HLAE gene polymorphism associated with susceptibility to kawasaki disease and formation of coronary artery aneurysms.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 60, No. 2, February 2009, pp 604–610 DOI 10.1002/art.24261 © 2009, American College of Rheumatology HLA–E Gene Polymorphism Associated With Susceptibility to Kawasaki Disease and Formation of Coronary Artery Aneurysms Y.-J. Lin,1 L. Wan,1 J.-Y. Wu,2 J. J.-C. Sheu,1 C.-W. Lin,3 Y.-C. Lan,3 C.-H. Lai,3 C.-H. Hung,3 Y. Tsai,1 C.-H. Tsai,1 T.-H. Lin,1 J.-G. Lin,3 K.-C. Hsueh,4 Y.-M. Huang,1 J.-S. Chang,4 and F.-J. Tsai1 Objective. Kawasaki disease (KD) is a pediatric systemic vasculitis of unknown cause for which a genetic influence is supposed. The purpose of this study was to identify possible genetic variants in the major histocompatibility complex (MHC) region that are associated with KD and the development of coronary artery aneurysms (CAAs) in a Taiwanese population. Methods. The 168 genetic variants covering the MHC locus were analyzed in an association study of a Taiwanese cohort of 93 KD patients and 680 unrelated healthy children matched for sex and age with the study patients. Results. Eleven single-nucleotide polymorphisms (SNPs) were associated with the occurrence of KD. The SNP located at the 3ⴕ-untranslated region of HLA–E (rs2844724) was highly associated (P < 1 ⴛ 10ⴚ7). In addition, the frequency of the C allele was higher in KD patients without CAAs than in controls (P < 0.001) due to a significantly increased frequency of the CC and CT genotypes. Plasma levels of soluble HLA–E were significantly higher in KD patients than in controls regardless of the presence of CAAs. Furthermore, there was a trend toward higher plasma levels of soluble HLA–E in KD patients with the CT and TT genotypes of the HLA–E gene polymorphism. Conclusion. Our results suggest that the HLA–E gene polymorphism may play a role in the pathogenesis of KD. Kawasaki disease (KD) is an acute, self-limited, and systemic vasculitis that is one of the leading causes of acquired heart disease in children (1–3). The vascular inflammation may cause the development of aneurysms and cardiac complications. Patients with these cardiovascular complications are at increased risk of developing ischemic heart disease, which may lead to myocardial infarction and sudden death (4). Although KD is a mysterious disease of unknown etiology and pathogenesis, it is believed to be caused by infectious agents, host immune dysregulation, and genetic susceptibility (5–8). Moreover, KD is overrepresented in Asian children (1,9–13). The annual incidence of KD in Taiwan is estimated to be 66/100,000 children, the third highest in the world after Japan and Korea (3,14). During the acute stage of KD, activation of vascular endothelial cells and increased serum levels of proinflammatory cytokines are involved in the occurrence of inflamed and injured vessels (15,16). The injured vascular tissues show subendothelial edema, vascular damage, gap formation, and fenestration of endothelial cells and contribute to the pathogenesis of this disorder (17,18). Human vascular endothelial cells process antigens and express class I and class II major histocompatibility complex (MHC) molecules and costimulatory Dr. Y.-J. Lin’s work was supported by China Medical University (grant CMU95-142), China Medical University Hospital (grant DMR-96-110), and the National Science Council, Taiwan (grant NSC94-2320-B-039-042). Dr. Wu’s work was supported by grants from the National Science and Technology Program for Genomic Medicine, National Science Council, Taiwan (National Genotyping Center grant NSC95-3112-B-001-011), and the Academia Sinica Genomic Medicine Multicenter Study. 1 Y.-J. Lin, PhD, L. Wan, PhD, J. J.-C. Sheu, PhD, Y. Tsai, PhD, C.-H. Tsai, MD, PhD, T.-H. Lin, MS, Y.-M. Huang, MS, F.-J. Tsai, MD, PhD: China Medical University Hospital, China Medical University, and Asia University, Taichung, Taiwan; 2J.-Y. Wu, PhD: Academia Sinica, Taipei, Taiwan; 3C.-W. Lin, PhD, Y.