Transcript EUMORPHIA Understanding human molecular physiology and

Challenges
•
Develop a series of mutant alleles for every gene in the mouse
genome
•
Develop and apply standardised phenotyping platforms to
determine the phenotypic consequences of each mutation
•
Identify models for the complete disease spectrum in the
human population
•
Translate functional information identified through mouse
models to the study of human genetic disease
Mouse Models for Human Disease
Challenges
•
Develop a series of mutant alleles for every gene in the mouse
genome
•
Develop and apply standardised phenotyping platforms to determine
the phenotypic consequences of each mutation
•
Identify models for the complete disease spectrum in the human
population
•
Translate functional information identified through mouse models to
the study of human genetic disease
Mouse Models for Human Disease
Mutagenesis in the Mouse
Phenotype-driven and gene-driven approaches
Gene
Driven
Phenotype
Driven
Mouse Models for Human Disease
•
•
•
•
Gene traps
Gene targeting
Gene driven ENU
RNAi
EUCOMM, Europe
European Conditional Mouse Mutagenesis
KOMP, US
Knock-out Mouse Project
• Ethylnitrosourea, ENU -
unbiased chemical mutagenesis
Mutagenesis in the Mouse
Phenotype-driven and gene-driven
Gene
Driven
Start with
a known
locus
Often make
a priori
assumptions
about
function of
gene
Unpredictable
phenotypes
Phenotype
Driven
Requires
identification
of mutated
gene
No
assumptions
about
underlying
pathways
Phenotype is
the starting
point
Mouse Models for Human Disease
ENU mutagenesis
• ENU - mutagenesis of male spermatogonial stem
cells
• Specific locus mutation rate of > 1 in 1,000 gametes
• Every 1,000 mice carry a new ENU hit at any locus
• A point mutagen
• Can deliver the full range of mutational effects - hypomorphs,
gain-of-function, dominant negative
Mouse Models for Human Disease
Value of ENU and other mutant models
- dissecting multifactorial disease
•
Require a range of mutational effects to study gene function
from knock-out to hypomorph to gain-of-function
•
All mouse mutations are contextual and deliver effects
depending upon genetic background
•
ENU mutations will often represent hypomorphic, partial lossof-function, alleles
•
ENU often explores alleles of moderate effect for genes that are
typical QTLs contributing to differences in traits between
mouse strains
Mouse Models for Human Disease
ENU mutagenesis
• G1 dominant genome-wide screens
• G3 recessive genome-wide screens
• Region-specific recessive screens using deletions or
balancers
• Modifier or sensitised screens
• Using ENU mutagenesis to identify mutations that enhance
or suppress phenotypic effects of other mutations
• Employing ENU on a genetic background sensitised to
developing the relevant phenotype
Mouse Models for Human Disease
Dominant
Genome
Wide
ENU
ENU
*
G0
G0
x
x
*
Inv
Brown
2001
G1 &* Balling
x
Curr. Op. Genet. Develop. 11: 268
G1
*
Recessive ENU
Genome
*
G0
Wide
G2
*
*
x
*
G3
x
x
*
ENU
G0
*
Targeted
Recessive Deletions
x
*
G2
*
G1
G1
*
*
test
*
*
*
x
Del
G2
*
G3
*
Brown & Hardisty *2003
lethal
test
carrier
Seminars Cell Dev. Biol. 14: 19
*
G1
EMS
*
Targeted
Recessive Inversions
*
carrier
uninformative
Dominant
Genome
Wide
ENU
G0
*
x
*
G1
*
• G1 dominant genome-wide screens
• G3 recessive genome-wide screens
Recessive ENU
Genome
*
G0
Wide
x
*
G1
EMS
x
*
*
G2
*
*
G3
*
*
*
G1
ENU
G0
*
x
Inv
G1
G2
*
*
x
x
*
*
G3
lethal
