inflammatory diseases (Asumalahti, Veal et al. 2002). The effect of the altered protein structure has been investigated in transgenic mice carrying either the wild-type allele or the risk allele (Elomaa et al. 2004). However, no phenotypic or histological changes were detected. First when comparing expression of genes, similarities to previous findings in psoriatic lesions was observed, such as upregulation of cytokeratins 6, 16 and 17. Also differences in expression of genes involved in terminal differentiation and formation of the cornified cell envelope was observed and the authors concluded that HCR might contribute to psoriasis development (Elomaa, Majuri et al. 2004). Whether this upregulation is a contributing factor or not remains to be investigated.
exon 1 exon 2
exon 1 exon 2
exon 6 exon 5 exon 4 exon 3 exon 2 exon 1
25234b p exon 2 exon 3 exon 1
4360 bp
PSORS1C2
PSORS1C1 CDSN HCR
POU5F1
Centromeric Telomeric
TCF19 PSORS1C3
HLA-C
Figure 8. Organisation of exons of PSORS1C1-3. The PSORS1C1 is indicated by grey shaded boxes, PSORS1C2 by striped and PSORS1C3 by dotted. The figure is not to scale.
sequence. When investigating the putative function of the product, weak similarity to Cu-Zn superdioxide dismutase proteins was identified. The shorter splice- isoform has a dissimilar N-terminus encoded by exon 1.
PSORS1C2 has a centromeric to telomeric direction and consists of two exons stretching 1.8 kb (figure 8). A transcript of 1185 bp was obtained for this sequence and it produces a putative protein of 134 amino acids. This protein sequence has two regions of low complexity and is rich in proline. This is a common feature of an activation domain in transcription factors. By comparison to other proteins and known domains there were indications of a N-terminus signal peptide located in the first 22 amino acids, a signal necessary for sorting of proteins, and supports a functional role for this sequence. We also cloned the murine and porcine orthologs, which show that PSORS1C2 is a highly conserved protein.
PSORS1C3 consists of three exons with a centromeric to telomeric orientation (figure 8). PSORS1C3 produces a transcript of 599 bp with the longest identified ORF being 58 amino acids. This putative protein does not show any similarity to known proteins or domains but the sequence is conserved between human, chimpanzee (Pan Troglodytes) and pig (Sus scrofa).
Expression patterns of PSORS1C1-3
Using RT-PCR on total RNA, expression was detected for all three genes in HaCaT cells, tonsil (only tested for PSORS1C3) and both normal and psoriasis skin. Next we investigated the expression by Northern blotting. For PSORS1C2, a strong band was detected in heart and skeletal muscle. For PSORS1C1, two different probes were used in order to separate the two isoforms identified. They did however generate the same pattern with weak expression consistently detected at 1.8 and 6 kb in heart, placenta, liver, skeletal muscle and pancreas. The larger identified band could indicate the presence of more exons, although no additional sequence was detected using RACE. During the characterization of ESTs for PSORS1C1 we found an alternative terminal exon spliced to
exon 1-3, however this exon did not reveal any signal neither by RT-PCR nor by Northern blot.
Different sizes for PSORS1C3 were also obtained. A 1 kb band in testis and pancreas and a 2 kb band in skeletal muscle were observed. This difference in size could result from alternative splicing. It may also indicate a larger gene than reported here, though no additional sequence was detected and no splice-variants were seen in the ESTs/RT-PCR sequencing results. It is quite common to find larger transcripts than expected on Northern blots. In addition, it is known that isoform expression can be organ and/or time-specific in up to 70% of genes (Lander et al. 2001) therefore one cannot rule out that other splice variants exist. In fact, regulation of mRNA stability has emerged as a major control point in gene expression and different splice variants appear to confer different stability on the mRNA level (Guhaniyogi and Brewer 2001; Kozak 2004).
With these analyses we were quite confident that all three candidates are active transcriptional units. They have the expected structure of a gene and are conserved between species, and transcripts have been identified in various tissues with RT-PCR and Northern blots. Although PSORS1C3 did not show any similarities to known proteins and functional domains, it may still be active on the RNA level. The experience in the field of RNA is evolving and functions at this level may be more important and developed than previously thought (Eddy 2001; Hirotsune et al. 2003; Mattick 2003).
Polymorphism detection and association with psoriasis
To investigate the presence of polymorphisms in PSORS1C1 and PSORS1C2 all exons were completely sequenced from both directions in all individuals. For PSORS1C3 polymorphism detection was performed in 87 patients and 50 controls and the additional genotyping of 131 patients and 77 controls was completed using pyrosequencing.
