Introduction

Psoriasis is a chronic immune-mediated skin disease, characterized by altered keratinocyte differentiation, angiogenesis and infiltration of inflammatory elements (T-lymphocytes, neutrophils and dendritic cells) into the dermis and the epidermis.¹ Familial recurrence of the disease is well documented, so that psoriasis is widely regarded as complex genetic trait, resulting from the interaction between environmental triggers and inherited disease susceptibility alleles.² Genome-wide linkage scans have unambiguously mapped a primary disease susceptibility locus (PSORS1) to the major histocompatibility complex (MHC),³ where refinement studies and resequencing efforts point to HLA-C as the most likely PSORS1 candidate.^{4, 5} More recently, the advent of genome-wide association scans has allowed the identification of several non-MHC susceptibility genes, including IL12B, IL23R, RNF114, TNFAIP3, TNIP1, IL4/IL13 and LCE3B/3C^{6, 7, 8, 9, 10, 11} Of these, IL12B and IL23R harbour variants that also confer susceptibility to Crohn's disease (CD)¹² and ankylosing spondylitis.¹³ Similarly, TNFAIP3 alleles have been associated with rheumatoid arthritis,¹⁴ systemic lupus erythematosus¹⁵ and type I diabetes.¹⁶ Similar instances of genes conferring susceptibility to multiple conditions (for example, PTPN2, IL2RA and IL2) have emerged from other genome-wide association scans,^{17, 18} highlighting unexpected connections between clinically distinct disorders.

In this context, our group has focused on the overlap between the genetic determinants for psoriasis and CD, based on the common inflammatory nature of these conditions, both of which are driven by the infiltration of CD4+ T-lymphocytes in epithelial tissues. Through the analysis of a well-characterized case-control resource, we have previously documented significant associations between psoriasis and three CD susceptibility alleles, mapping to chromosomes 1q24, 6p22 and 21q22.¹⁹ It is to be noted that the marker showing the strongest phenotypic effect is mapped to CDKAL1, a gene also associated with type II diabetes (TIID).^{20, 21, 22} All three loci were recently analysed in a North-American cohort and tentative support was obtained for the CDKAL1 association (P=0.06; one-sided P-value: 0.03).²³ With this study we provide significant replication data supporting the involvement of CDKAL1 in the pathogenesis of psoriasis. We also show that this association is independent from the allele conferring susceptibility to TIID. Finally we provide real-time PCR data, indicating that CDKAL1 is strongly expressed in CD4+ T-lymphocytes, a T-cell subset playing a major role in the pathogenesis of both psoriasis and CD.

Materials and methods

Patients

The United Kingdom case-control sample analysed in this study has been described elsewhere.¹⁹ Briefly, a total of 962 British patients of North-European descent were recruited through St John's Institute of Dermatology, London; Glasgow Western Infirmary; and the Dermatology Centre, University of Manchester. All patients were affected by an early onset of psoriasis vulgaris (disease onset occurring before 40 years of age). Control genotypes were obtained by querying the publicly available data generated by the Wellcome Trust Case-Control Consortium (WTCCC).¹⁸ Genotypes from the Collaborative Association Study of Psoriasis (CASP) were obtained from the Genetic Association Information Network (GAIN) Database, through dbGaP accession number phs000019.v1.p1. Primary keratinocytes were obtained from the skin of unaffected individuals, who were undergoing abdominoplasty at Guy's and St Thomas Hospital NHS Trust. All patients and controls gave their informed consent for participating in this project. The study was approved by the Guy's and St Thomas' Hospitals Ethics Committee, the Salford and Trafford Local Research Ethics Committee, and North Glasgow University Hospitals NHS Trust Local Research Ethics Committee.

Genotyping and direct sequencing

Single nucleotide polymorphism (SNP) rs10946398 was typed with a TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA, USA), run on a 7900HT fast real-time PCR system (Applied Biosystems). We expect the error rate for this assay to be less than 1%, as we have previously shown that our TaqMan genotypes are 99.7% concordant with those obtained by the WTCCC for the same markers.¹⁹

CDKAL1 RT-PCR products were sequenced with a BigDye Terminator cycle sequencing kit (Applied Biosystems) and loaded on an ABI 3730xl automated sequencer (Applied Biosystems). The sequence of the amplified products was compared with that of CDKAL1 full-length cDNA, using Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA).

