View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector ARTICLE

PSORS2 Is Due to Mutations in CARD14

Catherine T. Jordan,1 Li Cao,1 Elisha D.O. Roberson,1 Katherine C. Pierson,2 Chi-Fan Yang,3 Cailin E. Joyce,1 Caitriona Ryan,4 Shenghui Duan,1 Cynthia A. Helms,1 Yin Liu,5 Yongqing Chen,5 Alison A. McBride,6 Wuh-Liang Hwu,7 Jer-Yuarn Wu,3 Yuan-Tsong Chen,3 Alan Menter,4,8 Raphaela Goldbach-Mansky,5,8 Michelle A. Lowes,2,8 and Anne M. Bowcock1,*

Psoriasis is a common, immune-mediated genetic disorder of the skin and is associated with arthritis in approximately 30% of cases. Previously, we localized PSORS2 ( susceptibility locus 2) to chromosomal region 17q25.3-qter after a genome-wide linkage scan in a family of European ancestry with multiple cases of psoriasis and psoriatic arthritis. Linkage to PSORS2 was also observed in a Taiwanese family with multiple psoriasis-affected members. In caspase recruitment domain family, member 14 (CARD14), we identi- fied unique gain-of-function mutations that segregated with psoriasis by using genomic capture and DNA sequencing. The mutations c.349G>A (p.Gly117Ser) (in the family of European descent) and c.349þ5G>A (in the Taiwanese family) altered splicing between CARD14 exons 3 and 4. A de novo CARD14 mutation, c.413A>C (p.Glu138Ala), was detected in a child with sporadic, early-onset, gener- alized pustular psoriasis. CARD14 activates nuclear factor kappa B (NF-kB), and compared with wild-type CARD14, the p.Gly117Ser and p.Glu138Ala substitutions were shown to lead to enhanced NF-kB activation and upregulation of a subset of psoriasis-associated in keratinocytes. These genes included chemokine (C-C motif) ligand 20 (CCL20) and interleukin 8 (IL8). CARD14 is localized mainly in the basal and suprabasal layers of healthy skin epidermis, whereas in lesional psoriatic skin, it is reduced in the basal layer and more diffusely upregulated in the suprabasal layers of the epidermis. We propose that, after a triggering event that can include epidermal injury, rare gain-of-function mutations in CARD14 initiate a process that includes inflammatory cell recruitment by keratinocytes. This perpetuates a vicious cycle of epidermal inflammation and regeneration, a cycle which is the hallmark of psoriasis.

Introduction region 17q25.3. In both large, multiplex families, psoriasis segregated as an autosomal-dominant Mendelian trait Psoriasis is a poorly understood common inflammatory with high penetrance. disorder of the skin and other organs and affects approxi- To identify the familial mutations, we attempted associ- mately 2% of individuals of European descent.1 It can be ation studies and extensive resequencing of genes under associated with a chronic inflammatory psoriatic arthritis the linkage peak.17–19 Recently, we included targeted and in ~30% of affected individuals.2 Genome-wide associa- exome capture followed by NextGen sequencing of DNA tion studies (GWASs) have identified over 20 susceptibility from members of family PS1 and identified in caspase loci for psoriasis,3–11 but less than 20% of disease variance recruitment domain family, member 14 (CARD14 [MIM is explained.12,13 Additional low-risk loci, genetic interac- 607211])20 a mutation that segregated with psoriasis. We tions, or rare variants of large effect are thought to account then identified a second CARD14 mutation in the 17q25- for the remaining disease variance. It is difficult to identify linked Taiwanese family, and a young child with severe the latter, but they are expected to be observed in rare fami- generalized pustular psoriasis (PSORP [MIM 614204]) lies in which psoriasis segregates as a Mendelian trait. harbored a de novo mutation within CARD14. Wild-type Seventeen years ago, we mapped psoriasis susceptibility CARD14 activates nuclear factor-kappa B (NF-kB), and locus 2 (PSORS2 [MIM 602723]) to human chromosomal the psoriasis-associated missense mutations further upre- region 17q25-qter in a single large family (PS1) of Euro- gulate NF-kB activity. Compared with wild-type keratino- pean ancestry.14 The region of linkage spanned 3.5 Mb cytes, cultured keratinocytes from affected individuals from D17S784 to 17qter. Affected members had plaque with the mutations and a keratinocyte cell line transfected psoriasis, and approximately 30% also developed psoriatic with mutant CARD14 showed increased transcription of arthritis. Two additional genome-wide linkage scans genes encoding psoriasis-associated chemokines and cyto- further implicated PSORS2. A study of 224 sibling pairs kines such as interleukin 8 (IL8 [MIM 146930]), chemokine with psoriasis yielded a maximum LOD score of 2.09 (C-C motif) ligand 20 (CCL20 [MIM 601960]), and inter- with D17S802,15 and a five-generation psoriasis pedigree leukin 36, gamma (IL36G [MIM 605542]). CARD14 from Taiwan exhibited significant linkage to D17S928.16 peptides in the skin were almost exclusively localized in Both of these microsatellite loci map to chromosomal the epidermal keratinocytes, primarily in the basal layer

1Division of Human Genetics, Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA; 2Laboratory for Investigative Dermatology, The Rockefeller University, New York, NY 10065, USA; 3Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan; 4Psoriasis Research Institute, Baylor University Medical Center, Dallas, TX 75246, USA; 5National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, MD 20892, USA; 6National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; 7Depart- ments of Pediatrics and Medical Genetics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei 100, Taiwan 8These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.ajhg.2012.03.012. Ó2012 by The American Society of Human Genetics. All rights reserved.

