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Transcript of Mouse Models for Human Disease Challenges Develop a series of mutant alleles for every gene in the...
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
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 MousePhenotype-driven and gene-driven approaches
• Ethylnitrosourea, ENU -
unbiased chemical mutagenesisPhenotypeDriven
GeneDriven
• Gene traps• Gene targeting• Gene driven ENU• RNAi
EUCOMM, EuropeEuropean Conditional Mouse Mutagenesis
KOMP, USKnock-out Mouse Project
Mouse Models for Human Disease
Mutagenesis in the MousePhenotype-driven and gene-driven
PhenotypeDriven
GeneDriven
No assumptionsabout underlying pathways
Unpredictablephenotypes
Start with a known locus
Often make a priori assumptionsabout function of gene
Requires identification of mutated gene
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 loss-of-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
*
*
ENU
x
*
G0
G1
DominantGenome Wide
*
*
ENU
x
*x
*
*
*
*
*
*
G0
G1
G2 G1
G3
EMS
RecessiveGenome Wide
Inv
lethal test carrier
x
x
x
ENU
*
*
* *
**
*
G0
G1
G2
G3
Targeted Recessive - Inversions
Del
test carrier
x
x
ENU
*
*
* *
uninformative
G0
G1
G2
Targeted Recessive - Deletions
Brown & Balling 2001Curr. Op. Genet. Develop. 11: 268
Brown & Hardisty 2003Seminars Cell Dev. Biol. 14: 19
*
*
ENU
x
*
G0
G1
DominantGenome Wide
• G1 dominant genome-wide screens
*
*
ENU
x
*x
*
*
*
*
*
*
G0
G1
G2 G1
G3
EMS
RecessiveGenome Wide
• G3 recessive genome-wide screens
Inv
lethal test carrier
x
x
x
ENU
*
*
* *
**
*
G0
G1
G2
G3
Targeted Recessive - Inversions
• Region-specific recessive screens using deletions or balancers
*
*
ENU
x
*
*
*
ENU
x
*x
*
*
*
*
*
*
G0
G1
G0
G1
G2 G1
G3
EMS
DominantGenome Wide
RecessiveGenome Wide
Del
test carrier
x
x
ENU
*
*
* *
uninformative
G0
G1
G2
Targeted Recessive - Deletions
• Region-specific recessive screens using deletions or balancers
Mouse Models for Human Disease
ENU MutagenesisModifier Screens
X
F1 progeny
SCREENS
ENU
[carrying mutations in pathway of interest]
Screens insensitisedpathways
Mouse Models for Human Disease
*
ENU
x
*
G0
G1
rr
r
New dominant mutations*
r* r
Dominant Modifiers New Alleles
Progeny test
ENU MutagenesisModifier Screens - Dominant Enhancers
Mouse Models for Human Disease
X
F1 progeny
ENU MutagenesisGenome-wide screen for dominant mutations
SCREENS
ENU
Mouse Models for Human Disease
Screen for dominantphenotype
Mutagenise BALB/c males
Mate with C3H/He females
Visibleanomalies
SHIRPAtesting
Behaviouraltesting
BloodBiochemistry
Archive of embryos & sperm
Low resolution mapping (IVF used for
backcrosses into C3H)
Inheritance Testing
Data into Mutabase
Detailed analysis of selected mutants
New screens collaborators
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
Eumorphia Phenotype screens for mice
Developing an integrated platform
Mouse Models for Human Disease
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 protocolMuscle/Lower Motor Neurone
Body positionGaitPositional passivityWire manouvereRighting reflexMotor performanceSpontaneous activityTail elevationVisual placingLimb tonePassivityBalanceLocomotor ActivityLimb positionBody toneAbdominal toneUrination andDefecation
