Combined Haplotype Analysis of the Interleukin-19 and -20 Genes: Relationship to Plaque-Type Psoriasis

FULL PAPER Combined haplotype analysis of the interleukin-19 and -20 genes: relationship to plaque-type psoriasis

SKo˜ks1,3, K Kingo2,3,RRa¨tsep1,3, M Karelson2, H Silm2 and E Vasar1,3 1Department of Physiology, University of Tartu, Tartu, Estonia; 2Department of Dermatology and Venerology, University of Tartu, Tartu, Estonia; 3Centre of Molecular and Clinical Medicine, University of Tartu, Tartu, Estonia

There is increasing evidence to suggest that the newly discovered cytokines interleukin (IL)-19 and -20 have a role in the function of epidermis and in psoriasis. The genes encoding these cytokines locate into the genomic IL-10 region on human chromosome 1. The aim of the present study was to analyze whether single-nucleotide polymorphisms (SNPs) in these genes have an impact on the susceptibility for psoriasis. From pairwise linkage disequilibrium (LD) matrix of the IL-19 and -20 gene polymorphisms, what reflects the nonrandom association of alleles at these markers, it was apparent that IL-19 and -20 genes form one block of LD. We found that the HT3 CACCGGAA haplotype of the IL-19 and -20 genes was associated with an increased risk of psoriasis, reflecting its role in determining susceptibility to plaque-type psoriasis. Although association analysis of the IL-19 gene indicated that minor alleles of the IL-19 gene SNPs (rs2243188, rs2243169 and rs2243158) revealed protective effect to psoriasis and haplotype analysis of the IL-19 gene proved significant protective effect of the TGATA haplotype in case of late-onset disease, combined haplotype analysis of the IL-19 and -20 genes demonstrated that protective effect of the IL-19 gene is secondary to the susceptibility effect of the IL-20 gene. Genes and Immunity (2004) 5, 662–667. doi:10.1038/sj.gene.6364141 Published online 21 October 2004

Keywords: interleukin-19; interleukin-20; single-nucleotide polymorphism (SNP); haplotype analysis; psoriasis

