Transcript the Presentation

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 TCRNFB 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
IB
Ca2+
Calcineurin
CD80 (B7.1)
CD86 (B7.2)
CARD11(CARMA1)
Bcl10
MALT1
NFB
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