Spinocerebellar
Body positionGaitRighting reflexMotor performanceTail elevationVisual placingLimb toneBalanceLocomotor ActivityLimb positionBody toneAbdominal toneGrip strength
Sensory
Transfer arousalTouch escapeCorneal reflexAnalgesiaGaitVisual placingToe pinchLimb positionPinna reflexRighting reflex
Neuro-psychiatricBody positionTransfer arousalStartle responseBody toneFearAnxietyLearning/memorySpontaneous activityLocomotor ActivityTouch escapeRighting reflexIrritabilityVocalisationBizarre behaviourFood/water intakePositional passivityCatalepsyAggression, PPI
Autonomic
Palprebral closureTail elevationTemperatureHeart RatePilorectionSkin colourFood/water intakeUrinationStartle responseSalivationRespiration rateDefecation
Mouse Models for Human Disease
Harwell ENU programme
• 35,000 mice weaned, scored for visible phenotype
• 15,000 mice SHIRPA, 10,500 mice LMA, 10,500 PPI, 7,000
vision screens, 2,000 clinical chemistry…
• 1,500 abnormal phenotypes
• 376 inheritance tested
• 196 inherited mutations
• > 700 new mutations generated
• 100 mapped, 40 cloned
Focus on:
Neurological
Behavioural
Circadian
Deafness
Vision
Diabetes
Kidney stones
Alcohol preference
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
Tail4
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
Batface
Nanomouse
Mouse Models for Human Disease
ENU mutagenesis - Harwell
GENA Chr• 25 tornado, Todo 4• 29 kumba, Ku 14• 37 small 9• 38 sickly, Sic 7• 40 goth, Goth 2• 41 orbitor, Obt 4• 42 whitetoes and belly spot, Whto 7• 47 dizzy, Dz 4• 51 trembler-1H 11• 52 cyclone, Cyn 4• 53 small/imprinting 9• 57 trembler-2H 11• 60 eddy, Edy 4• 65 van gogh, Vng 5• 70 blind drunk, Bdr 2• 71 white feet and belly spot 1• 77 dark footpads 2, Dfp2 4• 78 saggy, Sagg 1• 104 jeff, Jf 17• 110 short head 1• 123 batface 17• 137 ferris, Ferr 4• 158 slalom, Slm 2• 171 metis 4• 175 spin cycle, Scy 15• 180 robotic, Rob 5
100 mutations mapped - 60% novel GENA Chr• 181 pardon, Pdo 19• 191 iris-corneal strands, Icst 2• 196 leda 4• 203 spag, Spag 15• 208 high startle 3• 232 lens-corneal adhesion 2, Leca2 2• 238 lens-corneal adhesion 1, Leca1 2• 239 waved5, Wa5 11• 241 low cholesterol 4• 243 low cholesterol 4• 246 retinal white spots, Rwhs 11• 251 late-onset deafness, Junb 3• 257 retinal arterial wiring, Raw 8• 263 high glucose 6• 265 lens-corneal adhesion 3, Leca3 2• 269 dilated pupils, Dilp1 5• 291 small vacuolar cataract, Svc 8• 295 Anne Boleyn, Anb 13• 300 Tommy, late-onset deafness 6• 303 short tail 17• 328 low ALP 4• 333 retinal vascular mass, Rvm 14• 336 retinal orange patches, Rorp 6• 348 high glucose 11• 368 lens-corneal adhesion 4, Leca4 2• 379 dilated pupils, Dilp2 X• 380 optic disc coloboma, Opdc 19• 389 high glucose 11• 396 high glucose 10
Novel phenotypesAlleles of existing phenotypes
Mouse Models for Human Disease
