title: Rapid screening for Japanese dysferlinopathy by fluorescent primer extension.
abstract: OBJECTIVE
  Mutations in the dysferlin gene cause limb-girdle muscular dystrophy (LGMD) 2B and Miyoshi myopathy (MM), which are collectively named dysferlinopathy. Dysferlinopathy is the most frequent type of LGMD in the Japanese population. Molecular genetic analysis is essential for the diagnosis of dysferlinopathy because of its variable immunohistochemical patterns of biopsied muscles, including patterns similar to normal controls. The analysis of the entire dysferlin gene however, is time-consuming and laborious; therefore a simple and rapid screening method to detect hot spot mutations in the dysferlin gene is essential for the diagnosis of dysferlinopathy.
  
  
  METHODS
  We previously showed that 4 mutations, c.937+1G>A, c.1566C>G, c.2997G>T and c.3373delG account for 50% of all the mutations identified in Japanese dysferlinopathy patients. We performed a one-tube multiplex PCR, followed by extension of primers for each mutation with a fluorescence-labeled dideoxynucleotide to screen the 4 hot spot mutations.
  
  
  RESULTS
  The multiplex primer-extension reaction was developed on samples of known mutations. The extension products were represented as 4 different peaks that corresponded to a mutated nucleotide on electropherogram. Using the developed screening method, we were able to detect mutations in these hot spots in 3 samples out of 8 clinically suspected LGMD2B/MM patients in only approximately 8 hours. These 3 cases were definitely diagnosed as LGMD2B/MM by exonic sequencing.
  
  
  CONCLUSIONS
  We have developed a simple and rapid screening method which could facilitate the definitive diagnosis of dysferlinopathy, contributing to an understanding of the genotype-phenotype correlations for dysferlinopathy.
authors.affiliation: Department of Neurology, Kawasaki Medical School, Kurashiki.
authors.forename: Saori
authors.initials: S
authors.lastname: Hayashi
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Ohsawa
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Takahashi
authors.forename: Naoki
authors.initials: N
authors.lastname: Suzuki
authors.forename: Tadashi
authors.initials: T
authors.lastname: Okada
authors.forename: Mitsue
authors.initials: M
authors.lastname: Rikimaru
authors.forename: Tatsufumi
authors.initials: T
authors.lastname: Murakami
authors.forename: Masashi
authors.initials: M
authors.lastname: Aoki
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Sunada
mesh_terms: D000293:Adolescent; D000368:Aged; D044466:Asian People / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D000073939:Dysferlin; D005260:Female; D005456:Fluorescent Dyes; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D049288:Muscular Dystrophies, Limb-Girdle / Q000175:diagnosis / Q000235:genetics*; D009154:Mutation; D013997:Time Factors; D055815:Young Adult
chemical_list: D017931:DNA Primers; C114839:DYSF protein, human; D000073939:Dysferlin; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D009124:Muscle Proteins