Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression.
Mutations that result in the loss of the protein dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires dysferlin. Based on this, we developed an in vitro assay to assess rescue of dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in dysferlin and produce sufficient functional dysferlin (approximately 15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for dysferlin-deficient patients harboring nonsense mutations.
Wang
Bingjing
B
University of Pennsylvania School of Medicine, Department of Physiology, B400 Richards Bldg., 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
Yang
Zhaohui
Z
Brisson
Becky K
BK
Feng
Huisheng
H
Zhang
Zhiqian
Z
Welch
Ellen M
EM
Peltz
Stuart W
SW
Barton
Elisabeth R
ER
Brown
Robert H
RH
Sweeney
H Lee
HL
D000818:Animals; D000831:Animals, Newborn; D001681:Biological Assay / Q000379:methods*; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D018389:Codon, Nonsense; D000073939:Dysferlin; D006801:Humans; D007038:Hypotonic Solutions; D008565:Membrane Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009124:Muscle Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D009997:Osmotic Pressure; D010069:Oxadiazoles / Q000494:pharmacology*; D052097:Quadriceps Muscle / Q000378:metabolism* / Q000473:pathology; D014162:Transfection
D018389:Codon, Nonsense; C114839:DYSF protein, human; C497505:Dysf protein, mouse; D000073939:Dysferlin; D007038:Hypotonic Solutions; D008565:Membrane Proteins; D009124:Muscle Proteins; D010069:Oxadiazoles; C515878:ataluren