784 The American Journal of Human Genetics 90, 784–795, May 4, 2012 of the epidermis. Compared with normal skin, psoriatic family (affected by c.349þ5G>A). PCR fragments included 0 0 skin showed reduced levels of CARD14 in the basal layer CARD14 exon 3 and 150 bp each of the 5 and 3 introns. Frag- but increased levels throughout the upper layers. We ments were digested by BamH1 and XbaI and were ligated into propose that, in the context of an inflammatory stimulus, the RHCglo vector (a gift from Dr. Tom Cooper at the Baylor keratinocytes harboring the CARD14 mutations can upre- College of Medicine) at the corresponding digest sites. All constructs were validated with DNA sequencing. Minigene gulate an inflammatory response via excessive activation constructs were used as described previously.25 Minigenes were of NF-kB-responsive genes and initiate the recruitment of transfected into human embryonic kidney (HEK) 293 cells. Total the inflammatory infiltrate seen in psoriasis. Our findings RNA was harvested after 24 hr. cDNA was synthesized with the resolve the identity of the elusive PSORS2 locus and iden- SuperScript II kit (Invitrogen) and the RTRHC oligonucleotide tify CARD14 as a regulator of skin inflammation. primer 50-GGGCTTTGCAGCAACAGTAAC-30. Using the TNIE4 50-AGGTGCTGCCGCCGGGCGGTGGCTG-30 and RSV5U 50-CAT 0 Subjects and Methods TCACCACATTGGTGTGC-3 primers, we sequenced the cDNA to evaluate the splicing of CARD14 exon 3 in the presence of the familial psoriasis mutations and in the presence of the reference Subjects CARD14 sequence. Sequencing primers were those described Family PS1 of European origin and the 17q25-linked Taiwanese 0 14,16 above as well as an internal exon 3 primer, 5 -ACTTGCTGGAT psoriasis-affected family are described elsewhere. The person 0 TTGCTGAAGAC-3 . with pustular psoriasis (individual 2192) was a 3-year-old child from Haiti. Her father, mother, and brother were unaffected, and Expression Plasmids there was no family history of psoriasis or any other autoimmune The pNFkB-luc plasmid (Clontech) contains the firefly luciferase disorder. Whole blood was obtained by venipuncture; skin and four tandem copies of the consensus NF-kB-binding samples were obtained by punch biopsy. Primary keratinocytes sequence fused to a TATA-like promoter. The pTAL-luc plasmid from family PS1 and classical-psoriasis cases were immortalized (Clontech) contains an enhancerless reporter with a minimal by infection with human papillomavirus E6/E7 DNA (a gift from TATA-like promoter and the firefly luciferase gene, and it was Dr. Denise Galloway at the Fred Hutchinson Center for Cancer used as a negative control. Research).21 Keratinocytes from individual 2192, who has pustular Two CARD14 cDNA clones, CARD14sh (coding for 740 amino psoriasis, and two foreskin samples used as controls were immor- acids; GenBank BC018142) and CARD14cl (coding for 434 amino talized without transformation as previously described.22 Proto- acids; RefSeq NM_052819) were used (Capital Biosciences). A full- cols were approved by local institutional review boards (IRBs). length (CARD14fl) construct was not available. Mutant forms of All subjects or their parents (if the subjects were minors) provided CARD14 were prepared from the CARD14sh construct (GenBank informed consent. BC018142) with the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Genomic Capture and Sequence Analysis Exome capture and targeted capture of the 17q25 linkage region NF-kB Luciferase Reporter Assay were performed with DNA from family PS1 and were followed HEK 293 cells were cultured under standard conditions and plated by NextGen sequencing. For exome sequencing, DNA from four at a density of 0.15 million cells per well in 12-well plates. Cells affected individuals was used. For targeted capture, pools of were cotransfected with (1) 0.5 mg wild-type CARD14sh or the genomic DNA from 14 affected and eight unaffected samples CARD14cl expression construct, (2) 0.5 mg pTAL-luc or pNF-kB- were used. Genomic libraries were prepared with the Illumina luc plasmid, and (3) 0.025 mg pGL4.70 Renilla reporter plasmid Paired-End Genomic DNA Sample Prep Kit. Genomic capture (Promega). In total, 1.025 mg of plasmid DNA was transfected was performed with Roche-Nimblegen SeqCap EZ Exome and into each cell. For cotransfections, 0.5 mg of each CARD14 Agilent SureSelect Target Enrichment kits. Captured DNA was construct was transfected into cells. Then, 0.05 mg of pGL4.70 sequenced on the Illumina Genome Analyzer II (GAIIx) for 76 and 0.5 mg of reporter plasmid were added for a total of 1.55 mg cycles (one lane per sample or pool). Both capture methods signif- plasmid DNA transfected into each cell. All transfections were per- icantly enriched the targeted regions (Table S1, available online). formed in triplicate. For stimulation with TNF-a (tumor necrosis factor alpha [encoded by TNF (MIM 191160)]), cells were treated Read Mapping and Sequence Analysis with 20 ng/ml TNF-a in culture medium. For TNF-a neutralization Read mapping, sequence analysis, and variant calling were per- experiments, cells were treated with indicated concentrations of 23 formed as described elsewhere. Known polymorphisms (variants TNF-a neutralizing antibody (Cell Applications). Cells were in dbSNP130 and eight previously sequenced HapMap individ- harvested 24 hr after transfection, and firefly luciferase activity 24 uals) were excluded. Candidate mutations in the 17q25 linkage was determined with the Dual-Luciferase Reporter Assay System region were those observed in at least three of the four exomes (Promega). We normalized firefly luciferase activity to Renilla lucif- sequenced and/or observed in the affected pool but not in the erase activity to control for transfection efficiency. Relative NF-kB unaffected pool. Mutations were manually annotated for amino luciferase activity for each sample was then calculated by back- acid changes and were Sanger sequenced for validation and for ground subtraction of pTAL luciferase activity and the subsequent checking segregation with disease (Table S2). division of the normalized NF-kB luciferase value by the back- ground pTAL luciferase value. Minigene Assay CARD14 minigenes were constructed by PCR amplification of Expression Profiling human genomic DNA from unaffected (wild-type) and affected HEK 001 cells (human-papillomavirus-16-transformed keratino- members of family PS1 (affected by c.349G>A) and the Taiwanese cytes) were transfected with wild-type CARD14sh or mutant

The American Journal of Human Genetics 90, 784–795, May 4, 2012 785 (c.349G>A [p.Gly117Ser] or c.413A>C [p.Glu138Ala]) CARD14sh 2 3 106((Ct 18S) (Ct CARD14)). We corrected for differences in trans- expression constructs. Cells were cultured for 24 hr, after which fection efficiency by normalizing expression levels in transfected time total RNA was isolated. Lesional and nonlesional skin biop- cells to levels of FLAG. Primers for the transcripts examined by sies from an affected member (GEN001) of family PS1 were ob- qRT-PCR are available upon request. tained from the Psoriasis Research Institute (Dallas, Texas), and Immunohistochemistry a lesional skin biopsy from child 2192, who has severe pustular Normal skin samples (n ¼ 3–4) and paired uninvolved and psoriasis, was obtained from the National Institute of Arthritis involved psoriatic samples (n ¼ 5–6) were obtained from Rockefel- and Musculoskeletal and Skin Diseases. Biopsies of normal skin ler University from individuals with mild-to-severe stable plaque (n ¼ 2) and moderate-to-severe lesional psoriasis skin (n ¼ 2) psoriasis. PS1 uninvolved and involved skin was collected and were obtained from Rockefeller University for global expression studied under a protocol approved by the IRB at Washington profiling. RNA was extracted from full-thickness skin biopsies. University. All frozen sections were stained with polyclonal rabbit Immortalized keratinocyte lines from uninvolved skin of affected anti-CARD14 (Sigma, 1:600). This antibody labels the internal members (K1-1 and K1-20) of family PS1 and from involved skin of coiled-coil domain of CARD14 and recognizes all known isoforms. classical psoriasis (K5-14, an HLA-Cw*0602-positive case) were Secondary biotin-labeled goat anti-rabbit antibodies (Vector cultured with or without stimulation by TNF-a, and total RNA Laboratories) were used. The staining signal was amplified with was isolated. Immortalized, nontransformed keratinocytes from avidin-biotin complex (Vector Laboratories) and developed with individual 2192 and from two foreskin samples were cultured in chromogen 3-amino-9-ethylcarbazole (Sigma Life Sciences). The medium only. For all samples described above, total RNA isolation negative control was stained with 1% serum in lieu of the primary was performed with the miRNeasy or RNeasy kit (QIAGEN). Global antibody. expression profiling was performed with HumanHT-12 v4 Expres- sion BeadChip (Illumina). For each BeadChip (Illumina), at least 0.2 mg of total RNA per sample was reverse transcribed, amplified, Results and labeled. Experiments were conducted in compliance with MIAME (minimum information about a microarray experiment) Exome- and Targeted-Sequencing Results guidelines. Raw and normalized expression data are deposited in Family PS1 is of European ancestry and has multiple the National Center for Biotechnology Information (NCBI) Gene cases of psoriasis and psoriatic arthritis.14 DNA of family Expression Omnibus (GEO) with accession number GSE36387. members was used for exome sequencing, targeted-capture For expression analyses, data were imported into R (v2.13.1) sequencing, and NextGen sequencing. Only the following with the BeadArray (v2.2.0) package. Raw data were used for anal- two -coding mutations were identified in the ~3.5 ysis. For single-sample-by-single-sample comparisons, probes that Mb linkage region and segregated with the disease (Tables did not show a detection p value < 0.05 in any sample were S1 and S2): c.349G>A (p.Gly117Ser) in CARD14 and excluded. The average signal, standard error, and number of beads c.365A>G (p.Tyr122Cys) in solute carrier family 26, were used for the computation of a two-sample, two-sided t test SLC26A11 assuming unequal variance. Fold-changes were calculated on member 11 ( [MIM 610117]) (Figures 1A and 1B CARD14 the basis of the average expression values. We adjusted p values and Table S2). Numbering of all mutations in with the Benjamini and Hochberg method to control for false this manuscript is based on RefSeq NM_024110.3. discovery. Numbering of the SLC26A11 mutation is based on RefSeq For group-wise comparisons, average expression values were NM_001166347.1. CARD14, also known as CARMA2, quantile normalized and log2 transformed. Contrasts for each encodes a member of the CARMA (caspase recruitment desired class comparison were created with Limma (v3.8.3). Probe domain [CARD]- and membrane-associated guanylate expression values were modeled as linear models, and statistics for kinase [MAGUK]) family; this member interacts with fold-changes and p values were calculated on these models with BCL10 and activates NF-kB.20 SLC26A11 encodes a the limma ‘‘eBayes’’ function. p values were adjusted as described sulfate/anion transporter.26 Both mutations were predicted above. to be potentially damaging by PolyPhen-2.027 and SIFT.28 CARD14 Transcript Isoform Analysis CARD14 cDNA Cloning and Sequencing Further Evidence of Mutations Predisposing SuperScript II (Invitrogen) was used for the generation of cDNA to Psoriasis from RNA derived from the involved skin of individual GEN001. Coding exons and flanking intronic DNA of CARD14 PCR-based cloning of the exon 2–4 region of CARD14 was per- and SLC26A11 were resequenced in affected members of formed according to standard protocols with exon-2- and exon- the Taiwanese psoriasis-affected family.16 This revealed 0 4-specific oligonucleotide primers (5 -AGGAGACACTGTGGGAG a c.349þ5G>A mutation in CARD14 but none in 0 0 0 ATGAT-3 for exon 2 and 5 -ATTGCTATAGTGCAGCGAGAGG-3 SLC26A11. Resequencing of 54 members of this family for exon 4). Plasmid inserts were sequenced with the same oligo- (21 affected, 20 unaffected, and 13 of unknown disease nucleotide primers. status) demonstrated that this mutation segregated with Quantitative RT-PCR psoriasis (Figure 1C). This family had previously been Quantitative RT-PCR (qRT-PCR) was performed in routine fashion on total RNA primed with random hexamer oligonucleotides. PCR sequenced for all genes under the linkage peak, and only reactions were performed with Power SYBR Green master mix (Life one other possible disease-causing variant, a mutation Technologies) on a 7900HT thermocycler (Life Technologies). upstream of the transcriptional start site of zinc finger Expression was normalized to 18S rRNA. Relative expression protein 750 (ZNF750 [MIM 610226]), had been identified levels were calculated according to the 2-DDCt method as follows: (discussed further below).19