Statistical analyses

Genotype quality controls (analysis of call rate and Hardy–Weinberg equilibrium) and ²-tests were implemented with PLINK. The genotypes of marker rs6908425 were also imputed with PLINK,²⁴ using CDKAL1 HapMap data as a reference and applying the following QC filters: call rate >90%, MAF1% and HWE P-value10^-4. The value of the INFO metric, which varies from 0 to 1 and measures the quality of imputed genotypes, was 0.92. The results of this analysis were validated with the MACH software,²⁵ which confirmed the presence of significant association between psoriasis and imputed rs6908425 genotypes (data not shown). Meta-analyses of the United Kingdom and CASP samples were implemented with Review Manager. The statistical power of the examined datasets was assessed with Genetic Power Calculator,²⁶ using published allele frequencies and odds ratios.

Expression studies

Multiple tissues and human blood fractions cDNA panels were purchased from Clontech (Mountain View, CA, USA). Both panels contain first-strand cDNA preparations obtained from pools of tissues/cells. Data provided by the manufacturer and based on the immuno-staining of relevant surface markers (CD4, CD8, CD19, CD25 and CD171), indicate that the purity of the lymphocyte populations in the blood fraction panel exceeds 95% for resting cells and 84% for activated cells.

Primary keratinocytes were grown in Epilife medium (Invitrogen, Portland, OR, USA) and were treated with 100 ng ml^-1 of each cytokine (all purchased from R&D Systems, Minneapolis, MN, USA) at passages 2–4. The cytokines bioactivity was verified by confirming their ability to induce the expression of known target genes (data not shown). About 24 h after cytokine treatment (5 h for interferon (IFN)-), cells were harvested, total RNA was extracted with TRI reagent (Sigma-Aldrich, St Louis, MO, USA) and cDNA was reverse transcribed using a Superscript first-strand synthesis kit (Invitrogen).

Transcripts were quantified by using TaqMan gene expression assays (Applied Biosystems), according to the manufacturer's protocol. Briefly, 2 l of cDNA were amplified in a final volume of 20 l, in the presence of primers and probes for CDKAL1 (Applied Biosystems assay id: Hs00214949_m1), PPIA (house-keeping gene encoding cyclophilin A; assay id: Hs99999904_m1) or RPLPO (house-keeping gene encoding large PO ribosomal protein; assay id: Hs99999902_m1). All real-time PCR reactions were carried out in duplicate, using the following cycling conditions: 15 min 95°C; (30 s 95°C, 1 in 60°C) 40. After amplification, relative CDKAL1 expression levels were derived using the Ct method.²⁷

Results

Validation of CDKAL1 association with psoriasis

In the first phase of this study, we sought access to the data generated by the CASP (an association scan of 1409 patients and 1436 controls of European ancestry¹⁰), with a view to validating the association between psoriasis and the CD loci on chromosomes 1q24, 6p22 and 21q22. Good quality SNP genotypes (call rate >90%, P-value for Hardy–Weinberg equilibrium >0.05) were available for markers rs12035082 (chromosome 1q24) and rs2836754 (chromosome 21q22), in 1323 cases and 1368 controls. As SNP rs6908425 (chromosome 6p22) was not present in the Perlegen array used by CASP, genotypes for this marker were imputed using PLINK. After association analysis, the CASP results were compared with those of our original study. As shown in Table 1, CDKAL1 SNP rs6908425 was the only marker generating convincing association in both the discovery and the replication sample. No evidence of heterogeneity was detected between the United Kingdom and CASP datasets (P=0.85; I²=0%) and a meta-analysis of the two studies yielded a P-value of 4 10^-6 (OR: 1.26; 95% CI: 1.15–1.41). A genotypic analysis of the combined resource (2579 cases vs 4306 controls) further confirmed the association between rs6908425 and psoriasis (P-value for Armitage trend test: 1.4 10^-8).

Table 1 - Association analysis of 1q24, 6p22 and 21q22 CD loci in two psoriasis datasets.

