SNP rs11652075 in the CARD14 Gene as a Risk Factor for Psoriasis (PSORS2) in a Spanish Cohort

Abstract

A recent genomic survey identified the association between a common single nucleotide polymorphism (SNP) at the CARD14 gene (SNP rs11652075; p.Arg820Trp) and psoriasis (Psor). Our aim was to replicate the association between this polymorphism and to determine whether other CARD14 variants could explain the association. A total of 400 Psor patients (mean age 47±15; 55% male) and 420 healthy controls (mean age 51±16; 56% male) all Caucasian were genotyped for rs11652075. The rs11652075 CC genotype was significantly associated with Psor in our population (p=0.003; odds ratios=1.59; 95% confidence intervals=1.16–2.19; statistical power >80). The sequencing of the whole CARD14 coding exons in a total of 15 patients did not identify other DNA variants that could explain this association. We did not find significant differences (allele/genotype frequencies) between the patients according to disease severity, presence of arthritis, onset of age, and family history of Psor. We confirmed the association between SNP rs11652075 at the CARD14 gene and Psor. The absence of other coding variants among our patients supported a direct role for this missense polymorphism on Psor risk.

Introduction

T he psoriasis (Psor) susceptibility locus 2 (PSORS2) was initially mapped to the human chromosome region 17q25-qter in a large family of European ancestry (Tomfohrde et al., 1994). Further genome-wide linkage scans confirmed this Psor susceptibility locus (Nair et al., 1997; Hwu et al., 2005). Recently, exome sequencing identified a mutation (p.Gly117Ser) in the caspase recruitment domain family, member 14 gene (CARD14) that segregated with the disease in a large PSORS2 family (Bertin et al., 2001; Jordan et al., 2012a). This and other CARD14 mutations lead to enhanced nuclear factor-kappa B (NF-κB) activation and upregulation of Psor-associated genes in keratinocytes; among others, genes that encoded cytokines and chemokines related to Psor such as IL8, CCL20, and IL36G (Jordan et al., 2012a). In the context of an inflammatory stimulus, keratinocytes harboring the CARD14 mutation could exhibit an increased NF-κB-response, which would, in turn, upregulate an inflammatory response through the recruitment of inflammatory mediators. Notably, CARD14 was found upregulated in a large-scale study of gene expression in Psor versus normal skin (Gudjonsson et al., 2010). The involvement of CARD14 on Psor risk was thus in agreement with genome-wide scans that revealed association of Psor with genes of the NF-κB pathways (Nair et al., 2009).

The sequencing of CARD14 in a large cohort of patients and controls identified several rare nucleotide changes only in the patients (Jordan et al., 2012b). Functional studies showed that some of the changes had an effect on NF-κB activation and gene transcription in keratinocytes. In addition to rare mutations, a common missense single-nucleotide polymorphism (SNP rs11652075, c.2458C>T, p.Arg820Trp) was associated with Psor (Jordan et al., 2012b). The association was only significant in some of the seven cohorts included in the study, but was confirmed by a large meta-analysis of three genome-wide association studies (Tsoi et al., 2012). In addition, evidence for an interaction between rs11652075 and HLA-Cw*0602 (PSORS1) in Psor risk was found in some cohorts.

To better define the association between rs11652075 on Psor, we genotyped a cohort of Spanish Caucasians. In addition, we sequenced the CARD14 coding sequence in a group of patients with the Psor-risk genotype.

Patients and Methods

Study subjects

A total of 400 Psor-patients (mean age 47±15; 55% male) and 420 healthy controls (mean age 51±16; 56% male) were recruited by dermatologists from Hospital Universitario Central Asturias (HUCA) between January 2007 and October 2012. All the patients and controls were Caucasians from the region of Asturias (Northern Spain, total population 1 million), and gave their written informed consent to participate in the study approved by the Ethics Committee of HUCA.

The main characteristics of most of these patients had been previously reported, and are summarized in Table 1 (Soto-Sánchez et al., 2010; Eiris et al., 2012). Ps was diagnosed based on clinical findings. The disease was defined as severe or nonsevere according to the psoriasis area and severity index (PASI): patients with a PASI score ≥10 were classified as severe, and those with a PASI <10 as nonsevere. Patients were considered to have an early onset of Psor if the onset of the disease was at any age ≤40 years, and a late onset of Psor if the onset was >40 years. Patients were considered to have familial Psor if they had at least one first- or second-degree affected relative. The existence of arthritis was assessed by a rheumatologist. All the patients and controls were genotyped for the Cw*0602 (PSORS1) and LCE3C_LCE3B-del (PSORS4) as reported (Tazi Ahnini et al., 1999; Coto et al., 2010).

Table 1.

