Kawasaki disease (KD) is the commonest cause of acquired heart disease among children in developed/industrialized countries. The incidence of KD varies between different countries, with Asian countries having a higher incidences than Western countries. Japan has the highest annual incidence in the world with 200 KD cases per 100,000 children under age 5 [1], [2], followed by Korea, with an annual incidence of 113.1 KD cases per 100,000 children under 5 years of age [3]. The incidence in Taiwan is 69 KD cases per 100,000 children under age 5 [4], [5], which ranks third after Japan and Korea. KD is an acute, self-limiting vasculitis of infants and children. Symptoms include prolonged fever that is unresponsive to antibiotics, polymorphous skin rash, swollen glands, red eyes, inflammation of the mouth, extensive rash, and swollen and red hands and feet. Characteristic peeling of the skin on fingers and toes occurs during convalescence [6]. Coronary aneurysms develop in 15%–25% of untreated children [6], [7]. The cause of this disease is currently unknown, although clinical and epidemiological features strongly suggest that an infectious agent triggers KD and genetic predisposition may underlie its etiology. It has been proposed that an inflammatory stimulus sets in motion a cascade of events that leads to host immune dysregulation in genetically predisposed individuals. During the acute stage of KD, infiltration of CD8-positive T cells and macrophages, activation of vascular endothelial cells, and increased serum levels of proinflammatory cytokines lead to inflammation and injury of blood vessels [8], [9]. Thus, identification of predisposing genetic factors would greatly facilitate understanding of the disease etiology and pathophysiology.

A number of genes that participate in immune-regulatory responses as well as cardiovascular-related loci have been extensively studied for an association with KD susceptibility or disease outcome using the candidate gene approach [10]. Several candidate genes have been examined in independent cohorts of the same or different ethnicity; however, the results are often conflicting. Most previous studies have been carried out on a single small cohort of KD patients, and findings were reported without validation in additional case-control sets. There have been few genome-wide studies of this disease. A genome-wide linkage analysis conducted with Japanese KD sib-pair samples revealed several linked regions, and led to the identification of a functional polymorphism in the ITPKC (inositol 1,4,5-triphosphate 3-kinase C) gene [11], [12]. A genome-wide association study (GWAS) conducted in an international cohort of Caucasian patients identified variants of genes with functions potentially related to inflammation, apoptosis, and cardiovascular pathology with NAALADL2 (rs17531088, p_combined = 1.13×10^-6 and ZFHX3 (rs7199343, p_combined = 2.37×10^-6 most significantly associated [13].

To identify novel mechanisms that might predispose patients to KD, we conducted a two-stage GWAS scan for KD in a Han Chinese population residing in Taiwan. In this paper, we present results of the first GWAS for KD in a Han-Chinese population, and identify new KD susceptibility loci that are involved in the immune response and are associated with increased risk of KD.

To the best of our knowledge, this study is the first report of a GWAS for KD conducted in a Han Chinese population. In this study, we identified novel candidates associated with KD susceptibility. The identification of these genes may provide new insights into the development of KD.

Three KD-associated SNPs—rs1873668, rs4243399, and rs16849083—are located in the same LD of chromosome 3q23 region, which contains three genes. The head-to-tail overlapping genes MRPS22 and COPB2 are located nearest to rs1873668 and rs4243399, whereas rs16849083 is located in the intronic region of PBP2. Mutations in MRPS22, a mitochondrial ribosomal protein, lead to antenatal mitochondrial disease, which presents with antenatal skin edema, hypotonia, cardiomyopathy, and tubulopathy [18]. PBP2 is thought to participate in the uptake and/or intracellular metabolism of vitamin A. The roles of NRPS22 and PBP2 have no obvious connection to the pathogenesis of KD. COPB2 constitutes the coat of nonclathrin-coated vesicles and is essential for Golgi budding and vesicular trafficking. A phosphoproteomic analysis of T cell receptor signaling, which identified proteins involved in receptor and membrane trafficking in endosomes and vesicles playing a role in T cell receptor signaling and critical for T cell activation [19]. The possibility of COPB2 involvement in T cell activation and KD pathogenesis will require further investigation.