-C. Lan, PhD, C.-H. Lai, PhD, C.-H. Hung, PhD, J.-G. Lin, MD, PhD: China Medical University, Taichung, Taiwan; 4K.-C. Hsueh, MD, J.-S. Chang, MD: China Medical University Hospital, Taichung, Taiwan. Drs. Y.-J. Lin and L. Wan contributed equally to this work. Address correspondence and reprint requests to J.-S. Chang, MD, or F.-J. Tsai, MD, PhD, Department of Medical Research, China Medical University Hospital, No. 2, Yuh Der Road, Taichung, Taiwan. E-mail: firstname.lastname@example.org or email@example.com. Submitted for publication February 12, 2008; accepted in revised form October 17, 2008. 604 HLA–E GENE POLYMORPHISM IN KD 605 Figure 1. Results of a single-nucleotide polymorphism (SNP) association study of the major histocompatibility complex (MHC) region on chromosome 6p21.3 in Taiwanese patients with Kawasaki disease (KD) and healthy individuals from the general population of Taiwan who were of Han Chinese ethnic background. Top, Map of the 168 SNPs located within 4 Mb of the MHC region (from 29,900,000 to 33,900,000 bp on chromosome 6) that were used for genotyping and SNP association analysis in 93 KD patients and 680 healthy individuals. Positions of HLA markers A, E, C, and B, tumor necrosis factor (TNF), complement C2, and HLA markers DRB1, DQB2, and DPB1 lying within this region are indicated. Bottom, Haplotype blocks for the 680 control subjects and 93 KD patients constructed according to the confidence interval approach using Haploview software (43). Red indicates linkage disequilibrium (D⬘ ⫽ 1, logarithm of odds [LOD] ⱖ2); white and blue indicate evidence of recombination (D⬘ ⬍ 1, LOD ⬍2 for white; D⬘ ⫽ 1, LOD ⬍2 for blue). molecules on their surface for presenting antigenic peptides to T cells and then initiating an acquired immune response (19,20). The roles of HLAs from the MHC region have been investigated in immune-mediated vascular diseases (21–32). However, HLAs that contribute to the pathogenesis of KD have been less well characterized. Genetic studies of HLA class I genes have demonstrated an association between the MICA gene and KD (30). The association between HLA class II genes and KD has also been investigated (27,28). However, no significant associations between either HLA–DRB1, DRB3, DQA1, DQB1, or DPB1 and KD have been demonstrated and none of them have proved clinically useful in terms of KD susceptibility. Thus, the MHC polymorphism data from case–control studies have not been conclusive. In the present study, we searched for genes that influence susceptibility to KD in Taiwanese children. A total of 168 polymorphic, evenly spaced common variations (target density 1 single-nucleotide polymorphism [SNP] for every 20 kb) in the MHC region were evaluated in 93 children with KD and in 680 unrelated healthy individuals. We investigated whether the identified gene polymorphisms were associated with KD or with the occurrence of coronary artery aneurysms (CAAs) in a case–control study. PATIENTS AND METHODS Study population. From 1998 to 2005, 93 individuals who attended the Department of Pediatrics, China Medical University Hospital in Taichung, and who fulfilled the diagnostic criteria for KD were identified and enrolled in this study (33–37). Every patient underwent regular echocardiography examinations, beginning during the acute stage of KD, at 2 months and 6 months after disease onset, and once a year thereafter. A CAA was identified when either the right coronary artery or the left coronary artery showed a dilated diameter of ⱖ3 mm in children younger than 5 years or ⱖ4 mm in children older than 5 years (38). The control group consisted of 680 healthy children randomly selected from the Han Chinese Cell and Genome Bank, in which 3,312 unrelated descendants of the Han Chinese were recruited based on their geographic distribution across Taiwan (39). Control subjects were matched for sex and age with the study patients. The estimated prevalence of KD is fewer than 1/1,000 children; therefore, it should be assumed that there were no KD cases in the control group. This study was approved by the Human Studies Committee of China Medical University Hospital, and informed consent was obtained from either the participants or their parents. 