Introduction increased in response to IL-20 and therefore this cytokine may modulate the inflammatory response in the skin.6 The molecular basis of the pathogenesis of psoriasis, the Furthermore, Blumberg et al6 have shown that over- chronic inflammatory skin disease, remains unclear, but expression of IL-20 under different promoters in trans- principal clinical features of psoriasis (inflammatory genic mice caused neonatal lethality with skin infiltrate and epidermal hyperproliferation with abnor- abnormalities, similar to those found in psoriatic skin. mal keratinocyte differentiation) appear to be driven IL-19 has been detected in immune cells, such as LPS- or mainly by various cytokines and chemokines released by GM-CSF-activated and resting monocytes, and at lower the activated, skin-homing pathogenic T-cell popula- level in resting and stimulated B cells.7,8 This cytokine tion.1–4 There is increasing evidence to suggest that binds to the type I IL-20R complex and modulates gene newly discovered cytokines interleukin-19 (IL-19) and expression in responsive cell types through activation of interleukin-20 (IL-20) have the role in the function of the STAT 1 and STAT 3 signal transduction pathway.9–11 epidermis and in psoriasis.5,6 Sharing the same receptor complex with IL-20 suggests IL-19 and -20 are members of the IL-10 family that that IL-19 may have partially overlapping biological were initially identified during a sequence database activities with IL-20. Moreover, in vitro data have proved search aimed to find potential IL-10 homologs.6,7 IL-20 that IL-19 acts as proinflammatory cytokine or mod- has been found to be preferentially expressed in ulator of the inflammatory response.12,13 Romer et al5 monocytes and its main targets are keratinocytes, where have confirmed the pathogenic role of described cyto- IL-20 binds type I IL-20R (IL-20Ra and -20Rb) and type II kines in psoriasis demonstrating the higher expression of IL-20R (IL-20Rb and -22R) complexes.6,8,9 Binding of the IL-19 and -20 and their receptors IL-20Ra and -20Rb in IL-20 in human HaCaT keratinocytic cell line results in involved psoriatic skin in contrast to uninvolved STAT 3 phosphorylation and activation of a promoter psoriatic skin. including STAT-binding sites.6 Microarray and RT-PCR Together with the genes encoding IL-10 and MDA-7, analyses in HaCaT cells have demonstrated that the IL-19 and -20 genes are found within a 200 kb region of expression of several genes involved in inflammation are chromosome 1 in q31–32 locus.6,7,14,15 Linkage to several common autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, have been Correspondence: Dr S Ko˜ks, Department of Physiology, University of 16–18 Tartu, 19 Ravila Street, 50411 Tartu, Estonia. E-mail: [email protected] detected in this locus. In addition, protective effect Received 22 June 2004; revised 27 August 2004; accepted 27 August of microsatellite marker IL-10.G9 allele 3 for familial 2004; published online 21 October 2004 psoriasis has been observed using the transmission/ IL-19 and -20 genes in psoriasis SKo˜ks et al 663 disequilibrium test (TDT) in persons with a positive onset psoriasis and type II phenotype as the prevalence family history of psoriasis.19 These results indicate that of minor allele at position rs2243168 was significantly locus q31–32 on chromosome 1 could be related to higher in controls than in patients with late-onset disease psoriasis susceptibility. (8.8 vs 3.3%, Po0.05) and in patients with type II In our previous study, we analyzed the frequency of phenotype (8.8 vs 2.5%, Po0.02). Owing to these reasons, single-nucleotide polymorphisms (SNPs) of the human rs2243158 and rs2243168 represent potential subtype IL-20 gene in an association case–control study involving specific markers. All other IL-19 gene SNPs resulted in 254 patients with plaque-type psoriasis and 148 unre- negative findings for both allele distributions when lated healthy volunteers. A significant association be- comparison between psoriatic patients and controls tween patients with psoriasis and the G allele at position was performed. À1053 and GAA haplotype was established.20 In the To test whether the individual protective effect of the present study, we attempted to clarify the role of IL-19 IL-19 polymorphisms, observed in single-marker asso- gene in predicting risk for psoriasis using study ciation analysis, depends on the haplotypic background population identical to the one used for IL-20 gene by which they are carried, LD and haplotype analyses of investigation. We analyzed seven SNPs of the IL-19 gene the IL-19 gene were executed. The pairwise LD matrix in patients with psoriasis and in healthy controls. showed that the nearly complete linkage disequilibrium Association and haplotype analysis of the IL-19 gene (D0 between 0.88 and 0.99) was present within the and combined haplotype analysis of the IL-19 and -20 polymorphisms of the IL-19 gene. We excluded SNP genes were performed. rs2243158 and SNP rs2243168 from the further haplotype analysis, because the minor allele frequencies of these polymorphisms were lower than 0.10. The presence of Results five haplotypes (HT1 CACCG, HT2 TGATA, HT3 CACTA, HT4 TAATA, and HT5 TACCG) with a In an initial study a database search to find the SNPs of frequency X1% was established. These most frequent the IL-19 gene (dbSNP, reference sequence NT_021877) haplotypes accounted for 97.7% of all haplotypes in the was performed. We chose the rs2243158, rs2243168, pooled samples. Psoriasis patients had a decreased rs2243191, rs2073186, rs2243174, rs2243188 and frequency of the HT2 TGATA haplotype compared to rs2243193 SNPs of the IL-19 gene. Frequency of minor controls, but the differences were not statistically allele, distance between SNPs and validation status of significant (P ¼ 0.09, OR 0.737, 95% CI 0.516–1.053). particular SNPs were taken as selection criteria (Figure 1). However, our data showed that HT2 TGATA haplotype Genotype distributions of the seven analyzed IL-19 was significantly more frequent in controls compared to gene polymorphisms had no deviation from Hardy– late-onset psoriasis group (P ¼ 0.05; OR 0.58, 95% CI Weinberg equilibrium. Allele frequencies of IL-19 SNPs 0.335–1.00). in controls and cases are reported in Table 1. Comparing In our previous study, we have demonstrated that the psoriatic patients with controls, patients with psoriasis nearly complete LD (D0 between 0.879 and 0.985) had a lower frequency of the SNP rs2243188 minor A occurred within the polymorphisms at positions À1053 allele (19.5 vs 26.0%, Po0.05), suggesting a protective (rs2981572), 1380 (rs2981573) and 1462 (rs2232360) of the effect of this minor A allele to psoriasis. Likewise, we IL-20 gene. As IL-19 gene maps close to the IL-20 gene on observed lower-representation of the A allele at this human chromosome 1q32, the measure of LD for all pairs position in patients with late-onset psoriasis (16.0 vs of IL-19 SNPs and IL-20 SNPs studied was implemented 26.0%, Po0.02) and in sporadic disease (19.3 vs 26.0%, Po0.05). Although there appeared to be fewer persons possessing the SNP rs2243188 A allele in the early onset Table 1 Results of association analysis of interleukin-19 gene and familial psoriasis group, the differences were not SNPs in plaque-type psoriasis statistically significant. SNP rs2243158 was associated with type II phenotype as the prevalence of minor allele SNP ID Polymorphism Allele frequency Allele frequency P-value at position rs2243158 was significantly higher in controls in controls (%) in cases (%) than in patients with type II phenotype (10.1 vs 4.1%, Po0.05) and SNP rs2243168 was associated with late- 1a 2b 1a 2b 1a 2b