*
test
• Region-specific recessive screens using
deletions or balancers
Targeted
Recessive Inversions
*
carrier
Dominant ENU
Genome
*
G0
Wide
x
*
G1
*
• Region-specific
recessive screens using
ENU
Recessive
deletions or balancers
GenomeG0
*
x
*
Wide
EMS
G1
x
*
ENU
G0
*
G2
*
G1
G1
*
*
test
G3
*
*
*
Targeted
Recessive Deletions
x
x
Del
G2
*
*
*
carrier
uninformative
ENU Mutagenesis
Modifier Screens
ENU
Screens in
sensitised
pathways
X
[carrying mutations
in pathway of interest]
F1 progeny
SCREENS
Mouse Models for Human Disease
ENU Mutagenesis
Modifier Screens - Dominant Enhancers
ENU
*
G0
r
r
x
r
*
New dominant mutations Dominant Modifiers New Alleles
G1
*
r
*
r
Progeny test
Mouse Models for Human Disease
ENU Mutagenesis
Genome-wide screen for dominant mutations
ENU
X
F1 progeny
SCREENS
Mouse Models for Human Disease
Mutagenise BALB/c males
Mate with C3H/He females
Screen for dominant
phenotype
Visible
anomalies
SHIRPA
testing
Behavioural
testing
Blood
Biochemistry
Data into Mutabase
Inheritance Testing
Low resolution mapping
(IVF used for
backcrosses into C3H)
Mouse Models for Human Disease
New screens
collaborators
Archive of embryos & sperm
Detailed analysis of
selected mutants
Challenges
•
Develop a series of mutant alleles for every gene in the mouse
genome
•
Develop and apply standardised phenotyping platforms to determine
the phenotypic consequences of each mutation
•
Identify models for the complete disease spectrum in the human
population
•
Translate functional information identified through mouse models to
the study of human genetic disease
Mouse Models for Human Disease
Eumorphia
Phenotype screens for mice
Developing an integrated
platform
•
•
•
High-throughput phenotypic assessment
- SHIRPA
Systematic screen for phenotypes based on protocol developed
by Irwin (1968)
Semi-quantitative assessment of muscle and lower
motorneuron, spinocerebellar, sensory, neuropsychiatric and
autonomic function
Simple and rapid screen incorporating approximately 40 tests
and lasting 10-15 minutes per mouse
Mouse Models for Human Disease
The SHIRPA screening protocol
Muscle/Lower Spinocerebellar Sensory
Motor Neurone
Neuropsychiatric
Body position
Body position
Gait
Gait
Righting reflex
Positional passivity
Motor performance
Wire manouvere
Tail elevation
Righting reflex
Visual placing
Motor performance
Limb tone
Spontaneous activity
Balance
Tail elevation
Locomotor Activity
Visual placing
Limb position
Limb tone
Body tone
Passivity
Abdominal tone
Balance
Grip strength
Locomotor Activity
Limb position
Body tone
Abdominal tone
Urination and
Defecation
Body position
Transfer arousal
Startle response
Body tone
Fear
Anxiety
Learning/memory
Spontaneous
activity
Locomotor Activity
Touch escape
Righting reflex
Irritability
Vocalisation
Bizarre behaviour
Food/water intake
Positional passivity
Catalepsy
Aggression, PPI
Mouse Models for Human Disease
Transfer arousal
Touch escape
Corneal reflex
Analgesia
Gait
Visual placing
Toe pinch
Limb position
Pinna reflex
Righting reflex
Autonomic
Palprebral
closure
Tail elevation
Temperature
Heart Rate
Pilorection
Skin colour
Food/water
intake
Urination
Startle
response
Salivation
Respiration
rate
Defecation
Harwell ENU programme
Focus on:
• 35,000 mice weaned, scored for visible phenotype
Neurological
• 15,000 mice SHIRPA, 10,500 mice LMA, 10,500 PPI,
7,000 vision screens, 2,000 clinical chemistry…
Behavioural
Circadian
• 1,500 abnormal phenotypes
