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DYSTROPHINOPATHIES: Duchenne

Early age
  Mild
  Myopathic groups
  Regeneration
  Severe
Later age
Dystrophin
Revertants

General features

DMD Muscle pathology: General features

  • Myopathy: Ongoing & Chronic
  • Molecular
    • Dystrophin staining
      • Screening Antibodies: Target Epitopes
        • Dys-3: N-terminus; Exons 10 to 12
        • Dys-1: Rod domain; Exons 26 to 30
        • 7G1: Rod domain; Exon 48 (Within deletion hotspot)
        • 6A9: Rod domain; Exon 50 (Within deletion hotspot)
        • Dys-2: C-terminus; Exon 79
          • Less useful: Few deletions in this regione
      • Duchenne MD clinical phenotype: Staining patterns
        • 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
        • 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.

Go to Becker muscular dystrophy pathology
Return to Dystrophinopathies.
Return to Neuromuscular syndromes
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References
1. J Gen Physiol 2022;15:e202213081

10/17/2022