ENU mutagenesis - Mutants ClonedLine # Chr• GENA6 bare patches, Bpa X Nsdhl• GENA29 kumba, Ku 14 Zic2• GENA37 small 9 Rasgfr1• GENA51 trembler-1H 11 Pmp22 • GENA53 small 9 Rasgfr1• GENA57 trembler-2H 11 Pmp22 • GENA104 jeff, Jf 17 to be reported• GENA123 batface, Bfc 9 to be reported• GENA158 slalom, Slm 2 Jag1 • GENA175 spin cycle, Scy 15 Celsr1• GENA180 robotic, Rob 5 Af4• GENA181 pardon, Pdo 19 Emx2 • GENA232 lens corneal adhesion 2 2 Pax6• GENA238 lens corneal adhesion 1 2 Pax6• GENA241 low cholesterol 4 Abca1• GENA243 low cholesterol 4 Abca1• GENA251 junbo, Junb 3 Evi1• GENA269 dilated pupils 1, Dilp1 5 Phox2b• GENA300 tommy, Tmy 6 Atp2b2• GENA328 low ALP 4 Akp2 • GENA348 type II diabetes 11 Gck• GENA368 lens corneal adhesion 4 2 Pax6• GENA370 trembler-3H 11 Pmp22• GENA371 no tail 17 T• GENA387 type II diabetes 11 Gck• GENA389 type II diabetes 11 Gck• GENA391 low cholesterol 4 Abca1• GENA396 type II diabetes 11 Gck• GENA450 lens cloudy 10 Mip• TAS1 belly spot 1 Pax3• TAS3 short tail 17 T• TAS9 crooked tail, Ka X to be reported
Mouse Models for Human Disease
ENU mutagenesis - Mutants ClonedLine # Chr• GENA6 bare patches, Bpa X Nsdhl• GENA29 kumba, Ku 14 Zic2• GENA37 small 9 Rasgfr1• GENA51 trembler-1H 11 Pmp22 • GENA53 small 9 Rasgfr1• GENA57 trembler-2H 11 Pmp22 • GENA104 jeff, Jf 17 to be reported• GENA123 batface, Bfc 9 to be reported• GENA158 slalom, Slm 2 Jag1 • GENA175 spin cycle, Scy 15 Celsr1• GENA180 robotic, Rob 5 Af4• GENA181 pardon, Pdo 19 Emx2 • GENA232 lens corneal adhesion 2 2 Pax6• GENA238 lens corneal adhesion 1 2 Pax6• GENA241 low cholesterol 4 Abca1• GENA243 low cholesterol 4 Abca1• GENA251 junbo, Junb 3 Evi1• GENA269 dilated pupils 1, Dilp1 5 Phox2b• GENA300 tommy, Tmy 6 Atp2b2• GENA328 low ALP 4 Akp2 • GENA348 type II diabetes 11 Gck• GENA368 lens corneal adhesion 4 2 Pax6• GENA370 trembler-3H 11 Pmp22• GENA371 no tail 17 T• GENA387 type II diabetes 11 Gck• GENA389 type II diabetes 11 Gck• GENA391 low cholesterol 4 Abca1• GENA396 type II diabetes 11 Gck• GENA450 lens cloudy 10 Mip• TAS1 belly spot 1 Pax3• TAS3 short tail 17 T• TAS9 crooked tail, Ka X to be reported
Mouse Models for Human Disease
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
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
DEPTH
BREADTH
BREADTH
BREADTH
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
+/+
Rob/+
Models of neurological diseasePronounced 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
• 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
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Models of alcoholismMutation in the GABAA 1 receptor gene leads to alcohol preference
Collaboration with Howard Thomas, St. Mary’s, Imperial College
• 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 1 receptor gene
• Mutation causes spontaneous channel
opening and reduced responses to GABA
• New gene involved in alcohol preference
Mouse Models for Human Disease
QTL mapping - IPGTT trait C57BL/6J x C3H F2 (C57BL/6J glucose intolerant)3 QTLs mappedChromosome 13 candidate gene Nnt (5 exon deletion)Toye et al. Diabetologia (2005) 48:675-686
Functional validation of NntsiRNA knockdown in Min6 insulin secreting cell lineTwo ENU alleles identified from ENU DNA archive
Models of type II diabetesNicotinamide Nucleotide Transhydrogenase (Nnt): a key role in insulin secretion
N68K
Wildtype, circles; heterozygotes, squares; homozygotes, triangles.
