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Early age   Mild   Myopathic groups   Regeneration   Severe Later age Dystrophin Revertants 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 More early in disease course Patterns Scattered Myopathic grouping Muscle fiber pathology "Delta lesions" Internal archetecture Often coarse (Immature) Ring fibers Immaturity (2C fiber type): Many fibers Chronic changes: Other tissues Endomysial connective tissue: Increased More with: Later pathology Fat accumulation Locations: Endomysial & Perimysial 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 46 (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 Stromal interaction molecule-1 (STIM1) Senses changes in luminal ER/SR Ca++ levels Orai1 Ca++ release-activated Ca++ channel in surface membrane SOCE trigger: Depletion of intracellular Ca++ stores

From: AG Engel Delta Lesions (Arrow) Description: Focal wedge-shaped lesions Locations: Subsarcolemmal regions of muscle fibers

H&E stain

VvG stain

NADH stain

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

H&E stain

Gomori trichrome stain

VvG stain

Myopathic grouping: Necrotic muscle fibers, clustered

H&E stain

NADH stain Necrotic muscle fibers are pale on NADH stain

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

VvG stain

VvG stain

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

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

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

H&E stain

H&E stain

Gomori trichrome stain

Gomori trichrome stain

VvG stain

ATPase pH 4.3 stain

Duchenne Muscular Dystrophy: Later Pathology (10 years)

H&E stain

H&E stain

VvG stain

Sudan stain

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

	H&E stain Endomysial connective tissue   Increased between fibers

ATPase pH 4.3 stain

Acid phosphatase stain

NADH stain

H&E stain

Gomori trichrome stain

NADH stain

NADH stain

Dystrophin staining

Normal	Duchenne Muscular Dystrophy:
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

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.