key: cord-000627-gvzs9vxr
authors: Yang, Ya-Ling; Chang, Wei-Pin; Hsu, Yu-Wen; Chen, Wei-Chiao; Yu, Hong-Ren; Liang, Chi-Di; Tsai, Yao-Ting; Huang, Ying-Hsien; Yang, Kuender D.; Kuo, Ho-Chang; Chang, Wei-Chiao
title: Lack of Association between CLEC5A Gene Single-Nucleotide Polymorphisms and Kawasaki Disease in Taiwanese Children
date: 2011-12-22
journal: J Biomed Biotechnol
DOI: 10.1155/2012/398628
sha: 
doc_id: 627
cord_uid: gvzs9vxr

Background. Kawasaki disease is characterized by systemic vasculitis of unknown etiology. Previous genetic studies have identified certain candidate genes associated with susceptibility to KD and coronary artery lesions. Host innate immune response factors are involved in modulating the disease outcome. The aim of this study was to investigate CLEC5A (C-type lectin domain family 5) genetic polymorphisms with regards to the susceptibility and outcome of KD. Methods. A total of 1045 subjects (381 KD patients and 664 controls) were enrolled to identify 4 tagging single-nucleotide polymorphisms (tSNPs) of CLEC5A (rs1285968, rs11770855, rs1285935, rs1285933) by using the TaqMan Allelic Discrimination Assay. The Hardy-Weinberg equilibrium was assessed in cases and controls, and genetic effects were evaluated by the chi-square test. Results. No significant associations were noted between the genotypes and allele frequency of the 4 CLEC5A tSNPs between controls and patients. In the patients, polymorphisms of CLEC5A showed no significant association with coronary artery lesion formation and intravenous immunoglobulin treatment response. Conclusions. This study showed for the first time that polymorphisms of CLEC5A are not associated with susceptibility to KD, coronary artery lesion formation, and intravenous immunoglobulin treatment response in a Taiwanese population.

Kawasaki disease (KD) is characterized by acute, febrile, and systemic vasculitis and was first described by Kawasaki et al. in 1974 [1] . In developed countries, KD is the leading cause of acquired heart diseases in children [2, 3] . KD occurs worldwide and particularly in Japan, Korea, and Taiwan and mainly affects children less than 5 years of age [4] [5] [6] . The most serious complication of KD is the occurrence of coronary artery lesions (CALs) [7, 8] . The prevalence of KD in children younger than 5 years is the highest in Japan, followed by Korea and Taiwan, and lowest in Europe. Previous studies have either failed to identify causative pathogens for KD or reported discrepant results [9] [10] [11] . Therefore, it is possible that a genetic background plays an important role in the pathogenesis of KD.

CLEC5A (C-type lectin domain family 5, member A; also known as myeloid DAP12-associating lectin (MDL-1)) Journal of Biomedicine and Biotechnology contains a C-type lectin-like fold similar to the naturalkiller T-cell C-type lectin domains and is associated with a 12-kDa DNAX-activating protein (DAP12) on myeloid cells [12] [13] [14] . Signaling via this complex constitutes a significant activation pathway in myeloid cells and plays an important role in immune defense. Recently, it has been demonstrated that CLEC5A acts as a signaling receptor for proinflammatory cytokine release, and that blockade of CLEC5A-mediated signaling attenuates the production of proinflammatory cytokines by macrophages infected with dengue virus [14] . In contrast, it has been demonstrated that MDL-1 stimulation induces a significant amount of RANTES and macrophage-derived chemokine (MDC) production in cooperation with signaling through TLR in mouse myeloid cells [15] . Furthermore, there is ample evidence that activation of peripheral blood monocytes/macrophages [16] [17] [18] , proinflammatory cytokines [16] , and the RANTES gene play a central role during acute KD [19, 20] . A persistent or increased expression of chemokine genes in the convalescent phase in patients is associated with coronary artery lesions [17, 19] . In addition, infiltration by the cells is notable in affected tissues in autopsy cases and in skin biopsy specimens from KD patients [21] .