rs2243158 G C 89.9 10.1 91.9 8.1 NS rs2243168 A T 91.2 8.8 93.3 6.7 NS rs2073186 C T 73.0 27.0 78.3 21.7 0.08 rs2243174 A G 76.7 23.3 81.1 18.9 NS rs2243188 C A 74.0 26.0 80.5 19.5 0.029* rs2243191 C T 74.0 26.0 78.0 22.0 NS rs2243193 G A 73.0 27.0 77.0 23.0 NS

Allele frequencies of the seven studied polymorphisms of the IL-19 gene were compared between the psoriatic patients (n ¼ 254) and the controls (n ¼ 148), P-values for allelic association were calcu- lated using Fisher’s exact test. The significance level for all Figure 1 Selected SNPs of IL-19 and -20 genes. 76 075 bp fragment statistical tests was 0.05. of human chromosome 1, locus 1q32. Selected SNPs are represented on the illustration by their cluster ID numbers in public Single *Po0.05 allelic differences compared to the controls. Nucleotide Polymorphism database. Coding regions (CDS) and a1—most frequent allele. mRNAs of respective genes are also shown in the illustration. b2—least frequent allele.

Genes and Immunity IL-19 and -20 genes in psoriasis SKo˜ks et al 664 in the present study. The pairwise LD matrix of the IL-19 individual IL-20 SNP effect at position À1053 (Po0.01, and -20 polymorphisms showed that IL-19 and -20 SNPs OR 2.548, 95% CI 1.379–4.706). Comparing psoriatic were in significant LD with each other (D0 between 0.78 patients with controls no significant association was and 0.99). observed concerning the other haplotypes. Protective Thereafter, eight-marker haplotype analysis with five effect of the IL-19 gene did not withstand the analysis SNPs across the IL-19 gene (rs2243191, rs2073186, after stratification for the known IL-20 susceptibility rs2243174, rs2243188 and rs2243193) and three SNPs factor. across the IL-20 gene (rs2981572, rs2981573 and In addition, the protective effect of the IL-19 TGATA rs2232360) was performed. We observed five major haplotype to late-onset psoriasis did not withstand after haplotypes (HT1 CACCGTAA, HT2 TGATAGGG, HT3 combined haplotype analysis of the IL-19 and -20 genes. CACCGGAA, HT4 CACTAGGG, HT5 TAATAGGG) The frequency of haplotype TGATAGGG among patients with a frequency X1% that account for 91.86% of all and controls did not differ significantly (P ¼ 0.34, OR possible marker combinations in the pooled samples. 0.722; 95% CI 0.367–1.422). Detailed results of the The frequencies for these haplotypes among patients and haplotype analysis of the IL-19 gene and combined controls and haplotype effects are presented in Table 2. haplotype analysis of the IL-19 and -20 gene among We found that patients with plaque psoriasis had a patients with late-onset disease and controls are given in higher frequency of the HT3 CACCGGAA haplotype Table 3. (Po0.01, OR 2.547, 95% CI 1.379–4.706), compared to control group. Likewise, the HT3 CACCGGAA haplotype was associated with an increased risk of early-onset Discussion psoriasis (Po0.02, OR 2.225, 95% CI 1.175–4.213) and late onset of disease (Po0.05, OR 2.467, 95% CI 1.1258–5.405), Chromosome 1 contains several genes regulating the familial psoriasis (Po0.02, OR 2.424, 95% CI 1.199–4.903) immune responses and IL-10 gene cluster of this and sporadic disease (Po0.01, OR 2.877, 95% CI 1.478– chromosome is a key regulator in a number of chronic 5.601). This association mainly reflects a significant pathological processes.21–24 The genes encoding IL-19

