Genome-wide association studies for human narcolepsy and other complex diseases

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1 ETHZ-JST Joint WS Sept , 2008 Genome-wide association studies for human narcolepsy and other complex diseases Katsushi Tokunaga Department of Human Genetics Graduate School of Medicine The University of Tokyo

2 GWAS Typing in Dept. Human Genetics, UT Type 2 diabetes Multi-institutional collaboration (PI: Kasuga M) GWAS based on 100K JSNPs; 939 (187/752) 3rd screening finished; 2,848 (1424/1424); 7 genes with 10 SNPs replication & high-density mapping; 8790 (4,378/4,412); new gene KCNQ1 (Yasuda et al. Nat Genet, E-pub Aug 2008) Narcolepsy Collaboration with Tokyo Inst. of Psychiatry (PI: Tokunaga K) GWAS by 500K SNPs; 611 (222/389) replication & high-density mapping; new susceptibility gene(s) (Miyagawa et al. in press) Multiple system atrophy Multi-institutional collaboration (PI: Tsuji S) GWAS by 500K SNPs finished; 680 (230/450) Panic disorder Multi-institutional collaboration (PI: Okazaki Y) GWAS by 500K SNPs finished; 650 (200/450) replication study; 1,200 (600/600) Alzheimer disease Multi-institutional collaboration (PI: Kuwano R) GWAS by 900K SNPs; 2,000 (1,000/1,000) and other diseases

3 Human Narcolepsy Sleep disorder, Incidence: % (25-30% in identical twins, 1-2% in siblings) HLA-DRB1*1501-DQB1*0602 haplotype: 100% in Japanese narcoleptics, while 10% in healthy Japanese (Juji et al. 1984; Matsuki et al. 1985) Decreased level of cerebrospinal orexin (hypocretin) in HLA-DQB1*0602 positive patients (Nishino et al. 2000) No variation in orexin, orexin receptor 1 & 2 genes associated with narcolepsy (Peyron et al. 2000) s=12; s(hla)=5 Multiple genetic factors in addition to HLA!

4 Genome-wide Association Study for Human Narcolepsy (Resistance gene NLC1A identified by GWAS using 23,000 microsatellite polymorphisms: Kawashima et al. Am.J.Hum.Genet. 2006) Narcoleptics (n=222), Controls (n=389) 500K SNP array (Affymetrix) Genotyping: GTYPE 4.1 (BRLMM algorism) Confidence score value: 0.5 Average call rates: Cases: Sty I: 98.0%, Nsp I: 97.9% Controls: Sty I: 96.1%, Nsp I: 97.3% Data cleaning: Call rate of each SNP: 95% in cases and controls Hardy-Weinberg s equilibrium: p in controls Minor allele frequency (MAF): 5% in all samples 249,113 SNPs, after data cleaning Miyagawa et al. (in press)

5 Association Mapping in the HLA Region - SNPs in the HLA region showed the strongest associations as expected - HLA-B TNF-A NOTCH4 HLA- DRB1 HLA- DQB1 HLA-DPB1 P value Miyagawa et al. (in press)

6 Genome-wide Association Data 8 HLA HLA Results of 249,133 SNPs -log 10 (P value) 6 4 (i) (ii) 2 Chr SNPs were selected for follow-up (i) α(significance level) = , for genome-wide SNPs (ii) α= , for SNPs in the vicinity of candidate genes and regions (Significance levels by the false positive report probability (FPRP) criterion) Miyagawa et al. (in press)

7 Replication Study Independent Japanese sample sets 1st set: 222 cases, 389 controls 2nd set: 159 cases, 190 controls One SNP(tentative ID: TM22.1) was replicated GWAS Replication MAF MAF OR p MAF MAF OR p (case) (cntr) (95%CI) (case) (cntr) (95%CI) x x10-4 ( ) ( ) Combined OR: 1.79 (95%CI: ) Combined p value: 4.4 x 10-7 Miyagawa et al. (in press)

8 High-Density Association Mapping P value TM 22.1 Gene X Gene Y R square The LD block includes two functionally interesting genes

