Large-scale Linkage Disequilibrium Mapping to Identify Rheumatoid Arthritis-associated Genes

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 ( λMZ ) • 12~62 • Relative risk to siblings (λsib) • 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 20th 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. Two Ways of Whole Genome LD Mapping～First stage～ Map-based Approach 7 6 2 8 1 3 5 4 9 Gene D Gene A Gene B Gene C Coding Gene-based Approach 8 9 2 3 1 7 4 6 5 Gene D Gene A Gene B Gene C

6. SNP distribution of RIKEN study

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

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 Not covered 8,263 8,381 12,890 Covered with block 4,509 RIEN project started Covered with SNPs not in block 2000 2001 2002 2003 2004 2005 50k 27,283Genes 40k 30k Gene 20k 10k

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

10. Major findings from SNP-based studies • 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 EffortMap of Human Genetic Variation Will Speed Search for Disease GenesBETHESDA, Md., Mon., Feb. 7, 2005 SNPs No. markers is increasing and a chart for LD-mapping is now available. Genome-scan kits are available.

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

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

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

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

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

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

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

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. Phylogeny and Ancestral Recombination Graph Association studies? Inter-species differences Nature Reviews Genetics3, 380-390 (2002); doi:10.1038/nrg795GENEALOGICAL TREES, COALESCENT THEORY AND THE ANALYSIS OF GENETIC POLYMORPHISMS Nature Reviews Genetics3, 380-390 (2002); doi:10.1038/nrg795GENEALOGICAL TREES, COALESCENT THEORY AND THE ANALYSIS OF GENETIC POLYMORPHISMS Intra-species differences

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 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 Lab of Functional Genomics IMS @ U of Tokyo Mark Lathrop Yukinori Okada Harumi Nagai

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