Journal of Human Genetics (2017) 62, 485–489 OPEN Official journal of the Japan Society of Human Genetics www.nature.com/jhg ORIGINAL ARTICLE Genome-wide association study using the ethnicity- specific Japonica array: identification of new susceptibility loci for cold medicine-related Stevens– Johnson syndrome with severe ocular complications Mayumi Ueta1,2,10,11, Hiromi Sawai2,10, Ryosei Shingaki3, Yusuke Kawai4, Chie Sotozono5, Kaname Kojima4, Kyung-Chul Yoon6, Mee Kum Kim7, Kyoung Yul Seo8, Choun-Ki Joo9, Masao Nagasaki4, Shigeru Kinoshita1 and Katsushi Tokunaga2,11 A genome-wide association study (GWAS) for cold medicine-related Stevens–Johnson syndrome (CM-SJS) with severe ocular complications (SOC) was performed in a Japanese population. A recently developed ethnicity-specific array with genome-wide imputation that was based on the whole-genome sequences of 1070 unrelated Japanese individuals was used. Validation analysis with additional samples from Japanese individuals and replication analysis using samples from Korean individuals identified two new susceptibility loci on chromosomes 15 and 16. This study might suggest the usefulness of GWAS using the ethnicity-specific array and genome-wide imputation based on large-scale whole-genome sequences. Our findings contribute to the understanding of genetic predisposition to CM-SJS with SOC. Journal of Human Genetics (2017) 62, 485–489; doi:10.1038/jhg.2016.160; published online 19 January 2017 INTRODUCTION significantly associated with HLA-B*44:03.9 In our first genome-wide Stevens–Johnson syndrome (SJS) is an acute inflammatory vesiculo- association study (GWAS), we analyzed SJS/TEN with SOC patients using bullous reaction of the skin and mucosa. It tends to involve the ocular the Affymetrix GeneChip Mapping 500 K array set (Affymetrix, Santa surface, oral cavity and genitals. In patients with extensive skin Clara, CA, USA). We found an association between prostaglandin E receptor detachment and a poor prognosis, the condition is called toxic 3 (PTGER3) and SJS/TEN with SOC.6,10 We subsequently documented epidermal necrolysis (TEN). Although these reactions are rare (annual that this association was stronger in CM-SJS/TEN patients with SOC than incidence 1–6 cases per 106 persons),1–3 the mortality rate is 3% for in all SJS/TEN patients with SOC.11 Moreover,weidentified an interaction SJS and 27% for TEN.4 Survivors often suffer severe sequelae such as with additive effects between HLA-A*02:06 and the high-risk genotypes vision loss due to severe ocular complications (SOC).5 PTGER3 rs1327464 GA or AA.11 A second GWAS using the Affymetrix About 40% of SJS/TEN patients diagnosed by dermatologists develop AXIOM Genome-Wide ASI 1 array (Affymetrix) identified the association SOC.2 Among SJS and TEN patients, especially those with SJS/TEN with between IKAROS family zinc-finger 1 (IKZF1) and CM-SJS/TEN with SOC SOC, cold medicines (CM), including multi-ingredient cold medications in the Japanese.7 These findings were indicative of a genetic predisposition and non-steroidal anti-inflammatory drugs, were identified as for CM-SJS/TEN with SOC. causative.2,6–9 We reported that in the Japanese, CM-related SJS/TEN A single-nucleotide polymorphism (SNP) array (Japonica Array, (CM-SJS/TEN) with SOC was strongly associated with HLA-A*02:06 and Toshiba, Japan) specifically designed for the genome-wide study of the 1Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; 2Department of Human Genetics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; 3Life Science Business Department, Healthcare Medical Business Promotion Division, Toshiba Corporation Healthcare Company, Tokyo, Japan; 4Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan; 5Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; 6Department of Ophthalmology, Chonnam National University, Gwangju, Korea; 7Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea; 8Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea and 9Department of Ophthalmology and Visual Science, Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea 10Contributed equally with the first author. 11These authors contributed equally to this work. Correspondence: Dr M Ueta, Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajiicho, Hirokoji, Kawaramachi, Kamigyoku, Kyoto 602-0841, Japan. E-mail: [email protected] or Dr K Tokunaga, Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo Bunkyoku, Tokyo 113-0033, Japan. E-mail: [email protected] Received 27 June 2016; revised 6 December 2016; accepted 7 December 2016; published online 19 January 2017 GWAS using Japonica array for Stevens–Johnson syndrome MUetaet al 486 Japanese population was developed by Kawai et al.12 Its haplotype GWAS, imputation with 1KJPN and validation analysis collection was constructed from whole-genome sequences of 1070 In the GWAS, we genotyped 808 samples from the same