However, no CLEC5A genetic association with KD has previously been reported. To gain further understanding of the genetic role of CLEC5A in the pathogenesis of KD, the aim of our study was to determine if any CLEC5A SNPs are associated with susceptibility to KD, CAL formation, or IVIG treatment response in Taiwanese children.

All study cases were children enrolled from Chang Gung Memorial Hospital, Kaohsiung Medical Center, between 2001 and 2009, who fulfilled the diagnostic criteria for KD. All patients were treated with IVIG (2 g/kg) and aspirin as per our previous studies [7, 8, 18] . This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. Blood samples were collected after informed consent was obtained from parents or guardians. CAL formation was defined as the internal diameter of the coronary artery measuring at least 3 mm (4 mm if the subject was over the age of 5 years) or the internal diameter of a segment at least 1.5 times that of an adjacent segment, as observed in echocardiography [8, 22] . IVIG responsiveness was defined as defervescence within 48 h after the completion of IVIG treatment and no recurrence of fever (temperature > 38 • C) for at least 7 days after IVIG with marked improvement or normalization of inflammatory signs [7, 8] .

Blood cells were subjected to DNA extraction by treating them first with 0.5% SDS lysis buffer and then protease K (1 mg/mL) for digestion of nuclear protein for 4 h at 60 • C. Total DNA was harvested by using a Gentra extraction kit followed by 70% alcohol precipitation as described in our previous report [23] . 

Susceptibility of KD. A total of 381 KD patients and 664 controls were included in this study ( Table 1 ). The distribution of CLEC5A genotypes was in accordance with the Hardy-Weinberg equilibrium for both cases and controls (Table 2) . However, none of the tSNPs was significantly associated with the genotype or allele frequency of the controls or KD patients under 3 genetic models (dominant, recessive, or allelic models) ( Table 2) . 

In this study, 37 patients (9.9%) had CAL formation and 49 patients (13.1%) had resistance to the initial IVIG treatment (Table 1) . However, no tSNPs were significantly associated with genotype or allele frequency in the KD patients with or without CAL formation (Table 3) .

Additionally, the CLEC5A polymorphisms tested in this study failed to show any significant associations with genotype or allele frequency in the KD patients who showed a response to IVIG treatment (Table 4 ). Table 1 ), CAL formation (Supplemental Table 2 ) and IVIG treatment response (Supplemental Table 3 ) in the KD patients. However, none was significantly associated with the phenotype.

The C-type lectin-like super domain (CTLD) family has diverse functions, and in particular, is important in innate immunity including nature killer (NK) function or pathogen recognition [25] . CLEC5A belongs to the Group V "NK cell receptors" family, and MDL-1 expression is upregulated in activated myeloid cells [26] and acts as a signaling receptor for proinflammatory cytokine and chemokine release [14] . Even though a number of reports have demonstrated that KD involves activation of a wide array of immunological elements such as T cells and macrophages [16] [17] [18] 27] , with the subsequent release of several cytokines [28] , only a few reports have addressed the role of lectin in the pathogenesis of KD. Several genetic associations with susceptibility to KD and CAL formation have been reported, but the results are inconsistent [29] [30] [31] [32] . Previous genetic association studies have indicated that the intronic SNP (rs28493229) of ITPKC, 1,4,5-trisphosphate 3-kinase C, reduces gene expression by altering splicing efficiency, and the C allele contributes to immune hyperreactivity in KD patients [29] . Recently, it has been demonstrated that rs28493229 is associated with susceptibility to KD and CAL formation [24, 29] . ITPKC is able to regulate the immune system via calcium-dependent NFAT pathways [29] . Similarly, previous studies have indicated that C-type lectin receptors (CLRs) are critical in the activation of the Syk-mediated NFAT signaling pathway [33] . In addition, CLEC5A has been shown to play a key role in host defense and to be involved in dengue virusmediated disease [14] . This finding suggests CLEC5A may be a potential target protein that involves calcium-dependent immune regulation and contributes to the development of coronary artery lesions. However, we did not find evidence to support a genetic role of CLEC5A in the pathogenesis of KD. Since we picked tagging SNPs from the HapMap database, only the tagging SNPs with a minor allele frequency of more than 10% were selected. Although our tSNP could capture majority of the underlying genetic variances with MAF > 10% across the CLEC5A gene, the rare causal genetic polymorphisms in CLEC5A may not have been detected in this study. Therefore, we cannot rule out or exclude rare causal genetic polymorphisms in CLEC5A. In addition, there are, at least, seventeen groups of CLRs in vertebrates. Indeed, it has been reported that mannose-binding lectin gene polymorphisms are associated with susceptibility to KD [34] and CAL formation [35] . Thus, large-scale DNA sequencing to CLR family is needed to better understand KD.