606 LIN ET AL Table 1. Genotype distributions of significant SNPs in the MHC regions in Taiwanese KD patients and controls* SNP Position Gene rs1611750 29922757 – rs410909 30057147 HCG9 rs2844724 30577169 HLA–E rs2517523 31134413 – rs1064190 31183094 C6orf15;CDSN;PSORS1C1 rs2844476 31689875 BAT2;AIF1 rs2269425 32231617 C6orf31;PPT2 rs1555115 32462498 BTNL2 rs2395161 32495730 – rs1383267 32941624 PSMB9 rs2076311 33253347 COL11A2 GG GT TT AA AC CC CC CT TT GG GA AA TT TG GG GG GA AA TT TC CC GG GC CC CC CA AA TT TC CC AA AC CC No. (%) of controls No. (%) of KD patients 6 (0.9) 103 (15.3) 565 (83.8) 18 (2.7) 187 (27.7) 470 (69.6) 76 (11.5) 299 (45.1) 288 (43.4) 131 (19.8) 348 (52.7) 181 (27.4) 138 (20.9) 318 (48.3) 203 (30.8) 108 (16.2) 307 (46.2) 250 (37.6) 20 (3.0) 235 (35.0) 416 (62.0) 1 (0.1) 33 (4.9) 635 (94.9) 0 (0.0) 46 (6.9) 622 (93.1) 96 (14.2) 320 (47.2) 262 (38.6) 33 (5.0) 229 (34.7) 397 (60.2) 0 (0) 6 (6.7) 84 (93.3) 1 (1.1) 16 (17.8) 73 (81.1) 15 (18.5) 58 (71.6) 8 (9.9) 30 (32.3) 38 (40.9) 25 (26.9) 33 (35.5) 34 (36.6) 26 (28.0) 1 (1.1) 51 (58.6) 35 (40.2) 8 (8.6) 35 (37.6) 50 (53.8) 2 (2.2) 3 (3.2) 88 (94.6) 1 (1.1) 6 (6.5) 86 (92.5) 4 (4.3) 48 (51.6) 41 (44.1) 11 (11.8) 32 (34.4) 50 (53.8) P OR (95% CI) 0.055 – 0.39 (0.17–0.92) 1 0.36 (0.05–2.72) 0.55 (0.31–0.97) 1 7.11 (2.90–17.38) 6.98 (3.28–14.88) 1 1.66 (0.93–2.95) 0.79 (0.46–1.35) 1 1.87 (1.07–3.26) 0.83 (0.49–1.43) 1 0.07 (0.01–0.49) 1.19 (0.75–1.88) 1 3.33 (1.39–7.95) 1.24 (0.78–1.96) 1 14.43 (1.30–160.82) 0.66 (0.20–2.18) 1 – 0.94 (0.39–2.27) 1 0.27 (0.09–0.76) 0.96 (0.61–1.50) 1 2.65 (1.26–5.56) 1.11 (0.69–1.78) 1 0.074 4.26 ⫻ 10⫺8 0.017 0.0061 0.0006 0.0173 0.012 0.027 0.029 0.029 * Genotype frequencies were determined by chi-square test using 2 ⫻ 3 contingency tables. P values less than 0.05 were considered significant. SNPs ⫽ single-nucleotide polymorphisms; MHC ⫽ major histocompatibility complex; KD ⫽ Kawasaki disease; OR ⫽ odds ratio; 95% CI ⫽ 95% confidence interval. SNP genotyping. A total of 201 SNPs from the dbSNP database at the National Center for Biotechnology Information were considered (40,41). The 201 SNPs were located in 4 Mb of the MHC region on chromosome 6p21.3; they included 9 classic HLA loci, 2 TAP genes, and 18 microsatellites (42). After excluding SNPs with a genotype call rate of ⬍0.85, a total of 168 of the 201 SNPs remained, and these were used in our study. The mean intermarker spacing was 21.5 kb, with a median of 15.7 kb, and a standard deviation of 21.3 kb. A summary of the SNP information, including the rs number, position, corresponding gene, and allele frequency, is available upon request from the corresponding author. Genomic DNA was extracted from peripheral blood leukocytes according to standard protocols (Genomic DNA kit; Qiagen, Chatsworth, CA). SNPs were genotyped using high-throughput matrix-assisted laser desorption ionization– time-of-flight (MALDI-TOF) mass spectrometry. Briefly, primers and probes were designed by using SpectroDesigner software (Sequenom, San Diego, CA). Multiplex polymerase chain reactions were performed, and unincorporated dNTPs were dephosphorylated with shrimp alkaline phosphatase (Hoffman-La Roche, Basel, Switzerland), followed by primer extension. The purified primer extension reaction product was spotted onto a 384-element silicon chip (SpectroChip; Sequenom) and analyzed in a Bruker Biflex III MALDI-TOF SpectroReader mass spectrometer (Sequenom). The resulting spectra were processed with SpectroTyper (Sequenom) software. Analysis of haplotype blocks. Based on the Haploview software, we used the Lewontin D⬘ measure to estimate the intermarker coefficient of linkage disequilibrium in both controls and KD patients (43). The confidence interval for linkage disequilibrium was estimated using a resampling procedure and was used to construct the haplotype blocks (44). Enzyme-linked immunosorbent assay (ELISA) for detection of soluble HLA–E. Concentrations of soluble HLA–E were quantified using an ELISA kit from USCN Life Science & Technology (Missouri City, TX; online at http://www.uscnlife.com/elisa/1257023105.html). ELISA was performed according to the manufacturer’s instructions. Briefly, plasma samples (100 l) were applied directly to wells. After 2 hours of incubation, the plasma samples were removed. Detection Reagent A (100 l) was then added and incubated for another 2 hours. After wash treatments, Detection Reagent B (100 l) was applied and incubated for 1 hour. After the final wash treatments, soluble HLA–E was then detected with the substrate and stop solutions. HLA–E GENE POLYMORPHISM IN KD 607 Table 2. Allele frequencies of significant SNPs in the MHC region in Taiwanese KD patients and controls* SNP Position Gene rs1611750 29922757 – rs410909 30057147 HCG9 rs2844724 30577169 HLA–E rs2517523 31134413 – rs1064190 31183094 C6orf15;CDSN;PSORS1C1 rs2844476 31689875 BAT2;AIF1 rs2269425 32231617 C6orf31;PPT2 rs1555115 32462498 BTNL2 rs2395161 32495730 – rs1383267 32941624 PSMB9 rs2076311 33253347 COL11A2 G T A C C T G A T G G A T C G C C A T C A C No. (%) of controls No. (%) of KD patients 115 (8.5) 1233 (91.5) 223 (16.5) 1127 (83.5) 451 (34.0) 875 (66.0) 610 (46.2) 710 (53.8) 594 (45.1) 724 (54.9) 523 (39.3) 807 (60.7) 275 (20.5) 1067 (79.5) 35 (2.6) 1303 (97.4) 46 (3.4) 1290 (96.6) 512 (37.8) 844 (62.2) 295 (22.4) 1023 (77.6) 6 (3.3) 174 (96.7) 18 (10.0) 162 (90.0) 88 (54.3) 74 (45.7) 98 (52.7) 88 (47.3) 100 (53.8) 86 (46.2) 53 (30.5) 121 (69.5) 51 (27.4) 135 (72.6) 7 (3.8) 179 (96.2) 8 (4.3) 178 (95.7) 56 (30.1) 130 (69.9) 54 (29.0) 132 (71.0) P OR (95% CI) 0.015 0.37 (0.16–0.85) 1 0.56 (0.34–0.93) 1 2.31 (1.66–3.21) 1 1.30 (0.95–1.76) 1 1.42 (1.04–1.93) 1 0.68 (0.48–0.95) 1 1.47 (1.03–2.08) 1 1.46 (0.64–3.33) 1 1.26 (0.59–2.71) 1 0.71 (0.51–0.99) 1 1.42 (1.01–2.00) 1 0.024 3.84 ⫻ 10⫺7 0.097 0.026 0.0237 0.0306 0.37 0.55 0.0425 0.044 * Allele frequencies were determined by chi-square test using 2 ⫻ 2 contingency tables. P values less than 0.05 were considered significant. SNPs ⫽ single-nucleotide polymorphisms; MHC ⫽ major histocompatibility complex; KD ⫽ Kawasaki disease; OR ⫽ odds ratio; 95% CI ⫽ 95% confidence interval. Statistical analysis. Categorical data were compared between groups using Fisher’s exact test, and continuous data (presented as the median and range) were compared with the use of 2-sample t-tests. Allelic association screening was performed using the Cochran-Armitage trend test for each SNP (45). RESULTS Association study of the MHC region. To identify KD susceptibility genes, a total of 168 SNPs within the MHC region (from 29,900,000 to 33,900,000 bp on chromosome 6) were genotyped in 93 Taiwanese patients with KD and in 680 healthy individuals from the general population of Taiwan who were of Han Chinese ethnic background for the SNP association study (Figure 1). Haplotype block profiles for the controls and the KD patients, as determined using Haploview software (43), are shown at the bottom of Figure 1. There were apparent differences in haplotype block structures. As shown in Tables 1 and 2, the genotype distributions and allele frequencies of 11 SNPs in the MHC region were statistically different in KD patients as compared with controls (P ⬍ 0.05). These SNPs were rs1611750, rs410909, rs2844724, rs2517523, rs1064190, rs2844476, rs2269425, rs1555115, rs2395161, rs1383267, and rs2076311. Among these 11 SNPs, the SNP located at the 3⬘-untranslated region (3⬘-UTR) of HLA–E (rs2844724) was found to be highly significantly associated with KD (P ⬍ 1 ⫻ 10⫺7) (Tables 1 and 2). A statistically significant difference in genotype frequency distribution was found in the KD patients as compared with the controls (P ⫽ 4.26 ⫻ 10⫺8) (Table 1). The frequencies of the CC and the CT genotypes were significantly higher in KD patients than in controls, with an odds ratio (OR) of 7.11 (95% confidence interval [95% CI] 