15 4 10 21 14 20 13 16 18 19 6 12 2 8 27 Materials and Methods Subjects n 25 1 Table 1 Profiles of subjects with Kawasaki disease  n Male/female 44/30 (60/40) Typical KD/atypical KD 65/9 (88/12) Family history of KD 3 (4.1) Recurrence history of KD 4 (5.4) Coronary complications 21 (28) Other complications of KD Gallbladder hydrops 14 (19) Pyuria 25 (34) Hepatopathy 38 (51) Arthritis 10 (14) KD, Kawasaki disease Genomic DNA was extracted by standard methods using the AccuPrep DNA extraction kit (Bioneer, Daejeon, Korea) from peripheral blood collected (4 ml) with ethylenediaminetetraacetic acid (EDTA) and kept at −20°C. HLA-A, -B, and -C Genotyping 5 17 26 2 The amplifications were performed in a My Cycler thermocycler (Bio-Rad, Hercules, USA). For amplification, 30 cycles were performed using the following steps: heating to 96°C for 1 min to denature the DNA; denaturation at 96°C for 25 s and at 70°C for 45 s; annealing and extension at 72°C for 30 s (for the first 5 cycles), 96°C for 25 s, 65°C 45 s, 72°C for 30 s (for the next 21 cycles); 96°C for 25 s, 55°C for 60 s, 72°C for 120 s (for the last 4 cycles); and a final 1 min extension at 72°C. The presence or absence of PCR products was determined after separation of the samples on a 1.5% agarose gel containing 0.5 μg/ml of ethidium bromide. HLA-DRB1 Genotyping 23 2 The amplifications performed in a My Cycler thermocycler (Bio-Rad, Hercules, USA). For amplification, 27 cycles were performed using the following steps: heating to 96°C for 3 min to denature the DNA; denaturation at 96°C for 20 s; annealing and extension at 66°C for 60 s (for the first 10 cycles), 62°C for 80 s (for the next 10 cycles), and 61°C for 120 s (for the last 7 cycles); and a final 10 min extension at 72°C. The presence or absence of PCR products was determined after separation of samples on a 1.5% agarose gel containing 0.5 μg/ml of ethidium bromide. Statistical Analysis p In this study, the data were not adjusted for multiple comparisons because the sample size was not large enough for multiple-comparison analysis correction. Results The Genotype Distribution and Allele Frequencies of HLA-A for the Patients With Kawasaki Disease and the Healthy Control Group p 2 Table 2 The allele frequencies of human leukocyte antigen-A (HLA-A) in the patients with Kawasaki disease and the control subjects HLA alleles n n n n Coronary complications n n n n HLA-A 01 5 (3.1) 5 (6.8) 2 (9.5) 3 (5.7) 02 85 (53.5) 43 (58.1) 12 (57.1) 31 (58.5) 03 5 (3.1) 3 (4.1) 0 (0.0) 3 (5.7) 11 31 (19.5) 21 (28.4) 6 (28.6) 15 (28.3) 24 57 (35.8) 33 (44.6) 12 (57.1) 21 (39.6) 26 25 (15.7) 6 (8.1) 4 (19.0) a 30 20 (12.6) 4 (5.4) 1 (4.8) 3 (5.7) 31 14 (8.8) 6 (8.1) 0 (0.0) 6 (11.3) 32 3 (1.9) 0 (0.0) 0 (0.0) 0 (0.0) 33 38 (23.9) 15 (20.3) 2 (9.5) 13 (24.5) 68 1 (0.6) 0 (0.0) 0 (0.0) 0 (0.0) KD, Kawasaki disease; RR, relative risk a p The Genotype Distribution and Allele Frequencies of HLA-B for the Patients With Kawasaki Disease and the Healthy Control Group p p p p 3 Table 3 The allele frequencies of human leukocyte antigen-B (HLA-B) in the patients with Kawasaki disease and the control subjects HLA alleles n n n n Coronary complications n n n n HLA-B 07 19 (11.9) 9 (12.2) 3 (14.3) 6 (11.3) 08 2 (1.3) 0 (0.0) 0 (0.0) 0 (0.0) 13 20 (12.6) 5 (6.8) 2 (9.5) 3 (5.7) 14 6 (3.8) 0 (0.0) 0 (0.0) 0 (0.0) 27 11 (6.9) 3 (4.1) 1 (4.8) 2 (3.8) 35 11 (6.9) a 4 (19.0) b 37 5 (3.1) 4 (5.4) 2 (9.5) 2 (3.8) 38 7 (4.4) 3 (4.1) 0 (0.0) 3 (5.7) 39 2 (1.3) 3 (4.1) 2 (9.5) 1 (1.9) 44 29 (18.2) 10 (13.5) 3 (14.3) 7 (13.2) 46 18 (11.3) 7 (9.5) 2 (9.5) 5 (9.4) 47 0 (0.0) 1 (1.4) 0 (0.0) 1 (1.9) 48 13 (8.2) 2 (2.7) 0 (0.0) 2 (3.8) 51 29 (18.2) 13 (17.6) 5 (23.8) 8 (15.1) 52 9 (5.7) 3 (4.1) 2 (9.5) 1 (1.9) 