1. SCANNING OF CANDIDATE GENES FOR THE SUSCEPTIBILITY OF KAWASAKI DISEASE IN THE HLA REGION Lee JK, Kim JJ, Kim S, Choi IH, Kim KJ, Hong SJ, Seo EJ, Yoo HW, Park IS Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul, Korea Abstract BACKGROUND: Kawasaki disease is an acute vasculitis of children under the age of 5 years that preferentially affects coronary arteries. The disease is characterized by persistent fever for at least 5 days, cervical lymphadenopathy, bilateral conjunctivitis, rash, inflammation of the mucous membranes, and skin rash. Treatment with intravenous immunoglobulin reduces systemic inflammation and prevents coronary artery lesions in Kawasaki disease. The information suggests the importance of immune response in the pathogenesis and disease susceptibility. METHODS: In this study, we investigated the possible association of human leukocyte antigen (HLA) region for the susceptibility of Kawasaki disease. We used HLA SNP chip (Illumina) which contains 2360 SNP markers covering HLA region with 2kb average spacing. In a HLA chip experiment, we used a total of 48 Kawasaki disease patients and 90 normal controls. RESULTS: In total, 5 candidate loci have been identified with significant level (p<0.01) in the initial association analysis of HLA chip data. However, only one candidate locus, which has not been reported previously, has been confirmed with significant association (odds ratio= 3.71, p= 0.001) in the replication study using 48 Kawasaki patients and 90 normal controls. Currently, genetic variants identified by resequencing in the HLA-G region are under investigation for the possible association with Kawasaki disease. CONCLUSIONS: Our initial study indicates that a new genetic locus in HLA region may play an important role for the susceptibility of Kawasaki disease. Introduction Kawasaki disease (KD) is an acute vasculitis of infants and young children, manifested by fever and signs of mucocutaneous inflammation. Furthermore, in the KD patients, coronary artery aneurysms develop in approximately 15 to 25% of untreated children and lead to ischemic heart disease and/or sudden death. However, intravenous infusion of high-dose immunoglobulin effectively reduces systemic inflammation and prevents coronary artery lesions in KD. Although its etiology is largely unknown, several lines of evidence suggest that immune response play a very important role for the development and treatment of. In regard to the genetic risk for KD, a genome-wide linkage analysis demonstrated an evidence for linkage to chromosome 12q24. Although it was recently reported that ITPKC functional polymorphism was associated with KD susceptibility, however, the genetic risk for KD was not well characterized yet. The major histocompatibility complex (MHC) region spans ~4 Mb on chromosome 6p21.3 and encodes over 160 genes, including the human leukocyte antigen (HLA) proteins, which are mainly involved in the immune system. It has been demonstrated that MHC region contribute to the majority of autoimmune and inflammatory disorders. In this study, we investigated the genetic association of MHC region for the association with KD by using a MHC Panel Set of 2,360 SNPs in the Korean population. Figure 1. Screening of HLA region for the association of Kawasaki disease. HLA genomic region was screened by using Illumina HLA SNP chip containing 2360 SNPs. A total of 48 Kawasaki disease DNA samples and 90 normal control DNA samples were used for the initial screening. Based on the association analysis under different genetic models (dominant, codominant or recessive), 5 candidate regions showing p-value < 0.01 were selected as potential association loci of Kawasaki disease. Among 5 candidate loci, candidate 2 showed the most significant signal for association. KD patientsNormal controlsAllele 1 vs. 2Co-dominantDominantRecessive GeneLocationdbSNP IDAllele11 a 1222MAF111222MAFOR (95%CI)P valueOR (95%CI)P valueOR (95%CI)P valueOR (95%CI)P value 1st OR2H2flanking_5’UTRSNP-1G>T341400.15453960.280.43(0.22-0.83)0.01570.40(0.20-0.80)0.00950.41(0.20-0.87)0.0199N/A0.9976 OR2H2flanking_3’UTRSNP-2G>A341400.15433960.290.42(0.22-0.80)0.01200.40(0.20-0.80)0.00950.39(0.19-0.83)0.0147N/A0.9975 OR2H2flanking_3’UTRSNP-3G>A341400.15453960.280.43(0.22-0.83)0.01570.40(0.20-0.80)0.00950.41(0.20-0.87)0.0199N/A0.9976 HLA-Gflanking_3’UTRSNP-4A>G1123140.53285390.391.74(1.06-2.87)0.04011.90(1.09-3.31)0.02261.52(0.68-3.41)0.31033.71(1.46-9.38)0.0057 HLA-Gflanking_3’UTRSNP-5A>C1222140.52285390.391.67(1.01-2.75)0.05901.79(1.04-3.09)0.03581.35(0.61-2.99)0.45193.71(1.46-9.38)0.0057 HLA-Gflanking_3’UTRSNP-6T>C1123140.53285390.391.74(1.06-2.87)0.04011.90(1.09-3.31)0.02261.52(0.68-3.41)0.31033.71(1.46-9.38)0.0057 HLA-Gflanking_3’UTRSNP-7C>T1123140.53285390.391.74(1.06-2.87)0.04011.90(1.09-3.31)0.02261.52(0.68-3.41)0.31033.71(1.46-9.38)0.0057 