786 The American Journal of Human Genetics 90, 784–795, May 4, 2012 Figure 1. Positional Cloning of PSORS2 and Mutations Segregating in Linked Families (A) Location of PSORS2 showing polymorphic microsatellites D17S784 and D17S982 and the locations of CARD14 and SLC26A11 and familial mutations. The following abbreviation is used: SGSH, N-sulfoglucosamine sulfohydrolase (MIM 605270). (B) The pedigree of family PS1 shows genotypes at mutated loci (phased as displayed) of CARD14 (c.349G>A; upper) and SLC26A11 (c.365A>G; lower). The unaffected woman with an asterisk recently developed psoriasis at 83 years of age. The affected male labeled GEN001 provided skin biopsies of uninvolved and involved (psoriatic plaque) skin used for this study. (C) The pedigree of 17q25-linked Taiwanese psoriasis-affected family shows genotypes of the c.349þ5G>A mutation in family members. Black-filled symbols indicate classical psoriasis. Gray-filled symbols indicate mild skin manifestation of psoriasis. Question marks indi- cate unknown disease status. (D) Both familial CARD14 mutations are located within the consensus splice donor sequence of exon 3. Chromatograms obtained after resequencing of this splice donor sequence in DNA of an affected member of each family are shown. The exon-intron junction is indi- cated by the vertical black line. The two mutations are boxed in red.

We also identified a de novo germline mutation Familial Psoriasis Mutations Alter Splicing of Exon 3 (c.413A>C [p.Glu138Ala]) in exon 4 of CARD14 in a The CARD14 nucleotides mutated in the two families and 3-year-old Haitian child (individual 2192) who had severe in the pustular case were highly conserved in vertebrates pustular psoriasis since the age of 6 months (Figures 2A– (Figure 2D). The amino acids altered in family PS1 and 2C). The mutation was predicted to be damaging by Poly- individual 2192 with pustular psoriasis were also highly Phen-2.027 and SIFT.28 The child had no family history of conserved (Figure S1). Although the p.Gly117Ser substitu- psoriasis or any other inflammatory disorder. She did not tion was a missense alteration, the fact that both familial harbor any mutation in SLC26A11 nor the interleukin 36 mutations lie in the splice donor sequence of exon 3 receptor antagonist (IL36RN [MIM 605507]), a gene suggests that they might affect splicing (Figure 1D). We recently described to be mutated in pustular psoriasis.32,33 investigated this with in vitro splicing assays (Figures 3A Neither the multiplex families nor the child with and 3B),25 which revealed that both familial mutations pustular psoriasis harbored the PSORS1 risk variant, HLA- lead to the use of a cryptic splice donor site 66 bp from Cw*0602,34 suggesting that CARD14 mutations are suffi- the start of intron 3 (AG/GTGCCC, Figures 3B and 3C). cient to lead to disease. None of these mutations were This caused an insertion of 22 amino acids into the detected in the sequences of the 1,000 Genomes Project,35 CARD14 peptide between exons 3 and 4 (Figure 3D). dbSNP130, or in eight previously exome-sequenced We were also interested in evaluating the effects of these HapMap individuals.24 They were also shown to be present mutations in vivo. Although RNA and cDNA from the skin in the psoriatic population at very low frequencies (<0.1% of affected members of the Taiwanese family were unavail- of cases) and to be even less frequent or absent in controls able, we obtained a skin biopsy and extracted cDNA from (see the accompanying paper36 in this issue of AJHG). the skin of an affected member (GEN001) of family PS1.

The American Journal of Human Genetics 90, 784–795, May 4, 2012 787 Figure 2. Detection of a De Novo CARD14 Mutation in an Individual with Pediatric Pustular Psoriasis and Conserva- tion of Mutated Nucleotides (A) Child 2192, who has pustular psoriasis, before treatment. Most of the body is covered with lesions. (B) Sequence traces for 2192 and her parents revealing a de novo CARD14 muta- tion, c.413A>C (p.Glu138Ala). (C) The pedigree of individual 2192 shows parental genotypes for de novo mutation c.413A>C (p.Glu138Ala). (D) Alignment of DNA sequences from the indicated species is shown for the segments of CARD14 exon 3, intron 3, and exon 4 harboring the CARD14 muta- tions described here. The mutations are shown in red and are marked with aster- isks. DNA and protein sequences were downloaded from the UCSC Genome Browser29 and were aligned with Clus- talW2.30,31 The genome builds used for each species are as follows: hg19 (human), panTro2 (chimp), rheMac2 (rhesus), mm9 (mouse), bosTau4 (cow), canFam2 (dog), monDom5 (opossum), fr2 (fugu), gasAcu1 (stickleback), and oryLat2 (medaka). cDNA sequencing with primers amplifying transcripts mine its effect as well as the contribution of the other between exons 2 and 4 of CARD14 from skin of this indi- mutant species to psoriasis pathogenesis. Nevertheless, vidual revealed the presence of low levels of a correctly additional functional studies (described below) indicated spliced mutant c.349A allele transcript (Figure S2A) and that the c.349A canonically spliced transcript itself has an isoform with splicing directly from exon 2 to exon 4 gain-of-function activity and is likely to contribute to (e.g., skipping of exon 3; Figure S2B). disease. RNA-Seq with RNA derived from normal skin or We performed further analyses of wild-type and mutant classical (nonfamilial) psoriatic skin only revealed correctly CARD14 transcripts from the exon 2–4 region by using spliced CARD14 transcripts between exons 3 and 4 (Figures RNA-Seq employing RNA from involved skin from S3 and S4 and Table S3). GEN001. This revealed that 47% of CARD14 transcripts harbored the wild-type c.349G allele and that 12% of tran- Effect of Mutations on CARD14 Function In Vitro scripts harbored the c.349A allele with correct splicing CARD14 encodes a 1,004 amino acid protein that activates between exons 3 and 4. 9% of transcripts harbored the NF-kB20 and is represented by several isoforms. Full-length 66 bp intronic insertion (predicted by the minigene assay) CARD14 (CARD14fl, also known as CARD14 isoform 1) between exons 3 and 4. We also identified 17% of the tran- encodes both an N-terminal CARD domain necessary scripts that were due to the skipping of exon 3 (presumably for activation of NF-kB and a C-terminal tripartite as a result of the c.349A mutation). Similar ratios of wild- MAGUK domain (PDZ/SH3/GUK).18 A shorter isoform, type and mutant transcripts of each type were seen after CARD14sh, encodes the CARD domain but not the tripar- PCR-based cloning and sequencing of cDNA spanning tite domain. Another isoform, CARDcl, lacks the CARD exons 2–4 (Figure S3 and Table S3). domain and the tripartite domain (these latter isoforms However, RNA-Seq revealed an additional RNA species lacking the tripartite domain are both known as isoform that could not be identified with PCR-based amplification 2; Figure S5). Interrogation of a human tissue panel of cDNA. This species extended from exon 3 into the revealed that CARD14sh was the most abundant isoform intron and terminated 838 bp downstream (Figure S3 in all tissues, including skin, in which it was expressed and Table S3). This mRNA isoform was confirmed to be (Table S4). from the sense strand of CARD14 (Figure S4). It was repre- We used luciferase reporter assays to test the effects of sented by ~15% of all transcripts and would encode a the familial and pustular-psoriasis substitutions on the peptide of an additional 59 amino acids after exon 3 and NF-kB-inducing activity of the abundant CARD14sh pro- a30UTR of 656 bp. This transcript would be predicted tein isoform. Expression constructs with the p.Gly117Ser to encode only the CARD domain. This peptide could ex- and p.Gly138Ala substitutions were generated, and an hibit constitutive CARD14-induced activation of NF-kB expression construct encoding CARD14cl, which lacks or exert a dominant-negative effect on wild-type CARD14. the CARD domain, served as a negative control for NF-kB Additional studies are needed if researchers are to deter- activation. A construct encoding the full-length CARD14fl

