Dysferlinopathies

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Dysferlinopathies
Myology Summer school June 2004
Guillaume Bassez, MD

• Nerve Muscle Group
• Créteil, France

Myology Institute, Salpêtrière, Paris

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Definition-nosology

• Molecular-based nosology
• Mutations in the dysferlin gene (DYSF) cause
  muscular dystrophies called dysferlinopathy
• which include 2 main clinical entities
• limb-girdle muscular dystrophy type 2B (LGMD2B)
  and
• Miyoshi myopathy (MM).

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Dysferlinopathies History

• Distal myopathy Miyoshi (1967, 1986)
• Locus 2p13.3
• DYSF gene mutation (Bashir et al Liu et al,
  1998)
• Type 2B limb girdle myopathy
• Firstly described in Palestinian families
  (Mahjneh et al, 1992)
• Chromosome 2p linked (Bashir et al, 1994)
• Both MM and LGMD phenotype in the same family
• (Illiaroshkin et al Weiler et al, 1996)

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Dysferlinopathies Epidemiology

• Geographical distribution
• MM identified in Japan
• LGMD (Palestinian, Lybian Jews)
• Prevalence
• Dysferlin mutation 1/3000 Lybian Jews (Argov et
  al, 2000)
• Most frequent distal myopathy (except
  Scandinavia)
• LGMD2B second cause of LGMD (Tagawa et al)
• Dysferlinopathies about 25 of unindentified
  muscular dystrophy

Various countries
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Dysferlinopathies Common traits

• AR inheritance
• Normal developmental milestones, sport possible
  prior to first symptoms
• Onset between 15 35 y (young adults)
• LL distal, proximo-distal, or proximal wk calf
  involvment
• UL biceps atrophy, moderate scapular involvment
• Facial, bulbar muscles spared
• Normal cardiac and respiratory function
• CK (10 to 123 N)
• Unspecific myopathic pattern, necrosis, no
  vacuoles
• Various severity

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Clinical Phenotypes

• Distal myopathy
• Posterior leg compartment (Miyoshi myop. MM)
• Anterior leg compartment
• Proximal myopathy  limb girdle  (LGMD2B)
• HyperCKaemia
• Polymyositis-like
• Exercise intolerance

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Case 1 30 y old man

• Consanguinous family
• No early symptoms
• Age at onset 19 y (running)
• Slow LL atrophy and weakness
• Climbing stairs difficulty at the age of 27.
• No UL symptoms
• Gastrocnemius MRC 2, other 3/4
• CK 5790 U (30 N)

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Case 1 CT scan
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Case 1 Muscle histology - Genetics

• Lower limb muscle biopsy
• Atrophy hypetrophy
• Necrosis
• Incresed collagen tissu
• Miyoshi myop.
• 2p linked
• Dysferlin gene mutation
• No muscle dysferlin expression

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• 1. Selective muscle involvment

MRC
Eymard, et al
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Miyoshi type myopathy French experience

• Partial brachial biceps atrophy

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Miyoshi myopathy follow-up study

• 17 MM patients
• Mean disease duration 11.8 y
• Mean Walton score 4.3

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• Follow-up study

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Walton
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0
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Age
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2p unlinked Miyoshi type myopathy phenotype

• 40 y old
• Onset distal LL
• Posterior leg involvment
• Further proximal wk
• Normal UL
• CK 2493 U

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Miyoshi type muscular dystrophy non 2p13-14 -
linked
Linssen et al, Neuromusc Disord (1998) 2/4
families showed linkage with a chromosome 10
marker
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Dysferlinopathies Clinical aspects

• Distal myopathy
• Posterior leg (Miyoshi myopathy)
• Anterior leg compartment
• Proximal myopathy  limb girdle  (LGMD2B)
• HyperCKaemia
• Polymyositis-like
• Exercise intolerance

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Dysferlinopathies Distal anterior compartment
leg myopathy

• Described in a spanish family (Illa et al, 1998)
• Early onset distal myopathy
• Clinical onset 14 to 28 y
• Anterior gt posterior leg wk
• Severe phenotype (wheelchair within 10 y)

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Dysferlinopathies Clinical aspects

• Distal myopathy
• Posterior leg (Miyoshi myopathy)
• Anterior leg compartment
• Proximal myopathy  limb girdle  (LGMD2B)
• HyperCKaemia
• Polymyositis-like
• Exercise intolerance