Table 2 Results of extended haplotype analysis of the IL-19 and -20 genes in patients with plaque-type psoriasis

Haplotype rs2073186 rs2243174 rs2243188 rs2243191 rs2243193 rs2981572 rs2981573 rs2232360 Controls Psoriasis Haplotypic OR P-value (n ¼ 148) patients (95% CI) (n ¼ 254)

HT1 C A C C G T A A 64.6 60.3 * HT2 T G A T A G G G 17.4 15.5 0.947 (0.639–1.403) 0.78 HT3 C A C C G G A A 5.6 13.5 2.548 (1.379–4.706) o0.01 HT4 C A C T A G G G 1.4 2.0 1.597 (0.420–6.073) 0.49 HT5 T A A T A G G G 2.4 0.8 0.342 (0.100–1.172) 0.09

The haplotype frequencies (%) and haplotypic ORs with their 95% CIs and P-values by comparison to the reference haplotype in psoriasis patients compared with controls are indicated. Haplotype frequencies and haplotype–phenotype associations were estimated using maximum likelihood method. *The haplotype combining the most frequent alleles at each site is chosen as the reference haplotype (CACCGTAA).

Table 3 Results of IL-19 haplotype analysis and extended haplotype analysis of the IL-19 and -20 genes in patients with late-onset psoriasis

Controls Late-onset psoriasis patients Haplotypic OR (95% CI) P-value (n ¼ 148) (n ¼ 74)

IL-19 haplotypes CACCG 70.7 77.3 * TGATA 21.8 13.4 0.581 (0.335–1.000) 0.05 TAATA 2.7 0.6 0.182 (0.022–1.501) 0.11 CACTA 1.0 3.2 2.690 (0.549–13.17) 0.22 TACCG 1.4 1.3 1.580 (0.277–8.999) 0.61

IL-19 and -20 combined haplotypes CACCGTAA 63.5 61.1 * TGATAGGG 16.7 11.8 0.722 (0.367–1.422) 0.34 CACCGGAA 5.9 14.2 2.467 (1.126–5.405) 0.02 TAATAGGG 2.7 0.7 0.260 (0.031–2.146) 0.21 TACCGTAA 2.4 0.7 0.318 (0.035–2.869) 0.31

The haplotype frequencies (%) and haplotypic ORs with their 95% CIs and P-values by comparison to the reference haplotype in late-onset psoriasis patients compared with controls are indicated. Haplotype frequencies and haplotype–phenotype associations were estimated using maximum likelihood method. *The haplotype combining the most frequent alleles at each site is chosen as the reference haplotype.