788 The American Journal of Human Genetics 90, 784–795, May 4, 2012 Figure 3. Familial CARD14 Mutations Alter Splicing of Exon 3 (A) The CARD14 exon 3 minigene constructs are wild-type, c.349G>A (mutation in the northern European PS1 family), and c.349þ5G>A (mutation in the Taiwanese psoriasis-affected family). The vertical line indicates the junction of exon 3 and the intron of CARD14. The following abbreviation is used: RSV, Rous sarcoma virus promoter. (B) Results of agarose gel electrophoresis after transfection of minigenes and isolation of cDNA. Mutant minigene PCR products were 60–70 bp larger than those of the wild-type construct. The following abbreviations are used: Non-tx, nontransfected; Vector, RHCglo25 vector alone; and WT, wild-type minigene. c.349G>A and c.349þ5G>A are the mutant minigenes. (C) Results of sequencing minigenes. Minigene c.349þ5G>A is shown. Both mutations altered splicing of exon 3 and led to the addition of 66 bp from intron 3 and splicing to exon 4 at a cryptic splice donor site (AG/GTGCCC). The following abbreviation is used: sTN1, chicken skeletal troponin I (RHCglo23 downstream gene fragment). (D) Consequence of altered splicing on CARD14. protein isoform was not available for subcloning into the Because TNF-a activates NF-kB pathways, it was impor- expression vector. tant to ascertain whether the increase in NF-kB activity The NF-kB assay revealed that compared with wild-type seen in some of our assays was due to TNF-a secreted by CARD14sh, both substitutions lead to increased levels of transfected cells. The addition of a neutralizing TNF anti- the luciferase reporter. The pustular-psoriasis substitution body did not have an effect on NF-kB reporter activity (p.Glu138Ala) led to a 7- to 9-fold increase in levels, and (Figure S6). These results support the disease-causing the PS1 p.Gly117Ser substitution led to a 3- to 4-fold potential of the familial and de novo pustular-psoriasis increase in levels (Figure 4B). CARD14cl failed to activate substitutions through activation of the NF-kB pathway. NF-kB, in agreement with previous studies.20,37 We also investigated the dominant nature of the substitutions Tissue-Specific Localization of CARD14 by cotransfecting wild-type and either p.Glu138Ala or To examine the localization of CARD14 in healthy and p.Gly117Ser CARD14sh constructs and performing the psoriatic skin, we stained normal and psoriatic skin NF-kB luciferase reporter assay. The resultant NF-kB samples with a polyclonal antibody specific to the coiled- activity was similar to that seen with the p.Glu138Ala coil domain of CARD14 (Figure 5). Staining of basal kerati- and p.Gly117Ser substitutions alone (Figure 4B), providing nocytes was observed in normal and uninvolved skin, and additional evidence of their gain-of function activity. decreased expression was seen in the upper layers of the Cotransfection of CARD14cl and CARD14sh caused skin, including the granular layer. This contrasted with NF-kB activity to be reduced by approximately 50% as involved skin, in which there was loss of labeling of basal a result of either a dilution of CARD14sh or a dominant- keratinocytes but labeling throughout the remainder of negative effect of CARD14cl (Figure 4B). the epidermis and of some dermal cells. There was reduced,

The American Journal of Human Genetics 90, 784–795, May 4, 2012 789 Figure 4. Location of CARD14 Alterations and Their Effect on Transcriptional Activation (A) The location of the familial alterations and the de novo substitution in CARD14 are shown relative to the key protein domains. The two familial alterations are shown by blue triangles; the pustular-psoriasis alteration is shown by the purple triangle. (B) NF-kB activation levels measured in HEK 293 cells transfected with one of four options: (1) CARD14sh alone, (2) the same construct harboring one of the rare variants shown, (3) CARD14cl, or (4) a combination of two constructs from options 1–3. The change in NF-kB activity relative to the background vector was determined for each variant (y axis) (see Subjects and Methods). Every data point repre- sents the average value of three replicates. Asterisks represent results from a two-tailed, unpaired student’s t test between the indicated construct and CARD14sh. The NF-kB activity induced by cotransfection of the p.Gly117Ser substitution and wild-type CARD14sh was not statistically significant from the NF-kB activity induced by CARD14sh alone (p ¼ 0.1) or p.Gly117Ser alone (p ¼ 0.7). (C) HEK 001 cells were transfected with wild-type or altered (c.349G>A [p.Gly117Ser] or c.413A>C [p.Glu138Ala]) CARD14sh, and qRT-PCR was performed so that the upregulation of CCL20, IL8, SOD2, and IL36G identified by global expression profiling could be confirmed. Asterisks represent results from a two-tailed, unpaired student’s t test between the indicated construct and CARD14sh. (D) Upregulation of those same transcripts was confirmed in primary keratinocytes of individual 2192 with pustular psoriasis (c.413A>C [p.Glu138Ala]). Expression in these primary keratinocytes was compared with that in two human foreskin keratinocyte samples (control 1 and control 2). For all qRT-PCR, expression levels were normalized to 18S by the 2-DDCt method. For the transformation of expression levels to nondecimal integers, all expression levels were multiplied by 100,000 before being plotted on graphs. Asterisks represent results from a two-tailed, unpaired student’s t test between the indicated control and the mutant (p.Glu138Ala). For (B–D), error bars represent the standard deviation of replicates, and *p % 0.05, **p % 0.01, and ***p % 0.001. The following abbreviations are used: NT, nontrans- fected; G117S, p.Gly117Ser; E138A, p.Glu138Ala; and Ctrl, control. patchy epidermal labeling in involved skin of GEN001 Effect of CARD14 Substitutions on Keratinocyte (Figures 5A and 5B and Figure S7). Although there might Gene Expression be a subset of dermal immune cells that express CARD14, CARD14 localizes strongly to epidermal keratinocytes (see keratinocytes are clearly a major source of CARD14. above), suggesting that the effect of the substitutions is Previous mRNA-expression studies have reported mediated at least in part through this cell type. We there- elevated levels (a 2.7-fold increase) of CARD14 in involved fore investigated the effect of wild-type and altered psoriatic skin versus uninvolved skin.38 We have con- CARD14 (p.Gly117Ser and p.Glu138Ala) on the transcrip- firmed this with qRT-PCR (not shown). However, our tome of psoriatic skin and keratinocytes separately. We per- data indicate that this apparent increase in gene expression formed global expression profiling of classical psoriatic might be due to an increase in the ratio of CARD14-posi- skin, skin from an affected member (GEN001) of family tive keratinocytes to CARD14-negative keratinocytes in PS1, and skin from individual 2192 with pustular psoriasis, involved skin rather than an increase in the number of and we compared transcript levels with those from normal CARD14 transcripts at the cellular level. skin. We also profiled HEK 001 immortalized keratinocytes

