Animal models for muscular dystrophy

Vincenzo NigroLaboratorio di genetica - Dipartimento di Patologia Generale, Seconda

Università degli Studi di NapoliTelethon Institute of Genetics and Medicine, Napoli

muscular dystrophy

• MD is a general term that describes a group of inherited and gradually debilitating myogenic disorders

• progressive muscle weakness affecting patients since from young age and can lead to early death

• pattern of inheritance can be X-linked recessive (DMD/BMD), autosomal dominant (LGMD1), or autosomal recessive (LGMD2)

• Some underlying genetic defects are well known, others are orphan diseases

• DMD Duchenne Muscular Dystrophy - 1/3,500 boysDMD Duchenne Muscular Dystrophy - 1/3,500 boysOnset -- Early childhood - about 2 to 6 yearsOnset -- Early childhood - about 2 to 6 yearsSymptoms -- Generalized Symptoms -- Generalized weaknessweakness and muscle wasting and muscle wasting affecting limb and trunk muscles first. Calves often enlargedaffecting limb and trunk muscles first. Calves often enlargedProgression -- Disease progresses slowly but will affect all Progression -- Disease progresses slowly but will affect all voluntary muscles. Survival possible beyond late twentiesvoluntary muscles. Survival possible beyond late twenties

• BMD Becker Muscular Dystrophy - 1/10,000 boysBMD Becker Muscular Dystrophy - 1/10,000 boysOnset -- Adolescence or adulthoodOnset -- Adolescence or adulthoodSymptoms -- Almost identical to Duchenne but often much Symptoms -- Almost identical to Duchenne but often much less severe. Can be significant heart involvementless severe. Can be significant heart involvementProgression -- Slower and more variable than Duchenne Progression -- Slower and more variable than Duchenne with survival well into mid to late adulthoodwith survival well into mid to late adulthood

dystrophin mutations

weakness

Proximal weakness: the most common site of weakness in a myopathic disorder

• Lower extremities– difficulty climbing stairs– arising from a low chair or toilet– getting up from a squatted position

• Upper extremities– trouble lifting objects over the head – brushing the hair

fatigue

• Much less useful “negative” symptom (non-specific)

• Many patients who complain of diffuse global

"weakness" or fatigue do not have a disorder of

muscle

• Abnormal fatigability after exercise:

– metabolic and mitochondrial myopathies

– define the duration and intensity

disease progression

• muscle tissue represent about 40% of the total body mass

• respiratory failure can be the cause of premature death as well as heart failure

• patients suffer from asymmetries in strength between reciprocal muscles that cause widespread joint and spine deformities requiring timely orthopaedic surgery

Distribution of muscle involvement

• CK (50x to 1.000x)

• LDH5, ALT, AST, aldolase

increase

• Clinical diagnosis of LGMD is

often made when disease has

no apparent X-linked

inheritance

• LGMD are classified as severe

(Duchenne-like) or mild

(Becker-like), depending on the

rate of progression and the age

of wheelchair confinement

Carrier of a balanced reciprocal X-autosome translocation

management management >> treatment treatment >> therapy therapy >> curecure

Animal models disease should be comparable to human Animal models disease should be comparable to human defectsdefects

Profoundly studied in all pathological characteristicsProfoundly studied in all pathological characteristics

Should allow a reliable prediction of the responseShould allow a reliable prediction of the response

The genetic basis of the disease The genetic basis of the disease should be the same as human diseaseshould be the same as human disease

Reiterate key hallmarks of the human Reiterate key hallmarks of the human diseasedisease

Animals commercially available, easy Animals commercially available, easy to maintainto maintain

Animal disease well characterized, Animal disease well characterized, with abundant literaturewith abundant literature

Robust phenotype that is reproducible Robust phenotype that is reproducible over generationsover generations

mdx (X-chr MD) mouseC57BL/10ScSn-Dmdmdx

• mdx is the best characterized mouse model for muscular dystrophy (>1,700 papers) since 1984

• mdx has a spontaneous nonsense mutation (stop) in exon 23 of the dystrophin gene and does not produce dystrophin

• absence of dystrophin reduces the DGC at the sarcolemma

mdx mouse may be DMD mouse?