In conclusion, this study showed for the first time that tSNPs of CLEC5A are not associated with susceptibility to KD, CAL formation, and IVIG treatment response in a Taiwanese population.

A new infantile acute febrile mucocutaneous lymph node syndrome (MLNS) prevailing in Japan

Kawasaki disease: infection, immunity and genetics

Kawasaki syndrome

Kawasaki disease in Korea

Epidemiologic features of Kawasaki disease in Taiwan

Increasing incidence of Kawasaki disease in Japan: Nationwide survey

Association of lower eosinophil-related T helper 2 (Th2) cytokines with coronary artery lesions in Kawasaki disease

Serum albumin level predicts initial intravenous immunoglobulin treatment failure in Kawasaki disease

Association between a novel human coronavirus and kawasaki disease

Lack of association between infection with a novel human coronavirus (HCoV), HCoV-NH, and Kawasaki disease in Taiwan

Lack of association between New Haven coronavirus and Kawasaki disease

Myeloid DAP12-associating lectin (MDL)-1 is a cell surface receptor involved in the activation of myeloid cells

CLEC5A (MDL-1) is a novel PU.1 transcriptional target during myeloid differentiation

CLEC5A is critical for dengue-virus-induced lethal disease

Expression and functional role of MDL-1 (CLEC5A) in mouse myeloid lineage cells

Immunological profile of peripheral blood lymphocytes and monocytes/macrophages in Kawasaki disease

Induction of MCP1, CCR2, and iNOS expression in THP-1 macrophages by serum of children late after kawasaki disease

Persistent monocytosis after intravenous immunoglobulin therapy correlated with the development of coronary artery lesions in patients with Kawasaki disease

Evidence for RANTES, monocyte chemotactic protein-1, and macrophage inflammatory protein-1β expression in Kawasaki disease

Production and expression of Gro-α and RANTES by peripheral blood mononuclear cells Isolated from patients with Kawasaki disease and measles

Class II major histocompatibility antigen expression on coronary arterial endothelium in a patient with Kawasaki disease

Kawasaki disease

Gene-gene and gene-environment interactions on IgE production in prenatal stage

Itpkc single nucleotide polymorphism associated with the kawasaki disease in a taiwanese population

How C-type lectins detect pathogens

The C-type lectin-like domain superfamily

Unique activation status of peripheral blood mononuclear cells at acute phase of Kawasaki disease

Advances in Kawasaki disease

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

ITPKC gene SNP rs28493229 and Kawasaki disease in Taiwanese children

Clinical implication of the C allele of the ITPKC gene SNP rs28493229 in kawasaki disease: association with disease susceptibility and BCG scar reactivation

Transforming growth factor-β signaling pathway in patients with Kawasaki disease

The SYK tyrosine kinase: a crucial player in diverse biological functions

Association of mannose-binding lectin gene polymorphisms with Kawasaki disease in the Japanese

Association of mannose-binding lectin genotype with cardiovascular abnormalities in Kawasaki disease

This study was partly supported by Grants from the National Science Council, Taiwan 

The authors declare that no competing interests exist.
