Post on 30-Dec-2015
Clinical Phenotype: Disease Signs & Symptoms
Rational treatment or prevention
Genotype
“Endophenotypes”
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 gene-gene interactions
MAN
MOUSE
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
Clinical and commercial translation: targetted development of new
therapeutics/preventions
Preclinical efficacy in mice with pathway defects like humans
Translation Gap: bridged by integrating next-gen sequencing and solid understanding of mechanism from Mendelian mouse mutants
MEDICINE
Fumbling for his recline button, Ted unwittingly instigates a disaster
The genome encodes the buttons and circuits of our immune system - we just need to know how to read the instructions
*
ENU treated C57BL/6 male C57BL/6 female
Random base substitutions in spermatogonial
stem cells
*
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
Systematic screening for recessive Mendelian syndromes in the immune system caused by single nucleotide variants
G0:
C57BL/6 female
Clinical Phenotype: eg susceptibility to infection with M tuberculosis or Poxvirus, allergic dermatitis,
disseminated lymphoid cancer
Preclinical model for treatment or prevention
“Endophenotypes”: eg altered blood cells in flow cytometry, altered antibody response
to immunization
Genotype: mutation mapped to chromosomal interval and identified by DNA sequencing
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
Plate 14062 Blood FACs bled 064/10 (05.03.10)
I think the technician forgot to scan following mouse:Sample 73 is ENU14Yaa:047:B6:G2 # 2
Mapping and identification of Mendelian mutations in the mouse circa 2009: legacy of the public mouse genome
project
- 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
Dopey
Mr T-lessMurdoch
Face
Redburst
Lochy
SanRoque
Rockstar
Wavy
Trembles
SocksPharlap
Unmodulated
Blobby
LeukSkywalker
Alberta
Eeyore
Winnie
Tipsy
Nephertiti
Tiny
Zippo
BuffyWillow
PollyWobbles
Plastic
T-wimp
B-blastBthy
Dwarf
Babe Vibes
Nessy
Flipper
Piebald
Senseless
Sweaty
Kenobi
Anarchy
PardonBotero
FatAussie Koy
Malewa
Fatso
Tilcara
6WT33
Anakan
Mozart
Tsavo
Armidilo
Thunder
Captain
Theoden
Jasmine
Xander
Pinky
HipsterT-Bird
Dorian
Lightning
Storm
Pengu
9B6 35 BDown
Jersey
MeioI
9B6 64
Delficio
PIAF
7B6148
Kenny
4AT32
Hoban
Fluoro
B-leg
Joey
LasVegas
8C63
Duane
Bata
Anubis
B52
8B6 27
Mal
Mr Hanky
KrustyPrimurus
11B658
1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 X Y
WachinRain
Rose
Collins
ThothSeshat
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
• 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
Am
ount
of
com
plem
ent
rece
ptor
on
B c
ell s
urfa
ce
Changes in antigen receptor density often indicate
abnormalilities in the BCR signalling pathways
Unmodulated variant breeds true as a simple Mendelian recessive trait
Control
Variant
Mean values for individual offspring from unmodulated affected mouse (un/un) crossed with heterozygous (un/+) carrier
Amount of antigen receptor on B cell surface
Am
ount
of
com
plem
ent
rece
ptor
on
B c
ell s
urfa
ce
Homozygous un/un mice make little antibody upon immunization, because their B cells proliferate poorly to BCR-stimulation
Th1 / IgG2a Th2 / IgG1
Rel
ativ
e am
ount
of
antib
ody
in s
erum
Unmodulated: point mutation in one protein-interaction domain of a MAGUK-family protein, CARD11 (Carma1)
* * * * * * * * *
Jesse Jun
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.
Example:MECHANISM STUDY: B cells from mice with inherited CARD11
mutations are selectively crippled for BCR activation of the IKK-NFkB pathway and proliferation
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
(eg. MYC)
PKCCARD11
Homozygous null mutations in CARD11 disrupt TCR-NFB signalling and cause immunodeficiency, but what about SNVs causing a quantitative decrease?
IKK
IB
NFB
PKC
CD80 (B7.1)
genes for cell growth and division
CD28
* * *
NFAT
Calcineurin
Ca2+
Y YY Y*
Y YY Y** *
*
LckFyn
Zap70
CD4 or
CD8
Y YY Y** *
*
Y YY Y** *
*
Y YY Y** *
*
TCR
MHC
Itk
CD86 (B7.2)
IL-2
IL-2R
JAKSTAT
CARD11(CARMA1)Bcl10MALT1
Ptpn22 (PEP)
Antigen
AireThymic epithelium
CD45
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
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.
Science 2008 319:1676-1679
L298Q unmodulated
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 gene-gene interactions
MAN
MOUSE
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
Clinical and commercial translation: targetted development of new
therapeutics/preventions
Preclinical efficacy in mice with pathway defects like humans
Translation Gap: bridged by integrating next-gen sequencing and solid understanding of mechanism from Mendelian mouse mutants
MEDICINE
IMPC GOAL:
A phenotyped mouse mutant for every gene
Genotyped & phenotyped mouse pedigrees: >3 homozygotes/mutation
ES cells with targetted null alleles
ENU-treated male mice with SNVs
Pedigree breeding, genotypic & phenotypic data acquisition & collation
1 deleterious mutation/pedigree ~70 deleterious mutations/pedigree
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
The John Curtin School of Medical Research,
The Australian National University, Australia’s Capital City, Canberra
Carola Vinuesa’s Group:
Di Yu
Vicki Athanasopoulos
Michelle Linterman
Diego Silva
Rob Rigby
ACRF, ARC, NHMRC,
The Wellcome Trust,
NIAID-NIH, JDRF, LLRF
Matthew Cook’s Group
Nick Simpson
Oxford & London:
Richard Cornall
Tess Lambe
Facundo Batista
Garvan Institute:
Rob Brink
Fabienne Mackay
Charles Mackay
John Sprent
Stuart Tangye
Tony Basten
Barbara FazekasANU Research
School of Chemistry:Gottfried Otting’s
Group:
Institute for Systems Biology:
Alan Aderem’s group
UCSF:
Art Weiss, Jason Cyster, Lewis Lanier