790 The American Journal of Human Genetics 90, 784–795, May 4, 2012 Figure 5. Distribution of CARD14 in Normal and Psoriatic Skin Representative images of normal and psoriatic (uninvolved and involved) skin labeled with a polyclonal antibody to the internal coiled-coil CARD14 domain that is shared by all known iso- forms. (A) Normal skin and classical uninvolved and involved psoriasis skin. (B) A normal-skin negative control and unin- volved and involved affected skin from indi- vidual GEN001 from family PS1. The epidermis is the darker-stained upper band (black arrow), and the dermis is the paler region below. The black line denotes the dermoepidermal junction; lesional skin is cut on a slight cross section, and the dermis is evident as islands projecting upwards into the epidermis. The scale bar repre- sents 100 mm. that had been transfected with a wild-type CARD14sh ways downstream of CARD14 can be similarly altered construct or a CARD14sh construct encoding one of the in classical, pustular, and familial psoriasis. Table S6 missense substitutions (p.Gly117Ser or p.Glu138Ala). We provides fold changes of transcripts that most strongly then looked for genes that were upregulated in psoriatic differentiate classical psoriasis from normal skin, as well skin and that were expressed at significantly higher levels as fold changes of the same transcripts in involved skin in the p.Glu138Ala and p.Gly117Ser transfectants than from GEN001 (PS1) and the child, 2192, with pustular in the wild-type-CARD14sh transfectants (Table S5). psoriasis. A number of transcripts consistently showed increased expression in psoriatic skin, upregulation after keratino- cytes were transfected with CARD14sh, and further Discussion upregulation in the presence of the p.Glu138Ala and p.Gly117Ser substitutions. This was confirmed with qRT- Here, rare, highly penetrant mutations in CARD14 have PCR (Figure 4C). For example, in the presence of the been shown to cause psoriasis, thus concluding a 17 year p.Glu138Ala and p.Gly117Ser substitutions, CCL20 was search for PSORS2.14 We identified two disease-causing upregulated 7.23 and 1.93 more, respectively, and IL8 CARD14 mutations in two multiplex psoriasis-affected was upregulated 4.63 and 1.63 more, respectively, than families (c.349G>A [p.Gly117Ser] in family PS1 of in the presence of wild-type CARD14sh. The p.Glu138Ala northern European ancestry and c.349þ5G>A in a family substitution increased expression of superoxide dismutase from Taiwan). In the European family, approximately 30% 2, mitochondrial (SOD2 [MIM 147460]) and IL36G of affected members also had psoriatic arthritis, indicating 4.13 and 1.83 more, respectively, than did wild-type that the c.349G>A (p.Gly117Ser) mutation might also CARD14sh (Table S5 and Figure 4C). We also confirmed contribute to inflammatory joint disease. Both of the upregulation of these transcripts in primary keratinocytes familial mutations disrupted splicing of CARD14 and from individual 2192 with pustular psoriasis (Figure 4D; lead to an in-frame insertion of 22 amino acids in vitro. primary keratinocytes from family PS1 were not available). The c.349G>A mutation was confirmed in vivo to lead to As a complement to these studies, we also profiled three multiple splice forms, including the 22 amino acid inser- immortalized keratinocyte lines before and after treatment tion predicted from the minigene assay, another with with TNF-a. Two lines were from uninvolved skin of skipping of exon 3, and a third with the addition of 59 affected members (K1-1 and K1-20) of family PS1, and amino acids from intron 3 and a subsequent termination the other was from involved skin of a classical-psoriasis- signal and 661 bp 30UTR. The latter is predicted to result affected individual (K5-14) who harbors the HLA-Cw*0602 in a truncated peptide with only the CARD domain. A risk allele. In all three immortalized keratinocyte lines, de novo mutation (c.413A>C [p.Glu138Ala]) in exon 4 TNF-a stimulation led to upregulation of the same tran- of CARD14 in a child with sporadic, severe, early-onset scripts described above. All of this provided evidence that pustular psoriasis and no family history or susceptibility after an inflammatory trigger, these transcripts are induced factors for psoriasis independently confirmed the disease- in keratinocytes of affected members of family PS1 and of causing potential of these mutations. Interestingly, exon classical-psoriasis-affected individuals (Table S5). 4 encodes part of the coiled-coil domain of the protein. It is also relevant that compared with those of normal A recessive mouse mutation in the coiled-coil domain of skin, the transcriptomes of involved skin from the a structurally related scaffold protein, CARD11, leads familial, pustular-psoriasis, and classical-psoriasis samples to an inflammatory skin phenotype resembling atopic exhibited very similar differences. This suggests that path- dermatitis. This suggests that the coiled-coil domains of

