Mapping spondyloarthritis susceptibility loci in B10.RIII mice

Integrating comparative genomics, transcriptomics, and functional approaches to identify genetic regions responsible for spondyloarthritis susceptibility in B10.RIII mouse model

1. Mapping spondyloarthritis susceptibility loci in B10.RIII mice Anu Shivalikanjli Postdoctoral Fellow Thomas Keane Mouse Genomes Project (MGP) 1

2. Mouse Genomes Project (MGP) MGP sequence variation catalog V5 52 strains ( PRJEB53906 ) 2

3. 3 Spondyloarthritis (SpA)

4. 4 Spondyloarthritis (SpA) - Genetics MHC Tumor Necrosis factor (TNF) Interleukin (IL)- 23/17 Furst & Louie, 2019

5. 5 Spondyloarthritis (SpA) - Mouse model SKG - BALB/c ZAP-70W163C mutant mice Zymosan - beta-glucan MHC Haplotype IL23 is systemically overexpressed B10.RIII- H2 r H2-T18 b /(71NS)SnJ JAX #000457

6. 6 B10.RIII- H2 r H2-T18 b /(71NS)SnJ JAX #000457 Research applications: Autoimmunity Inflammation MS Rheumatoid arthritis CIA and CAIA Spondyloarthritis - susceptible MHC-congenic B10.RIII mice Chr 17

7. 7 C57BL/10SnJ JAX # 000666 RIII/WySn MHC-congenic B10.RIII mice N8 ( Resistant to arthritis MHC Haplotype H2b MHC Haplotype H2r Susceptible to arthritis Extinct MHC Haplotype H2r B10.RIII- H2 r H2-T18 b /(71NS)SnJ JAX #000457

8. 8 C57BL/10SnJ JAX # 000666 RIII/WySn MHC-congenic B10.RIII mice N8 ( Resistant to arthritis MHC Haplotype H2b MHC Haplotype H2r Susceptible to arthritis Extinct IL-23 mc MHC Haplotype H2r Arthritis B10.RIII- H2 r H2-T18 b /(71NS)SnJ JAX #000457

9. 9 MHC-congenic B10.RIII and arthritis susceptibility Presence of two different loci on both the chromosome 10 and 17 could have a disease-promoting effect on arthritis

10. Framework Comparative genomics Whole-genome sequences Genetic experiments Backcrossing of mice Functional analysis RNA-Seq 10 03 01 02

11. Framework Comparative genomics Whole-genome sequences Genetic experiments Backcrossing of mice Functional analysis RNA-Seq 11 03 01 02

12. Whole-genome sequencing of 52 laboratory strains (MGP sequence variation catalog V5) 12 MHC Haplotype H2b Comparative genomics Identification of variants ‘private’ to B10.RIII genome C57BL/10SnJ JAX # 000666 MHC Haplotype H2r Resistant to arthritis Variants private to B10.RIII genome Susceptible to arthritis B10.RIII- H2 r H2-T18 b /(71NS)SnJ JAX #000457

13. Variants ‘private’ to B10.RIII genome w.r.t B10 Non-B10 genome 13 Findings from whole-genome sequence analysis of B10 and B10.RIII Non-B10 genomic clusters found on multiple chromosomes in B10.RIII genome Density of SNPs plotted for 1Mb chromosomal intervals Novel Previously known

14. 14 Findings from whole-genome sequence analysis of B10 and B10.RIII Multiple protein-coding genes in the non-B10 genomic clusters carry possible ‘ loss of function’ mutations in B10.RIII Variants ‘private’ to B10.RIII genome w.r.t B10 Possibly ‘loss of function’ mutations Density of SNPs plotted for 1Mb chromosomal intervals ‘Loss of function’’ - based on Ensembl VEP consequences and IMPACT ratings of HIGH and MODERATE effects on the gene-function

15. 15 Results - Comparative genomics B10.RIII genome contains multiple loci that differ from its B10 parent Multiple protein-coding genes in the non B10 genomic clusters carry p ossible ‘loss of function’ mutations Density of SNPs plotted for 1Mb chromosomal intervals Variants ‘private’ to B10.RIII genome w.r.t B10 Possibly ‘loss of function’ mutations Novel Previously known

16. Framework Comparative genomics Whole-genome sequences Genetic experiments Backcrossing of mice Functional analysis RNA-Seq 16 03 01 02

17. 17 C57BL/10J JAX #000665 Unmanipulated mice (not injected with minicircles) for a first pass analysis B lymphocytes Neutrophils Dendritic cells Macrophages γδ T cells CD4+ T cells CD8+ T cells CD4-CD8- DN T cells Bulk RNA-seq of Splenocytes MHC Haplotype H2b MHC Haplotype H2r Resistant to arthritis Susceptible to arthritis Spleen x 6 6 x B10.RIII- H2 r H2-T18 b /(71NS)SnJ JAX #000457 Joerg Ermann Jyotsna Soundararajan

