Responsible Genetic Factors for Vasculitis in Kawasaki Disease

Kawasaki disease (KD) is an acute febrile illness of early childhood that is characterized by high fever, bilateral conjunctivitis, redness of the oral mucosa, polymorphous skin rash, indurative edema of the hands and feet, and cervical lymphadenopathy (Kawasaki, 1967). The major pathological lesion of KD is vasculitis of small and medium-sized arteries (Amano et al., 1979). The coronary arteries are the most severely affected and coronary artery lesions (CALs) occur in 15–25% of untreated patients (Kato et al., 1975), making KD a leading cause of childhood acquired heart disease in developed countries. The fact that the peak incidence of KD is at 9–11 months of age, which coincides with the waning of maternal immunity, indicates that infections could contribute to the pathogenesis of KD. However, despite more than 40 years of intensive research, the causative microorganism of KD remains unknown. On the other hand, epidemiological studies have revealed a significant role of genetic components in host susceptibility to KD pathogenesis.

that the high incidence of KD in East Asian countries is due to the racial/ethnic genetic background rather than to geographic factors.

Individual susceptibility to KD
In Japan, a total of 20 nationwide biannual continued surveillances of KD have been carried out since 1970. Epidemiological evidence collected from these surveys suggested individual susceptibility to KD, which was mainly composed of multiple genetic factors. For example, sibling cases of KD have a ≥10 times higher incidence than expected (Fujita et al., 1989). In addition, parents of KD patients, who were affected by KD during their childhood, were observed 2 times more often than expected (Uehara et al., 2003).

Genetic studies of KD
KD is considered to be a multifactorial disease that is caused by the interplay of external and personal factors (Fig. 1). Thus, identification of the responsible genetic factors, which presumably determine an individual's susceptibility to KD, should provide clues to the pathogenesis of the disease. Furthermore, it could contribute to the development of novel clinical applications, such as a severity prediction method and new therapeutic measures.

Candidate gene studies
KD is an immune-mediated vasculitis syndrome; therefore, genes encoding proteins related to innate and acquired immune function or to vascular remodeling could be involved in its pathogenesis, response to treatment, and prognosis. Variations within such "candidate genes" have been extensively studied.

Human leukocyte antigen (HLA) genes
Genetic studies of KD were initially conducted by focusing on the HLA class I genes, and several serotypes of the HLA-B locus have been associated with KD in different ethnic groups (Table 1).

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Recently, significant associations of single nucleotide polymorphisms (SNPs) within the HLA-G and HLA-E genes were reported (J.J. Kim et al., 2008;. However, most of the previous studies, including those describing negative association results, were conducted by analyzing a relatively small number of cases and controls, while replication studies with larger cohorts have not been performed yet.

Non-HLA genes
Advances from the Human Genome and Hapmap projects have dramatically reduced the effort and cost of conducting genetic association studies of complex diseases, leading to a recent increase in the number of candidate gene studies, especially since 2005 (Table 2). Among the genes studied, tumor necrosis factor (TNF), which is a proinflammatory cytokine deeply related to the pathogenesis of KD, has been most frequently analyzed. Although many of these studies have failed to identify a statistically significant association, a systematic meta-analysis revealed a trend of association between the G allele of rs180629, which is located 308 bases upstream of the TNF gene, and KD (Ari-Ong et al., 2010).  Table 2. Association studies between polymorphisms of candidate genes and KD.
The association of these candidate SNPs and patient response to intravenous immunoglobulin (IVIG) therapy and the development of CALs has also been studied (Table 3).  Table 3. Association studies between candidate gene polymorphisms and KD-related phenotypes.

Genome-wide studies
In contrast to candidate gene studies, which are based on an assumption of the underlying cause of the disease, a strategy to identify disease-causing mutations or variations from the whole genome relies solely on positional information and was originally developed to map and identify the genes for Mendelian disorders. This genome-wide strategy has been adapted for complex diseases and has become the most reliable tool to identify diseaserelated genes following the completion of the Human Genome Project.

