Clinical Phenotype: Disease Signs & Symptoms

1. Genotype “Endophenotypes” Clinical Phenotype: Disease Signs & Symptoms Rational treatment or prevention

2. Mechanistic information: essential roles of individual genes; groups of genes clustered by pathway; systems biology understanding of gene-gene interactions Molecular, cellular & whole system consequences of mutations in individual genes Specific tests for endophenotypes: Cell-based or biomarker assays that can be applied to human samples Preclinical efficacy in mice with pathway defects like humans Clinical and commercial translation: targetted development of new therapeutics/preventions MAN Translation Gap: bridged by integrating next-gen sequencing and solid understanding of mechanism from Mendelian mouse mutants Next-gen resequencing to reveal family-specific variants: Statistical associations: Eg clusters of mutations in particular pathways MOUSE Translation gap MEDICINE

3. The genome encodes the buttons and circuits of our immune system - we just need to know how to read the instructions Fumbling for his recline button, Ted unwittingly instigates a disaster

4. Systematic screening for recessive Mendelian syndromes in the immune system caused by single nucleotide variants G0: ENU treated C57BL/6 male C57BL/6 female Random base substitutions in spermatogonial stem cells C57BL/6 female * G1 progeny: carry ~3000 single nucleotide substitutions spread randomly across paternal chromosomes: ~35 functional variants/G1 * G2progeny: Single nucleotide substitutions segregate G3 progeny: 12.5% of SNVs brought to homozygosity in each mouse

5. Genotype: mutation mapped to chromosomal interval and identified by DNA sequencing “Endophenotypes”: eg altered blood cells in flow cytometry, altered antibody response to immunization Clinical Phenotype: eg susceptibility to infection with M tuberculosis or Poxvirus, allergic dermatitis, disseminated lymphoid cancer Preclinical model for treatment or prevention

6. SSC FSC CD3 CD4 Tube 040: lowish CD4s & CD8s & higher %CD44hi in CD8s CD3 CD44 CD3 CD44 T Cells

7. CD3 B220 IgD IgM Tube 046: slightly higher IgM CD3 NK1_1 CD44 NK1_1 B Cells

8. CD44 IgE IgG2a IgE CD44 IgE IgG2a IgE Monocytes

10. Plate 14062 Blood FACs bled 064/10 (05.03.10) Comment: Mehmet : Sample 37,38,40 and 65 have reduced CD4 and CD8 T cells Sample 37 and 40 elevated IgE Chris Goodnow: Stains and gates look great. I worry that we’re not picking up IgE+ cells in most samples: gating or staining intensity problem? Tube 040: lowish CD4s & CD8s & higher %CD44hi in CD8s, higher IgE Tube 046: slightly higher IgM Tube 037: higher IgE I think the technician forgot to scan following mouse: Sample 73 is ENU14Yaa:047:B6:G2 # 2

11. Zippo Alberta Nephertiti Jasmine Mozart Armidilo Eddie Sweaty Xander Mapping and identification of Mendelian mutations in the mouse circa 2009: legacy of the public mouse genome project 1 2 3 4 5 6 7 8 9 10 Tsavo Rockstar Trembles 7B6148 LeukSkywalker Dopey BDown 9B6 35 Mr T-less Murdoch Delficio Kenobi Botero Tipsy Collins Face B-leg Pardon 9B6 64 4AT32 Wavy Redburst Pharlap Pinky PIAF Anarchy Fatso Lightning Pengu Socks Tiny Lochy Kenny LasVegas Jersey Blobby Fluoro Rain Polly Buffy Wachin SanRoque Wobbles Eeyore Fat Willow Storm MeioI Joey Koy Aussie Dorian 8C63 Tilcara Hoban Winnie Unmodulated Thoth Seshat Rose 8B6 27 Plastic Hipster 11B658 Anubis T-wimp T-Bird Malewa Mr Hanky Bata Vibes Bthy B-blast Captain Babe 6WT33 Duane Nessy Theoden Dwarf Thunder B52 Piebald Krusty Anakan Primurus Mal Flipper Senseless 11 12 13 14 15 16 17 18 19 X Y - Mapped over 95 strains with 77 having mutation identified (44 last 3 years) - Many available for researchers to study through the NHMRC Aust. Phenome Bank

12. Example #1: the value of integrating clinical correlations of DNA variants with understanding of mechanism from the mouse: Regulating NFkB activity and cell growth in normal, autoimmune and malignant B cells

13. Discovering new regulators of B cell growth by forward genetics: phenotypic screening of a library of mice segregating thousands of randomly-induced single nucleotide substitutions • Unmodulated  detected from B cell screen • Each dot represent one mouse. • Dashed lines  two SD • ~ 400 G3 animals Amount of complement receptor on B cell surface Amount of antigen receptor on B cell surface Changes in antigen receptor density often indicate abnormalilities in the BCR signalling pathways