SJS/TEN patients Japanese individuals.13 We posited that whole-genome imputation (n = 117) and the healthy Japanese controls (n = 691) used in our earlier study.7 with the Japonica array would increase the statistical power for We used the Japonica SNP array12 according to the manufacturer’s instructions. detecting associated variants of CM-SJS/TEN with SOC than was All genotyped samples passed the recommended sample quality control metric 4 possible with the AXIOM Genome-wide ASI 1 array we reported for the AXIOM arrays (dish QC 0.82); we excluded three control samples with an overall call rate o97%. We recalled the remaining 805 samples with previously.7 Genotyping Console 4.2.0.26 software (Affymetrix). In the current study, we performed GWAS using the Japonica array Because we had already performed identity-by-descent estimation and and imputation with the 1KJPN panel (Tohoku Medical Megabank principal component analysis for all 808 samples genotyped with the AXIOM Organization constructed the reference panel) to look for additional Genome-Wide ASI 1 array;7 all samples used in the current study were known genetic predispositions for CM-SJS/TEN with SOC in the Japanese to be from unrelated individuals and the effect of population stratification was population. negligible. A quantile–quantile plot of the distribution of test statistics for the comparison of genotype frequencies in our patients and controls showed MATERIALS AND METHODS that the inflation factor λ was 1.024 for all tested SNPs and decreased to Patients 1.021 when SNPs in the human leukocyte antigen (HLA) region (chr 6: This study was approved by the institutional review board of Kyoto Prefectural 29 645 038–33 360 787) were excluded (Supplementary Figure 1). University of Medicine (KPUM), the University of Tokyo and other collabor- For imputation, the clustering plots were classified by the Ps classification ating research institutes (Seoul National University College of Medicine, Yonsei function in the SNPolisher package (version 1.5.2; Affymetrix). SNPs that were University College of Medicine, Chonnam National University Medical School assigned 'recommended' by the Ps classification were retained. Excluded were and College of Medicine, The Catholic University of Korea in Korea). SNPs with a call rate o99.0%, a Hardy–Weinberg equilibrium (HWE) test We used the same samples from 117 Japanese individuals with CM-SJS/TEN result of Po0.0001 or a minor allele frequency (MAF) o0.5%. Prephasing was with SOC and 691 healthy Japanese volunteers as were used in our earlier conducted first with these SNPs using SHAPEIT (v.2.r644);15 the options were GWAS with the Affymetrix AXIOM Genome-Wide ASI 1 array.7 –burn 10, –prune 10 and –main 25. Genotype imputation was performed on SOC patients were defined as those with ocular sequelae such as severe dry the phased genotypes with IMPUTE2 (ver. 2.3.1)16 using a phased reference eye, trichiasis, symblepharon and conjunctival invasion into the cornea in the panel of 1070 healthy Japanese individuals (1KJPN panel).13 For IMPUTE2, the chronic stage,5 and as patients who manifested severe conjunctivitis with applied options were -Ne 2000, -k hap 1000, -k 120, -burnin 15, and -iter 50. pseudomembranes and epithelial defects on the ocular surface (cornea and/or After genome-wide imputation genotyped with the Japonica array, conjunctiva) in the acute stage.14 We have classified the patients who had taken we applied a SNP call rate ⩾ 95%, MAF ⩾ 1%, and HWE P ⩾ 0.001 for SNP CMs such as non-steroidal anti-inflammatory drugs and multi-ingredient cold quality control for data cleaning of the samples. The 6,714,496 SNPs and medications for a few–several days before the disease onset for common-cold insertions and deletions (indels) on autosomal chromosomes that passed the symptoms as CM-SJS/TEN. The specific drugs they used were not named by all quality control filters were used for GWAS. − patients (Supplementary Table 1). In Japan, doctors in hospital usually Confirmation of the genotypes at 6 candidate SNPs with Po10 7 in the prescribed CM such as non-steroidal anti-inflammatory drugs and acetamino- GWAS and the replication study using Korean samples was with the TaqMan phen with some antibiotics. However, we previously reported that the use of CM SNP genotyping assay (Applied Biosystems, Foster City, CA). We genotyped such as non-steroidal anti-inflammatory drugs and cold-remedies was signifi- extended sample sets of Japanese SJS patients (n = 138) and controls (n = 883) cantly associated with SJS/TEN with SOC, whereas the use of antibiotics was not to validate the GWAS results. Samples from Korean patients (n = 31) and associated.2 Detailed information on the SJS/TEN patients and the controls we controls (n = 110) were genotyped to replicate the GWAS results analyzed are shown in Supplementary Methods in the Online Repository.