A total of nine SNPs were detected in PSORS1C1, of which five associated to psoriasis. Of these a silent SNP, +39604 located in exon 2, showed the highest association and of the additional four only one changed an amino acid, SNP +26680. In
PSORS1C2 we identified four SNPs. Two of these encoded amino acid changes but did not associate to the disease. Only SNP +26276 showed significant difference between the study groups. In PSORS1C3, the polymorphism analysis revealed a total of 11 SNPs.
When comparing the frequencies in patients and controls three SNPs were not analyzed further, as they were rare variants. After pyrosequencing a fourth SNP was excluded from the study due to deviation from Hardy Weinberg equilibrium. Of the remaining seven SNPs, three located in exon 1 (+12019, +12072 and +12343) demonstrated significant association to the disease.
Association of HLA-Cw*0602 (paper II & III)
The strongest association detected was with HLA-Cw*0602. In the PSORS1C1/C2 study (paper II) HLA-Cw*0602 was present in 48% of the patients and 10% of the controls and in the PSORS1C3 study (paper III) it was even higher, 59% compared to 12%. In comparison to PSORS1C1/C2 we also investigated the association of the inferred HCR*WW allele detected in HCR (paper I) and the frequency of this allele differed significantly between patients and controls, however not as strong as with HLA-Cw*0602. When combining the two alleles HCR*WW and HLA-Cw*0602 the association appeared even stronger indicating the strong linkage disequilibrium between these variants. This could however, be due to observation bias. It has been demo nstrated that haplotypes shared between populations represent only a fraction of the total number of haplotypes observed (Crawford et al. 2004).
Testing for confounding factors
Knowing that HLA-Cw*0602 is a possible confound ing factor due to the high linkage disequilibrium in the region, we stratified the data to see the effect of this allele on the association. After stratification for all SNPs HLA-Cw*0602 still showed significant association, however when stratifying for HLA-Cw*0602 status only two SNPs from PSORS1C1 showed independent association to psoriasis (+39610 and +39604) both located in exon 2. The remaining SNPs were concluded to be associated to psoriasis due to linkage disequilibrium with HLA-Cw*0602. In a similar manner, we tested the effect
of the inferred haplotype HLA-Cw*0602-HCR*WW on the association of PSORS1C1 and PSORS1C2 and the same two SNPs in PSORS1C1 remained associated.
Although more information is necessary, the stratification shows that association of variations in these three genes are most likely due to their presence on the same haplotype as HLA-Cw*0602. In addition the associations detected are not near the levels obtained with HLA-Cw*0602, but for one SNP in PSORS1C1 (+39604), which also displayed independent association. Despite this, PSORS1C1 does not emerge as a strong candidate. The two HLA-Cw*0602-independent SNPs do not code for any amino acid changes and the exon in which they are located, does not appear to be translated.
However, if more splice variants exist, also indicated by the large fragment detected by Northern blot, there might be isoforms where exon 2 is part of an ORF. Another possibility is that this exon is involved in the regulation of the gene hence variations in it may have a potential effect on the function.
Continued studies of the putative function of all three genes are required in order to evaluate them as psoriasis genes and especially the functional role of PSORS1C3.
Today the understanding of the relationship between genetic information and biological function is rooted in the one gene - one protein hypothesis (Mattick and Gagen 2001).
With the emerging number of reports of genes presenting alternative splicing, and of non-coding RNAs performing a variety of tasks (gene silencing, catalysis, regulation of development), we may have to re-consider this hypothesis and not as easily discard sequences not behaving according to the existing dogma (Eddy 2001).
Searching for a candidate in the PSORS1 region has led to the identification and characterizatio n of several genes, including the four discussed above. The majority of these are significantly associated to psoriasis, but the strongest association that consistently is observed across populations is that of HLA-Cw*0602. The debate regarding its role in psoriasis has been ongoing and several arguments are used against the involvement of this gene in disease pathogenesis; low frequency of psoriasis in Cw*0602 positive individuals, more than 30% of psoriasis patients are negative for
HLA-Cw*0602, other candidate variants reach the same level of association as HLA-Cw*0602 and haplotypes carrying HLA-Cw*0602 associate more strongly to psoriasis than the allele itself (Jenisch et al. 1999; Asumalahti, Laitinen et al. 2000; Barker 2001; Elder, Nair et al. 2001; Veal, Capon et al. 2002).
Psoriasis is a multifactorial trait where several factors are likely to interact in the development of disease. Therefore it does not follow Mendelian inheritance patterns, a circumstance further complicated by heterogeneity. The high level of association seen with HLA-Cw*0602 is practically invariable and presence of this allele has been associated to severity of disease (Gudjonsson, Karason et al. 2002).
Additionally, homozygotes for HLA-Cw*0602 has been shown to have a higher risk of developing psoriasis (Gudjonsson et al. 2003). Considering that HLA-C plays an essential role in T cell biology, and T cells are involved in both the formation and maintenance of a psoriatic plaque, this molecule is very likely part of psoriasis aetiology and deserves thorough investigations.