Genes and Immunity IL-19 and -20 genes in psoriasis SKo˜ks et al 665 and -20 also locate into the genomic IL-10 region of the In the present study, we did not analyze functional human chromosome 1. Blumberg et al6 have demon- significance of the IL-19 and -20 haplotypes. Persons strated that IL-20 and its receptor complex play a part in with different haplotypes could have different IL-19 and epidermal function by regulating keratinocyte prolifera- -20 expression levels. Similar genetically determined tion and differentiation. IL-19 is proinflammatory cyto- differences have been shown nicely in case of IL-10.21 It is kine, which also may have role in the development of reasonable to expect that described haplotypes really psoriasis. In vitro data have suggested that IL-19 induces determine the differences in IL-19 and -20 expression IL-6 and TNF-a production and apoptosis in mono- levels. Supportive to this functional hypothesis is that we cytes.12 The involvement of the cytokines like IL-6 and described two haplotypes with opposite effects—one TNFa has been clearly demonstrated in the pathogenesis protective and another one for susceptibility. We suppose of psoriasis. In addition, focal suprapapillary epidermal that IL-19 TGATA haplotype might induce lower level of expression of IL-19 and -20 has been detected.5 These IL-19 expression than combined IL-19 and -20 haplotype data suggest that IL-19 and -20 may play a pathogenetic CACCGGAA. However, further studies are needed to role in psoriasis. Furthermore, the results of our previous support the functional role of the IL-19 and -20 genes in study proved an association of the IL-20 gene GAA the pathogenesis of psoriasis. haplotype in patients with plaque-type psoriasis.20 Based In conclusion, we established linkage disequilibrium on this knowledge, we hypothesized that association and of IL-19 and -20 genes and described five major haplotype analyses of the IL-19 gene and extended haplotypes. Moreover, we found that HT3 CACCGGAA haplotype analysis across a region encompassing the IL- is related to increased risk (OR 2.548) for psoriasis in 19 and -20 genes might indicate supplementary markers sample of unrelated patients and controls. We were not of disease susceptibility. able to prove the protective effect of the IL-19 gene in Association analysis of the IL-19 gene in the present context of extended haplotype analysis of the IL-19 and - study demonstrated that minor alleles of the IL-19 gene 20 genes in plaque-type psoriasis. Family-based studies SNPs (rs2243188, rs2243169 and rs2243158) revealed should be performed additionally to confirm the impact protective effect to psoriasis, especially to late-onset of IL-19 and -20 haplotypes in susceptibility to psoriasis. disease and type II phenotype. Protective cytokine loci have been shown to have a complex genetic basis in 25,26 several diseases. In psoriasis, certain HLA alleles Materials and methods with a protective effect have been identified.27 Moreover, IL-10.G polymorphism from the IL-10 promoter region Unrelated patients (n ¼ 254) with chronic plaque psor- has been identified to be protecting against psoriasis.19 iasis from the Department of Dermatology, University of Hensen et al28 have detected the existence of both Tartu, were divided into the subgroups according to the susceptible and protective loci in the chromosome age of disease onset and family history of psoriasis. 19p13 in patients with plaque-type psoriasis. Similarly, Patients with disease onset below the age of 40 years data of the present study in accordance with association were assigned as early-onset psoriasis (n ¼ 180), while analysis in our previous study reveal that in the patients with onset of disease at the age of 40 years and chromosome 1q32 region different loci have different later years were referred to as the late-onset disease effects in susceptibility to disease. (n ¼ 74). The mean age at early-onset disease group was Several studies confirm that single SNPs do not 20 years and the group included 94 male and 86 female represent the primary basis of the disease and rather subjects. The mean age at late-onset disease group was 53 SNP combination should be considered. From the pair- years and the group included 41 male and 33 female wise linkage disequilibrium (LD) matrix of the IL-19 gene patients. Patients were considered to have familial polymorphisms, what reflects the nonrandom association psoriasis if they had at least one first- or second-degree of alleles at these markers; it was apparent that the nearly relative with psoriasis (n ¼ 101), or to have sporadic complete linkage disequilibrium was present within the disease, if they had no affected relatives (n ¼ 153). polymorphisms of the IL-19 gene. Moreover, the pairwise Patients were regarded to have type I psoriasis, if they LD matrix of the IL-19 and -20 polymorphisms showed had early onset of disease and history with affected that IL-19 and -20 genes form one block of LD. Block of parents (n ¼ 87), or type II psoriasis, if they had late onset LD is a discrete chromosome region of high LD and low and sporadic form of disease (n ¼ 61). Caucasian healthy haplotype diversity, separated by possible hotspots of volunteers, living in Estonia, and free from the positive recombination and a breakdown of LD. family history of psoriasis, served as a control group The novelty of the present study lies in the extension of (n ¼ 148). The control group included 57 male and 91 the risk haplotype already described in IL-20 gene with female subjects. Individuals with a history of other new polymorphisms within the proximal IL-19 gene. dermatoses were not included in the control group. Estimating combined haplotype–phenotype association To detect the nucleotides at the specific positions we of IL-19 and -20 genes, we found that the HT3 applied the tetraprimer ARMS-PCR method.29 This CACCGGAA haplotype was associated with an in- method uses four different primers, two (the so-called creased risk of psoriasis, reflecting the possible role of inner primers) are allele specific and two (outer primers) this haplotype in determining susceptibility to plaque- are control primers. Primers were designed by using the type psoriasis. Although haplotype analysis of the IL-19 program at http://cedar.genetics.soton.ac.uk/public_ gene proved significant protective effect of the TGATA html/primer1.html. Primers we used for SNP detection haplotype in case of late-onset disease, combined are shown in Table 4. Each PCR reaction was carried out haplotype analysis of the IL-19 and -20 genes demon- in total volume of 20 ml, containing 100 ng of template strated that protective effect of the IL-19 gene is DNA, 20 pmol of each inner primer, 2 pmol of each outer secondary to the susceptibility effect of the IL-20 gene. primer, 0.2 mM dNTP and MgCl2 according to Table 4.