2.90–17.38) for the CC genotype and an OR of 6.98 (95% CI 3.28–14.88) for the CT genotype. The C allele frequency was significantly higher in KD patients as compared with controls (OR 2.31 [95% CI 1.66–3.21], P ⫽ 3.84 ⫻ 10⫺7) (Table 2). HLA–E polymorphism and occurrence of CAAs. As shown in Table 3, the frequency of the C allele of the HLA–E polymorphism was significantly higher in KD patients without CAA than in control subjects (OR 2.80 [95% CI 1.95–4.04], P ⬍ 0.001). A statistically significant difference in genotype frequency distribution was also found in KD patients without CAA as compared with controls (P ⬍ 0.001). The frequencies of the CC and CT genotypes were significantly higher in KD patients with- 608 LIN ET AL Table 3. Association of the HLA–E gene polymorphism in KD patients according to the presence or absence of CAA* Gene polymorphism HLA–E (rs2844724, at 3⬘-UTR) Allele C T Genotype CC CT TT KD patients with CAA KD patients without CAA No. (%) of controls No. (%) P OR (95% CI) No. (%) P OR (95% CI) 451 (34.0) 875 (66.0) 10 (33.3) 20 (66.7) 0.938 0.97 (0.45–2.09) 1 78 (59.1) 54 (40.9) ⬍0.001 2.80 (1.95–4.04) 1 76 (11.5) 299 (45.1) 288 (43.4) 0 (0.0) 10 (66.7) 5 (33.3) – 1.93 (0.65–5.70) 1 15 (22.7) 48 (72.7) 3 (4.6) 0.168 ⬍0.001 18.95 (5.35–67.14) 15.41 (4.75–50.03) 1 * Allele frequencies were determined by chi-square test using 2 ⫻ 2 contingency tables. Genotype frequencies were determined by chi-square test using 2 ⫻ 3 contingency tables. P values less than 0.05 were considered significant. KD ⫽ Kawasaki disease; CAA ⫽ coronary artery aneurysm; OR ⫽ odds ratio; 95% CI ⫽ 95% confidence interval; 3⬘-UTR ⫽ 3⬘-untranslated region. Figure 2. Detection of soluble HLA–E in plasma samples from 96 Taiwanese patients with Kawasaki disease (KD) and 93 healthy Taiwanese control subjects. Concentrations of soluble HLA–E in plasma samples from A, all KD patients, B, KD patients with or without coronary artery aneurysm (CAA), and C, KD patients with the CC, CT, and TT genotypes were compared with those in plasma from the healthy control subjects. Data are shown as box plots. Each box represents the 25th and 75th percentiles. Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent the median. Solid circles indicate outliers. P values were determined by Mann-Whitney U test. out CAA than in controls (OR 18.95 [95% CI 5.35– 67.14] for the CC genotype and OR 15.41 [95% CI 4.75–50.03] for the CT genotype). The allele and genotype frequencies were not statistically different in KD patients with CAA as compared with the controls. Plasma levels of soluble HLA–E. Plasma concentrations of soluble HLA–E were measured by ELISA in 96 patients with KD and in 93 healthy controls (Figure 2A). Levels of soluble HLA–E in plasma samples from KD patients were significantly higher than those in plasma samples from healthy controls (mean ⫾ SD 209.9 ⫾ 85.19 ng/ml versus 63.63 ⫾ 13.25 ng/ml; P ⬍ 0.0001). Plasma levels of soluble HLA–E were also analyzed in KD patients in relation to CAA formation because CAAs have been predicted to have possible functional correlations (Figure 2B). No significant difference between KD patients with CAA and those without CAA was observed (107.1 ⫾ 66.63 ng/ml versus 247.1 ⫾ 95.26 ng/ml; P ⫽ 0.412). Furthermore, plasma levels of soluble HLA–E in both groups of KD patients were significantly higher than those in the healthy controls. We also analyzed plasma levels of soluble HLA–E in relation to genotypes (Figure 2C). KD patients with the CT or TT genotype had significantly higher plasma levels of soluble HLA–E than the controls (227.2 ⫾ 79.45 ng/ml in KD patients with the CT genotype [P ⬍ 0.001] and 227.2 ⫾ 133.8 ng/ml in KD patients with the TT genotype [P ⬍ 0.005]). No significant difference between KD patients with the CC genotype and the control subjects was observed (P ⫽ 0.131). The number of KD patients with CAA was insufficient to compare plasma levels of soluble HLA–E with the genotype data. HLA–E GENE POLYMORPHISM IN KD DISCUSSION In this study, we used a mapping