title: Abnormal expression of dysferlin in skeletal muscle and monocytes supports primary dysferlinopathy in patients with one mutated allele.
abstract: BACKGROUND
  In some cases, a definitive confirmation of dysferlinopathy cannot be achieved by DNA test, because the mutation is detected in one allele only.
  
  
  METHODS
  DYSFERLIN expression in skeletal muscle and peripheral blood monocytes (PBM) was studied by Western blot in two unrelated adult patients. The comparative C(T) method (ΔΔC(T) ) was used to calculate relative changes in dysferlin mRNA determined from real-time quantitative PCR experiments. The dysferlin gene was studied by direct sequencing of cDNA and genomic DNA and by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis.
  
  
  RESULTS
  A comparable severe reduction in dysferlin was demonstrated in both skeletal muscle and PBM. The expression of dysferlin mRNA was significantly reduced. A novel mutation in exon 47 (c.5289G>C) of the dysferlin gene in the heterozygous state, causing an amino acid change (p.Glu1763Asp), was detected in both patients. The MLPA analysis did not reveal any deletion or duplication.
  
  
  CONCLUSIONS
  Dysferlin and/or dysferlin mRNA abnormalities are diagnostic for dysferlinopathy when mutational analysis detects a mutation in one allele only. Analysis of dysferlin mRNA can be helpful for distinguishing symptomatic heterozygotes from such patients.
authors.affiliation: Medical Faculty, Institute of Anatomy, University of Ljubljana, Ljubljana, Slovenia. marija.meznaric@mf.uni-lj.si
authors.forename: M
authors.initials: M
authors.lastname: Meznaric
authors.forename: L
authors.initials: L
authors.lastname: Gonzalez-Quereda
authors.forename: E
authors.initials: E
authors.lastname: Gallardo
authors.forename: N
authors.initials: N
authors.lastname: de Luna
authors.forename: P
authors.initials: P
authors.lastname: Gallano
authors.forename: M
authors.initials: M
authors.lastname: Fanin
authors.forename: C
authors.initials: C
authors.lastname: Angelini
authors.forename: B
authors.initials: B
authors.lastname: Peterlin
authors.forename: J
authors.initials: J
authors.lastname: Zidar
mesh_terms: D000328:Adult; D000483:Alleles; D015153:Blotting, Western; D004252:DNA Mutational Analysis; D000073939:Dysferlin; D005260:Female; D006579:Heterozygote; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D009000:Monocytes / Q000378:metabolism / Q000473:pathology*; D009124:Muscle Proteins / Q000235:genetics*; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology*; D049288:Muscular Dystrophies, Limb-Girdle / Q000175:diagnosis* / Q000235:genetics*; D010375:Pedigree; D017354:Point Mutation; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C114839:DYSF protein, human; D000073939:Dysferlin; D008565:Membrane Proteins; D009124:Muscle Proteins; D012333:RNA, Messenger