Introduction

The molecular basis of the pathogenesis of psoriasis, the chronic inflammatory skin disease, remains unclear, but principal clinical features of psoriasis (inflammatory infiltrate and epidermal hyperproliferation with abnormal keratinocyte differentiation) appear to be driven mainly by various cytokines and chemokines released by the activated, skin-homing pathogenic T-cell population.^{1, 2, 3, 4} There is increasing evidence to suggest that newly discovered cytokines interleukin-19 (IL-19) and interleukin-20 (IL-20) have the role in the function of epidermis and in psoriasis.^{5, 6}

IL-19 and -20 are members of the IL-10 family that were initially identified during a sequence database search aimed to find potential IL-10 homologs.^{6, 7} IL-20 has been found to be preferentially expressed in monocytes and its main targets are keratinocytes, where IL-20 binds type I IL-20R (IL-20R and -20R) and type II IL-20R (IL-20R and -22R) complexes.^{6, 8, 9} Binding of the IL-20 in human HaCaT keratinocytic cell line results in STAT 3 phosphorylation and activation of a promoter including STAT-binding sites.⁶ Microarray and RT-PCR analyses in HaCaT cells have demonstrated that the expression of several genes involved in inflammation are increased in response to IL-20 and therefore this cytokine may modulate the inflammatory response in the skin.⁶ Furthermore, Blumberg et al⁶ have shown that overexpression of IL-20 under different promoters in transgenic mice caused neonatal lethality with skin abnormalities, similar to those found in psoriatic skin. IL-19 has been detected in immune cells, such as LPS- or GM-CSF-activated and resting monocytes, and at lower level in resting and stimulated B cells.^{7, 8} This cytokine binds to the type I IL-20R complex and modulates gene expression in responsive cell types through activation of the STAT 1 and STAT 3 signal transduction pathway.^{9, 10, 11} Sharing the same receptor complex with IL-20 suggests that IL-19 may have partially overlapping biological activities with IL-20. Moreover, in vitro data have proved that IL-19 acts as proinflammatory cytokine or modulator of the inflammatory response.^{12, 13} Romer et al⁵ have confirmed the pathogenic role of described cytokines in psoriasis demonstrating the higher expression of IL-19 and -20 and their receptors IL-20R and -20R in involved psoriatic skin in contrast to uninvolved psoriatic skin.

Together with the genes encoding IL-10 and MDA-7, IL-19 and -20 genes are found within a 200 kb region of 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 detected in this locus.^{16, 17, 18} In addition, protective effect of microsatellite marker IL-10.G9 allele 3 for familial psoriasis has been observed using the transmission/disequilibrium test (TDT) in persons with a positive family history of psoriasis.¹⁹ These results indicate that locus q31–32 on chromosome 1 could be related to psoriasis susceptibility.

In our previous study, we analyzed the frequency of single-nucleotide polymorphisms (SNPs) of the human IL-20 gene in an association case–control study involving 254 patients with plaque-type psoriasis and 148 unrelated healthy volunteers. A significant association between patients with psoriasis and the G allele at position -1053 and GAA haplotype was established.²⁰ In the present study, we attempted to clarify the role of IL-19 gene in predicting risk for psoriasis using study population identical to the one used for IL-20 gene investigation. We analyzed seven SNPs of the IL-19 gene in patients with psoriasis and in healthy controls. Association and haplotype analysis of the IL-19 gene and combined haplotype analysis of the IL-19 and -20 genes were performed.

Results

In an initial study a database search to find the SNPs of the IL-19 gene (dbSNP, reference sequence NT_021877) was performed. We chose the rs2243158, rs2243168, rs2243191, rs2073186, rs2243174, rs2243188 and rs2243193 SNPs of the IL-19 gene. Frequency of minor allele, distance between SNPs and validation status of particular SNPs were taken as selection criteria (Figure 1).

Figure 1.

Selected SNPs of IL-19 and -20 genes. 76 075 bp fragment of human chromosome 1, locus 1q32. Selected SNPs are represented on the illustration by their cluster ID numbers in public Single Nucleotide Polymorphism database. Coding regions (CDS) and mRNAs of respective genes are also shown in the illustration.