The American Journal of Human Genetics 90, 784–795, May 4, 2012 791 CARD14 and related family members are important in Moreover, basal-level constitutively active phosphorylated regulating inflammation.39 NF-kB/RelA is found in uninvolved skin and is upregulated The identification of a CARD14 mutation in the Taiwa- in psoriasis plaques.42 Uncontrolled NF-kB activation has nese family settles a paradox that existed between the also been suggested as the inflammatory pathway in two multiplex psoriasis-affected families. The Taiwanese a recently published monogenic form of pustular psoriasis family harbors a variant (c.625A>C in the promoter of caused by mutations in IL36RN.32,33 Our findings suggest ZNF750) that was thought to cause psoriasis.19 However, that in psoriasis, NF-kB-pathway alterations implicated we identified no disease-causing mutation in ZNF750 in from GWASs can occur at the level of the keratinocyte as the PS1 family, and we found the ZNF750 c.625A>C well as at the level of the cells of the hematopoietic system. variant in only 2 out of 172 healthy Asian controls. Hence, Both of the missense substitutions reported here signifi- the CARD14 mutation rather than the ZNF750 variant is cantly increased NF-kB activation and led to upregulation probably causative for psoriasis in this family. of psoriasis-associated transcripts such as IL8 and CCL20. It is also worth noting that one PS1 family member who The pustular-psoriasis substitution also led to upregulation harbored the c.349G>A (p.Gly117Ser) mutation was of IL36G and SOD2. TNF-a stimulation of epidermal kerati- initially unaffected when ascertained in 199314 but devel- nocytes has previously been reported to lead to upregula- oped psoriasis 17 years later at 83 years old (indicated by tion of many genes, including IL8, CCL20, and SOD2, the asterisk in Figure 1B). It is unknown which precipi- harboring NF-kB binding sites in their promoters.43–45 tating factors contribute to this variable age of onset, but These transcripts are also notable because of the functions genetic background40 and environmental factors are likely of the they encode. IL8 is a well-known neutro- to play a role. Determining whether the SLC26A11 variant phil chemotaxin,46 and CCL20 is a chemotactic factor þ that cosegregated with psoriasis in family PS1 contributes for CCR6 immature dendritic cells and T cells. IL36G is to psoriasis or psoriatic arthritis independently needs a proinflammatory cytokine, and SOD2 is a potential anti- further investigation. necroptosis gene.47 All of these molecules have been A substantial proportion of individuals with psoriasis shown to be upregulated in keratinocytes in response to have no family history of the disease. This has been injury,48 suggesting that CARD14 mediates an important thought to be due to a requirement for environmental trig- conserved pathway that is triggered in response to disrup- gers and/or inheritance of multiple low-risk genetic factors. ted homeostasis (due to injury, infection, etc.) of the skin. Until now, evidence implicating de novo mutations as The effect of the pustular mutation on gene expression was being causative in psoriasis or other common disease for generally greater than that of the family PS1 mutation in which there is no family history has been lacking. How- transfected keratinocytes. This might reflect an increased ever, the early age of onset of severe generalized pustular severity in the presence of certain mutations such as psoriasis in a child with a de novo mutation indicates c.413G>A (p.Glu138Ala). However, it might be due in that such rare variants can account for some of these cases. part to the fact that we were unable to assay the combined The frequency of de novo mutations contributing to effect of the c.349G>A (p.Gly117Ser) mutation with all of common diseases such as psoriasis is of interest, and genes the in-frame, altered splice forms that occur as a result of harboring such variants are expected to be identified at an this mutation (and that are also predicted as a consequence increasing rate now that global sequencing of DNA from of the Taiwanese psoriasis mutation). Indeed, the relative parents and affected children is feasible. frequency of the splice forms might influence the severity The role of CARD14 in health and disease remains to be of disease as well as the effect of the missense p.Gly117Ser fully determined. It encodes a 1,004 amino acid protein substitution on NF-kB activation. that activates the transcription factor NF-kB20 and inhibits The expression alterations seen in keratinocytes with the apoptosis.37 It has not been implicated previously in psori- PS1 and pustular mutations allow us to propose a model of asis pathogenesis. CARD14 is expressed in the placenta and psoriasis pathogenesis. After an inflammatory trigger that mucosal surfaces,20,37,41 and, as demonstrated here, in the might include infection and/or epidermal injury,48 a subset keratinocytes of normal and inflamed skin. The localiza- of CARD14 substitutions that are found in psoriasis tion pattern of CARD14 differs between normal and patients induce enhanced activation of NF-kB. This leads involved lesional skin. We show that it is localized in the to pathologic levels of transcripts of some target genes, basal layer of the skin and is absent from the upper layers, including key chemokines implicated in psoriasis, such including the granular layer of normal skin, but the oppo- as IL8 and CCL20. We suggest that the release of these site is true of psoriatic skin. In psoriatic skin, CARD14 is inflammatory mediators leads to the recruitment and absent from the basal layer and is present in all suprabasal differentiation of inflammatory cells. Activated dendritic layers. This difference in expression pattern cannot be cells then produce IL-23, a critical factor for Th17 develop- easily explained. However, it highlights intrinsic differ- ment, and T cells produce IL17 and IL22, leading to further ences between keratinocytes of healthy and psoriatic skin. keratinocyte activation and epidermal hyperplasia.49 The Although variants in CARD14 have not been described activation of keratinocytes and expression of IL36G also previously in psoriasis, genetic alterations in components induce the activation of the NF-kB pathway in inflamma- of the NF-kB pathway are implicated from GWASs.5–11 tory cells and the further production of cytokines and

792 The American Journal of Human Genetics 90, 784–795, May 4, 2012 recently identified in some cases of pustular psoriasis.32,33 This protein directly opposes the activity of IL36g and also intersects with the NF-kB pathway. Determining whether and where activation pathways for CARD14 and IL36RN intersect requires further evaluation. Finally, our findings highlight the shortcomings of tradi- tional schemes for classifying human disease on the basis of clinical observations and underscore the utility of adapt- ing molecular classification schemes based on the physi- ology that is dysregulated in disease. In this instance, common plaque psoriasis, pustular psoriasis, and, possibly, psoriatic arthritis, represent different parts of a disease severity spectrum and might all be ascribed to mutations in the same gene (CARD14). Our findings also pave the way for novel therapeutic interventions for these diseases.

Supplemental Data

Figure 6. Proposed Model of Psoriasis Pathogenesis in the Pres- Supplemental Data include seven figures, six tables, and supple- ence of Psoriasis-Specific CARD14 Alterations mental references and can be found with this article online at After an inflammatory trigger, alterations in CARD14 induce activa- http://www.cell.com/AJHG. tion of NF-kB. This leads to the transcription of many genes, including key chemokines such as CCL20, IL8,andIL36G,impli- cated in psoriasis. The proteins encoded by these transcripts can Acknowledgments recruit immune cells involved in disease pathogenesis. Those cells, in turn, produce cytokines and chemokines that cause inflammation This research was supported by the following grants and lead to further keratinocyte activation and epidermal hyper- from the National Institutes of Health: AR050266 and plasia (see Discussion). Other proteins in the NF-kB pathway have 5RC1AR058681 (A.M.B.), T32AR007279 (E.D.O.R.), T32HL083822 been previously implicated in psoriasis and might be important in and T32GM07200 (C.T.J), AR060222 (M.A.L and K.C.P), and CARD14 signaling to NF-kB. These proteins include A20,50 TNIP1,8 T32HG000045 (C.E.J). R.G.M., Y.L., and Y.C. are supported by the and Act1,5,6,9 and these, in turn, implicate TRAF2, TRAF3, and TRAF6.37 IL36RN, recently found to be mutated in pustular psoriasis, Intramural Research Programs of the National Institute of Arthritis might also play a role in this pathway by inhibiting IL36g-induced and Musculoskeletal and Skin Diseases. A.A.M. was supported by immune cell activation.32,33 All of these events contribute to this the Intramural Research Programs of the National Institute of vicious cycle of inflammation and acanthosis seen in psoriasis. Allergy and Infectious Diseases. Additional funding came from the Academia Sinica and National Science Council (National Clinical Core, National Genotyping Core) of Taiwan. The authors chemokines. This creates a vicious cycle of inflammation thank the many individuals with psoriasis and the controls who and acanthosis that characterizes psoriasis (Figure 6). participated in this study. Mike Lovett provided helpful comments Notably, GWASs have identified psoriasis-associated alter- on the manuscript. The authors are indebted to the National ations at other loci harboring genes encoding members Psoriasis Foundation for continuing support during the course of of the NF-kB pathway. For example, variants near TNFAIP3 this study. (tumor necrosis factor, alpha-induced protein 3, [TNFAIP3 (MIM 191163)]) are associated with psoriasis. That gene Received: December 27, 2011 encodes the protein A20, which negatively regulates NF- Revised: February 29, 2012 kB activation by the CARD14-related peptides CARD11 Accepted: March 13, 2012 and CARD10.50 TNIP1 (TNFAIP3-interacting protein 1, Published online: April 19, 2012 [TNIP1 (MIM 607714)]) might also be involved in this process because it acts in concert with A20 and is downre- Web Resources gulated in psoriatic skin.8 Although future studies are The URLs for data presented here are as follows: needed for the dissection of the interaction between CARD14 and the NF-kB pathway, we know that CARD14- ClustalW2, http://www.ebi.ac.uk/Tools/msa/clustalw2/ induced activation of NF-kB is dependent on TRAF2 Microarray Gene Expression Data Society (MIAME), http://www. and might also require TRAF3 or TRAF6.37 Thus, one mged.org/Workgroups/MIAME_checklist.html NCBI Gene Expression Omnibus (GEO), http://www.ncbi.nlm. can hypothesize that activation of the CARD14-NF-kB nih.gov/geo/ pathway might be downstream of Act1, encoded by TRAF3IP2 NCBI Reference Sequence (RefSeq), http://www.ncbi.nlm.nih. (TRAF3-interacting protein 2, [TRAF3IP2 (MIM gov/RefSeq/ 607043)]), which harbors a variant associated with psoriasis Online Mendelian Inheritance in Man (OMIM), http://omim.org 5,6,9 and psoriatic arthritis. Alternatively, CARD14 might PolyPhen-2.0, http://genetics.bwh.harvard.edu/pph2/ be part of a parallel pathway leading to NF-kB activation. SIFT, http://sift.jcvi.org/ Loss-of-function amino acid substitutions in IL36RN were UCSC Genome Browser, http://genome.ucsc.edu/