We identified two KD-associated SNPs, rs149481 and rs27042, located in an intron of the ERAP1 gene. ERAP1 plays a critical role in trimming peptides to the optimal length for HLA class I presentation [20] and cleaving cell surface receptors for proinflammatory cytokines. It is ubiquitously expressed in every tissue, and is expressed at higher levels in the trachea, thymus, and lymph nodes (based on a National Center for Biotechnology Information [NCBI] expressed sequence tags profile). From a functional perspective, ERAP1 represent an excellent biological candidate for KD pathogenesis. ERAP1 trimming of peptides for HLA class I presentation could play a critical role in initiating the immune response. Furthermore, ERAP1 cleaves cell surface receptors for the proinflammatory cytokines IL-1 [21], IL-6 [22], and TNF [23], thereby downregulating their signaling. Genetic variants that alter the functioning of ERAP1 could, therefore, have proinflammatory effects through this mechanism. In addition to their association with KD, ERAP1 polymorphisms have been found to be associated with ankylosing spondylitis, an HLA class I–mediated autoimmune disease, and enthesitis, a common inflammatory arthritis characterized by axial skeletal inflammation [24]. We did not identify any significant SNPs in the HLA region in our GWAS, suggesting that augmentation of proinflammatory cytokine signaling through ERAP1 is more likely involved in KD. However, the association between HLA and KD remains inconclusive. Some SNPs located in HLA-A and HLA-B regions did show a nominal association (p<0.01) in our study. Determining whether HLAs play a role in KD susceptibility will require further investigation in a larger sample size using a traditional HLA genotyping method. Clarifying a potential association between HLA and KD will also help to elucidate the involvement of an ERAP1 downstream mechanism in KD.

We found that a cluster of SNPs located in chromosome 14 immunoglobulin heavy chain variable regions exhibited the strongest association in the joint analysis. The heavy chain molecule is a major contributor to the generation of immunoglobulin diversity and specificity. Therefore, in addition to cell-mediated immunity, antibody-mediated immunity may be critical in KD pathogenesis. IgA plasma cells infiltrate inflamed tissues, including coronary arteries, in acute KD [25], [26], and oligoclonal KD antibodies bind to an antigen in acute KD-inflamed ciliated bronchial epithelium [27]. Specific immunoglobulins produced by particular heavy chain recombinants following a KD antigen trigger could play a role in KD pathogenesis. Identification of these specific antibodies could lead to the elucidation of disease-relevant mechanisms and potential treatments.

We also performed a haplotype analysis, identifying two haplotypes with p-values <10⁻⁵. The significance of these two haplotypes, however, was mainly contributed by single SNPs showing strong associations in single-point analyses. Also, the haplotype frequencies were less than 0.05. Accordingly, we could not exclude the possibility that the significant p-values and the small differences between the haplotype frequencies of cases and controls might have resulted from estimation errors, given the small sample sizes. Additional analyses with larger numbers of subjects are warranted.

The major limitation of this study is that the low sample size, which reflected the difficulties inherent in recruiting patients with such a rare disease, could only reach a statistical power of 0.78 to detect a disease allele with a frequency of 0.10 and a relative risk of 3.0, assuming a disease incidence of 0.01% under an additive model. Also, the small sample size is underpowered to detect very small effects. With the additional replication sample, the joint analysis marginally strengthened some of the SNPs in the GWAS. Many of the significantly associated SNPs in the discovery phase were either insignificant or of marginal significance in the replication phase. This raises the possibility of false positives, a problem common to all GWAS. Replication of the findings in large, well-powered independent samples is crucial if this problem is to be overcome, and will likely require a multicenter collaboration. Such an increase in the size of the study cohorts would also make it possible to detect smaller effects. In addition, we found a higher proportion of male KD patients than controls in our study because of the higher incidence of KD in males. Although it is unclear whether and how this might have affected the results, it could have led to some degree of bias. Several genes implicated in previous candidate gene studies were not shown to have −log10 (p_best) values greater than 4 in our studies, probably due to difference in the relative importance of particular genetic variants in determining susceptibility to complex diseases among different ethnic groups or ethnicity-linked haplotype structures. However, some of the SNPs located in the candidate gene regions did show a KD association with p-values <0.01. Further replication with a larger sample size and different populations would be helpful in evaluating the involvement of these genes in the susceptibility of KD.

In summary, we conducted a GWAS for KD in a Han Chinese population. Ten SNPs located in three novel loci were found to be associated with KD in this population. The novel KD risk loci contained genes that have been implicated in T cell receptor signaling, proinflammatory cytokine regulation, as well as antibody-mediated immune responses. Our results suggest that antigen initiation, uncontrolled inflammation, and antibodies are involved in the pathogenesis of KD, findings that may provide new directions for future studies. In particular, further characterization of causative variants and elucidating their mechanisms of action may lead to the identification of novel diagnostic and therapeutic targets for KD.