strategy focusing on the MHC region and identified a SNP that contributes to KD susceptibility in Taiwanese children of Han Chinese ethnic background. We observed a significant association between the HLA–E gene polymorphism and the occurrence of cardiac artery aneurysm in KD patients. We further showed that plasma levels of soluble HLA–E were significantly higher in patients with KD and that there was a trend toward higher plasma levels of soluble HLA–E in both CAA subgroups of KD patients than in the healthy controls. Furthermore, higher plasma levels of soluble HLA–E in KD patients with CT and TT genotypes of the HLA–E gene polymorphism were also noted. Our results suggest that polymorphism of the HLA–E gene may play a role in the pathogenesis of KD. Our genetic association study showed that the frequencies of alleles and genotypes with 1 or 2 copies of the C allele were significantly higher in KD patients than in controls. Furthermore, this polymorphism was associated with KD patients without CAA. These results suggest that the HLA–E gene polymorphism is involved in disease susceptibility and progression. Individuals with KD who have 1 or 2 copies of the C allele tend not to develop CAA. Therefore, it is logical to assume that the CC or CT genotype of the 3⬘-UTR of the HLA–E gene polymorphism may affect serum/plasma levels of soluble HLA–E or the development of CAA in KD patients, although the influence of this SNP on the production of soluble HLA–E by vascular endothelial cells remains unknown. A possible explanation for the influence of the 3⬘-UTR may be because specific sequences in the 3⬘-UTR of RNA, together with stabilizing and destabilizing proteins, determine the messenger RNA stability and, consequently, the level of expression of proteins (46–48). Our studies showed that regardless of the presence of CAA, KD patients had significantly higher levels of soluble HLA–E than did healthy controls. Furthermore, KD patients with the CT and TT genotypes of this gene polymorphism appeared to have higher soluble HLA–E levels. HLA–E is a known ligand of CD94/ natural killer cell receptor group 2-A (NKG2-A) and CD94/NKG2-C, which are expressed on natural killer cells, a subset of T cells, and vascular endothelial cells (49–51). Recent studies have shown that the expression of soluble HLA–E may have important implications in the pathogenesis of immune-mediated vascular diseases (51). It is also believed that HLA–E has regulatory functions in both the innate and adaptive immune 609 responses. KD is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis (1,52). The vascular endothelium is a functional barrier between the vessel wall and the bloodstream, and endothelial cell damage or vascular injury leads to the expression and release of HLA–E molecules (51). Taken together, these data suggest that HLA–E molecules may be involved in the pathogenesis of KD. In conclusion, we have shown that susceptibility to the development of KD is associated with genetic predisposition in Taiwanese children of Han Chinese ethnic background. Genetic polymorphism in the MHC region, particularly the HLA–E gene, is associated with susceptibility to KD. AUTHOR CONTRIBUTIONS Dr. F.-J. Tsai had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. Hsueh, Chang, F.-J. Tsai. Acquisition of data. Sheu, T.-H. Lin, Huang. Analysis and interpretation of data. Y.-J. Lin, Wan, Y. Tsai, J.-G. Lin. Manuscript preparation. Y.-J. Lin, C.-W. Lin, Lai, Hung, C.-H. Tsai. Statistical analysis. Wu, Lan. REFERENCES 1. Burns JC, Glode MP. Kawasaki syndrome. Lancet 2004;364: 533–44. 2. Kawasaki T. Kawasaki disease: a new disease? Acta Paediatr Taiwan 2001;42:8–10. 3. Chang LY, Chang IS, Lu CY, Chiang BL, Lee CY, Chen PJ, et al, and the Kawasaki Disease Research Group. Epidemiologic features of Kawasaki disease in Taiwan, 1996-2002. Pediatrics 2004; 114:e678–82. 4. Kato H, Sugimura T, Akagi T, Sato N, Hashino K, Maeno Y, et al. Long-term consequences of Kawasaki disease: a 10- to 21-year follow-up study of 594 patients. Circulation 1996;94:1379–85. 