14. Unmodulated variant breeds true as a simple Mendelian recessive trait Control Amount of complement receptor on B cell surface Variant Amount of antigen receptor on B cell surface Mean values for individual offspring from unmodulated affected mouse (un/un) crossed with heterozygous (un/+) carrier

15. Homozygous un/un mice make little antibody upon immunization, because their B cells proliferate poorly to BCR-stimulation Relative amount of antibody in serum Th1 / IgG2a Th2 / IgG1

16. * * * * * * * * * Unmodulated: point mutation in one protein-interaction domain of a MAGUK-family protein, CARD11 (Carma1) Jesse Jun

17. Example:MECHANISM STUDY: B cells from mice with inherited CARD11 mutations are selectively crippled for BCR activation of the IKK-NFkB pathway and proliferation Jun et al 2003. “Identifying the MAGUK protein Carma-1 as a central regulator of humoral immune responses and atopy by genome wide mouse mutagenesis”. Immunity 18:751-762.

18. Building up mechanistic information from knockouts, point mutants, knock-downs, etc:essential roles of individual genes; groups of genes clustered by pathway; …towards a systems-level understanding of how human alleles of different genes might interact CARD11 PKC (eg. MYC)

19. Y Y * * Y Y Y Y Y Y * * * Y * Y Y Y * * Y Y * * * Y Y * * Y Y * * Y Y * * Homozygous null mutations in CARD11 disrupt TCR-NFB signalling and cause immunodeficiency, but what about SNVs causing a quantitative decrease? Aire Thymic epithelium Antigen MHC CD80 (B7.1) CD4 CD86 (B7.2) TCR or e e g d CD8 z Itk CD45 Lck CD28 Fyn PKC Zap70 Ptpn22 (PEP) I B k CARD11(CARMA1) Ca2+ Bcl10 MALT1 Calcineurin NF B k * * * IKK IL-2 NFAT IL-2R genes for cell growth and division STAT JAK

20. Quantitative decrease in CARD11 signalling preserves T cell activation but diminishes Treg formation, resulting uncontrolled formation of TH2 cells, hyper-IgE, and mast cell dermatitis John Altin, Matthew Cook, Jesse Jun

21. Mouse-to-Man-to-Medicine: translating knowledge of pathways controlling B cell proliferation and resequencing of carefully phenotyped clinical samples L298Q unmodulated Science 2008 319:1676-1679 Robertson MJ et al. 2007. J Clin Oncol 25:1741-6. Phase II study of enzastaurin, a protein kinase C beta inhibitor, in patients with relapsed or refractory diffuse large B-cell lymphoma. Morschhauser F et al. 2008. Ann Oncol 19:247-253. A phase II study of enzastaurin, a protein kinase C beta inhibitor, in patients with relapsed or refractory mantle cell lymphoma.

22. Mechanistic information: essential roles of individual genes; groups of genes clustered by pathway; systems biology understanding of gene-gene interactions Molecular, cellular & whole system consequences of mutations in individual genes Specific tests for endophenotypes: Cell-based or biomarker assays that can be applied to human samples Preclinical efficacy in mice with pathway defects like humans Clinical and commercial translation: targetted development of new therapeutics/preventions MAN Translation Gap: bridged by integrating next-gen sequencing and solid understanding of mechanism from Mendelian mouse mutants Next-gen resequencing to reveal family-specific variants: Statistical associations: Eg clusters of mutations in particular pathways MOUSE Translation gap MEDICINE

23. IMPC GOAL: A phenotyped mouse mutant for every gene Genotyped & phenotyped mouse pedigrees: >3 homozygotes/mutation Pedigree breeding, genotypic & phenotypic data acquisition & collation 1 deleterious mutation/pedigree ~70 deleterious mutations/pedigree ES cells with targetted null alleles ENU-treated male mice with SNVs

24. The John Curtin School of Medical Research, The Australian National University, Australia’s Capital City, Canberra Chris Goodnow’s group Adrian Liston Owen Siggs Katrina Randall Jesse Jun Lina Tze Anselm Enders Keisuke Horikawa Charis Teh Sally Mapp Gerard Hoyne Zuopeng Wu Aust Phenomics Network Ed Bertram Belinda Whittle Carola Vinuesa’s Group: Di Yu Vicki Athanasopoulos Michelle Linterman Diego Silva Rob Rigby Matthew Cook’s Group Nick Simpson Garvan Institute: Rob Brink Fabienne Mackay Charles Mackay John Sprent Stuart Tangye Tony Basten Barbara Fazekas ACRF, ARC, NHMRC, The Wellcome Trust, NIAID-NIH, JDRF, LLRF Oxford & London: Richard Cornall Tess Lambe Facundo Batista ANU Research School of Chemistry:Gottfried Otting’s Group: Institute for Systems Biology: Alan Aderem’s group UCSF: Art Weiss, Jason Cyster, Lewis Lanier