Deafness
• 376 inheritance tested
Vision
• 196 inherited mutations
Diabetes
Kidney stones
Alcohol
preference
• > 700 new mutations generated
• 100 mapped, 40 cloned
Nolan et al. Nature Genetics 2000
Mouse Models for Human Disease
Phenotype Classes
 Pigment
31
 Skin and hair texture
21
 Growth
31
 Craniofacial
12
 Digits/limbs
3
 Tail
4
 Clinical chemistry
13
 Type II diabetes, dyslipidemias, bone & liver disease
 Vestibular/Deafness
21
 Eye/Vision
24
 Neurological/Behavioural
56
Mouse Models for Human Disease
Detection of visible mutations
Dominant spotting
Microphthalmia
Mouse Models for Human Disease
Batface
Nanomouse
ENU mutagenesis - Harwell
100 mutations mapped - 60% novel
GENA
Chr
• 25
• 29
• 37
• 38
• 40
• 41
• 42
• 47
• 51
• 52
• 53
• 57
• 60
• 65
• 70
• 71
• 77
• 78
• 104
• 110
• 123
• 137
• 158
• 171
• 175
• 180
4
14
9
7
2
4
7
4
11
4
9
11
4
5
2
1
4
1
17
1
17
4
2
4
15
5
tornado, Todo
kumba, Ku
small
sickly, Sic
goth, Goth
orbitor, Obt
whitetoes and belly spot, Whto
dizzy, Dz
trembler-1H
cyclone, Cyn
small/imprinting
trembler-2H
eddy, Edy
van gogh, Vng
blind drunk, Bdr
white feet and belly spot
dark footpads 2, Dfp2
saggy, Sagg
jeff, Jf
short head
batface
ferris, Ferr
slalom, Slm
metis
spin cycle, Scy
robotic, Rob
Mouse Models for Human Disease
GENA
Chr
• 181
• 191
• 196
• 203
• 208
• 232
• 238
• 239
• 241
• 243
• 246
• 251
• 257
• 263
• 265
• 269
• 291
• 295
• 300
• 303
• 328
• 333
• 336
• 348
• 368
• 379
• 380
• 389
• 396
19
2
4
15
3
2
2
11
4
4
11
3
8
6
2
5
8
13
6
17
4
14
6
11
2
X
19
11
10
pardon, Pdo
iris-corneal strands, Icst
leda
spag, Spag
high startle
lens-corneal adhesion 2, Leca2
lens-corneal adhesion 1, Leca1
waved5, Wa5
low cholesterol
low cholesterol
retinal white spots, Rwhs
late-onset deafness, Junb
retinal arterial wiring, Raw
high glucose
lens-corneal adhesion 3, Leca3
dilated pupils, Dilp1
small vacuolar cataract, Svc
Anne Boleyn, Anb
Tommy, late-onset deafness
short tail
low ALP
retinal vascular mass, Rvm
retinal orange patches, Rorp
high glucose
lens-corneal adhesion 4, Leca4
dilated pupils, Dilp2
optic disc coloboma, Opdc
high glucose
high glucose
Alleles of existing phenotypes
Novel phenotypes
ENU mutagenesis - Mutants Cloned
Line #
• GENA6
• GENA29
• GENA37
• GENA51
• GENA53
• GENA57
• GENA104
• GENA123
• GENA158
• GENA175
• GENA180
• GENA181
• GENA232
• GENA238
• GENA241
• GENA243
• GENA251
• GENA269
• GENA300
• GENA328
• GENA348
• GENA368
• GENA370
• GENA371
• GENA387
• GENA389
• GENA391
• GENA396
• GENA450
• TAS1
• TAS3
• TAS9
Mouse Models for Human Disease
bare patches, Bpa
kumba, Ku
small
trembler-1H
small
trembler-2H
jeff, Jf
batface, Bfc
slalom, Slm
spin cycle, Scy
robotic, Rob
pardon, Pdo
lens corneal adhesion 2
lens corneal adhesion 1
low cholesterol
low cholesterol
junbo, Junb
dilated pupils 1, Dilp1
tommy, Tmy
low ALP
type II diabetes
lens corneal adhesion 4
trembler-3H
no tail
type II diabetes
type II diabetes
low cholesterol
type II diabetes
lens cloudy
belly spot
short tail
crooked tail, Ka
15
11
11
11
11
Chr
X
14
9
11
9
11
17
9
2
Celsr1
5
19
2
2
4
4
3
5
6
4
Gck
2
11
17
Gck
Gck
4
Gck
10
1
17
X
Nsdhl
Zic2
Rasgfr1
Pmp22
Rasgfr1
Pmp22
to be reported
to be reported
Jag1
Af4
Emx2
Pax6
Pax6
Abca1
Abca1
Evi1
Phox2b
Atp2b2
Akp2
Pax6
Pmp22
T
Abca1
Mip
Pax3
T
to be reported
ENU mutagenesis - Mutants Cloned
Line #
• GENA6
• GENA29
• GENA37
• GENA51
• GENA53
• GENA57
• GENA104
• GENA123
• GENA158
• GENA175
• GENA180