CARD14 rs11652075 (c.2458C>T, p.Arg820Trp) Genotype Frequencies (%) in Psor Patients and Healthy Controls

	CARD14	CARD14	CARD14
	TT	CT	CC
Controls (n=420)	107 (25)	224 (53)	89 (21)
Patients (n=400)	70 (17)	210 (53)	120 (30)
PASI<10 (n=241)	44 (63)	123 (59)	74 (62)
PASI≥10 (n=159)	26 (37)	87 (41)	46 (38)
Arthritis (n=96)	18 (26)	44 (21)	34 (28)
No arthritis (n=304)	52 (74)	166 (79)	86 (72)
Early onset (n=283)	50 (71)	146 (70)	87 (73)
Late onset (n=117)	20 (29)	64 (30)	33 (27)
Familial (n=192)	33 (47)	101 (48)	58 (48)
Nonfamilial (n=208)	37 (53)	109 (52)	62 (52)
CW6+(n=161)	27 (39)	87 (41)	47 (39)
CW6−(n=239)	43 (61)	123 (59)	73 (61)
LCE3 del-del (n=157)	30 (43)	82 (39)	45 (38)
LCE3 ins (n=243)	40 (57)	128 (61)	75 (62)

For each CARD14 genotype, we also indicated the number of patients relative to the PASI score, presence of arthritis, Psor onset of age, family history of Psor, and the Cw6 and LCE genotypes.

CARD14-CC, patients versus controls: p=0.003, OR=1.59; 95% CI=1.16–2.19.

LCE3 ins=del/ins+ins/ins genotypes.

Psor, psoriasis; PASI, psoriasis area and severity index; OR, odds ratios; CI, confidence intervals.

All the data are available in an individual anonymized form upon request to the corresponding author.

CARD14 SNP genotyping

SNP rs11652075 was genotyped through a real-time TaqMan assay (assay id. C_7964277_10; Applied BioSystems, www.appliedbiosystems.com). SNPs rs3813063 (intron 14) and rs11653893 (intron 17) were genotyped through polymerase chain reaction (PCR) amplification followed by restriction fragment length polymorphism (RFLP) (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/dna). To confirm the accuracy of these methods, we confirmed the genotypes in several individuals through sequencing of PCR fragments.

CARD14 sequencing

We sequenced the 21 coding CARD14 exons in 15 patients who were rs11652075 CC. Exon 4, which the previous study identified as a mutational hotspot, was sequenced in an additional group of 15 patients. Briefly, genomic DNA was PCR-amplified with primer pairs (Supplementary Table S2), and the fragments were purified and sequenced with BigDye chemistry in an automated ABI3130xl equipment (Applied Biosystems). Sequences were compared with the reference CARD14 sequence in the Ensembl database (accession numbers ENSG00000141527 and ENST00000573882; www.ensembl.org).

Statistical analysis

The χ² test was used to compare the genotype and allele frequencies between the groups, and to determinate whether the genotype frequencies deviated from the Hardy–Weinberg equilibrium. The odds ratios (OR) and their 95% confidence intervals (CI) were also calculated. The Student's t-test was used to compare the quantitative data between the groups. We performed multivariate logistic regression analysis to adjust for risk factors. The interaction between CARD14 and the CW6 and LCE genotypes was determined through linear regression using the SPSS software. Linkage disequilibrium (LD) and frequencies of the haplotypes defined by a pair of loci were calculated with the Cubic exact solutions for the estimation of pairwise haplotype frequencies (Cubex; www.oege.org/software/cubex). A Bonferroni corrected cutoff of p≤0.01 was considered statistically significant.

Results

In Table 1, we summarized the CARD14 rs11652075 genotype frequencies in patients and controls. The genotype frequencies did not deviate from the Hardy–Weinberg equilibrium in patients and controls (p>0.05; Supplementary Table S3). The rs11652075 CC genotype was significantly associated with Psor in our population (p=0.003; OR=1.59; 95% CI=1.16–2.19; statistical power >80). We did not find differences between the genotypes for the frequency of severe Psor (PASI score ≥10), early onset of disease (≤40 years), presence of arthritis, or family history of Psor (Table 1).

In addition to rs11652075, patients and controls were genotyped for the common rs3813063 and rs11653893 SNPs in introns 14 and 17, respectively. Nominally, the two CARD14 variants were also significantly associated with Psor in our population (Supplementary Table S3). The haplotype count showed that the two SNPs were in total (rs11653893) or almost total LD (rs3813063; D′=0.98). In this way, the association between the two polymorphisms could be explained by LD with rs11652075.

Patients and controls were also genotyped for the Cw*0602 allele and the LCE3C_LCE3B deletion, two genetic risk factors for psor. The LCE-del/del genotype was significantly associated to Psor in our cohort (p=0.015; OR=1.43, 95% CI=1.10–1.90). As expected, the CW6 allele was strongly associated with Psor (p<0.001; OR=23.40, 95% CI=14.94–36.82) (Supplementary Table S4). The LCE-del/del and CW6-positive frequencies did not differ between the rs11652075 genotypes (Table 1). No significant interaction was observed between the CARD14 and CW6 genotypes (p>0.05). Moreover, the rs11652075 remained significantly associated to Psor in the multiple linear regression, including CW6 as a covariate (p=0.045; OR=1.50, 95% CI=1.01–2.28). This suggested that in our population, the association between the CARD14 SNP and Psor was not conditioned on HLA-CW6. However, the comparison was underpowered because it was based on only 25 CW6-positive controls (Supplementary Table S4). The same independent effect of the rs11652075 was observed when the LCE genotype was included as a covariate (p=0.021; OR=1.59, 95% CI=1.07–2.37).