Full table

The CDKAL1 SNP associated with TIID does not confer susceptibility to psoriasis or CD

The data generated by the HapMap Consortium indicate that rs6908425 is not correlated with the CDKAL1 markers that have been associated with TIID (rs10946398, rs9465871, rs7767391; r²0.04 for all), suggesting that the two association signals are completely independent. This does not exclude the possibility that TIID associated SNPs may confer susceptibility to psoriasis, independently from rs6908425. To explore this hypothesis, we analysed the SNP yielding the strongest association with TIID (rs10946398), in 962 United Kingdom psoriatic cases and 2938 unrelated controls. We also examined the data generated by the CASP study for SNP rs9460546 (r² with rs10946398: 1.0). Although our calculations indicated that the statistical power of the combined dataset exceeded 90%, we failed to detect any significant association between rs10943698 and psoriasis (P=0.60). We next queried the data generated by the Wellcome Trust Case Control Consortium,¹⁸ to assess whether rs10943698 was associated with CD. Once more, the analysis of a sizeable resource (1747 cases and 2938 controls) failed to generate any evidence for association (P=0.84). Taken together, these data document the presence of allelic heterogeneity at the CDKAL1 locus, where rs6908425 shows an association with psoriasis and CD that is completely independent from that between rs10946398 and TIID.

CDKAL1 transcripts are virtually absent from skin keratinocytes, but are clearly expressed in immune cells

The function of the CDKAL1 protein is currently unknown and only limited information is available on CDKAL1 gene expression, as previous studies have mostly focused on tissues, which are relevant to the pathogenesis of diabetes.^{20, 22} We therefore carried out a comprehensive analysis of CDKAL1 transcript levels, by real-time PCR analysis of cDNAs from 20 tissues and cell types. In agreement with published data, we observed strong CDKAL1 expression in skeletal muscle and weaker signals in the pancreas, the liver and the kidney (Figure 1). Surprisingly, very low transcript levels were observed in some of the tissues affected by CD (the small intestine and the colon) and psoriasis (skin keratinocytes). Conversely, CDKAL1 was abundantly expressed in immune cells, with the strongest signal seen in CD4+ T-lymphocytes and CD19+ B-lymphocytes (Figure 1).

Figure 1.

Real-time PCR analysis of CDKAL1 gene expression. Top panel: Transcript levels were quantified in 20 tissues and cell types, using the expression of PPIA as an internal control. The CDKAL1/PPIA ratio for the brain was set as a baseline value, to which all transcript levels were normalized. Skel. Muscle: skeletal muscle; small intest: small intestine; CD4+: CD4+ T-lymphocytes; CD8+: CD8+ T-lymphocytes; CD19+: CD19+ B-lymphocytes. Bottom panel: Transcript levels were quantified in cDNAs obtained from pools of resting and activated lymphocytes, using the expression of PPIA as an internal control. The CDKAL1/PPIA ratio for CD4+ T-lymphocytes was set as a baseline value, to which all transcript levels were normalized. Error bars refer to technical duplicates in real-time PCR experiments. CD4+: resting CD4+ T-lymphocytes; Ac CD4+: activated CD4+ T-lymphocytes; CD8+: resting CD8+ T-lymphocytes; Ac CD8+: activated CD8+ T-lymphocytes; CD19⁺: resting CD19+ B-lymphocytes; Ac CD19+: activated CD19+ B-lymphocytes. ^*P<0.05; ^**P<0.01.

Full figure and legend (72K)

The information available on the Ensembl genome browser indicates the existence of four CDKAL1 transcript isoforms (the full length cDNA, two transcripts lacking portions of the gene untranslated regions and a shorter cDNA lacking exons 4 and 13), which can all be detected by the exon 10 probe used for our real-time PCR experiments. To determine whether our expression analysis had detected the full-length CDKAL1 transcript, the isoform lacking exons 4 and 13, or both, we designed RT-PCR primers that would amplify both transcripts, by annealing to exons 1 and 16. After electrophoresis of RT-PCR products, we consistently observed a single amplification band, whose molecular weight and sequence matched those of the full-length CDKAL1 transcript (Supplementary Figure 1). This led us to conclude that the short CDKAL1 isoform was not expressed in any of the examined tissues, so that the real-time PCR signals we had previously observed were because of the presence of transcripts that included all of the CDKAL1 coding exons.

Given the fundamental role played by lymphocytes and keratinocytes in the pathogenesis of psoriasis, we decided to further investigate CDKAL1 expression in these cell types. We first analysed cDNA from primary keratinocytes obtained from healthy donors and treated with a panel of pro-inflammatory cytokines (IFN, IFN, TNF, IL-17, IL-22 and IL-23), to assess whether any of these molecules could induce CDKAL1 gene expression. We failed to observe up-regulation of gene expression by any of these cytokines (Supplementary Figure 2).

