Animal models for muscular dystrophy

Vincenzo Nigro
Laboratorio di genetica - Dipartimento di Patologia Generale, Seconda
Università degli Studi di Napoli
Telethon 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
 dystrophin mutations

• DMD Duchenne Muscular Dystrophy - 1/3,500 boys

Onset -- Early childhood - about 2 to 6 years
Symptoms -- Generalized weakness and muscle wasting
affecting limb and trunk muscles first. Calves often enlarged
Progression -- Disease progresses slowly but will affect all
voluntary muscles. Survival possible beyond late twenties

• BMD Becker Muscular Dystrophy - 1/10,000 boys

Onset -- Adolescence or adulthood
Symptoms -- Almost identical to Duchenne but often much
less severe. Can be significant heart involvement
Progression -- Slower and more variable than Duchenne
with survival well into mid to late adulthood
 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 > treatment > therapy > cure

Animal models disease should be comparable to human defects

Profoundly studied in all pathological characteristics
Should allow a reliable prediction of the response
 The genetic basis of the disease should be
the same as human disease
 Reiterate key hallmarks of the human
disease
 Animals commercially available, easy to
maintain
 Animal disease well characterized, with
abundant literature
 Robust phenotype that is reproducible over
generations
 mdx (X-chr MD) mouse
C57BL/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
but
• 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

mdx mouse is a bad DMD mouse model

 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 = retinal
Dp140 = central nervous system
and kidney
Dp116 = Schwann cells
Dp71 = 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
 hDMD mouse mRNA
RT-PCR analyses using either mouse- or human-specific
primers show correct transcription of the human DMD
gene in muscle tissue
 hDMD mouse IF

expression of human dystrophin in skeletal muscle detected

by IF using the human-specific Ab MANDYS106
Dys2 reacts with both human and mouse dystrophin
 Online Mendelian Inheritance in Animals (OMIA)
is a database of genes, inherited disorders and traits in more than 135
animal species (other than human and mouse, which have their own
resources). The database contains textual information and references, as
well as links to relevant PubMed and Gene records at the NCBI

Dog Cow Cat Pig Horse Sheep Chicken Goat Rabbit Other TOTAL

Total Phenes 489 376 280 215 193 186 179 70 49 456 2581

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

Phenes ch. at the molecular

68 43 24 13 15 17 22 7 3 28 246
level
Potential models for human
224 126 136 70 97 68 38 25 29 172 1029
disease
Potential model in other
280 229 215 149 135 131 72 59 39 385 1757
animals
 Simple search for: "muscular dystrophy"

10 records found
OMIA 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 dominant
LGMD1A 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 recessive
LGMD2A 15q15 calpain 3 (Richard, 1995)
LGMD2B 2p13.2 dysferlin (Bashir, Liu, 1998)
LGMD2C 13q12 g-sarcoglycan (Noguchi, 1995)
LGMD2D 17q21.33 a-sarcoglycan (Roberds, 1994)
LGMD2E 4q12 b-sarcoglycan (Bonnemann, Lim, 1995)
LGMD2F 5q33 d-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
N
protein aa MW expression ex. chrom
a-sarcoglycan 387 50 muscle 10 17q12
e-sarcoglycan 413 52 ubiquit. 12 7q21
C

g-sarcoglycan 291 35 muscle 8 13q12

C
d-sarcoglycan 290 35 muscle 9 5q33
z-sarcoglycan 299 36 brain 9 8p22

N b-sarcoglycan 318 43 muscle 6 4q12

alternative sarcoglycan complexes

a b g d

e b z d

e b g d
 K.O. of the e-sarcoglycan gene

We deleted exons 6-9 of e-sarcoglycan encoding Cys-

rich and transmembrane domains

Hind III Eco RV Hind III Spe I

1.3 kb 9.5 kb vector

b gal NEO

Hind III Eco RV Hind III Spe I

intron 5 e-SG intron 9 e-SG

target locus
ex. 6 exx. 7- 9

Hind III Eco RV Hind III Spe I

intron 5 e-SG b gal NEO intron 9 e-SG

K.O.
 Mice heterozygous for the deletion in the
e-sarcoglycan gene (+/-) are smaller at birth
and show tremors in the posterior legs

(+/-)

(+/+)
 cardiomyopathy hamsters (886 items)

• 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
 Analysis of d-sarcoglycan gene in the
cardiomyopathic hamster

• strong reduction (>30 times) of

d-sarcoglycan mRNA
• identification of the true first exon
(24kb upstream) which is deleted
 d-sarcoglycan deletion is identical
in all cardiomyopathic hamster
strains all over the world
 PATHOLOGIC STAGES IN BIO 14.6
HAMSTERS LIFE
Stages of the disease Clinical Effects

20-30 days Tongue calcifications

30-40 days Cardiac lesions
40-50 days Muscular necrotic lesions
100 days Myocardial hypertrophy
150 days Strong myocardial hypertrophy

Fibrosis and calcifications in heart and

180 days
muscular tissues

7-12 months Heart failure

14 months Death
wt BIO14.6 rescue