PSORS1C1intronSNP-8A>G331410.17414270.140.44(0.24-0.83)0.01390.42(0.22-0.80)0.00890.38(0.18-0.80)0.01020.25(0.03-2.11)0.2041 SKIV2LintronSNP-9C>T172650.38552780.241.91(1.12-3.27)0.02491.86(1.09-3.18)0.02302.87(1.38-5.93)0.00461.19(0.37-3.87)0.7700 SKIV2LintronSNP-10G>A172650.38552780.241.91(1.12-3.27)0.02491.86(1.09-3.18)0.02302.87(1.38-5.93)0.00461.19(0.37-3.87)0.7700 TAP2flanking_3’UTRSNP-11T>C301350.243248100.380.52(0.30-0.91)0.02840.51(0.29-0.91)0.02210.33(0.16-0.68)0.00290.93(0.30-2.90)0.9007 2nd OR2H2flanking_5’UTRSNP-1G>T29960.24453960.280.79(0.44-1.43)0.52970.81(0.46-1.42)0.45840.52(0.24-1.09)0.08402.21(0.67-7.30)0.1931 HLA-Gflanking_3’UTRSNP-4A>G142090.44285390.391.22(0.72-2.04)0.54711.28(0.73-2.24)0.38950.94(0.43-2.04)0.86672.38(0.87-6.52)0.0911 HLA-Gflanking_3’UTRSNP-5A>C1419110.47285390.391.34(0.80-2.24)0.32621.39(0.80-2.41)0.24120.97(0.45-2.10)0.93403.00(1.14-7.91)0.0263 PSORS1C1intronSNP-8A>G221830.28414270.310.86(0.49-1.51)0.69650.86(0.48-1.55)0.62060.80(0.39-1.65)0.54500.89(0.22-3.62)0.8699 SKIV2LintronSNP-9C>T221660.32552780.241.49(0.85-2.61)0.21731.40(0.83-2.37)0.20541.57(0.76-3.25)0.22321.62(0.52-4.99)0.4021 TAP2flanking_3’UTR SNP-1 1 T>C19 60.353248100.380.90(0.53-1.52)0.78600.89(0.51-1.54)0.67320.73(0.35-1.52)0.39391.26(0.43-3.73)0.6725 Combined OR2H2flanking_5’UTRSNP-1G>T632360.19453960.280.59(0.36-0.97)0.04910.61(0.37-0.99)0.04410.46(0.25-0.84)0.01170.98(0.30-3.15)0.9686 HLA-Gflanking_3’UTRSNP-4A>G2543230.49285390.391.47(0.97-2.23)0.08851.55(1.00-2.41)0.05041.19(0.63-2.26)0.59093.04(1.32-7.02)0.0090 HLA-Gflanking_3’UTRSNP-5A>C2641250.49285390.391.50(0.99-2.28)0.06931.54(1.00-2.37)0.05091.15(0.61-2.17)0.67403.36(1.47-7.68)0.0041 PSORS1C1intronSNP-8A>G553240.22414270.310.62(0.39-1.00)0.06440.62(0.38-1.01)0.05330.55(0.30-0.99)0.04570.55(0.15-1.93)0.3471 SKIV2LintronSNP-9C>T3942110.35552780.241.70(1.08-2.69)0.03031.64(1.05-2.56)0.03042.14(1.18-3.86)0.01201.39(0.53-3.64)0.5000 TAP2flanking_3’UTRSNP-11T>C4932110.293248100.380.68(0.44-1.06)0.11150.69(0.44-1.06)0.09260.48(0.27-0.88)0.01691.09(0.44-2.70)0.8584 Table 1. Summary of screening results of MHC region for the association with Kawasaki disease and replication study of 5 candidate loci a 11, 12 and 22 represent homozygote for the major allele, heterozygote and homozygote for the minor allele, respectively. Logistic regression analysis was used for calculating odds ratios (95% confidence interval) and corresponding P values for each SNP site. KD, Kawasaki disease; MAF, minor allele frequency; OR, odds ratio; CI, confidence interval, N/A; not available Figure 4. SNP map of HLA-G locus. The SNP map of HLA-G region was shown. All the SNP identified by resequencing were shown in the Figure. The LD block and r 2 map of HLA-G locus were also presented. Materials and Methods Subjects and genomic DNA isolation The 90 control samples in the initial screening were members of the general Korean population derived from cohort samples. In addition, several Korean individuals with KD and healthy control subjects were recruited at Asan Medical Center in Korea. This protocol conformed to the ethical guidelines of the Institutional Review Board. All the parents of the patients gave written informed consent. Genomic DNA was isolated from whole blood with a PureGene kit (Gentra Systems). SNP genotyping and resequencing SNP genotyping was performed using the Illumina GoldenGate BeadExpress system (San Diego, CA, USA) on the MHC Panel Set containing 2,360 SNPs in the MHC region. For the initial screening, a total of 48 children with KD and 90 healthy adults were recruited. To replicate findings with respect to the initial significant SNPs, additional 48 children with KD were used for SNP genotyping by direct sequencing of PCR products using ABI3730 sequencers (Applied Biosystems) according to standard protocols. In order to identify all genetic variants in the HLA-G locus, resequencing method was also used by direct sequencing of 12 cases and 12 normal control DNA samples. Statistical analysis Statistical analysis of case-control study was performed using Haplotyper and/or Haploview program. The association and Hardy-Weinberg equilibrium were examined with the Chi-squared (χ 2 ) test and Fisher’s exact test. General statistical analysis was performed with SPSS 12.0 (SPSS Inc., Chicago, Illinois, USA) and statistical significance was inferred at a two- tailed value of P<0.05. Figure 2. Replication study of 5 candidate loci for the association with Kawasaki disease. Potential 5 candidate loci were investigated by replication study using new patient DNA samples (N=48) and same control DNA samples (N=90). Among 5 loci, only 1 locus (HLA-G locus) was replicated with significance (p-value= 0.026). Figure 3. Association and LD map of HLA-G locus. Using the SNP genotype data obtained in the initial screening of MHC SNP chip, we analyzed the LD block structure with the Haploview program. The significantly associated SNPs were located in the HLA-G locus.