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Dysferlinopathy LGMD2B

• Early proximal
• No scapular winging (compared to calpain
  deficiency)
• Knee flexor gt extensor involvment
• Simultaneous proximal distal wk possible

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Patient L, born 1979

• Origine marocaine
• No neuromuscular disorder in family
• No consanguinity
• 1995 16 y, difficulty for jumping
• Rapid worsening LL gt axe gt UL
• 1996 calf wasting
• 1997 tigh weakness
• 1998 upper limb weakness
• 2000 2 cannes, walking distance 10 m

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• investigations
• CK 8795 UI,
• SR 14 mm
• No auto-antibody (anti-nuclear, anti-DNA)
• EMG myopathic
• 2 muscle biopsies polymyositis
• Therapy
• Corticostéroïde transient amélioration
• azathioprine
• cyclophosphamide
• still worsening

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Clinical data (23 y)

• UL
• abduction lt90
• scapular weakness
• No scapula alata
• biceps en boule
• no facial, pharyngeal involvement

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• LL
• Diffuse and prononced weakness and wasting
• axial weakness

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Mr L
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Genetic screening for mutation
N. Levy
SSCP
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Phenotype variability proximal or distal
weakness
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Dysferlinopathies Clinical aspects

• Distal myopathy
• Posterior leg (Miyoshi myopathy)
• Anterior leg compartment
• Proximal myopathy  limb girdle  (LGMD2B)
• Hyper-CK-aemia
• Polymyositis-like
• Exercise intolerance

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High serum level of creatine kinase

•  Asymptomatic prelude to distal myopathy 
• Galassi et al, Muscle Nerve (1987)
• 3 patients (2 unrelated families)
• Idiopathic hyper-CK-aemia
• Distal myop symptoms within 5 to 9 y
• Asymptomatic myopathy

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Dysferlinopathies Clinical aspects

• Distal myopathy
• Posterior leg (Miyoshi myopathy)
• Anterior leg compartment
• Proximal myopathy  limb girdle  (LGMD2B)
• HyperCKemia
• Polymyositis-like
• Exercise intolerance

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Dysferlinopathy Muscular dystrophy with
inflammation

• Polymyositis as initial diagnosis 8 cases (in
  Salpêtrière exp.)
• Corticosteroids and immunosuppressive therapies
  always ineffective
• Dysf gene mutation identified (Am J Med Genet,
  2000)
• Immunohistochemical analysis different pattern
  from idiopathic inflammatory myopathies
  (PM/IBM/DM)
• no diffuse MHC I labelling, CD3 infiltrates
  close to necrotic f

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Dysferlinopathies Clinical aspects

• Distal myopathy
• Posterior leg (Miyoshi myopathy)
• Anterior leg compartment
• Proximal myopathy  limb girdle  (LGMD2B)
• HyperCKaemia
• Polymyositis-like
• Exercise intolerance

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Exercise intolerance

• 41 y woman
• 10 years duration of lower limb pain after
  exercise
• No clinical atrophy/weakness
• High permanent CK elevation
• No significant CT-scan hypodensities
• Normal extensive metabolic investigations

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Dysferlinopathy temporal clinical variability
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Dysferlinopathies Histology
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Dysferlinopathies Diagnosis
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1. Immunohistochemistry
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2. Western Blot analysis
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Anderson et al, 2000
dysf
calp
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Dysferlin expression in muscular dystrophies

• Piccolo et al, Ann Neurol (2000)
• Reduced sarcolemmal expression
• Accumulation in the cytoplasm of fast fibers
• in 20 dystrophic muscle from DMD, BMD, 50 from
  LGMD2C-F

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• Immunostaining patterns classified to 4 groups.
• Normal shows clear membrane staining similar to
  normal control muscles (a),
• Negative is defect of the immunoreaction (b),
• Faint reveals apparently weak immunoreaction at
  the plasma membrane compared to the control
  muscles (c),
• Abnormal shows cytoplasmic accumulation of
  immunopositive materials or positive/negative
  mosaic staining patterns (d).
• From K. Tagawa et al. / Journal of the
  Neurological Sciences 211 (2003) 2328

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Dysferlinopathies frequencyFanin, et al
Neurology 2001

• Combined Dysferlin Calpain-3 WB analysis
• Total 407 muscle biopsies
• Dysferlin deficiency 6.5
• Absent 35
• Severely reduced 65
• LGMD phenotype 1
• Distal myopathy phenotype 60
• Incidence of secondary dysferlin, calpain-3
  deficiency ?