54 23 (14.5) 10 (13.5) 3 (14.3) 7 (13.2) 55 3 (1.9) 4 (5.4) 1 (4.8) 3 (5.7) 56 2 (1.3) 0 (0.0) 0 (0.0) 0 (0.0) 57 2 (1.3) 1 (1.4) 0 (0.0) 1 (1.9) 58 15 (9.4) 5 (6.8) 0 (0.0) 5 (9.4) 59 4 (2.5) 3 (4.1) 0 (0.0) 3 (5.7) 60 11 (6.9) 7 (9.5) 2 (9.5) 5 (9.4) 61 23 (14.5) 13 (17.6) 6 (28.6) 7 (13.2) 62 29 (18.2) 14 (18.9) 1 (4.8) 13 (24.5) 67 4 (2.5) 2 (2.7) 1 (4.8) 1 (1.9) 71 7 (4.4) 1 (1.4) 0 (0.0) 1 (1.9) 75 2 (1.3) c 0 (0.0) d KD, Kawasaki disease; RR, relative risk a p b p c p d p The Genotype Distribution and Allele Frequency of HLA-C for the Patients With Kawasaki Disease and the Healthy Control Group p p 4 Table 4 The allele frequencies of human leukocyte antigen-C (HLA-C) in the patients with Kawasaki disease and the control subjects HLA alleles n n n n Coronary complications n n n n HLA-Cw 01 59 (37.1) 22 (29.7) 6 (28.6) 16 (30.2) 02 0 (0.0) 1 (1.4) 0 (0.0) 1 (1.9) 04 21 (13.2) 11 (14.9) 2 (9.5) 9 (17.0) 05 6 (3.8) 1 (1.4) 0 (0.0) 1 (1.9) 06 25 (15.7) 10 (13.5) 5 (23.8) 5 (9.4) 07 44 (27.7) 20 (27.0) 6 (28.6) 14 (26.4) 08 29 (18.2) 10 (13.5) 3 (14.3) 7 (13.2) 09 23 (14.5) a 5 (23.8) b 10 39 (24.5) 24 (32.4) 5 (23.8) 19 (35.8) 12 11 (6.9) 4 (5.4) 2 (9.5) 2 (3.8) 14 34 (21.4) 18 (24.3) 7 (33.3) 11 (20.8) 15 8 (5.0) 1 (1.4) 1 (4.8) 0 (0.0) KD, Kawasaki disease; RR, relative risk a p b p The Genotype Distribution and the Allele Frequencies of HLA-DRB1 for the Patients With Kawasaki Disease and the Healthy Control Group p p p 5 Table 5 The allele frequencies of human leukocyte antigen-DRB1 (HLA-DRB1) in the patients with Kawasaki disease and the control subjects HLA alleles n n n n Coronary complications n n n n HLA-DRB1 01 29 (18.2) 8 (10.8) 1 (4.8) 7 (13.2) 03 7 (4.4) 1 (1.4) 0 (0.0) 1 (1.9) 04 48 (30.2) 30 (40.5) 6 (28.6) a 07 26 (16.4) 8 (10.8) 1 (4.8) 7 (13.2) 08 32 (20.1) 14 (18.9) 3 (14.3) 11 (20.8) 09 28 (17.6) 12 (16.2) b 5 (9.4) 10 4 (2.5) 4 (5.4) 2 (9.5) 2 (3.8) 11 10 (6.3) 8 (10.8) c 3 (5.7) 12 18 (11.3) 11 (14.9) 2 (9.5) 9 (17.0) 13 32 (20.1) 10 (13.5) 3 (14.3) 7 (13.2) 14 21 (13.2) 10 (13.5) 3 (14.3) 7 (13.2) 15 35 (22.0) 20 (27.0) 5 (23.8) 15(28.3) 16 4 (2.5) 2 (2.7) 1 (4.8) 1 (1.9) KD, Kawasaki disease; RR, relative risk a p b p c p Discussion 22 11 24 28 Our data were confined to Korean children and focused on patients who had Kawasaki disease with CC. Therefore, further research on the effect of the family history and recurrence of Kawasaki disease in association with HLA polymorphisms is needed. 14 1 3 12 14 16 19 Review of the medical literature on Kawasaki disease and HLA shows that there is a trend toward an association between the HLA-B loci and Kawasaki disease. However, to date, there is no confirmed relationship to a particular locus. It is likely that the HLA-B locus is not the only locus associated with Kawasaki disease. This can be the case because of its functional variation or the potential effects from other related genes around the HLA-B locus. The HLA-B locus and other associated genes might be useful genetic markers for Kawasaki disease. 9 p p p 7 2 8 Interpopulation discrepancies of the frequencies of HLA alleles make generalization of results difficult. Matched population profiles are needed for disease association studies because ethnic differences can provide altered disease associations. In this study, we compared HLA alleles of Kawasaki disease patients with known polymorphic loci of HLA genes in healthy Korean adults. The results we report on the polymorphisms of HLA genes in Kawasaki disease patients may be limited to the Korean population. However, this information may be helpful in future studies on HLA genetic polymorphisms in Kawasaki disease. Further studies with a larger sample size are needed for confirmation of our findings.