Collaboration with Fran Ashcroft, Physiology, Oxford
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Mouse Models for Human Disease
Curtin et al. Current Biology, 2003
Models of neural tube developmentSpin 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
Mouse Models for Human Disease
Models of kidney diseaseENU 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
100m
100m
Collecting ducts
Renal cortex
von Kossa reaction for detection of calcification
Collaboration with Raj Thakker, Oxford
Mouse Models for Human Disease
Models of Otitis MediaMutations Jeff and Junbo provide first genetic models of otitis media
• 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
• Jeff - novel gene; suggestive association in association studies in human population
MEC
MEC
TB
Jbo/+ +/+
MEC
Jbo/+ Jbo/+
13DAB
180DAB
Mouse Models for Human Disease
Models of neurodegenerative diseaseENU-induced dynein mutation (Loa) modifies Huntington’s disease
HD/+; Loa/+HD/+; +/+ HD/+; Loa/+HD/+; +/+ HD/+; Loa/+HD/+; +/+
Age (weeks)
2220181614121086
Cum Survival
1.2
1.0
.8
.6
.4
.2
0.0
GENOTYPE
HD/+; +/+
HD/+; Loa/+
Motor perfomance (Rotarod)
0
50
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150
200
5 7 9 11 13 15 17
Age (weeks)
latency to fall (s)
HD/+; Loa/+
HD/+; +/+
+/+; Loa/+
Dynein mutation decreased survival andaccelerated the appearance of motor dysfunction and inclusion formation in HD mice
Ravikumar & Acevedo et al. Nature Genetics, 2005Collaboration with Dept. Medical Genetics, Cambridge
• Huntington’s onset and progression are
enhanced by the Loa mutation and
autophagosome-lysosome fusion is
impaired
• 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
Mouse Models for Human Disease
xG0
G1
HD Tg
Dominant Modifiers
ENU MutagenesisModifier Screens - Dominant Enhancers
Loa HD TgHD TgLoa
Loa
Mouse Models for Human Disease
* x
*
G0
G1
HD Tg
New dominant mutations*
*
Dominant Modifiers New Alleles
Progeny test
ENU MutagenesisModifier Screens - Dominant Enhancers
HD Tg HD TgHD Tg
Mouse Models for Human Disease
XBALB/c C3H
ENU
Sperm archiveDNA archive
Mutant identification
Recovery and examination of mutants
New archive: 5,000
dHPLC, TGCE, Cel1
Coghill et al.Nature Genetics2002
ENU gene-driven screensCreation of parallel archives of DNA and sperm
IVF
Mouse Models for Human Disease
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Number of DNAs screened
Probability of n or more alleles
4 or more alleles
3 or more alleles
2 or more alleles
1 or more alleles
Probabilities of finding n or more mutant alleles in varying numbers of DNAs from offspring of ENU
mutagenised male mice
Assumptions1. Mutation rate of 1/1000 per locus2. Mutation detection rate of 90%
Mouse Models for Human Disease
Basepairs screened Number of individuals Total Mbp Mutations Amino acid Rederived by
exon screened screened screened found change IVF
1 Retn* 344 (3/3) 2230 0.77 1 0
2 Kir 6.2 1172 (1/1) 5302 6.21 3 1 + stop 1
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 1
6 Capn10 1305 (8/12) 4224 5.51 6 3
7 Tectb* 582 (4/10) 2230 1.3 1 0
8 Cx26* 681 (1/1) 2230 1.52 3 3 2
9 Foxf2 1340 (2/2) 2230 2.98 2 1 1
10 Sfrp5 277 (1/3) 1920 0.53 1 Stop 1
11 Sfrp2 502 (1/3) 3072 1.54 2 2 2
12 Sfrp1 421 (1/3) 3072 1.29 1 1 1
13 Mro 658 (4/7) 5072 3.34 8 4 + 2splice site 5
14 Foxf2 294 (3/16) 3072 0.9 3 0
15 Rgs2 635 (4/5) 3072 1.95 2 2 1
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 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
Gene
Coghill et al. Nature Genetics 2002Quwalid et al. Mammalian Genome 2004
Predicted: 1 functional change every 2.38MbObserved: 1 potential functional change every 1.82Mb
Mouse Models for Human Disease
Mutagenesis in the MousePhenotype-driven and gene-driven approaches
• Ethylnitrosourea, ENU -
unbiased chemical mutagenesisPhenotypeDriven
GeneDriven
• Gene traps• Gene targeting• Gene driven ENU• RNAi
EUCOMM, EuropeEuropean Conditional Mouse Mutagenesis
KOMP, USKnock-out Mouse Project
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