• mdx shows signs of MD during first 6 weeks of life which results in an increase of the newly differentiated myofibers

• it has muscle regeneration with an expansion of the satellite cell population and muscle hypertrophy

• Centralized nuclei (50-60%), heterogeneity in fiber size

• Necrosis at early stages, but decreases after 60 days

• Plasma creatine kinase is 5.000-12.000 U/L

• the most affected muscle (diaphragm) reproduces the degenerative changes of MD

4 weeks of age, soleus muscle

C57 mdx

butbut

mdx mouse is a bad DMD mouse model

• Fibrosis is only in diaphragm

• Absolute muscle force of limb muscles remains similar to unaffected mice

• Lifespan is shorter but no so much (-19% in males)

• it has muscle regeneration with an expansion of the satellite cell population and muscle hypertrophy

• mdx lacking the muscle-specific transcription factor MyoD or myocyte nuclear factor (expressed in the satellite cells) show more severe MD

Double mouse mutants

utrophin/dystrophin

• Utrophin is a developmentally regulated protein, an autosomal homologue to dystrophin

• utrophin is overexpressed when dystrophin is absent

• the utrn−/−/mdx mice are severely affected • reduced lifespan• severe muscle weakness with joint contractures,

growth retardation, and cardiomyopathy• the phenotype is ameliorated by skeletal-muscle

specific expression of utrophin

Dp260 = retinalDp140 = central nervous system

and kidneyDp116 = Schwann cellsDp71 = high levels, but not in

muscle

mdx52 mouse

• mdx52 is dystrophin KO mouse

• mdx has a deletion in exon 52 of the dystrophin gene and does not produce dystrophin

• in contrast to mdx, this mouse cannot produce also Dp260(ret) and Dp140(CNS), maintaining Dp116 (S) and Dp71

• it is very similar to mdx mouse with the absence of dystrophin that reduces the DGC at the sarcolemma, but has no cardiomyopathy

mdx2cv-5cv mice

• they were generated by chemical mutagenesis using N-ethyl-nitrosurea

• mdx 2cv lacks dys, Dp260

• mdx 3cv lacks dys, Dp260, Dp140, Dp116 and Dp71

• mdx 4cv lacks dys, Dp260 and Dp140

• mdx 5cv lacks only dystrophin

• phenotypes are very similar to mdx mouse and no phenotype worsening

targeted inactivation of Dp71 only

• there is a mouse that cannot produce Dp71 only

• it has normal phenotype

Dystrophin revertant fibres and transcripts in mdx mouse muscle

Fall et al. Genetic Vaccines and Therapy 2006 4:3

the "humanized" hDMD mouse

• “humanised” DMD (hDMD) mice carry an integrated and functional copy of the full-length human DMD gene

• it serves to test the “exon skipping strategy” that is a sequence-specific therapeutic approach

human sequence-specific DMD exon skipping in vivo

• the hDMD mouse model allows the direct testing of human-specific AONs and target sequences in a mouse experimental background

• the induction of specific skipping of the hDMD exons 44, 46, and 49, whilst the endogenous mouse transcripts are not affected [Bremmer-Bout et al., Mol. Ther. 2004]

• this underlines that AONs, based upon specific design, can be highly sequence-specific small molecule drugs.

antisense-induced exon skipping

RT-PCR analyses using either mouse- or human-specific primers show correct transcription of the human DMD

gene in muscle tissue

hDMD mouse mRNA

expression of human dystrophin in skeletal muscle detected by IF using the human-specific Ab MANDYS106

Dys2 reacts with both human and mouse dystrophin

hDMD mouse IF

Dog Cow Cat PigHorse

Sheep

Chicken

GoatRabbit

Other

TOTAL

Total Phenes 489 376 280215

193 186 179 70 49 456 2581

Single-locus phenes 131 75 47 35 29 68 72 10 13 68 573

Phenes ch. at the molecular level

68 43 24 13 15 17 22 7 3 28 246

Potential models for human disease

224 126 136 70 97 68 38 25 29 172 1029

Potential model in other animals

280 229 215 149 135 131 72 59 39 385 1757

Online Mendelian Inheritance in Animals Online Mendelian Inheritance in Animals (OMIA)(OMIA) is a database of genes, inherited disorders and traits in is a database of genes, inherited disorders and traits in more than 135 animal species (other than human and mouse, more than 135 animal species (other than human and mouse, which have their own resources). The database contains textual which have their own resources). The database contains textual information and references, as well as links to relevant PubMed information and references, as well as links to relevant PubMed and Gene records at the NCBIand Gene records at the NCBI

