ITPKC functional polymorphism associated with Kawasaki disease susceptibility and formation of coronary artery aneurysms

Abstract

Kawasaki disease is a pediatric systemic vasculitis of unknown etiology for which a genetic influence is suspected. We identified a functional SNP (itpkc_3) in the inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) gene on chromosome 19q13.2 that is significantly associated with Kawasaki disease susceptibility and also with an increased risk of coronary artery lesions in both Japanese and US children. Transfection experiments showed that the C allele of itpkc_3 reduces splicing efficiency of the ITPKC mRNA. ITPKC acts as a negative regulator of T-cell activation through the Ca2+/NFAT signaling pathway, and the C allele may contribute to immune hyper-reactivity in Kawasaki disease. This finding provides new insights into the mechanisms of immune activation in Kawasaki disease and emphasizes the importance of activated T cells in the pathogenesis of this vasculitis.

Figure 1

Results of SNP screening of chromosome 19 and structure of the linkage disequilibrium (LD) block in Japanese individuals showing SNPs significantly associated with Kawasaki disease. (a) Maximum lod score plot of affected sib-pair analysis conducted on 78 Japanese families. MLS, maximum lod score. (b) Case-control association analysis of 1,222 SNPs in 94 individuals with Kawasaki disease and 564 controls. × and y axes indicate the position from the p terminus of the chromosome and −log of P value for allele frequency comparison, respectively. The three most significant SNPs are marked by red dots. (c) Genes oriented q terminus to p terminus are in upper row, with genes in the opposite orientation shown below. Arrowheads indicate the position of SNPs significantly associated with Kawasaki disease: red arrowheads indicate the original three SNPs found by association studies, and blue arrowheads indicate the six SNPs from resequencing that were in LD with original three SNPs.

Figure 2

Comparison of relative mRNA expression of ITPKC in different tissues and cell lines. (a) Quantitative RT-PCR was carried out on RNA extracted from different human tissues, and the results were normalized to β-actin transcripts. RNA from both resting PBMCs and PBMCs stimulated with ionomycin (iono) and PMA was also analyzed. Results are mean ± s.d. of triplicate assays. (b) Expression pattern of ITPK isoforms in leukemic cell lines and PBMCs. Bars indicate relative mRNA copy number of ITPKA (yellow), ITPKB (red) and ITPKC (black), respectively. Expression was evaluated both in resting state and activated state. NS, no stimulation.

Figure 3

Allele-specific transcript quantification of ITPKC in PBMC. (a) Genomic organization of the genes. Exons of NUMBL, ADCK4, ITPKC, FLJ41131 and SNRPA are shown with purple, green, red, blue and black filled boxes, respectively. Positions of the SNPs within the genes are indicated by open triangles. Haplotypes of volunteers based on their genotype at numbl_6, adck4_14, itpkc_3, itpkc_14 and flj41131_3 (frequency > 1%) are shown. The G allele in itpkc_14 creates a SmaI site. (b,c) ASTQ showing decreased undigested transcript of ITPKC associated with haplotype III. *Two-tailed P value by Welch's t-test.

Figure 4

Negative regulatory role of ITPKC expression in stimulated Jurkat cells. (a) Plasmid constructs used for transfection: pNFAT contains three tandem repeats of the NFAT/AP1 (N/A) binding sites driving luciferase expression in the pGL3-Basic vector; pITPKC contains the CMV promoter driving expression of ITPKC cloned into pcDNA3.1(+); pITPKC shRNA contains the human U6 promoter substituted for the CMV promoter in pcDNA3.1(+) driving expression of a short hairpin RNA (shRNA) targeting ITPKC mRNA; pControl shRNA contains the human U6 promoter driving expression of a random shRNA. (b,c) Effects of ITPKC overexpression on luciferase activity (b) or IL-2 expression (c) in cells transfected with constructs in a. (d–h) Effect of ITPKC knockdown by transfection of shRNA. (d–f) Specific knockdown of ITPKC by shRNA. (g) Effect of ITPKC knockdown on luciferase expression mediated by the NFAT/AP1 binding sites in the IL2 promoter. (f) Effect of ITPKC knockdown on IL-2 expression. Results are mean ± s.d. of quadruplicate assays in c and triplicate assays in a,b,d–h. *Two-tailed P > 0.1, **two-tailed P < 0.05, by Student's t-test.

Figure 5

Reduced splicing efficiency of intron 1 and reduced ITPKC transcript abundance mediated by the itpkc_3 C allele. (a) Plasmids were constructed for the G allele, the C allele and the G allele with a mutation in the 5′ splice site (pSDM) as a negative control for luciferase activity. SNPs and a mutation are represented by underlined red and blue text, respectively. Positions of the primers for RT-PCR analysis are indicated by small arrows. (b) The function of itpkc_3 was evaluated by luciferase assay in transfected and stimulated Jurkat cells. Data represent mean ± s.d. of quintuplicate assays. *Two-tailed P < 0.02 by Student's t-test. (c) PCR of spliced and unspliced transcripts with or without an RT step. Representative gel image of five independent experiments is shown. **Mean ratio of fluorescent intensity corresponding to spliced and unspliced transcripts. ***Two-tailed P value by Student's t-test.