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Genotype “Endophenotypes” Clinical Phenotype: Disease Signs & Symptoms Rational treatment or prevention Translation Gap: bridged by integrating next-gen sequencing and solid understanding of mechanism from Mendelian mouse mutants MOUSE Molecular, cellular & whole system consequences of mutations in individual genes Mechanistic information: essential roles of individual genes; groups of genes clustered by pathway; systems biology understanding of genegene interactions MAN Next-gen resequencing to reveal family-specific variants: Statistical associations: Eg clusters of mutations in particular pathways Translation gap 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 MEDICINE Clinical and commercial translation: targetted development of new therapeutics/preventions 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 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 G2 progeny: Single nucleotide substitutions segregate G3 progeny: 12.5% of SNVs brought to homozygosity in each mouse * 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 SSC CD3 FSC CD4 CD3 Tube 040: lowish CD4s & CD8s & higher %CD44hi in CD8s CD3 CD44 CD44 T Cells CD3 IgD B220 IgM CD3 Tube 046: slightly higher IgM CD44 NK1_1 NK1_1 B Cells CD44 IgG2a IgE CD44 IgE IgG2a IgE IgE Monocytes 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 I think the technician forgot to scan following mouse: Sample 73 is ENU14Yaa:047:B6:G2 # 2 Tube 046: slightly higher IgM Tube 037: higher IgE Mapping and identification of Mendelian mutations in the mouse circa 2009: legacy of the public mouse genome project 1 2 3 Rockstar Dopey Murdoch Mr T-less Face Redburst Lightning Lochy Rain Storm Bthy SanRoque Dorian Zippo Trembles 9B6 35 Botero Collins Wavy Pengu Wachin Nephertiti Plastic 8B6 27 T-wimp Anubis B-blast Babe Duane Dwarf 4 B52 5 6 7B6148 7 8 Tsavo 9 10 LeukSkywalker BDown Delficio Kenobi Tipsy B-leg Alberta 4AT32 9B6 64 Pharlap PIAF Pinky Fatso Socks Tiny Kenny Fluoro Jersey Blobby Polly Buffy Eeyore Fat Willow Joey Koy MeioI Aussie Tilcara Winnie Hoban Unmodulated Seshat Thoth Jasmine Pardon Anarchy LasVegas Wobbles 8C63 Rose 11B658 Mozart Malewa T-Bird Armidilo Vibes Mr Hanky Bata Captain 6WT33 Sweaty Eddie Nessy Theoden Piebald Thunder Krusty Anakan Primurus Mal Xander Flipper Senseless Hipster 11 12 13 14 15 16 17 18 19 - 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 X Y 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 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 Amount of complement receptor on B cell surface • Unmodulated detected from B cell screen • Each dot represent one mouse. • Dashed lines two SD • ~ 400 G3 animals Amount of antigen receptor on B cell surface Changes in antigen receptor density often indicate abnormalilities in the BCR signalling pathways Amount of complement receptor on B cell surface Unmodulated variant breeds true as a simple Mendelian recessive trait Control 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 Relative amount of antibody in serum Homozygous un/un mice make little antibody upon immunization, because their B cells proliferate poorly to BCR-stimulation Th1 / IgG2a Th2 / IgG1 Unmodulated: point mutation in one protein-interaction domain of a MAGUK-family protein, CARD11 (Carma1) Jesse Jun * * * * * * * * * 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. 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) Homozygous null mutations in CARD11 disrupt TCRNFB signalling and cause immunodeficiency, but what about SNVs causing a quantitative decrease? Aire Thymic epithelium Antigen CD4 or CD8 eg ed Y Y *Y Y * * CD45 MHC TCR Lck Y Y Y Y*Y * Y *Y *Y* * *Y Y * z Y Y Y Y Y Y * * * * * * Itk CD28 Fyn Ptpn22 (PEP) Zap70 * * * NFAT genes for cell growth and division PKC IB Ca2+ Calcineurin CD80 (B7.1) CD86 (B7.2) CARD11(CARMA1) Bcl10 MALT1 NFB IKK IL-2 IL-2R STAT JAK 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 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. Translation Gap: bridged by integrating next-gen sequencing and solid understanding of mechanism from Mendelian mouse mutants MOUSE Molecular, cellular & whole system consequences of mutations in individual genes Mechanistic information: essential roles of individual genes; groups of genes clustered by pathway; systems biology understanding of genegene interactions MAN Next-gen resequencing to reveal family-specific variants: Statistical associations: Eg clusters of mutations in particular pathways Translation gap 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 MEDICINE Clinical and commercial translation: targetted development of new therapeutics/preventions 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 ES cells with targetted null alleles ~70 deleterious mutations/pedigree ENU-treated male mice with SNVs The John Curtin School of Medical Research, The Australian National University, Australia’s Capital City, Canberra Chris Goodnow’s group Carola Vinuesa’s Group: Adrian Liston Di Yu Owen Siggs Vicki Athanasopoulos Katrina Randall Michelle Linterman Jesse Jun Diego Silva Lina Tze Rob Rigby Anselm Enders Matthew Cook’s Group Keisuke Horikawa Nick Simpson Charis Teh Garvan Institute: Sally Mapp Rob Brink Gerard Hoyne Fabienne Mackay Zuopeng Wu Aust Phenomics Network Ed Bertram Belinda Whittle Charles Mackay ACRF, ARC, NHMRC, The Wellcome Trust, John Sprent Stuart Tangye NIAID-NIH, JDRF, LLRF Tony Basten 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: Barbara Fazekas Art Weiss, Jason Cyster, Lewis Lanier
