Transcript Slide 1

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
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
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 1:14,0001:20,000 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
LGMD1B
LGMD1C
LGMD1D
LGMD1E
LGMD1F
LGMD1G
5q31.2
1q21
3p25.3
6q22
7q35
7q31.1
4p21
myotilin (Hauser, 2000)
lamin A/C (Bonne, 1999)
caveolin 3 (Minetti, 1997)
?
?
?
?
Autosomal recessive
LGMD2A
LGMD2B
LGMD2C
LGMD2D
LGMD2E
LGMD2F
LGMD2G
LGMD2H
LGMD2I
LGMD2J
LGMD2K
LGMD2L
LGMD2M
LGMD2N
LGMD2O
15q15
2p13.2
13q12
17q21.33
4q12
5q33
17q12
9q33.1
19q13.3
2q24.3
9q34.1
9q31
1p34.1
14q24
11p13-p12
calpain 3 (Richard, 1995)
dysferlin (Bashir, Liu, 1998)
g-sarcoglycan (Noguchi, 1995)
a-sarcoglycan (Roberds, 1994)
b-sarcoglycan (Bonnemann, Lim, 1995)
d-sarcoglycan (Nigro, 1996)
telethonin (Moreira, 2000)
TRIM 32 (Frosk, 2002)
FKRP (Brockington, 2001)
titin (Udd, 2002)
POMT1 (Balci, 2005)
fukutin (Godfrey, 2006)
POMGnT1 (Clement, 2008)
POMT2 (Biancheri, 2007)
? (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
e-sarcoglycan
387
413
50
52
muscle
ubiquit.
10
12
17q12
7q21
C
g-sarcoglycan
d-sarcoglycan
z-sarcoglycan
291
290
299
35
35
36
muscle
muscle
brain
8
9
9
13q12
5q33
8p22
N
b-sarcoglycan
318
43
muscle
6
4q12
C
alternative sarcoglycan complexes
a b g
d
e
e
b g
d
b z
d
K.O. of the e-sarcoglycan gene
We deleted exons 6-9 of e-sarcoglycan encoding Cysrich and transmembrane domains
Hind III
Hind III Eco RV
Spe I
1.3 kb
9.5 kb
b gal
intron 5 e-SG
NEO
Hind III
Eco RV
Hind III
Hind III
Hind III Eco RV
intron 5 e-SG
b gal
NEO
Spe I
intron 9 e-SG
exx. 7- 9
ex. 6
vector
target locus
Spe I
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
180
days
Fibrosis and calcifications
muscular tissues
7-12
months
Heart failure
14
months
Death
in
heart
and
wt
BIO14.6
rescue