title: Novel mutations in patients with McArdle disease by analysis of skeletal muscle mRNA.
abstract: OBJECTIVE
  To identify pathogenic mutant alleles of the PYGM gene in "genetic manifesting heterozygous" patients with McArdle disease-that is, those in whom we could only find a sole mutant allele by genomic DNA analysis.
  
  
  METHODS
  We studied four unrelated patients. PCR-RFLP, gene sequencing, and muscle cDNA analysis were performed to search for mutations in the PYGM gene. The effects of the mutations were evaluated by in silico analysis, and gene expression was assessed by real-time polymerase chain reaction (PCR).
  
  
  RESULTS
  Patient 1 was a compound heterozygous for the p.G205S missense mutation and for a novel "in frame" mutation, p.Q176_M177insVQ, resulting from a retention of six nucleotides from the 3'-end sequence of intron 4. Patient 2 was heterozygous for the common nonsense mutation p.R50X, and for a 1094 bp, c.1969+214_2177+369del mutation, spanning from intron 16 to intron 17 sequences. Furthermore, mRNA expression level was dramatically reduced consistent with nonsense mediated decay. Patient 3 was heterozygous for the p.R50X substitution, and patient 4 was heterozygous for the relatively common private Spanish mutation p.W798R. These two patients harboured a heterozygous exonic synonymous variant, p.K215K. Quantification of gene transcripts in patient 3 revealed a drastic decrease in the relative expression of the gene, which strongly supports the possibility of nonsense mediated decay.
  
  
  CONCLUSIONS
  Our results indicate that skeletal muscle cDNA studies in "genetic manifesting heterozygous" patients with McArdle disease are prone to identify their second mutant allele.
authors.affiliation: Centro de Investigación, Hospital Universitario 12 de Octubre, Avda Córdoba s/n, 28041 Madrid, Spain.
authors.forename: I
authors.initials: I
authors.lastname: García-Consuegra
authors.forename: J C
authors.initials: JC
authors.lastname: Rubio
authors.forename: G
authors.initials: G
authors.lastname: Nogales-Gadea
authors.forename: J
authors.initials: J
authors.lastname: Bautista
authors.forename: S
authors.initials: S
authors.lastname: Jiménez
authors.forename: A
authors.initials: A
authors.lastname: Cabello
authors.forename: A
authors.initials: A
authors.lastname: Lucía
authors.forename: A L
authors.initials: AL
authors.lastname: Andreu
authors.forename: J
authors.initials: J
authors.lastname: Arenas
authors.forename: M A
authors.initials: MA
authors.lastname: Martin
mesh_terms: D000328:Adult; D003198:Computer Simulation; D015870:Gene Expression; D024982:Glycogen Phosphorylase, Muscle Form / Q000235:genetics* / Q000378:metabolism; D006012:Glycogen Storage Disease Type V / Q000235:genetics* / Q000473:pathology; D006579:Heterozygote; D006801:Humans; D008297:Male; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D012333:RNA, Messenger; D024982:Glycogen Phosphorylase, Muscle Form