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DYSF gene

• Encompasses 55 exons
• Spanning over 150 kb of genomic DNA
• Large variety of mutations
• No mutational hot-spots
• No common mutation (except in inbred population)
• DNA-based molecular diagnosis not common
• Protein analysis approach needed

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Dysferlin protein

• 230 kD protein product
• Not homologous to any known mammalian gene
• Homology with vesicule specific fertilization
  factor fer-1 of C. elegans
• Early developmental expression (5 wk)
• Novel membrane protein of unknown function
• Interacting with caveolin 3

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Dysferlin is located in the muscle plasma
membrane
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Cellular localization ultrastructure
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Animal model SJL-Dysf mouse
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Mutations identifiées dans le gène DYSF
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Analyse des mutations dans la population
françaiseN. Levy
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Dysferlinopathies phenotype in French population

• 55 patients
• inclusion criteria no or faint immunoréactivity
  (traces)

Karine NGuyen, N. Levy
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various geographic origin
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Age of onset young adult
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dysferline deficiency phénotype ?
3 principal phénotypic groups
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Follow-up, prognosis
Walton Score
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misdiagnosis
polymyositis 27.7
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Dysferlin Recent findings
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Dysferlin analyses in a large group of Japanese
muscular dystrophy patientsTagawa et al, Journal
of the Neurological Sciences July 2003

• A total of 107 unrelated Japanese patients
  biopsies, including
• 53 unclassified LGMD,
• 28 MM
• 26 other neuromuscular disorders (ONMD)
• deficiency of dysferlin protein (by both IHC and
  MMW) in
• 75 of MM patients
• 19 of LGMD
• 19 of dysferlin-deficient patients had 3370G-T
  missense mutation
• patients with homozygous 3370G-T mutation showed
  milder clinical phenotypes.

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Genotype-phenotype correlation
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Genotype-phenotype correlation (2)Miyoshi
myopathy Relationship to phenotypeT. Takahashi,
Neurology, June 2003

• 25 Japanese patients with MM.
• 16 different mutations in 20 patients with MM
• 10 were novel
• 4 mutations (C1939G, G3370T, 3746delG, and
  4870delT) are relatively more prevalent in this
  population, accounting for 60 of the mutations
  in this study.
• G3370T mutation was associated with milder forms
  of MM and the G3510A mutation was associated with
  a more severe form.

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Mutations in Japanese patients are distributed
along the entire length of the gene.T.
Takahashi, Neurology, June 2003
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Dysferlinopathies pathophysiologyDavis DB, et
al. J Biol Chem 2002 June

• Muscle fiber injury mechanism ?
• Contains six C2 domains
• Found in a variety of membrane-associated
  proteins
• Implicated in calcium, phospholipid, and
  protein-binding
• Mutated dysferlin C2A domain reduced
  calcium-sensitive phospholipid binding
• Abnormal calcium-regulated phospholipid binding
  may lead to defective maintenance and repair of
  muscle membranes

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Bensal et al, Nature 2003
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Defective membrane repair in dysferlin-deficient
muscular dystrophyBensal et al, Nature 8 MAY
2003

• sub-sarcolemmal accumulations of vesicles in
  dysferlin-null muscle.
• dysferlin-deficient muscle fibres are defective
  in Ca2-dependent sarcolemma resealing.
• disruption of the muscle membrane repair
  machinery is responsible for dysferlin-deficient
  muscle degeneration,
• important new basic cellular mechanism of
  membrane resealing in human disease.

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Sarcolemme repair mechanism
N.J. Lennon et al,
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Conclusions

• Various clinical phenotypes (in addition to
  typical Miyoshi myopathy)
• Dysferlin sarcolemmal distribution, but not an
  integral component of DGC
• Role in maintenance of muscle membranes by
  phospholipid binding ?
• Transmembrane proteine involved in membrane
  fusion ?
• Novel pathogenic mechanism muscle membrane
  repair defect

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Acknoledgements
Bruno Eymard
Karine NGuyen Nicolas Levy

• Institut de Myologie Salpêtrière, Paris

La Timone, Marseille

France Leturcq
Cochin, Paris