We next focused our attention on lymphocytes and carried out real-time PCR analyses of T- and B-cells that had been activated, with well-characterized mitogens (concavalin A for CD4+ lymphocytes, phytohaemoagglutinin for CD8⁺ lymphocytes and pokeweed mitogen for CD19+ lymphocytes, all used for 72 h). The comparison with resting cells showed that CDKAL1 is markedly downregulated in CD4+, CD8+ and CD19+ proliferating lymphocytes (Figure 1). To exclude the possibility that these data might reflect a differential expression of the housekeeping gene (PPIA) in stimulated vs non-stimulated cells, we also measured the transcription of RPLPO (ribosomal protein, large PO) mRNA. The amplification of this additional reference gene confirmed our previous results, indicating that CDKAL1 is genuinely downregulated in proliferating lymphocytes (Supplementary Figure 3).

Discussion

The identification of shared genetic determinants for clinically distinct disorders is an emerging theme underlying the results of recent genome-wide association scans.²⁸ With this study, we sought to dissect the molecular genetic basis of one such observation of overlap between disease susceptibility loci. CDKAL1 was identified as a putative susceptibility gene for TIID and CD by the Wellcome Trust Case Control Consortium.¹⁸ Both associations were extensively replicated by follow-up studies,^{20, 22, 29} independent scans²¹ and analyses of related phenotypes (for example, pancreatic -cell function for TIID³⁰ and ulcerative colitis for CD³¹). Our group subsequently identified a significant association between CDKAL1 and psoriasis, in a sizeable cohort of British descent.¹⁹ Here, we provide an independent validation for CDKAL1 involvement in psoriasis, through the analysis of data generated by the CASP study. CDKAL1 is, thus, the third CD gene (after IL23R and IL12B) to be convincingly implicated in psoriasis susceptibility. This suggests a pathogenetic mechanism, whereby risk alleles at shared disease loci may account for the increased incidence of psoriasis among CD patients.^{32, 33}

Our results also show that the CDKAL1 SNP conferring susceptibility to TIID is not associated with psoriasis or CD, thus validating the presence of two independent association signals at this locus. It is to be noted that the psoriasis associated SNP (rs6908425), the TIID associated SNP (rs10946398) and the various markers that are in LD with them (rs4712528, rs7748720 for rs6908425; rs9460546, rs9565871 for rs10946398; r²>0.8 for all) all map to CDKAL1 intron 5, suggesting that this gene region may contain important regulatory elements. This notion is consistent with the annotation data available through the University of California at Santa Cruz (UCSC) genome browser, which shows the presence of evolutionary conserved regions in proximity of both rs6908425 and rs10946398. In this context, it is tempting to speculate that the two SNPs may confer susceptibility to clinically distinct disorders, by affecting regulatory elements that drive CDKAL1 expression in different cell types. It should be noted that the existence of CDKAL1 tissue-specific enhancers has already been demonstrated for a conserved segment of intron 9, which can drive the expression of a lacZ reporter in the mouse foetal brain (data generated by Lawrence Berkley National Laboratory and available through VISTA whole genome enhancer browser³⁴).

As the function of the CDKAL1 protein is unknown, we analysed the gene expression pattern in a wide range of tissues and cell types. Although our real-time PCR analysis confirmed previous reports of high CDKAL1 expression in the skeletal muscle, it also generated a number of novel and significant findings. CDKAL1 transcripts were virtually absent from primary keratinocytes, the small intestine and the colon. Conversely, gene expression was clearly detected in immune cells. A very strong signal was observed in CD4⁺ T-lymphocytes, which are crucial to the pathogenesis of both psoriasis and CD. CDKAL1 was also found to be markedly downregulated in activated T- and B-cells, providing additional support to the notion that its gene product has an important role in the biology of lymphocytes. Further studies will now be required to dissect the molecular interactions underpinning CDKAL1 function in the immune system.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Massimo Mangino for his contribution to the statistical analyses and John Mee for providing access to primary keratinocytes. Funding support for the CASP study was provided by the National Institutes of Health and the genotyping of samples was implemented through the Genetic Association Information Network (GAIN). Samples and associated phenotype data for the CASP study were provided by Goncalo Abecasis. We acknowledge the use of genotype data from the British 1958 Birth Cohort DNA collection, funded by the Medical Research Council (grant G0000934) and The Wellcome Trust (grant 068545/Z/02). This work was also supported by a PhD studentship from The Psoriasis Association (MQ) and grants from the British Skin Foundation (FC) and the Medical Research Council (JNB, RCT). Funding: British Skin Foundation, The Psoriasis Association, Medical Research Council

Supplementary Information accompanies the paper on Genes and Immunity website (http://www.nature.com/gene)