Genes and Immunity IL-19 and -20 genes in psoriasis SKo˜ks et al 666 Table 4 Primers and conditions for tetra-ARMS-PCR used for IL-19 genotyping

0 0 2+ Genetic Primers (5 –3 ) Tm Annealing Mg Amplicon size polymorphism temperature (mM)

rs2243158 G/C Forward inner primer (C allele): GGT GGA TCC ACC CAG CAA ACC TTC AC 721C681C 2.5 487 bp—control Reverse inner primer (G allele): TTT TAT TCA GGT GGA TAA GAG GAA ATG GTC 661C 290 bp—G allele Forward outer primer: GCC ACA GCT CTC AGG AAA GTG ACC TAA G 691C 253 bp—C allele Reverse outer primer: CCA GCA TCT GGA ACA TCA TAG CCA TAC A 691C rs2243168 A/T Forward inner primer (T allele): GGA AGT TGC CAA GCT GCC CTC TAT CT 691C601C 3.5 328 bp—control Reverse inner primer (A allele): CAA TAA GGA GCT AGG GGA AGA AGC CGA T 691C 215 bp—T allele Forward outer primer: AGA AGG GTA AGA GAA TGA GAA GCG GTG G 691C 167 bp—A allele Reverse outer primer: TGG TTT TTG ATG TTT GCC CCT GAA ATA A 691C rs2073186 C/T Forward inner primer (C allele): AGG TGC TCA GAG GGG ACA GGA TTG CC 731C651C 2.5 295 bp—control Reverse inner primer (T allele): ACC TTC CAA AAT TAC CCC CAA GCC CCA 731C 209 bp—C allele Forward outer primer: TGT TAG GGC ACG CTA GTG TCC CAG GGA TA 731C 139 bp—T allele Reverse outer primer: TCC TTT GGG TCA CAA ACC TGG TCA CCT C 731C rs2243174 A/G Forward inner primer (A allele): TGG TGC TGT TCT TAC AAT GGA CAC CA 681C601C 2.5 407 bp—control Reverse inner primer (G allele): GAT CTT GTC ACT GGC TTT CCT GCC TAA C 681C 272 bp—G allele Forward outer primer: TTG GAA TCC CTG GGA GGA ATT AAA GAA G 681C 189 bp—A allele Reverse outer primer: AGA ATA CTT CCC AGG ACT GGA GGA GCT C 681C rs2243188 C/A Forward inner primer (C allele): TGG GGA AGA TGG AAG ATG AGA GGT AGA CCC 721C651C 2.5 366 bp—control Reverse inner primer (A allele): AGG AGG CTG AAG GCC TGG CAC CCT TCT 761C 238 bp—A allele Forward outer primer: AAT GTC ACT TCT CAT GTG GGG AGG CAG GA 741C 185 bp—C allele Reverse outer primer: AAT GAT GGA GAT GGG GAG CCC AGG AGA T 741C rs2243191 C/T Forward inner primer (C allele): CTG GAT TAA TAA GAA TCA TGA AGT AAT TTC 591C601C 2.5 436 bp—control Reverse inner primer (T allele): ATA CAG GTT CCT TGT CAT CAA GCT GCG A 701C 279 bp—T allele Forward outer primer: CCT GCG AGG AAA ATA ATA TTG AGT CTG T 641C 215 bp—C allele Reverse outer primer: CAC AGT AGA CTT TTT GGC TAT TTT CAA GC 641C rs2243193 G/A Forward inner primer (A allele): AAG GGC TGC CTT CCC ATC TAA TTT ATT TTA 671C601C 2.5 414 bp—control Reverse inner primer (G allele): TCA CAT CAC AGA CAT GGA CTA TAT GAC GTC 671C 281 bp—G allele Forward outer primer: GGA ACC TGT ATA GTG ATC CAG GGA TGA A 671C 193 bp—A allele Reverse outer primer: CTG TCA GAA ACA CCC TGT CCT CAG TCT T 671C