18. 18 Bulk RNA-seq B10 and B10.RIII in the eight immune cell types Differentially expressed genes - whole genome Upregulated in B10.RIII Downregulated in B10.RIII DE genes with log fold change (LFC) > 0.5 and significant at 10% FDR

19. 19 Bulk RNA-seq B10 and B10.RIII in the eight immune cell types Enrichment of differentially expressed genes in the identified non-B10 genome in B10.RIII Upregulated in B10.RIII Downregulated in B10.RIII DE genes with log fold change (LFC) > 0.5 and significant at 10% FDR

20. 20 Bulk RNA-seq B10 and B10.RIII in the eight immune cell types Enrichment of differentially expressed genes in the identified non-B10 genome in B10.RIII

21. Framework Comparative genomics Whole-genome sequences Genetic experiments Backcrossing of mice Functional analysis RNA-Seq 21 03 01 02

22. F1 Backcrossing (B10.RIII x B10) 22 SpA severity is independent of Chr 10 ( Fig. A ) Chr 15 and Chr 17 genomic clusters are required for IL-23 induced arthritis phenotype ( Fig. B and C ) Joerg Ermann Jyotsna Soundararajan Haplotype r No clinically significant symptoms Arthritic development Arthritic development

23. Framework 23 Comparative genomics Whole-genome sequences Genetic experiments Backcrossing of mice Functional analysis RNA-Seq 03 01 02 Known QTLs in arthritis

24. 24 Known QTLs for arthritis B10.RIII - CIA, CAIA DBA/1J - CIA DBA/2J - PGIA BALB/cJ - PGIA MRL/Ipr - lpr arthritis

25. Framework Comparative genomics Whole-genome sequences Genetic experiments Backcrossing of mice Functional analysis RNA-Seq 25 03 01 02 Known QTLs in arthritis

26. 26 Conclusions Upregulated in B10.RIII Downregulated in B10.RIII B10.RIII genome contains multiple loci that differ from its B10 parent SpA severity is independent of Chr 10 Chr 15 and Chr 17 underlie the incidence and severity of IL-23 induced arthritis in B10.RIII mice DE genes with log fold change (LFC) > 0.5 and significant at 10% FDR Density of SNPs plotted for 1Mb chromosomal intervals

27. 27 Conclusions Upregulated in B10.RIII Downregulated in B10.RIII B10.RIII genome contains multiple loci that differ from its B10 parent SpA severity is independent of Chr 10 Chr 15 and Chr 17 underlie the incidence and severity of IL-23 induced arthritis in B10.RIII mice DE genes with log fold change (LFC) > 0.5 and significant at 10% FDR Density of SNPs plotted for 1Mb chromosomal intervals

28. 28 Suppresses the synthesis of IFN-α/β and proinflammatory TNFα Ilrun - Chr 17 Inflammation and lipid regulator with UBA-like and NBR1-like domains Ambrose et al ., 2018 C6 = Ilrun (prev. C6orf106)

29. 29 Contains missense mutation found only in B10.RIII Ilrun - Chr 17 Upregulated in B10.RIII in dendritic cell type (LFC ~ 2) Has a human ortholog (Chr 6)and is highly conserved across species Inflammation and lipid regulator with UBA-like and NBR1-like domains

30. 30 Contains deleterious missense mutations - shared with DBA, BALB strains (arthritis models) Baiap3 - Chr 17 Downregulated in B10.RIII in γδ T, CD8+, CD4-CD8- DN T cells (LFC ~ 2) BAI1-associated protein 3

31. 31 Lymphocyte antigen 6 complex (Lyc61, Ly6e) on Chr 15 Human GWS arthritis loci Future work - candidates

32. 32 Conclusions B10.RIII genome contains multiple loci that differ from its B10 parent SpA severity is independent of Chr 10 Chr 15 and Chr 17 underlie the incidence and severity of IL-23 induced arthritis in B10.RIII mice Ilrun , Baiap3 (Chr 17) and Ly6 (Chr15) Density of SNPs plotted for 1Mb chromosomal intervals Upregulated in B10.RIII Downregulated in B10.RIII DE genes with log fold change (LFC) > 0.5 and significant at 10% FDR

33. 33 Acknowledgements Ermann Laboratory, Harvard Medical School Joerg Ermann Jyotsna Soundararajan Jackson Laboratory Laura Reinholdt Anne Czechanski Wellcome Sanger Institute David Adams Elizabeth Anderson EMBL-EBI Thomas Keane Mohab Helmy Claudia Perez Calles Instituto Gulbenkian de Ciência (IGC) Jingtao Lilue

34. Mapping spondyloarthritis susceptibility loci in B10.RIII mice Anu Shivalikanjli Postdoctoral Fellow Thomas Keane Mouse Genomes Project (MGP) 34