• GENA181
• GENA232
• GENA238
• GENA241
• GENA243
• GENA251
• GENA269
• GENA300
• GENA328
• GENA348
• GENA368
• GENA370
• GENA371
• GENA387
• GENA389
• GENA391
• GENA396
• GENA450
• TAS1
• TAS3
• TAS9
Mouse Models for Human Disease
bare patches, Bpa
kumba, Ku
small
trembler-1H
small
trembler-2H
jeff, Jf
batface, Bfc
slalom, Slm
spin cycle, Scy
robotic, Rob
pardon, Pdo
lens corneal adhesion 2
lens corneal adhesion 1
low cholesterol
low cholesterol
junbo, Junb
dilated pupils 1, Dilp1
tommy, Tmy
low ALP
type II diabetes
lens corneal adhesion 4
trembler-3H
no tail
type II diabetes
type II diabetes
low cholesterol
type II diabetes
lens cloudy
belly spot
short tail
crooked tail, Ka
15
11
11
11
11
Chr
X
14
9
11
9
11
17
9
2
Celsr1
5
19
2
2
4
4
3
5
6
4
Gck
2
11
17
Gck
Gck
4
Gck
10
1
17
X
Nsdhl
Zic2
Rasgfr1
Pmp22
Rasgfr1
Pmp22
to be reported
to be reported
Jag1
Af4
Emx2
Pax6
Pax6
Abca1
Abca1
Evi1
Phox2b
Atp2b2
Akp2
Pax6
Pmp22
T
Abca1
Mip
Pax3
T
to be reported
Mutations and phenotypes
•
Known phenotypes in known or novel
genes i.e. alleles of existing mutations for
which the gene may or may not have been
characterised
DEPTH
BREADTH
 Novel phenotypes in known genes, for
which there is some prior functional
annotation
 Novel phenotypes in novel genes, for which
there is no prior functional annotation
Mouse Models for Human Disease
BREADTH
BREADTH
Challenges
•
Develop a series of mutant alleles for every gene in the mouse
genome
•
Develop and apply standardised phenotyping platforms to determine
the phenotypic consequences of each mutation
•
Identify models for the complete disease spectrum in the human
population
•
Translate functional information identified through mouse models to
the study of human genetic disease
Mouse Models for Human Disease
Models of neurological disease
Pronounced cerebellar Purkinje cell defect in the Robotic mouse
+/+
• Ataxic mouse identified in
Harwell neurological screen
• Mutation in Af4 - transcription
factor
• Expression in brain localised
to Purkinje cells
Rob/+
• Af4 knock-out - defects in B
and T-cell development
Isaacs et al. J. Neurosci., 2003
Collaboration with Kay Davies, Oxford
Mouse Models for Human Disease
Models of alcoholism
Mutation in the GABAA b1 receptor gene leads to alcohol preference
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
• Identified Alco22 line from Harwell ENU
mutagenesis screen for alcohol preference imbibes 75-80% of their total fluid intake as
10% ethanol
• Mutation in the GABAA b1 receptor gene
• Mutation causes spontaneous channel
opening and reduced responses to GABA
• New gene involved in alcohol preference
Collaboration with Howard Thomas, St. Mary’s, Imperial College
Mouse Models for Human Disease
Models of type II diabetes
Nicotinamide Nucleotide Transhydrogenase (Nnt): a key role in insulin secretion
QTL mapping - IPGTT trait
C57BL/6J x C3H F2 (C57BL/6J glucose intolerant)
3 QTLs mapped
Chromosome 13 candidate gene Nnt (5 exon deletion)
Toye et al. Diabetologia (2005) 48:675-686
Functional validation of Nnt
siRNA knockdown in Min6 insulin secreting cell line
Two ENU alleles identified from ENU DNA archive
20
35
**
**
15
**
10
5
1.6
30
25
*
*
*
*
20
15
10
**
**
Insulin (μg/l)
**
**
Glucose (mmol/l)
Glucose (mmol/l)
25
N68K
1.2
0.8
**
**
0.4
**
**
5
0
0.0
0
0
30
60
Time (mins)
120
0
10
20
30
0
Time (mins)
10
20
Time (mins)
Wildtype, circles; heterozygotes, squares; homozygotes, triangles.
