximately 2000 bp of its putative promoter, and 2 kb downstream from the 30 end in 48 psoriasis vulgaris individuals and 48 matched healthy controls. We also aimed to cover the genes with evenly spaced SNPs, avoiding gaps larger than 5 kb within a gene. Then we detected some public polymorphisms by BLAST search of the National Center for Biotechnology Information SNP database.

Assay of IL-15 30 UTR It is accepted that some variants in 30 UTRs may be responsible for complex disease, and they are more likely to be involved in RNA stability or regulation of translation (possibly through binding of micro RNAs). We performed luciferase assays to evaluate whether the SNPs at the IL-15 30 UTR influence expression. As there was no difference distribution in cases and controls, the Hap2 was excluded for further analysis. 30 UTRs of human IL-15 containing the three kinds of haplotypes, Hap1, Hap3, and Hap4, were subcloned www.jidonline.org 2549

X-J Zhang et al. Interleukin-15 Gene Polymorphism and Psoriasis Vulgaris

into the pGL3 promoter luciferase vector (Promega, Madison, WI). The target segments corresponding to Hap1, Hap3, and Hap4 were amplified by PCR using genotype-known genomic DNAs as templates; primers for this procedure were the upstream primer 50 -GCtctagaTAGACATAACAAAACACTC-30 and downstream primer 50 - GCtctagaATTGCAATTTCTTTATTTCA-30 (lower case including XbaI restriction site). PCR products were digested with XbaI (New England Biolabs). Colonies were screened by direct PCR using the forward primer IL-15 30 UTR s-f 50 -CGTCGCCAGTCAAGTAAC AA-30 and the reverse primer IL-15 30 UTR s-r 50 -TGGTTTGTCCAA ACTCATCA-30 , and just the colonies with right insert direction can get productions. After sequencing, we get the positive colonies those called pGL-Hap1, pGL-Hap3, and pGL-Hap4. The positive plasmids DNA isolated with a QIAGENs plasmid Midi kit (Qiagen, Hilden, Germany). We found IL-15 has high expression level in leukocyte, so the Jurkat cell line was selected. The plasmids were transfected into Jurkat cells by the Superfectant transfection reagent (Qiagen). All cells were collected 48 hours later after transient transfection and the lysate was analyzed for luciferase activity with Microlumet Plus LB 96 V (Berthold Technologies, Bad Wildbach, Germany) using luciferase assay reagent (Promega, TM046) following the manufacturer’s protocol. At least three independent experiments of transfection were performed.

Statistical analysis LD pattern was characterized by calculating pairwise D0 values using the Linkage Disequilibrium Analyzer 1.0 software (Ding et al., 2003). Haploview 3.2 was used to generate the LD plot (Barrett et al., 2005) (http://www.broad.mit.edu/mpg/haploview/). All the genotyping data were tested for Hardy–Weinberg equilibrium. Case–control association analysis was performed by using both SNP genotypes and haplotypes. Genotype frequency difference between cases and controls was tested using a two-sided Pearson’s w2 test. Haplotype-based association analysis was performed using the program PHASE 2.0 (Stephens et al., 2001), where haplotypes were first constructed from unphased genotypes using the EM algorithm, and haplotype frequency difference between cases and controls was then tested by permutation of 1000 times. A P-value less than 0.05 was considered statistically significant. Statistical analysis was performed with the SPSS 10.0 software.

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The authors state no conflict of interest.

Lee YA, Ruschendorf F, Windemuth C, Schmitt-Egenolf M, Stadelmann A, Nurnberg G et al. (2000) Genome wide scan in German families reveals evidence for a novel psoriasis-susceptibility locus on chromosome 19p13. Am J Hum Genet 67:1020–4

ACKNOWLEDGMENTS

Macdonald P (2001) Diversity in translational regulation. Curr Opin Cell Biol 13:326–31

CONFLICT OF INTEREST

This work was supported by grants from the key program of National Natural Science Foundation of China (30530670), the general program of National Natural Science Foundation of China (30371296), National Key Project Grant for Basic Research from Chinese Ministry of Science and Technology (2004CB518605), Chinese Ministry of Education Grant (2003), and Key Project Grant for Basic Research from Shanghai Science and Technology Committee (04DJ14003). We are most grateful to all the families who willingly participated in this study, which made this study possible.

SUPPLEMENTARY MATERIAL Table S1. PCR primer sequences used for the IL-15 gene mutation analysis and SNP identified. Table S2. Information for the novel identified SNPs in IL-15 gene.

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