Simple search for: "muscular dystrophy"

10 records foundOMIA 000679 Muscular dystrophy in Gallus gallus (chicken)

Sub-type: Abnormal muscle; AM Genes: WWP1

OMIA 000679 Muscular dystrophy in Canis familiaris (dog) OMIA 000679 Muscular dystrophy in Ovis aries (sheep)

OMIA 000679 Muscular dystrophy in Meleagris gallopavo (turkey) OMIA 000679 Muscular dystrophy in Mustela lutreola (European mink)

OMIA 000679 Muscular dystrophy in Felis catus (cat) OMIA 001081 Muscular dystrophy, Duchenne and Becker types in Felis catus (cat)

Genes: DMD OMIA 001081 Muscular dystrophy, Duchenne and Becker types in Canis familiaris (dog)

Sub-type: X-linked muscular dystrophy OMIA 000681 Muscular dystrophy, dysphagia-associated in Canis familiaris (dog)

OMIA 000828 Progressive muscular dystrophy in Mustela lutreola (European mink European mink)

Golden retriever dog with muscular dystrophy (GRMD)

GRMD arises from a mutation in the acceptor splice site of intron 6 of the dystrophin gene

Skipping of exon 7 disrupts the mRNA reading frame and results in premature termination of translation

Golden retriever dog with muscular dystrophy (GRMD)

complete absence of the dystrophin, early and severe muscle degeneration with reduction of motility and walking ability

Death usually occurs at about 1 year of age as a result of failure of respiratory muscles

dystrophic Golden Retriever dog

• gradual weakness and loss of muscle mass

• development of contractures, skeletal deformities

• significant phenotypic variability among litters

Spitz dogs

• Becker-like dystrophy with a truncated form of dystrophin was recently identified in a family of Japanese Spitz dogs

LGMD forms

• LGMD have a highly variable onset and progression, but the unifying theme is the proximal muscle involvement

• The a. dominant forms (LGMD1) are generally milder and relatively rare representing less than 10% of all LGMD

• The a. recessive forms (LGMD2) are much more common, having a cumulative prevalence of 1:14,000-1:20,000 with some differences among countries, depending on the carrier distribution and the degree of consanguinity

• There are, however, at least 25% of families who can be excluded from any known locus and 40% of typical LGMD cases with no mutation in any known gene

Autosomal dominantLGMD1A 5q31.2 myotilin (Hauser, 2000)LGMD1B 1q21 lamin A/C (Bonne, 1999)LGMD1C 3p25.3 caveolin 3 (Minetti, 1997)LGMD1D 6q22 ?LGMD1E 7q35 ?LGMD1F 7q31.1 ?LGMD1G 4p21 ?

Autosomal recessiveLGMD2A 15q15 calpain 3 (Richard, 1995)LGMD2B 2p13.2 dysferlin (Bashir, Liu, 1998)LGMD2C 13q12 -sarcoglycan (Noguchi, 1995)LGMD2D 17q21.33 -sarcoglycan (Roberds, 1994)LGMD2E 4q12 -sarcoglycan (Bonnemann, Lim, 1995)LGMD2F 5q33 -sarcoglycan (Nigro, 1996)LGMD2G 17q12 telethonin (Moreira, 2000)LGMD2H 9q33.1 TRIM 32 (Frosk, 2002)LGMD2I 19q13.3 FKRP (Brockington, 2001)LGMD2J 2q24.3 titin (Udd, 2002)LGMD2K 9q34.1 POMT1 (Balci, 2005)LGMD2L 9q31 fukutin (Godfrey, 2006)LGMD2M 1p34.1 POMGnT1 (Clement, 2008)LGMD2N 14q24 POMT2 (Biancheri, 2007)LGMD2O 11p13-p12 ? (Jarry, 2007)