Collaboration with Fran Ashcroft, Physiology, Oxford
Mouse Models for Human Disease
*
*
30
Models of neural tube development
Spin cycle (Scy), Crash (Crsh) mutant alleles reveal new player in PCP
• Spin cycle mutation recovered as
part of vestibular screen at
Harwell
• Planar cell polarity defect of hair
cells in inner ear
• Mutation in Celsr1 - seven pass
transmembrane cadherin receptor
• Reveals new player in PCP in
mammals
• Investigation of PCP in neural
tube formation
Curtin et al. Current Biology, 2003
Mouse Models for Human Disease
Models of kidney disease
ENU mutations with kidney stone disease
•
Nephrolithiasis (kidney stones) is a
common disorder affecting ~12% men
and ~5% women by the seventh decade
•
Hypercalciuria associated with ~60%
cases
•
Identified three mouse lines with
autosomal dominant nephrocalcinosis
from X-ray collection - and resurrrected
from archive
•
Mutations mapped to chromosome 11
(GENA 406) and chromosome 17 (GENA
408). Refine mapping and candidate gene
sequencing in progress
Renal cortex
100m
Collecting ducts
100m
von Kossa reaction
for detection of
calcification
Collaboration with Raj Thakker, Oxford
Mouse Models for Human Disease
Models of Otitis Media
Mutations Jeff and Junbo provide first genetic models of otitis media
13DAB
TB
MEC
MEC
Jbo/+
+/+
• Recovered from deafness screen, two
mutants show a conductive deafness due to
a chronic middle ear inflammatory disease
in the absence of any other pathology
• Strong genetic component to susceptibility
to OM in human population - but no genes
known
• Junbo - missense mutation in the Evi1
transcription factor; related pathways in
vitro have been implicated in control of
mucin transcription
180DAB
MEC
• Jeff - novel gene; suggestive association in
association studies in human population
Jbo/+
Mouse Models for Human Disease
Jbo/+
Models of neurodegenerative disease
ENU-induced dynein mutation (Loa) modifies Huntington’s disease
• Huntington’s onset and progression are
enhanced by the Loa mutation and
autophagosome-lysosome fusion is
impaired
1.2
1.0
.8
.6
.4
GENOTYPE
.2
HD/+; +/+
0.0
HD/+; Loa/+
6
8
10
12
14
16
18
20
22
Age (weeks)
Motor perfomance (Rotarod)
latency to fall (s)
200
150
HD/+; Loa/+
100
HD/+; +/+
+/+; Loa/+
50
0
5
7
9
11
13
15
Age (w eeks)
HD/+; +/+
HD/+; Loa/+
17
• Clearance of aggregate-prone proteins is
crucial for the development of
proteinopathies and autophagy is one of
the main pathways involved in clearance
• This work provides a mechanistic link
between dynein mutations and inclusion
formation in motor neuron diseases
Dynein mutation decreased survival and
accelerated the appearance of motor
dysfunction and inclusion formation in HD
mice
Mouse Models for Human Disease
Ravikumar & Acevedo et al. Nature Genetics, 2005
Collaboration with Dept. Medical Genetics, Cambridge
ENU Mutagenesis
Modifier Screens - Dominant Enhancers
G0
G1
Loa
Loa
HD Tg
x
Loa
HD Tg
Dominant Modifiers
Mouse Models for Human Disease
HD Tg
ENU Mutagenesis
Modifier Screens - Dominant Enhancers
*
G0
G1
*
HD Tg
x
HD Tg
*
HD Tg
HD Tg
*
New dominant mutations Dominant Modifiers New Alleles
Progeny test
Mouse Models for Human Disease
ENU gene-driven screens
Creation of parallel archives of DNA and sperm
ENU
BALB/c X C3H
Coghill et al.
