Rare and common variants in CARD14, encoding an epidermal regulator of NF-kappaB, in psoriasis

Abstract

Psoriasis is a common inflammatory disorder of the skin and other organs. We have determined that mutations in CARD14, encoding a nuclear factor of kappa light chain enhancer in B cells (NF-kB) activator within skin epidermis, account for PSORS2. Here, we describe fifteen additional rare missense variants in CARD14, their distribution in seven psoriasis cohorts (>6,000 cases and >4,000 controls), and their effects on NF-kB activation and the transcriptome of keratinocytes. There were more CARD14 rare variants in cases than in controls (burden test p value = 0.0015). Some variants were only seen in a single case, and these included putative pathogenic mutations (c.424G>A [p.Glu142Lys] and c.425A>G [p.Glu142Gly]) and the generalized-pustular-psoriasis mutation, c.413A>C (p.Glu138Ala); these three mutations lie within the coiled-coil domain of CARD14. The c.349G>A (p.Gly117Ser) familial-psoriasis mutation was present at a frequency of 0.0005 in cases of European ancestry. CARD14 variants led to a range of NF-kB activities; in particular, putative pathogenic variants led to levels >2.5× higher than did wild-type CARD14. Two variants (c.511C>A [p.His171Asn] and c.536G>A [p.Arg179His]) required stimulation with tumor necrosis factor alpha (TNF-α) to achieve significant increases in NF-kB levels. Transcriptome profiling of wild-type and variant CARD14 transfectants in keratinocytes differentiated probably pathogenic mutations from neutral variants such as polymorphisms. Over 20 CARD14 polymorphisms were also genotyped, and meta-analysis revealed an association between psoriasis and rs11652075 (c.2458C>T [p.Arg820Trp]; p value = 2.1 × 10(-6)). In the two largest psoriasis cohorts, evidence for association increased when rs11652075 was conditioned on HLA-Cw*0602 (PSORS1). These studies contribute to our understanding of the genetic basis of psoriasis and illustrate the challenges faced in identifying pathogenic variants in common disease.

Figure 1

CARD14 Protein Domains and Locations of Amino Acid Substitutions Missense variants identified in CARD14 by resequencing are shown relative to key protein domains. Red outlining indicates increased NF-kB activation by a variant (relative to wild-type); green outlining indicates reduced activation. Gold outlining indicates variants that showed increased NF-kB activation only in response to TNF-α stimulation. SNPs and variants also identified in dbSNP135, which includes data from the 1,000 Genomes Project and the National Heart, Lung, and Blood Institute (NHLBI) and National Human Genome Research Institute (NHGRI) Exome Project, are indicated with blue triangles. A polymorphism, rs114688446, was observed at the same site as p.Ser200Asn, but a different substitution, p.Ser200Ile, was detected. SNPs used for meta-analysis are indicated with gray triangles.

Figure 2

Meta-Analyses of rs11652075 and p.Ser200Asn across Six Case/Control Cohorts of European Ancestry The results of the fixed- (A) and random-effects (B) meta-analyses for rs11652075 (c.2458C>T [p.Arg820Trp]) and the fixed-effects meta-analysis for p.Ser200Asn (c.599G>A) (C) are shown. The random-effects meta-analysis for p.Ser200Asn was identical to that of the fixed-effects meta-analysis and is therefore not pictured. Forest plots indicate the direction of effect, relative weight, and confidence interval for the odds ratio of this SNP in each cohort. The number of cases and controls successfully genotyped in each cohort is shown, and the meta-analysis OR and p value are listed below each plot. The following abbreviations are used: OR, odds ratio; and C.I., confidence interval.

Figure 3

Effect of Wild-Type and Altered CARD14 on NF-kB Activation HEK 293 cells were transfected with the construct that codes for CARD14sh, the same construct harboring one of the rare variants shown, or a construct that codes for CARD14cl and lacks the CARD domain. NF-kB activity was determined by measuring relative luciferase activity. Transfection efficiency was controlled for by first normalizing all values to Renilla expression, and activity of the empty background vector, pTAL-luc, was controlled for by adjusting the values. Change in NF-kB activity relative to the background vector was determined for each variant (y axis, NF-kB activity). Every data point represents the average value of three replicates. Error bars represent the standard deviation of replicates. For experiments involving TNF-α stimulation, treated cells were exposed to 20 ng/ml TNF-α in culture media for 24 hr. Asterisks show results from two-tailed, unpaired student's t tests comparing NF-kB activation induced by the indicated variant to either that of unstimulated cells with CARD14sh (A) or that of TNF-α-stimulated cells with CARD14sh (B). ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, and ^∗∗∗p ≤ 0.001.

Figure 4

Heat Map Generated after Transcriptome Analysis of Wild-Type and Mutant CARD14 Transfectants Cells were transfected with CARD14cl, wild-type CARD14sh, or variant CARD14 expression constructs. After 24 hr, RNA was extracted and used for the interrogation of Illumina bead arrays. A subset of differentially expressed genes was used for clustering of CARD14 variants (see Subjects and Methods).