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DYSTROPHINOPATHIES: Duchenne

Early age
  Mild
  Myopathic groups
  Regeneration
  Severe
Later age
Dystrophin
Revertants
Post gene transfer

General features

DMD Muscle pathology: General features

  • Myopathy: Ongoing & Chronic
    • Fiber sizes: Varied
    • Small fibers: Round
    • Hypercontracted (opaque) muscle fbers
      • Sizes: Often large
    • Necrosis & Regeneration of muscle fibers
    • Muscle fiber pathology
    • Chronic changes: Other tissues
  • Molecular
    • Dystrophin staining
      • Screening Antibodies: Target Epitopes
        • N-terminus
          • Dys-3: vs Exons 10 to 12
        • Rod domain
          • 7G1: vs Exon 46 (Within deletion hotspot)
          • 6A9: vs Exon 50 (Within deletion hotspot)
          • Dys-1: vs Exons 26 to 30
        • C-terminus
          • Dys-2: vs Exon 79
          • Less useful
            • Few deletions in this region
            • May be reduced in other myopathies
      • Duchenne MD clinical phenotype: Staining patterns
        • DMD Muscle
          • Dystrophin: Absent staining for all epitopes
          • Utrophin: Normal (Absent on muscle fibers) or Increased staining
          • nNOS: Often absent with Exon 45-51 hotspot deletions
          • Sarcoglycans: Often reduced in Duchenne MD
        • Schwann cells: May stain for dystrophin with mutations proximal to Intron 55
      • Becker MD clinical phenotype: Staining patterns
        • Overall reduced staining for all antibodies
        • Relative reduction in intensity of staining for some dystrophin epitopes
        • Complete loss of some dystrophin epitopes
      • Dystrophin female carriers
        • Non-necrotic, dystrophin-negative muscle fibers
    • Other sarcolemma-related proteins
      • Sarcoglycans: Reduced
      • Aquaporin 4: Reduced
      • Laminin-α2: Normal or Increased
      • Utrophin: Increased
      • β-Spectrin: Normal
    • Calcium 1
      • Increased in myoplasm
      • Pathomechanisms of Ca++ increase in DMD muscle fibers
        • Enhanced Ca++ leak from sarcoplasmic reticulum (SR)
          • Through oxidized Ryanodine receptor type 1 (RyR1) Ca++ release channels
          • Hypernitrosylation of cysteine residues in RyR1
          • FKBP1A dissociation from RyR1
          • Destabilization of RyR1 channel closed state
          • Increased RyR1-dependent SR Ca++ leak
          • Uncontrolled production of reactive species
            • Oxygen (ROS)
            • Nitrogen (RNS)
          • Reduced activity of SR/ER Ca++ ATPase(SERCA) (Ca++pump)
        • Excessive extracellular Ca++ trans-sarcolemmal influx
          • Micro-tears: Delta lesions
          • Abnormal Ca++-permeable ion channels
            • Enhanced store-operated Ca2+ entry (SOCE)
            • Related molecules
            • SOCE trigger: Depletion of intracellular Ca++ stores
 
From: AG Engel
Delta Lesions (Arrow)
Description: Focal wedge-shaped lesions
Locations: Subsarcolemmal regions of muscle fibers

DMD
  Muscle fibers
    Size: Varied
    Shape: Small fibers round or polygonal
    Internal nuclei
    Hypercontracted
    Necrosis
  Endomysial connective tissue: Increased between muscle fibers
  Fat: Increased in endomysium & preimysium

H&E stain

DMD
  Endomysial connective tissue: Increased between muscle fibers

VvG stain


NADH stain
DMD: Immature muscle fibers
  Coarse internal architecture on NADH (Above)
  2C fibers: Intermediate staining on ATPase pH 4.3 (Below)

ATPase pH 4.3 stain

Duchenne Muscular Dystrophy: Early Pathology



H & E stain
Muscle fibers
  Sizes: Varied; Small fibers are rounded or polygonal; Occasional hypertrophic fiber
  Necrosis (Left): Fibers scattered & in small groups
  Myopathic groups (Below): Clusters of small necrotic (Black arrow) & regenerating (White arrow) muscle fibers
  Internal nuclei: Occasional
Endomysial connective tissue: Normal to mildly increased
Perimysium: Early replacement by fat


Acid phosphatase stain
Acid phosphatase + cells
  Endomysium: Small Scattered cells
  Necrotic muscle fibers: Clusters of phagocytic cells
Ring fibers (Arrow)

Acid phosphatase stain
Acid phosphatase + cells
  Necrotic muscle fibers: Clusters of phagocytic cells




DMD: Muscle Fiber Necrosis

Necrotic Fibers: Scattered; Varied stages

H&E stain

Gomori trichrome stain
Necrotic Fibers: Scattered; Replaced by cells

VvG stain

Myopathic Grouping

Muscle fiber Stages
  Necrosis
  Regeneration
    Early
    Late

Myopathic grouping: Necrotic muscle fibers, clustered


H&E stain
Necrosis, Early stage
  Necrotic muscle fibers: Pale stained
  Macrophages: Early invasion of muscle fibers

NADH stain
Necrotic muscle fibers are pale on NADH stain

Myopathic grouping: Clusters of very small regenerating muscle fibers & histiocytic cells