Nature Genetics
2002
DNA archive
Sperm archive New archive: 5,000
IVF
Mutant identification
dHPLC, TGCE, Cel1
Mouse Models for Human Disease
Recovery and
examination of
mutants
Probabilities of finding n or more mutant alleles in
varying numbers of DNAs from offspring of ENU
mutagenised male mice
1
0.9
0.8
0.7
0.6
0.5
4 or more alleles
0.4
3 or more alleles
0.3
2 or more alleles
0.2
1 or more alleles
0.1
Probability of n
or more alleles
0
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Number of DNAs screened
Assumptions
1. Mutation rate of 1/1000 per locus
2. Mutation detection rate of 90%
Mouse Models for Human Disease
Basepairs screened Number of individuals Total Mbp Mutations
Amino acid
Rederived by
IVF
Gene
exon screened
screened
screened
found
change
1
Retn*
344 (3/3)
2230
0.77
1
0
2
Kir 6.2
1172 (1/1)
5302
6.21
3
1 + stop
3
Cav 3
456 (2/2)
1487
0.68
0
0
4
Bscl2
965 (6/10)
3072
2.96
3
0
5
QTL-gene
854 (5/21)
3072
2.62
3
2
6
Capn10
1305 (8/12)
4224
5.51
6
3
7
Tectb*
582 (4/10)
2230
1.3
1
0
3
3
2
2
1
1
Coghill
Genetics
8
Cx26*et al. Nature
681 (1/1)
2230 2002
1.52
9
Foxf2 et al.
1340
(2/2)
2230
2.98
Quwalid
Mammalian
Genome
2004
1
1
10
Sfrp5
277 (1/3)
1920
0.53
1
Stop
1
11
Sfrp2
502 (1/3)
3072
1.54
2
2
2
Predicted:
1 functional
change
every1.29
2.38Mb1
12
Sfrp1
421 (1/3)
3072
1
13
Mro
658 (4/7)
5072
3.34
8
4 + 2splice site
Observed:
1 potential
functional
change
every
1.82Mb
1
5
14
Foxf2
294 (3/16)
3072
0.9
3
0
15
Rgs2
635 (4/5)
3072
1.95
2
2
16
IKBB
774 (3/7)
4224
3.23
1
1
17
AFC3
114 (1/21)
6454
0.73
0
18
new Jeff
693 (7/22)
4224
2.93
2
1
19
Uba52
398 (3/4)
4224
1.68
3
0
20
KYIP1
399 (2/ )
4224
1.68
2
2
2
21
Zic 5
686 (2/ )
5760
3.95
5
3
2
22
P55
375 (2/16)
4224
1.58
0
23
RG18
167 (1/ )
3200
0.53
1
1 (stop)
1
24
GRS2
308 (2/ )
3200
0.98
2
2
2
25
X30B11
124 (1/ )
2400
0.29
2
2
2
26
Rwhs
316 (2/ )
5088
1.60
4
4
4
27
Hsp20
1790
4224
7.56
2
1
1
28
Cited2
816 (2/ )
4224
3.44
2
1
1
101027
64.28
65
41
31
Mouse Models for Human Disease
1
1
Mutagenesis in the Mouse
Phenotype-driven and gene-driven approaches
Gene
Driven
Phenotype
Driven
Mouse Models for Human Disease
•
•
•
•
Gene traps
Gene targeting
Gene driven ENU
RNAi
EUCOMM, Europe
European Conditional Mouse Mutagenesis
KOMP, US
Knock-out Mouse Project
• Ethylnitrosourea, ENU -
unbiased chemical mutagenesis
Challenges
•
Develop a series of mutant alleles for every gene in the mouse
genome
•
Develop and apply standardised phenotyping platforms to determine
the phenotypic consequences of each mutation
•
Identify models for the complete disease spectrum in the human
population
•
Translate functional information identified through mouse models to
the study of human genetic disease
Mouse Models for Human Disease