9 Risk Allele Associated with Lower Expression Levels of Genes X and Y Gene X Gene Y P < t-test P < 0.01 t-test TT TC C: risk allele TT TC mrna expression level of each gene in peripheral leukocytes was measured by real-time quantitative PCR. Miyagawa et al. (in press)

10 Association Studies in Different Populations Population No. Cases/ maf maf OR P allele No. Controls cases controls Japanese 381 / x10-7 Korean* 115 / USA Caucasian* 388 / USA African* 86 / *Collaboration with Prof. Mignot, Stanford Univ. Meta-analysis: OR: 1.63, P = 5.9 x 10-8 Common risk gene, but significant ethnic differences in frequencies!! Miyagawa et al. (in press)

11 Gene X A rate-controlling enzyme of long-chain fatty acid β-oxidation in muscle mitochondria Several reports including knock-out mice data have suggested interaction between fatty acid β-oxidation and the gene X related system in sleep regulation Gene Y Participate in the choline pathway leading to phosphatidylcholine biosynthesis # acetylcholine: REM and wake promoting neurotransmitter increasing narcolepsy symptoms (Nishino et al., 1997)

12 Summary SNP-based GWAS for Human Narcolepsy SNP-based genome-wide association study was first performed and >30 candidate SNPs were detected Association of a SNP (TM22.1) was replicated and the combined data showed a genome-wide significant association: P = , OR = 1.79 TM22.1 or an allele in strong LD was primary for the association A meta-analysis in Japanese, Korean, USA Caucasian and African provided P = , OR = 1.63 Two functionally relevant genes are located near the SNP High risk allele was associated with lower mrna expression levels of the genes

13 Nature Genetics, QuickTimeý Dz TIFFÅiîÒàèkÅj êlí ÉvÉçÉOÉâÉÄ Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇ žç½ç ÇÕïKóvÇ-ÇÅB QuickTimeý Dz TIFF ÅiîÒà èkåj êlí ÉvÉçÉOÉâÉÄ Ç Ç±ÇÃÉsÉNÉ` ÉÉǾå ÇÈÇžÇ½Ç Ç ÕïKóvÇ -Ç ÅB

14 Multistage Genome-wide Association Study For Type 2 Diabetes based on JSNPs 1st Screening Sample Set patients 100K SNPs by Invader Assay 2nd Screening 3rd Screening High-Density Mapping Sample Set patients 752 controls Sample Set patients 672 controls Set ,424 patients 1,424 controls 2,880 SNPs by Invader Assay 201 SNPs by SSP-PCR-FCS 10 SNPs in 7 genes (3 SNPs in KCNQ1) by SSP-PCR-FCS TaqMan

15 Association Data between KCNQ1 and Type 2 Diabetes in Different Population Samples 1st Screening Replication set 1 Replication set 2 All Japanese (n=8,790) (SNP rs ) P value 6.7 x x x x Chinese Korean All East Asian (n=13,173) European All samples (n=19,930) 1.3 x x x x x Odds Ratio

16 Population Differences of Type 2 Diabetes Genes Gene Population odds p minor allele ratio frequency TCF7L2 European x / 0.25 Japanese* x / 0.02 *Miyake et al. J Hum Genet (2008) KCNQ1 European x / 0.05 Japanese x / 0.40 Yasuda et al. Nat Genet (2008) FTO Caucasian x / 0.36 Japanese* no association *Horikawa et al. J Clin Endocrinol Metab (2008) Common risk gene but differences in frequencies!!

17 Conclusion Genome-wide association study (GWAS) is a productive strategy for identifying common susceptibility genes to complex diseases. But there are still many more genes to be identified. Collection of sample sets for replication study and ethnic comparison is crucial. GWAS in Asian populations serves opportunity to identify new susceptibility genes.

18 Acknowledgements Taku MIYAGAWA, Minae KAWASHIMA, Mihoko SHIMADA, Jun OHASHI, Nao NISHIDA (University of Tokyo, Graduate School of Medicine) Yutaka HONDA, Makoto HONDA (Tokyo Institute of Psychiatry and Neuropsychiatric Research Institute) Emmanuel MIGNOT, Ling LIN, Juliette FARACO (Stanford University, School of Medicine) Collaborators for multi-institutional disease studies, especially Kazuki YASUDA, Masato KASUGA (International Medical Center of Japan, Research Institute)

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