The reaction buffer and Taq polymerase (AmpliTaq DNA 0182128s02 (PARNH2128), by the Estonian Science Polymerase, Applied Biosystems, Foster City, CA, USA) Foundation Grants No. 5712 and 5688 and by the Centre were added according to the manufacturer’s guidelines. of Molecular and Clinical Medicine Grant VARMC-TIPP. To increase PCR reaction specificity, we applied touch- down cycles: initial denaturation at 951C for 2 min followed by 10 cycles of 1 min denaturation at 951C, annealing at 101C higher than annealing temperature (in References Table 4) for 1 min (decreasing by 11C per cycle) and extension at 721C for 1 min. The following 25 cycles were 1 Bos JD, De Rie MA. The pathogenesis of psoriasis: immuno- performed at appropriate annealing temperature, fol- logical facts and speculations. Immunol Today 1999; 20: 40–46. lowed by final extension at 721C for 10 min. PCR 2 Prinz JC. The role of T cells in psoriasis. JEADV 2003; 17: products were analyzed by gel electrophoresis using 257–270. 3 Griffiths CEM. The immunological basis of psoriasis. JEADV 2% agarose gel. To control the tetraprimer ARMS-PCR 2003; 17 (Suppl 2): 1–5. method and to validate the polymorphisms, direct 4 Gudjonsson JE, Johnston A, Sigmundsdottir H, Valdimarsson sequencing of incidental DNA samples were performed, H. Immunopathogenic mechanisms in psoriasis. Clin Exp using ABI Genetic Analyzer 310. Results of the tetra- Immunol 2004; 135: 1–8. primer ARMS-PCR were completely consistent with the 5 Romer J, Hasselager E, Norby PL, Steiniche T, Thorn Clausen results of the direct sequencing. J, Kragballe K. Epidermal overexpression of interleukin-19 Statistical analysis of the genotype–phenotype associa- and -20 mRNA in psoriatic skin disappears after short-term tions was performed using GENEPOP Version 3.3 soft- treatment with cyclosporine a or calcipotriol. J Invest Dermatol ware. For haplotype analysis, THESIAS software was 2003; 121: 1306–1311. 6 Blumberg H, Conklin D, Xu W et al. Interleukin 20: discovery, used. Pairwise LD was estimated by a log-linear model receptor identification, and role in epidermal function. Cell and the extent of disequilibrium was expressed in terms of 2001; 104: 9–19. standardized D’ characteristic. Haplotype analysis was 7 Gallagher G, Dickensheets H, Eskdale J et al. Cloning, performed using maximum likelihood method for estimat- expression and initial characterization of interleukin-19 ing simultaneously haplotype frequencies and haplotype– (IL-19), a novel homologue of human interleukin-10 (IL-10). phenotype association as described by Tregouet et al.30 Genes Immun 2000; 1: 442–450. 8 Wolk K, Kunz S, Asadullah K, Sabat R. Cutting edge: immune cells as sources and targets of the IL-10 family members? J Immunol 2002; 168: 5397–5402. Acknowledgements 9 Dumoutier L, Leemans C, Lejeune D, Kotenko SV, Renauld JC. Cutting edge: STAT activation by IL-19, IL-20 and mda-7 This study was supported by the target-based funding through IL-20 receptor complexes of two types. J Immunol from the Estonian Ministry of Education Grant No. 2001; 167: 3545–3549.