Dy/dy dy2J/dy2J

• Two mouse models for laminin-α2 deficiency were identified in the Jackson Laboratories (http://www.jax.org/)

• dy/dy (dystrophia-muscularis) mouse• allelic dy2J/dy2J mouse• Both mice are models for merosin-deficient Congenital

MD (CMD1A)• Neither of these mouse models exhibits a complete

deficiency of laminin α2 chain

alfa-syntrophin -/- mouse

• no defect in muscle

• nNOS and aquaporin-4 are displaced, like in mdx

• aquaporin KO are also normal

• nNOS KO are normal

• nNOS(-/-)/mdx are = mdx

alfa-dystrobrevin -/- mouse

• maintain the expression of DGC at the sarcolemma

• mild muscular dystrophy but not yet in humans

• is affected DGC complex signaling?

• nNOS and aquaporin-4 are displaced, like in mdx

• aquaporin KO are also normal

SARCOGLICANOPATHY

Mutation in any

of the

sarcoglycan

genes

produces a

secondary loss

of the other

components

LGMD2C, 2D, 2E, 2F with sarcoglycan gene mutations

• Mutation in any of the sarcoglycan genes produces a

phenotype very similar to DMD/BMD

• Onset between 7-19 years, variable progression with some

patients that never loss deambulation and other that are

more severely affected, also with identical mutations

• Atrofic muscular dystrophy. Calf hypertrophy. It may involve

distal muscles. Little shoulder girdle involvement. Heart is

seldom involved. CK is very high

sarcoglycan family members

proteinprotein aaaa MWMW expression expression ex. ex. chrom chrom

-sarcoglycan-sarcoglycan 387387 5050 musclemuscle 1010 17q1217q12

-sarcoglycan 413 52 ubiquit. 12 7q21

-sarcoglycan -sarcoglycan 291291 3535 musclemuscle 88 13q1213q12

-sarcoglycan-sarcoglycan 290290 3535 musclemuscle 99 5q335q33

-sarcoglycan 299 36 brain 9 8p22

-sarcoglycan -sarcoglycan 318318 4343 musclemuscle 66 4q124q12

CC

NN

CC

NN

alternative sarcoglycan complexes

NEO

intron 5 -SG

1.3 kb 9.5 kb

Hind III Spe I

gal

ex. 6intron 9 -SG

exx. 7- 9

intron 5 -SG

Hind III Spe I

intron 9 -SG

Hind III Spe I

Hind III

Hind III

Hind III

Eco RV

Eco RV

NEO gal

Eco RV

K.O. of the -sarcoglycan gene

K.O.K.O.

target locustarget locus

vectorvector

We deleted exons 6-9 of -sarcoglycan encoding Cys-rich and transmembrane domains

Mice heterozygous for the deletion in the -sarcoglycan gene (+/-) are smaller at birth

and show tremors in the posterior legs

(+/-)(+/-)

(+/+)(+/+)

• The BIO14.6 hamster was generated in 1962, when Homburger fixed by repeated inbreeding a spontaneous trait of Syrian hamster characterized by muscular dystrophy and cardiomyopathy

• This animal model is one of the most studied models for inherited dilated cardiomyopathy and muscular dystrophy

• Its lifespan is shortened to 10-13 months, because the heart dilation progresses to an ejection fraction below 30-35% that causes heart failure

cardiomyopathy hamsters (886 items)

Analysis of -sarcoglycan gene in the cardiomyopathic hamster

• strong reduction (>30 times) of

-sarcoglycan mRNA

• identification of the true first exon

(24kb upstream) which is deleted

-sarcoglycan deletion is identical

in all cardiomyopathic hamster

strains all over the world

Death14 months

Heart failure7-12 months

Fibrosis and calcifications in heart and muscular tissues

180 days

Strong myocardial hypertrophy150 days

Myocardial hypertrophy100 days

Muscular necrotic lesions40-50 days

Cardiac lesions30-40 days

Tongue calcifications20-30 days

Clinical EffectsStages of the disease

PATHOLOGIC STAGES IN BIO 14.6 HAMSTERS LIFE

BIO14.6 rescuewt