Linkage study
The first genome-wide study for KD was conducted by our group (Y. Onouchi et al., 2007). In this study, 399 microsatellite marker alleles that were shared identical by descent between 78 affected KD sib-pairs were analyzed, and 10 chromosomal regions linked with the disease were identified (Fig. 2).
Results from an affected sib-pair study (Y. Onouchi et al., 2007). Arrows indicate those chromosomal regions with a maximum LOD score (MLS) >1.0.

Linkage disequilibrium mapping
We narrowed down the candidate regions identified in the sib-pair study with a casecontrol association study using "tagging" SNPs. Chunks of genomic regions containing the associated tagging SNPs were extensively analyzed by re-sequencing and a further association study. We identified a SNP that was associated with KD in both the Japanese and US populations (Y. Onouchi et al., 2008) (Table 4). The SNP, rs28493229, is located in intron 1 of the inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) gene, which catalyzes the phosphorylation of inositol 1,4,5-trisphosphate (IP3 The at-risk allele of rs28493229 (C) reduces the splicing efficiency of ITPKC (Fig. 3). Transcripts with an unspliced intron are not properly translated because of premature termination. An increase in the number of such immature transcripts might lead to reduced ITPKC activity. IP3 is a second messenger molecule of the Ca 2+ /NFAT pathway in a wide variety of cells and, in mammals, 3 iso-enzymes (ITPKA, ITPKB, and ITPKC) have been identified with the same enzyme activity. Knockdown and overexpression experiments of ITPKC in the Jurkat cell line result in increased and decreased NFAT activity, respectively, as well as the expression of interleukin 2 mRNA. These findings highlighted the importance of the Ca 2+ /NFAT pathway in the pathogenesis of KD. The association with the at-risk SNP allele was higher in KD patients with CAL than in those without CAL in both the Japanese and US populations. The same trend was also observed in KD patients in the US who responded poorly to intravenous immunoglobulin (IVIG) therapy. Two replication studies for the association of this SNP to KD, one negative and one positive, have been reported in the Taiwanese population (Chi et al., 2010;M.T. Lin et al., 2011). In the positive report, the SNP was also associated with the reactivation of previous BCG inoculation sites (M.T. Lin et al., 2011).

Positional candidate gene analysis
From the candidate region of chromosome 4, we identified the susceptibility gene via a different approach. The caspase-3 gene, which is located at 185.8 Mb, close to the linkage peak at 184.9 Mb, was focused on and studied as a positional candidate gene (Y. . Multiple SNPs around the gene in linkage disequilibrium were associated with KD in the Japanese and US Caucasian populations. The functional SNP (rs72689236 G/A) was located in the 5′-untranslated region of the gene, and the risk allele (A) reduces the enhancer activity around the SNP to which NFATc2 is related (Table 5, Fig. 4). Caspase-3 is an effector caspase with a central role in apoptosis. T cells from caspase-3deficient mice have a reduced susceptibility to activation-induced cell death (Woo et al, 1998). It was also reported that caspase-3 cleaves Nfatc2 as a substrate (W. Wu et al, 2006). Transient anergy of peripheral T cells in the convalescent phase of KD, which has been www.intechopen.com documented in a couple of reports, is suggestive because the NFAT-driven expression of caspase-3 in T cells is related to T cell anergy (Macián et al., 2002). Currently, only one replication study has examined the association between rs72689236 and KD (Kuo et al, 2011). Although not statistically significant, the same trend of association was observed in the Taiwanese population. Notably, in this report, the SNP was associated with increased risk for IVIG resistance and CAL formation.

Genome-wide association studies (GWAS)
Today, GWAS using platforms by which 5.0 × 10 5 to 1 × 10 6 SNPs can be genotyped at a time have become commonplace for the analysis of complex disorders. GWAS for KD have been performed in 3 different ethnic groups: Caucasian, Korean, and Taiwanese (Burgner et al., 2009; J.J. Tsai et al., 2011). The number of subjects and SNPs analyzed in each study are summarized in Table 6. Although many candidate SNPs were identified, no locus was repeatedly associated with KD in these studies ( Table 7). Considering that none of these associations fulfilled the genome-wide level significance threshold, further validation of the association at each locus within the same populations is essential.