5. Newburger JW, Takahashi M, Beiser AS, Burns JC, Bastian J, Chung KJ, et al. A single intravenous infusion of gamma globulin as compared with four infusions in the treatment of acute Kawasaki syndrome. N Engl J Med 1991;324:1633–9. 6. Tse SM, Silverman ED, McCrindle BW, Yeung RS. Early treatment with intravenous immunoglobulin in patients with Kawasaki disease. J Pediatr 2002;140:450–5. 7. Nagashima M, Matsushima M, Matsuoka H, Ogawa A, Okumura N. High-dose gammaglobulin therapy for Kawasaki disease. J Pediatr 1987;110:710–2. 8. Kato H, Koike S, Yamamoto M, Ito Y, Yano E. Coronary aneurysms in infants and young children with acute febrile mucocutaneous lymph node syndrome. J Pediatr 1975;86:892–8. 9. Bronstein DE, Dille AN, Austin JP, Williams CM, Palinkas LA, Burns JC. Relationship of climate, ethnicity and socioeconomic status to Kawasaki disease in San Diego County, 1994 through 1998. Pediatr Infect Dis J 2000;19:1087–91. 10. Holman RC, Curns AT, Belay ED, Steiner CA, Schonberger LB. Kawasaki syndrome hospitalizations in the United States, 1997 and 2000. Pediatrics 2003;112:495–501. 11. Gardner-Medwin JM, Dolezalova P, Cummins C, Southwood TR. Incidence of Henoch-Schönlein purpura, Kawasaki disease, and rare vasculitides in children of different ethnic origins. Lancet 2002;360:1197–202. 610 12. Royle JA, Williams K, Elliott E, Sholler G, Nolan T, Allen R, et al. Kawasaki disease in Australia, 1993-95. Arch Dis Child 1998;78: 33–9. 13. Schiller B, Fasth A, Bjorkhem G, Elinder G. Kawasaki disease in Sweden: incidence and clinical features. Acta Paediatr 1995;84: 769–74. 14. Park YW, Han JW, Park IS, Kim CH, Yun YS, Cha SH, et al. Epidemiologic picture of Kawasaki disease in Korea, 2000-2002. Pediatr Int 2005;47:382–7. 15. Lin CY, Lin CC, Hwang B, Chiang B. Serial changes of serum interleukin-6, interleukin-8, and tumor necrosis factor ␣ among patients with Kawasaki disease. J Pediatr 1992;121:924–6. 16. Matsubara T, Furukawa S, Yabuta K. Serum levels of tumor necrosis factor, interleukin 2 receptor, and interferon-␥ in Kawasaki disease involved coronary-artery lesions. Clin Immunol Immunopathol 1990;56:29–36. 17. Leung DY, Geha RS, Newburger JW, Burns JC, Fiers W, Lapierre LA, et al. Two monokines, interleukin 1 and tumor necrosis factor, render cultured vascular endothelial cells susceptible to lysis by antibodies circulating during Kawasaki syndrome. J Exp Med 1986;164:1958–72. 18. Leung DY, Cotran RS, Kurt-Jones E, Burns JC, Newburger JW, Pober JS. Endothelial cell activation and high interleukin-1 secretion in the pathogenesis of acute Kawasaki disease. Lancet 1989; 2:1298–302. 19. Danese S, Dejana E, Fiocchi C. Immune regulation by microvascular endothelial cells: directing innate and adaptive immunity, coagulation, and inflammation. J Immunol 2007;178:6017–22. 20. Choi J, Enis DR, Koh KP, Shiao SL, Pober JS. T lymphocyteendothelial cell interactions. Annu Rev Immunol 2004;22:683–709. 21. Pay S, Simsek I, Erdem H, Dinc A. Immunopathogenesis of Behçet’s disease with special emphasize [sic] on the possible role of antigen presenting cells. Rheumatol Int 2007;27:417–24. 22. Durrani K, Papaliodis GN. The genetics of AdamantiadesBehçet’s disease. Semin Ophthalmol 2008;23:73–9. 23. Kato S, Kimura M, Tsuji K, Kusakawa S, Asai T, Juji T, et al. HLA antigens in Kawasaki disease. Pediatrics 1978;61:252–5. 24. Kaslow RA, Bailowitz A, Lin FY, Koslowe P, Simonis T, Israel E. Association of epidemic Kawasaki syndrome with the HLA–A2, B44, Cw5 antigen combination. Arthritis Rheum 1985;28:938–40. 25. Maclaren N, Skordis N. Is Kawasaki HLA associated? Prog Clin Biol Res 1987;250:475–84. 26. Chang CC, Hawkins BR, Kao HK, Chow CB, Lau YL. Human leucocyte antigens in southern Chinese with Kawasaki disease [letter]. Eur J Pediatr 1992;151:866. 27. Barron KS, Silverman ED, Gonzales JC, St Clair M, Anderson K, Reveille JD. Major histocompatibility complex class II alleles in Kawasaki syndrome—lack of consistent correlation with disease or cardiac involvement. J Rheumatol 1992;19:1790–3. 