1. Large-scale Linkage Disequilibrium Mapping to Identify Rheumatoid Arthritis-associated Genes Ryo Yamada IMS U of Tokyo Tokyo Japan 1st International Symposium on Key Issues on Infectious Diseases June 5 2007 Grand Hilton Hotel Seoul Korea

2. Rheumatoid Arthritis  World-wide distribution with prevalence from 0.6% to 1.0%  Woman-dominant  Complex etiology Genetic components Environmental components  Destructive polyarthritis  Extra-articular involvement  Autoimmunity

3. Genetics and Genetic Analysis of Rheumatoid Arthritis  Twin and family studies Relative risk to monozygotic twin ( λ MZ )  12~62 Relative risk to siblings (λ sib )  2~17 HLA locus explains 1/3-1/2 of total genetic components. There are multiple non-HLA genes.  Multiple linkage studies  Many candidate-approach studies

4. SNP-based large scale LD- mapping The first stage  From the end of 20 th century to 2003  Human genome sequence was being completed  SNPs and LD were being characterized  High-throughput SNP genotyping platforms were being developed The second stage  From 2003 ～  HapMap project offered variation map for whole- genome study  Multiple commercial genotyping packages are available  Significant reduction in the typing cost

5. 9 8 7 6 5 4 1 2 3 1 23 7 6 98 4 5 Coding Gene-based Approach Map-based Approach Gene A Gene B Gene A Gene B Gene D Gene C Gene D Two Ways of Whole Genome LD Mapping ～ First stage ～

6. SNP distribution of RIKEN study

7. SNPs Samples Replication-based analysis SNPs Samples Stage 1 Stage 2 One-Stage Design Joint analysis SNPs Samples Stage 1 Stage 2 Two-Stage Design Michael Boehnke : Design Considerations in Large Scale Genetic Association StudiesDesign Considerations in Large Scale Genetic Association Studies HapMap Tutorials 836 vs. 658 two-stage joint screening

8. 12,890 / 21,153 genes 12,890 （ 60.9% ） genes were evaluated with block/SNPs No. SNPs per gene and density of SNPs 5.0±6.4 /gene 0.2±0.3 /kb No. coding genes in autosomal chromosomes ： 21,153 Covered with SNPs not in block Covered with block Not covered 4,509 8,381 8,263 12,890 Gene 200520002001200220032004 10k 20k 40k 30k 50k RIEN project started 27,283Genes

9. Japanese, Korean : RA-specific Caucasians : SLE RA and other autoimmunities Japanese : RA Caucasian : IBD Japanese : RA and other autoimmunities Major findings from SNP-based studies

10.  Japanese study (RIKEN) PADI4 : RA  Post-translational enzyme to produce targets of the most RA-specific autoantibodies. SLC22A4 /A5 : RA & Crohn  Ergothioneine or carnitine transporter expressed in hematologic lineages. FCRL3 : RA, SLR & AITD  Fc receptor homolog on B-cell membrane  US study (A.Begovich et al.) PTPN22 : T1DM, SLE, RA & AITD  Lymphoid-specific intracellular phophatase

11. Validation with meta-analysis

12. MΦ Anti-oxydant transporter

13. Multiple Genes and Multiple Diseases

15. Summary  Coding gene-based SNP-LD mapping identified multiple RA- susceptible genes with functional variants.  Some genes are associated with multiple autoimmume diseases.

16. What will change in the next stage of LD-mapping?  SNP markers  Genes  Genotyping technologies and further developments  SNPs and other polymorphisms  Larger study scale and the associated problems in analysis  What are the genetic “ association ” studies?

17. International HapMap Consortium Expands Mapping Effort Map of Human Genetic Variation Will Speed Search for Disease Genes BETHESDA, Md., Mon., Feb. 7, 2005 PopulationsCEUCHB+JPNYRI Total Non-Redundant3,904,2183,936,4823,846,092 Total QC+ SNPs4,871,1274,881,4414,774,448 Total Gentyped SNPs6,838,9236,799,2386,798,546 No. markers is increasing and a chart for LD-mapping is now available. Genome-scan kits are available. SNPs

18. Central Dogma and DNA Variations and their functionality ＤＮ Ａ mRNA Peptide Transcription Translation Transcription initiation point Transcription termination point Splicing and mRNA maturation Translation initiation point Codon triplets Translation termination point Variations Post-translational peptide modifications Molecules Genes

19. SusceptibleNon-susceptible DNA-mRNA-Protein relation is not straight, but comparison between DNA variations and phenotype variations bypassing mRNA/proteins simplifies the analysis structure. Genome Transcriptome Proteome Metabolome Phenome All-or-non simplest case Genes

20. Another big world of heritable items (genes) Non-coding RNA x 23,000 in mammals Functional RNA-genes Non-coding genes Genes

21. Coding gene Non-coding-gene DNA Functional RNA Effects on transcription Effects on translation Effects on phenotypes Genes

22. Genotyping technologies and further developments  100K marker-panel  250K marker-panel  500K marker-panel  … 1M marker-panel … … Whole genome typing of all samples Typing

23. Jennifer L. Freeman et al. Genome Res. 2006; 16: 949-961 Other polymorphisms SNPs CNVs

24. Biased sampling Even sampling Sampling from a structured population Large study-associated problems

25. P 値昇順プロット P値P値 P-value Markers Large study-associated problems Many significant results when samples are biased with population structure.

26. Species are distinct categorical phenotypes with many genetic differences. Species-specific genetic factors are difficult to nail down. Association studies?

27. Human individuals are unique but phenotypic variations are less distinct than species. Intra-species genetic variations are much less than inter-species, but still difficult to identify association between phenotypic and genetic variations. Association studies?

28. Nature Reviews Genetics 3, 380-390 (2002); doi:10.1038/nrg795 GENEALOGICAL TREES, COALESCENT THEORY AND THE ANALYSIS OF GENETIC POLYMORPHISMS Inter-species differences Intra-species differences Phylogeny and Ancestral Recombination Graph Association studies?

29. Genetic Approach and Molecular Validation  Genetic Approach Dissection of combined effects of multiple variations in many genes on phenotypes in human.  Molecular Validation Molecular assays to reveal causative links between genetic variations and phenotype manifestation.  Genetic Validation with Controlled Interventions Transgenic animals are also used for validation of findings from genetic studies in human.

30. Lab of Functional Genomics IMS @ U of Tokyo Mark Lathrop Yukinori Okada Harumi Nagai Lab for Rheumatic Diseases @ SRC RIKEN Kazuhiko Yamamoto Akari Suzuki Yuta Kochi Kenichi Shimane Keiko Myozen Kyoko Kobayashi Miyako Ohtake Emi Kanno Yoshinobu Hayashi Keiko Komakine Yusuke Nakamura CGM @ Kyoto U Fumihiko Matsuda Koichiro Ohmura Meiko Takahashi Alexandre Vasilescu Victor Renault Masao Yamaguchi Katsura Hirosawa Kenei Ohigashi Akiko Yoshizumi Miki Kokubo Shoko Matsubara

31. Time course Triggering Event Disease Manifest ations Diag nosi s Clinical F/U Birth No observation In pre-clinical phase RNA, proteins and others DNA DNA-analyses Data is Simple and Fixed throughout the Life. No quantitative, chronological or spatial variation is present for DNA polymorphisms. Time course Birt RNA, proteins and othersDNA