The American Journal of Human Genetics 90, 784–795, May 4, 2012 793 References tibility mapped to the distal end of human 17q. Science 264, 1141–1145. 1. Lowes, M.A., Bowcock, A.M., and Krueger, J.G. (2007). Patho- 15. Nair, R.P., Henseler, T., Jenisch, S., Stuart, P., Bichakjian, C.K., genesis and therapy of psoriasis. Nature 445, 866–873. Lenk, W., Westphal, E., Guo, S.W., Christophers, E., Voorhees, 2. Nograles, K.E., Brasington, R.D., and Bowcock, A.M. (2009). J.J., and Elder, J.T. (1997). Evidence for two psoriasis suscepti- New insights into the pathogenesis and genetics of psoriatic bility loci (HLA and 17q) and two novel candidate regions arthritis. Nat. Clin. Pract. Rheumatol. 5, 83–91. (16q and 20p) by genome-wide scan. Hum. Mol. Genet. 6, 3. Capon, F., Bijlmakers, M.J., Wolf, N., Quaranta, M., Huffmeier, 1349–1356. U., Allen, M., Timms, K., Abkevich, V., Gutin, A., Smith, R., 16. Hwu, W.L., Yang, C.F., Fann, C.S., Chen, C.L., Tsai, T.F., Chien, et al. (2008). Identification of ZNF313/RNF114 as a novel Y.H., Chiang, S.C., Chen, C.H., Hung, S.I., Wu, J.Y., and Chen, 17 psoriasis susceptibility gene. Hum. Mol. Genet. , 1938– Y.T. (2005). Mapping of psoriasis to 17q terminus. J. Med. 1945. Genet. 42, 152–158. 4. Cargill, M., Schrodi, S.J., Chang, M., Garcia, V.E., Brandon, R., 17. Helms, C., Cao, L., Krueger, J.G., Wijsman, E.M., Chamian, F., Callis, K.P., Matsunami, N., Ardlie, K.G., Civello, D., Catanese, Gordon, D., Heffernan, M., Daw, J.A., Robarge, J., Ott, J., et al. J.J., et al. (2007). A large-scale genetic association study (2003). A putative RUNX1 binding site variant between confirms IL12B and leads to the identification of IL23R as SLC9A3R1 and NAT9 is associated with susceptibility to 80 psoriasis-risk genes. Am. J. Hum. Genet. , 273–290. psoriasis. Nat. Genet. 35, 349–356. 5. Ellinghaus, E., Ellinghaus, D., Stuart, P.E., Nair, R.P., Debrus, S., 18. Speckman, R.A., Wright Daw, J.A., Helms, C., Duan, S., Cao, L., Raelson, J.V., Belouchi, M., Fournier, H., Reinhard, C., Ding, J., Taillon-Miller, P., Kwok, P.Y., Menter, A., and Bowcock, A.M. et al. (2010). Genome-wide association study identifies a psori- (2003). Novel immunoglobulin superfamily gene cluster, 42 asis susceptibility locus at TRAF3IP2. Nat. Genet. , 991–995. mapping to a region of human chromosome 17q25, linked 6. Hu¨ffmeier, U., Uebe, S., Ekici, A.B., Bowes, J., Giardina, E., to psoriasis susceptibility. Hum. Genet. 112, 34–41. Korendowych, E., Juneblad, K., Apel, M., McManus, R., Ho, P., 19. Yang, C.F., Hwu, W.L., Yang, L.C., Chung, W.H., Chien, Y.H., et al. (2010). Common variants at TRAF3IP2 are associated Hung, C.F., Chen, H.C., Tsai, P.J., Fann, C.S., Liao, F., and with susceptibility to psoriatic arthritis and psoriasis. Nat. Chen, Y.T. (2008). A promoter sequence variant of ZNF750 42 Genet. , 996–999. is linked with familial psoriasis. J. Invest. Dermatol. 128, 7. Liu, Y., Helms, C., Liao, W., Zaba, L.C., Duan, S., Gardner, J., 1662–1668. Wise, C., Miner, A., Malloy, M.J., Pullinger, C.R., et al. 20. Bertin, J., Wang, L., Guo, Y., Jacobson, M.D., Poyet, J.L., Srini- (2008). A genome-wide association study of psoriasis and vasula, S.M., Merriam, S., DiStefano, P.S., and Alnemri, E.S. 4 psoriatic arthritis identifies new disease loci. PLoS Genet. , (2001). CARD11 and CARD14 are novel caspase recruitment e1000041. domain (CARD)/membrane-associated guanylate kinase 8. Nair, R.P.,Duffin, K.C., Helms, C., Ding, J., Stuart, P.E.,Goldgar, (MAGUK) family members that interact with BCL10 and acti- D., Gudjonsson, J.E., Li, Y., Tejasvi, T., Feng, B.J., et al; Collabo- vate NF-kappa B. J. Biol. Chem. 276, 11877–11882. rative Association Study of Psoriasis. (2009). Genome-wide 21. Kaur, P., McDougall, J.K., and Cone, R. (1989). Immortaliza- scan reveals association of psoriasis with IL-23 and NF-kappaB tion of primary human epithelial cells by cloned cervical carci- pathways. Nat. Genet. 41, 199–204. noma DNA containing human papillomavirus type 16 E6/E7 9. Strange, A., Capon, F., Spencer, C.C., Knight, J., Weale, M.E., open reading frames. J. Gen. Virol. 70, 1261–1266. Allen, M.H., Barton, A., Band, G., Bellenguez, C., Bergboer, 22. Chapman, S., Liu, X., Meyers, C., Schlegel, R., and McBride, J.G., et al; Genetic Analysis of Psoriasis Consortium & the A.A. (2010). Human keratinocytes are efficiently immortalized Wellcome Trust Case Control Consortium 2. (2010). A by a Rho kinase inhibitor. J. Clin. Invest. 120, 2619–2626. genome-wide association study identifies new psoriasis 23. Harbour, J.W., Onken, M.D., Roberson, E.D., Duan, S., Cao, L., susceptibility loci and an interaction between HLA-C and Worley, L.A., Council, M.L., Matatall, K.A., Helms, C., and ERAP1. Nat. Genet. 42, 985–990. Bowcock, A.M. (2010). Frequent mutation of BAP1 in metasta- 10. Stuart, P.E., Nair, R.P., Ellinghaus, E., Ding, J., Tejasvi, T., sizing uveal melanomas. Science 330, 1410–1413. Gudjonsson, J.E., Li, Y., Weidinger, S., Eberlein, B., Gieger, 24. Ng, S.B., Turner, E.H., Robertson, P.D., Flygare, S.D., Bigham, C., et al. (2010). Genome-wide association analysis identifies A.W., Lee, C., Shaffer, T., Wong, M., Bhattacharjee, A., Eichler, three psoriasis susceptibility loci. Nat. Genet. 42, 1000–1004. E.E., et al. (2009). Targeted capture and massively parallel 11. Sun, L.D., Cheng, H., Wang, Z.X., Zhang, A.P., Wang, P.G., Xu, sequencing of 12 human exomes. Nature 461, 272–276. J.H., Zhu, Q.X., Zhou, H.S., Ellinghaus, E., Zhang, F.R., et al. 25. Singh, G., and Cooper, T.A. (2006). Minigene reporter for (2010). Association analyses identify six new psoriasis suscep- identification and analysis of cis elements and trans factors tibility loci in the Chinese population. Nat. Genet. 42, 1005– affecting pre-mRNA splicing. Biotechniques 41, 177–181. 1009. 26. Vincourt, J.B., Jullien, D., Amalric, F., and Girard, J.P. (2003). 12. Vineis, P., and E Pearce, N. (2011). Genome-wide association Molecular and functional characterization of SLC26A11, studies may be misinterpreted: Genes versus heritability. a sodium-independent sulfate transporter from high endothe- Carcinogenesis 32, 1295–1298. lial venules. FASEB J. 17, 890–892. 13. Chen, H., Poon, A., Yeung, C., Helms, C., Pons, J., Bowcock, 27. Adzhubei, I.A., Schmidt, S., Peshkin, L., Ramensky, V.E., A.M., Kwok, P.Y., and Liao, W. (2011). A genetic risk score Gerasimova, A., Bork, P., Kondrashov, A.S., and Sunyaev, S.R. combining ten psoriasis risk loci improves disease prediction. (2010). A method and server for predicting damaging PLoS ONE 6, e19454. missense mutations. Nat. Methods 7, 248–249. 14. Tomfohrde, J., Silverman, A., Barnes, R., Fernandez-Vina, 28. Kumar, P., Henikoff, S., and Ng, P.C. (2009). Predicting the M.A., Young, M., Lory, D., Morris, L., Wuepper, K.D., Stastny, effects of coding non-synonymous variants on protein func- P., Menter, A., et al. (1994). Gene for familial psoriasis suscep- tion using the SIFT algorithm. Nat. Protoc. 4, 1073–1081.