Genes and Immunity IL-19 and -20 genes in psoriasis SKo˜ks et al 667 10 Pletnev S, Magracheva E, Kozlov S et al. Characterization 20 Kingo K, Koks S, Nikopensius T, Silm H, Vasar E. Polymorph- of the recombinant extracellular domains of human isms in the interleukin-20 gene: relationships to plaque-type interleukin-20 receptors and their complexes with psoriasis. Genes Immun 2004; 5: 117–121. interleukin-19 and interleukin-20. Biochemistry 2003; 42: 21 Crawley E, Kay R, Sillibourne J, Patel P, Hutchinson I, Woo P. 12617–12624. Polymorphic haplotypes of the interleukin-10 50 flanking 11 Parrish-Novak J, Xu W, Brender T et al. Interleukins 19, 20, and region determine variable interleukin-10 transcription and 24 signal through two distinct receptor complexes. Differences are associated with particular phenotypes of juvenile rheu- in receptor–ligand interactions mediate unique biological matoid arthritis. Arthritis Rheum 1999; 42: 1101–1108. functions. J Biol Chem 2002; 227: 47517–47523. 22 Lim S, Crawley E, Woo P, Barnes PJ. Haplotype associated 12 Liao Y-C, Liang W-G, Chen F-W et al. IL-19 induces with low interleukin-10 production in patients with severe production of IL-6 and TNF-a and results in cell apoptosis asthma. Lancet 1998; 352: 113. through TNF-a. J Immunol 2002; 169: 4288–4297. 23 Howell WM, Turner SJ, Bateman AC, Theaker JM. IL-10 13 Kotenko SV. The family of IL-10-related cytokines and their promoter polymorphisms influence in cutaneous malignant receptors: related, but to what extent? Cytokine Growth Factor melanoma. Genes Immun 2001; 2: 25–31. Rev 2002; 13: 223–240. 24 Shin HD, Wincler C, Stephens JC et al. Genetic restriction of 14 Eskdale J, Kube D, Tesch H, Gallagher G. Mapping of the HIV-1 pathogenesis to AIDS by promoter alleles of IL-10. Proc human IL-10 gene and further characterization of the 5-prime Nat Acad Sci USA 2000; 97: 14467–14472. flanking sequence. Immunogenetics 1997; 46: 120–128. 25 Sivalingam SP, Yoon KH, Koh DR, Fong KY. Single-nucleotide 15 Jiang H, Lin JJ, Su ZZ, Goldstein NI, Fisher PB. Subtraction polymorphisms of the interleukin-18 gene promoter region in hybridization identifies a novel melanoma differentiation- rheumatoid arthritis patients: protective effect of AA geno- associated gene, mda-7, modulated during human melanoma type. Tissue Antigens 2003; 62: 498–504. differentiation, growth and progression. Oncogene 1995; 11: 26 Smith AJ, Keen LJ, Billingham MJ et al. Extended haplotypes 2477–2486. and linkage disequilibrium in the IL1R1-IL1A-IL1B-IL1RN 16 Johanneson B, Lima G, von Salome J et al. A major gene cluster: association with knee osteoarthritis. Genes Immun susceptibility locus for systemic lupus erythematosus 2004; 5: 451–460. maps to chromosome 1q31. Am J Hum Genet 2002; 71: 27 Contu L, Orru S, Carcassi C et al. A psoriasis vulgaris 1060–1071. protective gene maps close to the HLA-C locus on the EH18.2- 17 Martinez A, Pascual M, Pascual-Salcedo D, Balsa A, Martin J, extended haplotype. Tissue Antigens 2004; 64: 43–57. de la Concha EG. Genetic polymorphisms in Spanish 28 Hensen P, Windemuth C, Huffmeier U et al. Association scan rheumatoid arthritis patients: an association and linkage of the novel psoriasis susceptibility region on chromosome 19: study. Genes Immun 2003; 4: 117–121. evidence for both susceptible and protective loci. Exp Dermatol 18 Barrera P, Faure S, Prud’homme JF et al. European genetic 2003; 12: 490–496. study on rheumatoid arthritis: is there a linkage of the 29 Ye S, Dhillon S, Ke X, Collins AR, Day IN. An efficient interleukin-1 (IL-1), IL-10 or IL-4 genes to RA? Clin Exp procedure for genotyping single nucleotide polymorphisms. Rheumatol 2001; 19: 709–714. Nucleic Acids Res 2001; 29: E88–8. 19 Hensen P, Asadullah K, Windemuth C et al. Interleukin-10 30 Tregouet DA, Barbaux S, Escolano S et al. Specific haplotypes promoter polymorphism IL10.G and familial early onset of the P-selectin gene are associated with myocardial infarc- psoriasis. Br J Dermatol 2003; 149: 381–385. tion. Hum Mol Genet 2002; 11: 2015–2023.

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