We sequenced the CARD14 coding exons (plus at least 10 flanking intronic nucleotides) from 15 patients who were rs11652075 CC. We found several previously reported polymorphisms (Table 2). Only one was a missense change (p.Arg547Ser, rs2066964). Among the intronic variants, SNP rs11653893 was in the consensus acceptor site of intron 17 (ivs −4 A/G). However, a bioinformatic online analysis (www.fruitfly.org/cgi-bin/seq_tools/splice.pl) showed that this nucleotide change would not result in pre-mRNA splicing (Supplementary Table S5).

Table 2.

CARD14 Variants Found After Sequencing 15 Patients Who Were rs11652075 CC (MAF, T=0.49)

SNP ID	Exon/intron	D′ (genotypes)	MAF
rs4889990 G/A	Ex 4; c.633G>A (p.E211E)	Nd (3 AG+7 GG)	A=0.37
rs4889991 G/A	Int4; c.675+26A>G	Nd (2 AG+6 AA)	G=0.17
rs2066964 G/C	Ex 12; c.1641G>C (p.R547S)	Nd (3 CC+7 GG)	C=0.40
rs8069255 T/A	Int15; c.2219+14T>A	1 (all AA)	A=0.49
rs4889996 A/G	Int 17; c.2398+30A>G	1 (all GG)	G=0.49
rs4889836 T/C	Int17; c.2398+42T>C	1 (all CC)	C=0.49
rs11653893 A/G	Int 17; c.2399−4A>G	1 (all AA)	A=0.49

Exon 4 was sequenced in a total of 30 patients. Nucleotide changes are numbered relative to the cDNA sequence (transcript id. ENST00000573882; www.ensembl.org). The reported LD value (D′) between each variant and rs11652075 in Caucasians and the genotypes found among our sequenced cases (parenthesis) are also indicated.

Nd, nondetermined; MAF, minor allele frequencies among Caucasians; SNP, single-nucleotide polymorphism; LD, linkage disequilibrium.

Discussion

We found a significant association between the CARD14 rs11652075 CC genotype and Psor. The association was independent of the Cw*0602 and the LCE3C_LCE3B del/del genotypes, two recognized genetic risk factors for Psor (Nair et al., 2006; De Cid et al., 2009; Coto et al., 2011). Because rs11652075 is a missense change (p.R820W), it is plausible that an effect on protein function could explain the observed association. Amino acid 820 is in the C-terminal guanylate kinase domain of the CARD14 protein, which would relay external signals to the cellular milieu (Bertin et al., 2001). In this way, the protein with Trp at amino acid 820 could increase the levels of NF-κB and, in turn, of transcripts associated with Psor. It is, however, possible that this SNP is in LD with other functional variants that would be responsible for the observed association. To address this issue, we sequenced the CARD14 coding exons from 15 patients with the risk genotype. Among the identified variants, the only missense change was p.Arg547Ser (SNP rs2066964), which the previous studies showed would not alter significantly the NF-κB activation levels (Jordan et al., 2012a). None of the intronic variants was predicted to affect pre-mRNA splicing. Together, the data were in agreement with the results from a large-scale resequencing that found rare CARD14 functional variants in Psor patients, but not a common polymorphism in LD with rs11652075 that could explain the reported association (Jordan et al., 2012a).

The effect of CARD14 points to the importance of Psor risk on genes that encode proteins involved in skin innate host defense mechanisms. In addition to genes implicated in the NF-κB pathways (such as CARD14), candidate genes involved in interferon-mediated antiviral responses and macrophage activation have been linked to Psor through genome-wide association studies (Tsoi et al., 2012).

Finally, CARD14 encodes a protein engaged in signaling pathways that are targeted by biological therapies, such as anti-TNF antibodies (Chong and Wong, 2007; Gudjonsson et al., 2012). In this context, the CARD14 variants linked to Psor risk could be candidates to test the pharmacogenetic response to these therapeutic agents (O'Rielly and Rahman, 2011).

In conclusion, our data supported a direct linkage between the common CARD14 rs11652075 variant (p.R820W) and the risk of developing Psor. In spite of its effect on Psor risk, this SNP did not affect disease severity, onset of age, or the risk for arthritis in our population.

Footnotes

Acknowledgment

This work was partially funded by ABBVIE.

Disclosure Statement

The authors state no conflict of interests. This article has not been submitted elsewhere and all the authors have seen and approved the final version and the submission.

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