Common variants in CASP3 confer susceptibility to Kawasaki disease

Abstract

Kawasaki disease (KD; OMIM 611775) is an acute vasculitis syndrome which predominantly affects small- and medium-sized arteries of infants and children. Epidemiological data suggest that host genetics underlie the disease pathogenesis. Here we report that multiple variants in the caspase-3 gene (CASP3) that are in linkage disequilibrium confer susceptibility to KD in both Japanese and US subjects of European ancestry. We found that a G to A substitution of one commonly associated SNP located in the 5' untranslated region of CASP3 (rs72689236; P = 4.2 x 10(-8) in the Japanese and P = 3.7 x 10(-3) in the European Americans) abolished binding of nuclear factor of activated T cells to the DNA sequence surrounding the SNP. Our findings suggest that altered CASP3 expression in immune effecter cells influences susceptibility to KD.

Figure 1.

Linkage disequilibrium (LD) structure of the CASP3 locus and association of the variants with KD in Japanese subjects. Pairwise LD plots with 46 variants distributed across the 36 kb region in and surrounding CASP3 are illustrated using Haploview software. Values for r² were calculated using genotype data from Japanese control samples (n = 1031). Blue horizontal bars under SNP IDs represent LD blocks defined by Gabriel's rule. The genomic organization of CASP3 and the coiled-coil domain containing 111 (CCDC111; only 5′ part is shown) is illustrated with blue and gray boxes representing the exons. Arrows under the gene names indicate the orientation of transcription. The position and the negative log of the P-values from the genetic association study (637 KD cases and 1031 controls; allelic frequency comparison) for each variant tested are shown by vertical bars in the upper panel. Threshold for statistical significance (P = 0.001) was indicated by a gray horizontal line in the upper panel.

Figure 2.

Functional analyses of the G and A alleles of rs72689236. (A) Single or four tandem copies of oligonucleotides for the G allele and A allele of rs72689236 were cloned upstream of the SV40 promoter in the PGL3 luciferase reporter vector and transfected into Jurkat cells (left), PBMCs (middle) and CD3⁺ peripheral T cells (right; single assay). Data represent mean ± SEM of triplicate assays for Jurkat and PBMCs. (B) Effect of cyclosporine A on enhancer activity of rs72689236 G allele. (C) Transcriptional activity of CASP3 promoter with different alleles of rs72689236. Data represent mean ± SEM of quintuplicate assays. (D) EMSA was performed using nuclear extracts from PBMCs stimulated with ionomycin and PMA. Oligonucleotides corresponding to the A allele (lanes 1–3) and to the G allele (lanes 4–6) were used as probes. Binding reaction was performed with no specific competitor and with excess amounts (×100) of either unlabelled G or A allele oligonucleotides. n.s., non specific bands. (E) Binding of NFATs to the rs72689236 G allele was assessed by EMSA using nuclear extracts from PBMCs treated with cyclosporine A in addition to ionomycin and PMA (lane 1), competition assay using oligonucleotides containing an NFAT binding sequence from the human IL-2 promoter or its mutant (lanes 3 and 4) and a supershift assay with antibodies against NFATc1 (lane 5), NFATc2 (lane 6) and their isotype controls (lanes 7 and 8).

Figure 3.

Allele-specific transcript quantification (ASTQ) of CASP3 in PBMC. (A) Genomic structure of CASP3 gene and location of the two SNPs (rs72689236 and rs6948). An amplicon of PCR was indicated by a horizontal bar. Haplotypes for the two SNPs with frequencies larger than 2.5% in the Japanese population were shown. (B) Comparison of relative expression level of CASP3 mRNA from different haplotypes. Ratio of digested and undigested PCR products from genomic DNA and cDNA of PBMCs stimulated with PMA and ionomycin from healthy volunteers heterozygous (left panel; n = 8) and homozygous (right panel; n = 5) at rs72689236. *two-tailed P = 3.0 × 10⁻⁷, **two-tailed P = 0.40 by Student's t-test.