Mouse Models for Human Disease Challenges Develop a series of mutant alleles for every gene in the mouse genome Develop and apply standardised phenotyping

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







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


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


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


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


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

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ENU

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G0

G1

DominantGenome Wide

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ENU

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G0

G1

G2 G1

G3

EMS

RecessiveGenome Wide

Inv

lethal test carrier

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x

ENU

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G0

G1

G2

G3

Targeted Recessive - Inversions

Del

test carrier

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ENU

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uninformative

G0

G1

G2

Targeted Recessive - Deletions

Brown & Balling 2001Curr. Op. Genet. Develop. 11: 268

Brown & Hardisty 2003Seminars Cell Dev. Biol. 14: 19

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ENU

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G0

G1

DominantGenome Wide

• G1 dominant genome-wide screens

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ENU

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G0

G1

G2 G1

G3

EMS


• G3 recessive genome-wide screens

Inv

lethal test carrier

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x

ENU

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G0

G1

G2

G3

Targeted Recessive - Inversions


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ENU

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ENU

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G0

G1

G0

G1

G2 G1

G3

EMS

DominantGenome Wide


Del

test carrier

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ENU

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uninformative

G0

G1

G2

Targeted Recessive - Deletions



ENU MutagenesisModifier Screens

X

F1 progeny

SCREENS

ENU

[carrying mutations in pathway of interest]

Screens insensitisedpathways


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ENU

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G0

G1

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New dominant mutations*

r* r

Dominant Modifiers New Alleles

Progeny test

ENU MutagenesisModifier Screens - Dominant Enhancers


X

F1 progeny

ENU MutagenesisGenome-wide screen for dominant mutations

SCREENS

ENU


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






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


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


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


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


Detection of visible mutations

Dominant spotting

Microphthalmia

Batface

Nanomouse


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


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


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


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







+/+

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


QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

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


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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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


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


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


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

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GENOTYPE

HD/+; +/+

HD/+; Loa/+

Motor perfomance (Rotarod)

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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


xG0

G1

HD Tg

Dominant Modifiers


Loa HD TgHD TgLoa

Loa


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G0

G1

HD Tg

New dominant mutations*

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Dominant Modifiers New Alleles

Progeny test


HD Tg HD TgHD Tg


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


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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%


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


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






Documents

Mouse Models for Human Disease Challenges Develop a series of mutant alleles for every gene in the mouse genome Develop and apply standardised phenotyping