794 The American Journal of Human Genetics 90, 784–795, May 4, 2012 29. Kent, W.J., Sugnet, C.W., Furey, T.S., Roskin, K.M., Pringle, 40. Prots, I., Skapenko, A., Wendler, J., Mattyasovszky, S., Yone´, T.H., Zahler, A.M., and Haussler, D. (2002). The human C.L., Spriewald, B., Burkhardt, H., Rau, R., Kalden, J.R., Lipsky, genome browser at UCSC. Genome Res. 12, 996–1006. P.E., and Schulze-Koops, H. (2006). Association of the IL4R 30. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., single-nucleotide polymorphism I50V with rapidly erosive Higgins, D.G., and Thompson, J.D. (2003). Multiple sequence rheumatoid arthritis. Arthritis Rheum. 54, 1491–1500. alignment with the Clustal series of programs. Nucleic Acids 41. Blonska, M., and Lin, X. (2011). NF-kB signaling pathways Res. 31, 3497–3500. regulated by CARMA family of scaffold proteins. Cell Res. 31. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., 21, 55–70. McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., 42. Lizzul, P.F., Aphale, A., Malaviya, R., Sun, Y., Masud, S., Dom- Wilm, A., Lopez, R., et al. (2007). Clustal W and Clustal X brovskiy, V., and Gottlieb, A.B. (2005). Differential expression version 2.0. Bioinformatics 23, 2947–2948. of phosphorylated NF-kappaB/RelA in normal and psoriatic 32. Marrakchi, S., Guigue, P., Renshaw, B.R., Puel, A., Pei, X.Y., epidermis and downregulation of NF-kappaB in response to Fraitag, S., Zribi, J., Bal, E., Cluzeau, C., Chrabieh, M., et al. treatment with etanercept. J. Invest. Dermatol. 124, 1275– (2011). Interleukin-36-receptor antagonist deficiency and 1283. generalized pustular psoriasis. N. Engl. J. Med. 365, 620–628. 43. Stein, B., and Yang, M.X. (1995). Repression of the inter- 33. Onoufriadis, A., Simpson, M.A., Pink, A.E., Di Meglio, P., leukin-6 promoter by estrogen receptor is mediated by NF- Smith, C.H., Pullabhatla, V., Knight, J., Spain, S.L., Nestle, kappa B and C/EBP beta. Mol. Cell. Biol. 15, 4971–4979. F.O., Burden, A.D., et al. (2011). Mutations in IL36RN/IL1F5 44. Banno, T., Gazel, A., and Blumenberg, M. (2005). Pathway- are associated with the severe episodic inflammatory skin specific profiling identifies the NF-kappa B-dependent tumor disease known as generalized pustular psoriasis. Am. J. Hum. necrosis factor alpha-regulated genes in epidermal keratino- Genet. 89, 432–437. cytes. J. Biol. Chem. 280, 18973–18980. 34. Elder, J.T.; Cluster 17 Collaboration. (2005). Fine mapping of 45. Banno, T., Gazel, A., and Blumenberg, M. (2004). Effects of the psoriasis susceptibility gene PSORS1: A reassessment of tumor necrosis factor-alpha (TNF alpha) in epidermal kerati- risk associated with a putative risk haplotype lacking HLA- nocytes revealed using global transcriptional profiling. J. Cw6. J. Invest. Dermatol. 124, 921–930. Biol. Chem. 279, 32633–32642. 35. 1000 Genomes Project Consortium. (2010). A map of human 46. Sticherling, M., Sautier, W., Schro¨der, J.M., and Christophers, genome variation from population-scale sequencing. Nature E. (1999). Interleukin-8 plays its role at local level in psoriasis 467, 1061–1073. vulgaris. Acta Derm. Venereol. 79, 4–8. 36. Jordan, C.T., Cao, L., Roberson, E.D.O., Duan, S., Helms, C.A., 47. Thapa, R.J., Basagoudanavar, S.H., Nogusa, S., Irrinki, K., Nair, R.P., Duffin, K.C., Stuart, P.E., Goldgar, D., Hayashi, G., Mallilankaraman, K., Slifker, M.J., Beg, A.A., Madesh, M., et al. (2012). Rare and Common Variants in CARD14, Encod- and Balachandran, S. (2011). NF-kappaB protects cells from ing an Epidermal Regulator of NF-kappaB, in Psoriasis. Am. J. gamma interferon-induced RIP1-dependent necroptosis. Hum. Genet. 90, in press. Published online April 19, 2012. Mol. Cell. Biol. 31, 2934–2946. 10.1016/j.ajhg.2012.03.013. 48. Kennedy-Crispin, M., Billick, E., Mitsui, H., Gulati, N., Fujita, 37. Scudiero, I., Zotti, T., Ferravante, A., Vessichelli, M., Vito, P., H., Gilleaudeau, P., Sullivan-Whalen, M., Johnson-Huang, and Stilo, R. (2011). Alternative splicing of CARMA2/ L.M., Suarez-Farinas, M., and Krueger, J.G. (2012). Human CARD14 transcripts generates protein variants with differen- Keratinocytes’ Response to Injury Upregulates CCL20 and tial effect on NF-kB activation and endoplasmic reticulum Other Genes Linking Innate and Adaptive Immunity. J Invest stress-induced cell death. J. Cell. Physiol. 226, 3121–3131. Dermatol. 132, 105–113. Published online September 1, 2011. 38. Sua´rez-Farin˜as, M., Lowes, M.A., Zaba, L.C., and Krueger, J.G. 10.1038/jid.2011.262. (2010). Evaluation of the psoriasis transcriptome across 49. Johnston, A., Xing, X., Guzman, A.M., Riblett, M., Loyd, C.M., different studies by gene set enrichment analysis (GSEA). Ward, N.L., Wohn, C., Prens, E.P., Wang, F., Maier, L.E., et al. PLoS ONE 5, e10247. (2011). IL-1F5, -F6, -F8, and -F9: a novel IL-1 family signaling 39. Jun, J.E., Wilson, L.E., Vinuesa, C.G., Lesage, S., Blery, M., system that is active in psoriasis and promotes keratinocyte Miosge, L.A., Cook, M.C., Kucharska, E.M., Hara, H., antimicrobial peptide expression. J. Immunol. 186, 2613– Penninger, J.M., et al. (2003). Identifying the MAGUK protein 2622. Carma-1 as a central regulator of humoral immune responses 50. Stilo, R., Varricchio, E., Liguoro, D., Leonardi, A., and Vito, P. and atopy by genome-wide mouse mutagenesis. Immunity (2008). A20 is a negative regulator of BCL10- and CARMA3- 18, 751–762. mediated activation of NF-kappaB. J. Cell Sci. 121, 1165–1171.

The American Journal of Human Genetics 90, 784–795, May 4, 2012 795