Clinical implementation of genetic findings
The standard treatment for KD is a combination of oral aspirin and high-dose IVIG (Z. Onouchi & Kawasaki, 1999). While the majority of patients respond to this therapy, around 15% are resistant and require additional IVIG or alternative drugs to prevent the development of CAL. As the etiology and pathophysiology of KD are largely unknown, the mechanism of action of these therapies on the disease is not fully understood. The identification of genetic factors that influence patient response to therapy might provide an insight to this problem.

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We investigated the association between the functional SNPs in the ITPKC and caspase-3 genes with IVIG unresponsiveness and CAL formation, and found that patients with at least 1 susceptible allele at both SNPs had 2.7-2.9 times higher risk for these unfavorable events (manuscript submitted). Considering that ITPKC and caspase-3 are possibly negative regulators of the Ca 2+ /NFAT pathway (Fig. 5), hyperactivation of the pathway might underlie a more severe clinical manifestation of the disease. From this point of view, Cyclosporine A, an immunosuppressant drug which potently suppresses the activity of T cells by targeting calcineurin, a key molecule of the Ca 2+ /NFAT pathway (Fig. 5), may be a good option for refractory cases of KD. In Japan, a study to investigate the tolerability, safety, and efficacy of Cyclosporine A for KD has been started.

Conclusion
Many candidate variations for susceptibility to KD have been reported from candidate gene and genome-wide studies; however, most of the findings from these studies are not robust and have yet to be confirmed by replication studies. Considering the modest odds ratios observed in recent GWAS for complex disorders, discovery studies should be conducted with much larger cohorts. Replication studies in different ethnic groups should be designed with careful attention to their power to detect a significant association and, most importantly, to the difference in the linkage disequilibrium structure. As the majority of KD patients are infants and children and the disease incidence is low, especially in countries outside East Asia, there are limitations in collecting subjects. In an attempt to overcome this difficulty, several consortia have been formed. Those engaged in KD research hope to unravel the mystery of this vasculitis and save these children from damage to their heart. We wish to contribute to this important mission by identifying the underlying genetic components of this disease.
Distribution and incidence of the vascular lesions. Japanese Circulation Journal, Vol.43, No.8, pp. 741-748, (August 1979) Pediatric Cardiology, Vol.30, No.3, pp. 331-335, (April 2009). ISSN 0172-0643 Ban, JY.; Yoon, KL. & Chung, JH. (2010). Association between polymorphisms of matrix metalloproteinase 11 (MMP-11) and Kawasaki disease in www.intechopen.com the Korean population. Life Sciences, Vol.86, No.19-20, pp. 756-759, (May 2010). ISSN 0024-3205 Biezeveld, MH.;Kuipers, IM.;Geissler, J.;Lam, J.;Ottenkamp, JJ.;Hack, CE. & Kuijpers, TW. (2003). Association of mannose-binding lectin genotype with cardiovascular abnormalities in Kawasaki disease. The Lancet, Vol.361, No.9365, pp. 1268-1270, (April 2003 This book represents the culmination of the efforts of a group of outstanding experts in vasculitis from all over the world, who have endeavored to devote their work to this book by keeping both the text and the accompanying figures and tables lucid and memorable. Here, you will find an amalgam between evidencebased medicine to one based on eminence, through an exciting combination of original contributions, structured reviews, overviews, state-of the-art articles, and even the proposal of novel pathogenetic models of disease. The book contains contributions on the etiology and pathology of vasculitis, the potential role of endothelial cells and cytokines in vascular damage and repair as well as summaries of the latest information on several primary and secondary vasculitis syndromes. It also covers selected topics such as organ-specific vasculitic involvement and quality of life issues in vasculitis. The editor and each of the authors invite you to share this journey through one of the most exciting fields of the medicine, the world of Vasculitis.