Full figure and legend (32K)

Genotype distributions of the seven analyzed IL-19 gene polymorphisms had no deviation from Hardy–Weinberg equilibrium. Allele frequencies of IL-19 SNPs in controls and cases are reported in Table 1. Comparing psoriatic patients with controls, patients with psoriasis had a lower frequency of the SNP rs2243188 minor A allele (19.5 vs 26.0%, P<0.05), suggesting a protective effect of this minor A allele to psoriasis. Likewise, we observed lower-representation of the A allele at this position in patients with late-onset psoriasis (16.0 vs 26.0%, P<0.02) and in sporadic disease (19.3 vs 26.0%, P<0.05). Although there appeared to be fewer persons possessing the SNP rs2243188 A allele in the early onset and familial psoriasis group, the differences were not statistically significant. SNP rs2243158 was associated with type II phenotype as the prevalence of minor allele at position rs2243158 was significantly higher in controls than in patients with type II phenotype (10.1 vs 4.1%, P<0.05) and SNP rs2243168 was associated with late-onset psoriasis and type II phenotype as the prevalence of minor allele at position rs2243168 was significantly higher in controls than in patients with late-onset disease (8.8 vs 3.3%, P<0.05) and in patients with type II phenotype (8.8 vs 2.5%, P<0.02). Owing to these reasons, rs2243158 and rs2243168 represent potential subtype specific markers. All other IL-19 gene SNPs resulted in negative findings for both allele distributions when comparison between psoriatic patients and controls was performed.

Table 1 - Results of association analysis of interleukin-19 gene SNPs in plaque-type psoriasis.

Full table

To test whether the individual protective effect of the IL-19 polymorphisms, observed in single-marker association analysis, depends on the haplotypic background by which they are carried, LD and haplotype analyses of the IL-19 gene were executed. The pairwise LD matrix showed that the nearly complete linkage disequilibrium (D' between 0.88 and 0.99) was present within the polymorphisms of the IL-19 gene. We excluded SNP 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 five haplotypes (HT1 CACCG, HT2 TGATA, HT3 CACTA, HT4 TAATA, and HT5 TACCG) with a frequency 1% was established. These most frequent haplotypes accounted for 97.7% of all haplotypes in the pooled samples. Psoriasis patients had a decreased frequency of the HT2 TGATA haplotype compared to controls, but the differences were not statistically significant (P=0.09, OR 0.737, 95% CI 0.516–1.053). However, our data showed that HT2 TGATA haplotype was significantly more frequent in controls compared to late-onset psoriasis group (P=0.05; OR 0.58, 95% CI 0.335–1.00).

In our previous study, we have demonstrated that the nearly complete LD (D' between 0.879 and 0.985) occurred within the polymorphisms at positions -1053 (rs2981572), 1380 (rs2981573) and 1462 (rs2232360) of the IL-20 gene. As IL-19 gene maps close to the IL-20 gene on human chromosome 1q32, the measure of LD for all pairs of IL-19 SNPs and IL-20 SNPs studied was implemented in the present study. The pairwise LD matrix of the IL-19 and -20 polymorphisms showed that IL-19 and -20 SNPs were in significant LD with each other (D' between 0.78 and 0.99).

Thereafter, eight-marker haplotype analysis with five SNPs across the IL-19 gene (rs2243191, rs2073186, rs2243174, rs2243188 and rs2243193) and three SNPs across the IL-20 gene (rs2981572, rs2981573 and rs2232360) was performed. We observed five major haplotypes (HT1 CACCGTAA, HT2 TGATAGGG, HT3 CACCGGAA, HT4 CACTAGGG, HT5 TAATAGGG) with a frequency 1% that account for 91.86% of all possible marker combinations in the pooled samples. The frequencies for these haplotypes among patients and controls and haplotype effects are presented in Table 2. We found that patients with plaque psoriasis had a higher frequency of the HT3 CACCGGAA haplotype (P<0.01, 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 psoriasis (P<0.02, OR 2.225, 95% CI 1.175–4.213) and late onset of disease (P<0.05, OR 2.467, 95% CI 1.1258–5.405), familial psoriasis (P<0.02, OR 2.424, 95% CI 1.199–4.903) and sporadic disease (P<0.01, OR 2.877, 95% CI 1.478–5.601). This association mainly reflects a significant individual IL-20 SNP effect at position -1053 (P<0.01, OR 2.548, 95% CI 1.379–4.706). Comparing psoriatic patients with controls no significant association was observed concerning the other haplotypes. Protective effect of the IL-19 gene did not withstand the analysis after stratification for the known IL-20 susceptibility factor.