28. Fildes N, Burns JC, Newburger JW, Klitz W, Begovich AB. The HLA class II region and susceptibility to Kawasaki disease. Tissue Antigens 1992;39:99–101. 29. Huang FY, Chang TY, Chen MR, Hsu CH, Lee HC, Lin SP, et al. Genetic variations of HLA-DRB1 and susceptibility to Kawasaki disease in Taiwanese children. Hum Immunol 2007;68:69–74. 30. Huang Y, Lee YJ, Chen MR, Hsu CH, Lin SP, Sung TC, et al. Polymorphism of transmembrane region of MICA gene and Kawasaki disease. Exp Clin Immunogenet 2000;17:130–7. 31. Szyld P, Jagiello P, Csernok E, Gross WL, Epplen JT. On the Wegener granulomatosis associated region on chromosome 6p21.3. BMC Med Genet 2006;7:21. 32. Miyashita R, Tsuchiya N, Yabe T, Kobayashi S, Hashimoto H, Ozaki S, et al. Association of killer cell immunoglobulin-like receptor genotypes with microscopic polyangiitis. Arthritis Rheum 2006;54:992–7. LIN ET AL 33. Newburger JW, Takahashi M, Gerber MA, Gewitz MH, Tani LY, Burns JC, et al. Diagnosis, treatment, and long-term management of Kawasaki disease: a statement for health professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, American Heart Association [published erratum appears in Pediatrics 2005;115:1118]. Pediatrics 2004;114:1708–33. 34. Falcini F. Kawasaki disease. Curr Opin Rheumatol 2006;18:33–8. 35. Kim S, Dedeoglu F. Update on pediatric vasculitis. Curr Opin Pediatr 2005;17:695–702. 36. Wu SF, Chang JS, Wan L, Tsai CH, Tsai FJ. Association of IL-1Ra gene polymorphism, but no association of IL-1␤ and IL-4 gene polymorphisms, with Kawasaki disease. J Clin Lab Anal 2005;19: 99–102. 37. Wu SF, Chang JS, Peng CT, Shi YR, Tsai FJ. Polymorphism of angiotensin-1 converting enzyme gene and Kawasaki disease. Pediatr Cardiol 2004;25:529–33. 38. Akagi T, Rose V, Benson LN, Newman A, Freedom RM. Outcome of coronary artery aneurysms after Kawasaki disease. J Pediatr 1992;121:689–94. 39. Hung SI, Chung WH, Liou LB, Chu CC, Lin M, Huang HP, et al. HLA-B*5801 allele as a genetic marker for severe cutaneous adverse reactions caused by allopurinol. Proc Natl Acad Sci U S A 2005;102:4134–9. 40. Sherry ST, Ward MH, Kholodov M, Baker J, Phan L, Smigielski EM, et al. dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 2001;29:308–11. 41. Sherry ST, Ward M, Sirotkin K. Use of molecular variation in the NCBI dbSNP database. Hum Mutat 2000;15:68–75. 42. Walsh EC, Mather KA, Schaffner SF, Farwell L, Daly MJ, Patterson N, et al. An integrated haplotype map of the human major histocompatibility complex. Am J Hum Genet 2003;73:580–90. 43. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21: 263–5. 44. Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, et al. The structure of haplotype blocks in the human genome. Science 2002;296:2225–9. 45. Schaid DJ, Jacobsen SJ. Biased tests of association: comparisons of allele frequencies when departing from Hardy-Weinberg proportions. Am J Epidemiol 1999;149:706–11. 46. Amrani N, Sachs MS, Jacobson A. Early nonsense: mRNA decay solves a translational problem. Nat Rev Mol Cell Biol 2006;7: 415–25. 47. Behm-Ansmant I, Kashima I, Rehwinkel J, Sauliere J, Wittkopp N, Izaurralde E. mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons. FEBS Lett 2007;581:2845–53. 48. Khajavi M, Inoue K, Lupski JR. Nonsense-mediated mRNA decay modulates clinical outcome of genetic disease. Eur J Hum Genet 2006;14:1074–81. 49. Braud VM, Allan DS, O’Callaghan CA, Soderstrom K, D’Andrea A, Ogg GS, et al. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 1998;391:795–9. 50. Lee N, Llano M, Carretero M, Ishitani A, Navarro F, Lopez-Botet M, et al. HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proc Natl Acad Sci U S A 1998;95: 5199–204. 51. Coupel S, Moreau A, Hamidou M, Horejsi V, Soulillou JP, Charreau B. Expression and release of soluble HLA-E is an immunoregulatory feature of endothelial cell activation. Blood 2007;109:2806–14. 52. Burns JC. Commentary: translation of Dr. Tomisaku Kawasaki’s original report of fifty patients in 1967. Pediatr Infect Dis J 2002;21:993–5.