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Background: Coronary artery lesions (CAL) are a serious complication of Kawasaki disease (KD). The increased serum E-selectin level during the acute phase of KD and the association of E-selectin gene (SELE) polymorphisms with the prevalence of coronary artery disease in adults suggest a possible association between SELE polymorphisms and the development of CAL in KD patients.
Methods: The subjects consisted of 177 KD patients, including 59 with and 118 without CAL, and 305 healthy controls. Two single nucleotide polymorphisms (SNP) of SELE, 98G>T (rs1805193) and Ser128Arg (rs5361), were genotyped by direct sequencing and the high-resolution melting curve method, respectively. The allele distributions were assessed using the chi-squared test.
Results: There were no significant differences in the T allele frequency at 98G>T between KD patients and controls (1.4% vs 1.0%, P= 0.55) or between KD patients with and without CAL (1.7% vs 1.3%, P= 0.77). Similarly, there were no differences in the distribution of the C allele (128Arg) at Ser128Arg between KD patients and controls (4.5% vs 3.4%, P= 0.40) or between KD patients with and without CAL (4.2% vs 4.7%, P= 0.86).
Conclusion: Although no association was detected between these SELE polymorphisms and the prevalence of KD or the development of CAL, this may have been due to the study limitations, including a low frequency of the minor alleles and a small sample size. A larger-scale association study is needed in order for a definitive conclusion to be made as to whether these SNP are associated with susceptibility to KD or not.
Kawasaki disease (KD) is an acute, self-limiting systemic vasculitis syndrome that mainly affects small and medium-sized arteries, and is found primarily in infants and young children.1 The most important cause of morbidity and mortality in KD is coronary artery lesions (CAL) that occur in 15–25% of untreated patients with KD.2,3 The etiology of KD has not been fully elucidated, but it is thought to be a multifactorial disease involving both genetic and environmental factors in its onset and development.4
To date, a number of genetic association studies have been performed with KD patients for genes mainly involving the immune system or inflammatory reactions. For example, 27 genes encoding cytokines (e.g. interleukin [IL]-4, IL-6, IL-10, tumor necrosis factor [TNF]) or surface antigens (e.g. CD14, CD40, Fcgamma receptor [FCGR]2A, FCGR3A) are listed in relation to KD on the Genetic Association Database (http://geneticassociationdb.nih.gov/cgi-bin/index.cgi). Inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) acts as a negative regulator of T-cell activation.4,5 Recently, a genome-wide association study (GWAS) demonstrated an association of an ITPKC polymorphism with KD, which confers both an increased risk of KD and an increased risk of CAL formation.5
The immune system is markedly activated during the acute phase of KD, accompanied by infiltration of neutrophils, lymphocytes and macrophages into the vascular wall.6 Selectins facilitate the adhesion of inflammatory cellular components to the activated vascular endothelium, and promote localized vascular inflammation.7 Among the selectins, E-selectin is considered to be the most important for the pathophysiology of coronary artery diseases, because its expression is limited on activated endothelial cells upon stimulation with inflammatory cytokines.8 Interestingly, the soluble E-selectin level has been reported to increase during the acute phase of KD.9 It was also reported that higher plasma E-selectin levels might predict the likelihood of CAL in patients with KD.10
A base substitution from adenine (A) to cytosine (C) in the coding region (561A>C) of the E-selectin gene (SELE) results in an amino acid exchange from serine to arginine at codon 128 (Ser128Arg). The polymorphism has been known to increase the ligand-binding function of the protein11 and to be associated with early-onset atherosclerosis.12 Furthermore, another base substitution of the gene from guanine (G) to thymine (T) in the 5′-untranslated region (98G>T) is associated with severe coronary artery diseases in adults.13 Although CAL contribute greatly to the morbidity and mortality of KD, the association of SELE polymorphisms with KD has not yet been investigated.
We hypothesized that either or both the 98G>T or Ser128Arg polymorphism of the SELE gene may be associated with KD and, in particular, with the predisposition to the development of CAL. We herein report the results of a genetic association study between these SELE polymorphisms and the prevalence of KD or the development of CAL among KD patients. We also report the use of a new method for rapid and accurate genotyping: high-resolution melting curve (HRM) analysis.
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Kawasaki disease is a systemic immune-mediated vasculitis syndrome that often involves the coronary arteries.1–3 Endothelial dysfunction, followed by the infiltration of leukocytes from the circulating blood into the vascular wall, plays a crucial part in the progression to vasculitis. Leukocyte rolling and tethering along the endothelial surface is the first step in their adhesion to endothelial cells, and this is mediated by the selectin family of adhesion molecules.6 The selectin family includes three molecules: P-, L-, and E-selectins. E-selectin is expressed on endothelial cells after activation mediated by inflammatory cytokines such as IL-1 and TNF-α.6 Moreover, the soluble E-selectin level has been reported to increase during the acute phase of KD9 and to predict the likelihood of CAL in patients with KD.10 The Ser128Arg polymorphism of SELE, located in the epidermal growth factor (EGF)-like domain of the protein,16 is functional in that it increases the protein's ligand affinity.11 Therefore, it is reasonable to hypothesize that E-selectin polymorphisms may influence the prevalence of KD or the development of CAL in KD patients.
The present study examined the possible correlation between the SELE polymorphisms and KD, including either the prevalence of KD or the development of CAL in KD patients. To the best of our knowledge, this is the first report to study the possible association between E-selectin polymorphisms and KD. We investigated two SNP, 98G>T and Ser128Arg, which have been shown to be associated with an increased risk of coronary artery diseases in adults, but we did not find any association between these SNP and KD or the development of CAL in KD patients. The sample size, however, might not have been of sufficient size to investigate the SNP, because their minor allele frequencies were too low in the present subjects. Therefore, we cannot totally exclude the possibility that either of these SELE polymorphisms may influence the pathogenesis of KD, because the association might have been missed due to the limited sample size. Further trials are necessary to determine whether the 98G>T and/or Ser128Arg polymorphisms in SELE are associated with an increased risk of KD or for CAL in KD patients.
In the present study we used a recently established HRM method for Ser128Arg genotyping that has the advantages of simplicity of procedure, low cost and high accuracy. It may not work for all SNP, given that we failed to perform 98G>T genotyping by this method using several primer sets. But whenever feasible, it is worth setting up the HRM method for any SNP analysis.
Finally, there has been a trend to apply GWAS,17,18 which can assess hundreds to thousands of SNP per individual, to determine the genetic relationships with many diseases or traits. Although GWAS are powerful and usually do not require an understanding of the pathological mechanism of the disease in advance, it is well known that some statistical data mining processes such as Bonferroni correction, may restrict the identification of important pathophysiologically plausible SNP.19,20