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

Full table

In addition, the protective effect of the IL-19 TGATA haplotype to late-onset psoriasis did not withstand after combined haplotype analysis of the IL-19 and -20 genes. The frequency of haplotype TGATAGGG among patients and controls did not differ significantly (P=0.34, OR 0.722; 95% CI 0.367–1.422). Detailed results of the haplotype analysis of the IL-19 gene and combined haplotype analysis of the IL-19 and -20 gene among patients with late-onset disease and controls are given in Table 3.

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.

Full table

Discussion

Chromosome 1 contains several genes regulating the immune responses and IL-10 gene cluster of this chromosome is a key regulator in a number of chronic pathological processes.^{21, 22, 23, 24} The genes encoding IL-19 and -20 also locate into the genomic IL-10 region of the human chromosome 1. Blumberg et al⁶ have demonstrated that IL-20 and its receptor complex play a part in epidermal function by regulating keratinocyte proliferation and differentiation. IL-19 is proinflammatory cytokine, which also may have role in the development of psoriasis. In vitro data have suggested that IL-19 induces IL-6 and TNF- production and apoptosis in monocytes.¹² The involvement of the cytokines like IL-6 and TNF has been clearly demonstrated in the pathogenesis of psoriasis. In addition, focal suprapapillary epidermal expression of IL-19 and -20 has been detected.⁵ These data suggest that IL-19 and -20 may play a pathogenetic role in psoriasis. Furthermore, the results of our previous study proved an association of the IL-20 gene GAA haplotype in patients with plaque-type psoriasis.²⁰ Based on this knowledge, we hypothesized that association and haplotype analyses of the IL-19 gene and extended haplotype analysis across a region encompassing the IL-19 and -20 genes might indicate supplementary markers of disease susceptibility.

Association analysis of the IL-19 gene in the present study demonstrated that minor alleles of the IL-19 gene SNPs (rs2243188, rs2243169 and rs2243158) revealed protective effect to psoriasis, especially to late-onset disease and type II phenotype. Protective cytokine loci have been shown to have a complex genetic basis in several diseases.^{25, 26} In psoriasis, certain HLA alleles with a protective effect have been identified.²⁷ Moreover, IL-10.G polymorphism from the IL-10 promoter region has been identified to be protecting against psoriasis.¹⁹ Hensen et al²⁸ have detected the existence of both susceptible and protective loci in the chromosome 19p13 in patients with plaque-type psoriasis. Similarly, data of the present study in accordance with association analysis in our previous study reveal that in the chromosome 1q32 region different loci have different effects in susceptibility to disease.

Several studies confirm that single SNPs do not represent the primary basis of the disease and rather SNP combination should be considered. From the pairwise linkage disequilibrium (LD) matrix of the IL-19 gene polymorphisms, what reflects the nonrandom association of alleles at these markers; it was apparent that the nearly complete linkage disequilibrium was present within the polymorphisms of the IL-19 gene. Moreover, the pairwise LD matrix of the IL-19 and -20 polymorphisms showed that IL-19 and -20 genes form one block of LD. Block of LD is a discrete chromosome region of high LD and low haplotype diversity, separated by possible hotspots of recombination and a breakdown of LD.

The novelty of the present study lies in the extension of the risk haplotype already described in IL-20 gene with new polymorphisms within the proximal IL-19 gene. Estimating combined haplotype–phenotype association of IL-19 and -20 genes, we found that the HT3 CACCGGAA haplotype was associated with an increased risk of psoriasis, reflecting the possible role of this haplotype in determining susceptibility to plaque-type psoriasis. Although 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.