VvG stain
Myopathic groups: Clusters of small muscle fibers in similar, early, stage of regeneration or immaturity

VvG stain
Myopathic groups: Clusters of small muscle fibers in similar, early, stage of regeneration or immaturity

H & E stain

ATPase, pH 4.3

NADH


Clusters of small cells
  Many stain for Acid phosphatase

Acid phosphatase

Acid phosphatase


H & E stain

Myopathic Grouping: Late


H & E stain
Myopathic grouping: Intermediate-sized, Immature muscle fibers in clusters

H & E stain
Immature fibers are small & basophilic
 

ATPase, pH 4.3
Immature fibers are: Small &
  2C (Intermediate staining)

Alkaline phosphatase
Immature small fibers have:
  Cytoplasmic staining


NADH
Immature fibers have coarse cytoplasmic staining

NADH
 


VvG
Immature small fibers have coarse cytoplasmic staining

Immature muscle fibers: Many

Myopathic group, Early DMD Alkaline phosphatase + immature muscle fibers Many 2C muscle fibers
Staining properties of immature muscle fibers includes:
  1. H & E (left): Basophilic fibers
  2. Alkaline phosphatase positive (center)
  3. 2C fibers: Intermediate staining on ATPase pH 4.3 (right)

Duchenne Muscular Dystrophy: Severe at age 2 years


H&E stain
DMD
  Endomysial connective tissue: Increased
  Muscle fibers
    Necrosis & Regeneration
    Size: Varied

H&E stain
 
H&E stain
DMD
  Endomysial connective tissue: Increased
  Muscle fibers
    Necrosis & Regeneration
    Size: Varied


Gomori trichrome stain

Gomori trichrome stain

VvG stain

DMD
  Many intermediate-stained muscle fibers

ATPase pH 4.3 stain

Duchenne Muscular Dystrophy: Later Pathology (10 years)

Endomysial connective tissue
Fat replacement
Fiber types
Internal architecture
Necrotic fibers


H&E stain
DMD: Later pathology
  Fiber size: Varied
  Endomysial connective tissue: Increased
  Fat replacement of muscle: Prominent

H&E stain

VvG stain


Sudan stain
Replacement of Muscle fibers & Perimysial connective tissue by Fat (Dark black)

Sudan stain


H&E stain
Endomysial connective tissue: Increased
Fiber size: Variable
Small fibers: Rounded
Large or hypercontracted muscle fibers: Scattered
Necrotic fibers (Arrow): Scattered

DMD: 5 yrs
H&E stain
DMD: 10 yrs

Endomysial connective tissue
  Increased between fibers

Muscle fiber size: Varied
Small & Large fibers: May be are either type (I = Dark; II = Light)
Many type IIC fibers: Smaller size; Intermediate stain

ATPase pH 4.3 stain

DMD late: Few necrotic muscle fibers

Acid phosphatase stain

Muscle fiber internal architecture: Coarse

NADH stain
DMD: Ring Fibers

H&E stain

Gomori trichrome stain

NADH stain
DMD: Ring fibers

NADH stain

Dystrophin staining

Normal
Normal dystrophin
Duchenne Muscular Dystrophy:
Absent dystrophin Absent dystrophin; 1 revertant
Normal dystrophin staining
around the rim of muscle fibers.
Absent dystrophin: Duchenne muscular dystrophy
Left: No staining around the rim of any muscle fibers
Right: No staining of most muscle fibers
  One "revertant" fiber with dystrophin staining.
  Revertant fibers reflect a somatic mutation allowing dystrophin expression

Normal: Dystrophin present near surface of muscle fibers

Dys1 stain
Duchenne MD: Dystrophin absent from surface of muscle fibers

Dys1 stain

Muscle from Duchenne MD male with large area of revertant muscle fibers

Muscle histology (Above): One fascicle (Arrow) has more normal muscle fibers & connective tissue amid otherwise myopathic muscle
Dystrophin staining (Below): Muscle fibers in preserved fascicle are revertants with dystrophin present around their rim
    Some revertant muscle fibers are present in myopathic regions as well.

Western blot: Dystrophin from dystrophinopathies

     
      from Novocastra

Lane 1: Becker dystrophy; Dystrophin has reduced abundance but normal size.
Lane 2: Becker dystrophy; Dystrophin has reduced size and abundance.
Lane 3: Normal; Dystrophin has normal size and amount.
Lane 4: Duchenne dystrophy; Almost no protein is present.
Lane 5: Duchenne outlier; Dystrophin has severely reduced abundance.

DMD muscle: After dystrophin mini-gene transfer


Dys3 antibody
  Stains dystrophin N-terminal epitope present on transferred mini-protein
  Present, irregularly, on surfaces of minority of muscle fibers
    Most dystrophin positive muscle fibers are small & intermediate sized

Dys2 antibody
  Stains dystrophin C-terminal epitope not present on transferred mini-protein
  Staining is generally not present on surfaces of muscle fibers

Go to Becker muscular dystrophy pathology
Return to Dystrophinopathies.
Return to Neuromuscular syndromes
Return to Neuromuscular home page

References
1. J Gen Physiol 2022;15:e202213081

2/25/2025