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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 100m Collecting ducts 100m 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