In the present study, we did not analyze functional significance of the IL-19 and -20 haplotypes. Persons with different haplotypes could have different IL-19 and -20 expression levels. Similar genetically determined differences have been shown nicely in case of IL-10.²¹ It is reasonable to expect that described haplotypes really determine the differences in IL-19 and -20 expression levels. Supportive to this functional hypothesis is that we described two haplotypes with opposite effects—one protective and another one for susceptibility. We suppose that IL-19 TGATA haplotype might induce lower level of IL-19 expression than combined IL-19 and -20 haplotype CACCGGAA. However, further studies are needed to support the functional role of the IL-19 and -20 genes in the pathogenesis of psoriasis.

In conclusion, we established linkage disequilibrium of IL-19 and -20 genes and described five major haplotypes. Moreover, we found that HT3 CACCGGAA is related to increased risk (OR 2.548) for psoriasis in sample of unrelated patients and controls. We were not able to prove the protective effect of the IL-19 gene in context of extended haplotype analysis of the IL-19 and -20 genes in plaque-type psoriasis. Family-based studies should be performed additionally to confirm the impact of IL-19 and -20 haplotypes in susceptibility to psoriasis.

Materials and methods

Unrelated patients (n=254) with chronic plaque psoriasis from the Department of Dermatology, University of Tartu, were divided into the subgroups according to the age of disease onset and family history of psoriasis. Patients with disease onset below the age of 40 years were assigned as early-onset psoriasis (n=180), while patients with onset of disease at the age of 40 years and later years were referred to as the late-onset disease (n=74). The mean age at early-onset disease group was 20 years and the group included 94 male and 86 female subjects. The mean age at late-onset disease group was 53 years and the group included 41 male and 33 female patients. Patients were considered to have familial psoriasis if they had at least one first- or second-degree relative with psoriasis (n=101), or to have sporadic disease, if they had no affected relatives (n=153). Patients were regarded to have type I psoriasis, if they had early onset of disease and history with affected parents (n=87), or type II psoriasis, if they had late onset and sporadic form of disease (n=61). Caucasian healthy volunteers, living in Estonia, and free from the positive family history of psoriasis, served as a control group (n=148). The control group included 57 male and 91 female subjects. Individuals with a history of other dermatoses were not included in the control group.

To detect the nucleotides at the specific positions we applied the tetraprimer ARMS-PCR method.²⁹ This method uses four different primers, two (the so-called inner primers) are allele specific and two (outer primers) are control primers. Primers were designed by using the program at http://cedar.genetics.soton.ac
.uk/public_html/primer1.html. Primers we used for SNP detection are shown in Table 4. Each PCR reaction was carried out in total volume of 20 l, containing 100 ng of template DNA, 20 pmol of each inner primer, 2 pmol of each outer primer, 0.2 mM dNTP and MgCl₂ according to Table 4. The reaction buffer and Taq polymerase (AmpliTaq DNA Polymerase, Applied Biosystems, Foster City, CA, USA) were added according to the manufacturer's guidelines. To increase PCR reaction specificity, we applied touchdown cycles: initial denaturation at 95°C for 2 min followed by 10 cycles of 1 min denaturation at 95°C, annealing at 10°C higher than annealing temperature (in Table 4) for 1 min (decreasing by 1°C per cycle) and extension at 72°C for 1 min. The following 25 cycles were performed at appropriate annealing temperature, followed by final extension at 72°C for 10 min. PCR products were analyzed by gel electrophoresis using 2% agarose gel. To control the tetraprimer ARMS-PCR method and to validate the polymorphisms, direct sequencing of incidental DNA samples were performed, using ABI Genetic Analyzer 310. Results of the tetra-primer ARMS-PCR were completely consistent with the results of the direct sequencing.

Table 4 - Primers and conditions for tetra-ARMS-PCR used for IL-19 genotyping.

Full table

Statistical analysis of the genotype–phenotype associations was performed using GENEPOP Version 3.3 software. For haplotype analysis, THESIAS software was used. Pairwise LD was estimated by a log-linear model and the extent of disequilibrium was expressed in terms of standardized D' characteristic. Haplotype analysis was performed using maximum likelihood method for estimating simultaneously haplotype frequencies and haplotype–phenotype association as described by Tregouet et al.³⁰

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Acknowledgements

This study was supported by the target-based funding from the Estonian Ministry of Education Grant No. 0182128s02 (PARNH2128), by the Estonian Science Foundation Grants No. 5712 and 5688 and by the Centre of Molecular and Clinical Medicine Grant VARMC-TIPP.