title: A sensitive assay for measuring SMN mRNA levels in peripheral blood and in muscle samples of patients affected with spinal muscular atrophy.
abstract: Different therapeutic strategies are currently evaluated in spinal muscular atrophy (SMA) that are aimed at increasing full-length (FL) mRNA levels produced from the SMN2 gene. Assays measuring SMN mRNA levels are needed. We have developed a sensitive, comparative assay based on multiplex fluorescent reverse-transcription polymerase chain reaction (RT-PCR) that can measure, in the same reaction, the levels of SMN mRNA with and without exon 7 sequences as well as those of total SMN mRNA. This assay allows to calculate directly the ratios of FL SMN mRNA to SMN mRNA without exon 7 (Delta7). We have used this assay to compare the levels of SMN transcripts in the blood of 75 unrelated normal subjects and of 48 SMA patients, and in muscle samples of 8 SMA patients. The SMN1 and the SMN2 genes produced very similar levels of total mRNA. Levels of transcripts lacking exon 7 were linearly dependent on the number of SMN2 copies, both in SMA patients and in controls. In patients, FL mRNA levels correlated with SMN2 copy number. A significant but weaker inverse correlation was also observed between FL or Delta7 mRNA levels and disease severity, but patients with three SMN2 copies and different SMA types displayed similar mRNA levels. A significantly higher FL to Delta7 ratio was measured in blood cells than in skeletal muscle (0.80+/-0.18 versus 0.47+/-0.11). This assay can be used as a sensitive biomarker for monitoring the effects of various drugs in forthcoming clinical trials of SMA.
authors.affiliation: Department of Genetics, Rouen University Hospital and INSERM U614, Institute for Biomedical Research, University of Rouen, Rouen, France.
authors.forename: Myriam
authors.initials: M
authors.lastname: Vezain
authors.forename: Pascale
authors.initials: P
authors.lastname: Saugier-Veber
authors.forename: Judith
authors.initials: J
authors.lastname: Melki
authors.forename: Annick
authors.initials: A
authors.lastname: Toutain
authors.forename: Eric
authors.initials: E
authors.lastname: Bieth
authors.forename: Marie
authors.initials: M
authors.lastname: Husson
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Pedespan
authors.forename: Louis
authors.initials: L
authors.lastname: Viollet
authors.forename: Isabelle
authors.initials: I
authors.lastname: Pénisson-Besnier
authors.forename: Séverine
authors.initials: S
authors.lastname: Fehrenbach
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Bou
authors.forename: Thierry
authors.initials: T
authors.lastname: Frébourg
authors.forename: Mario
authors.initials: M
authors.lastname: Tosi
mesh_terms: D001483:Base Sequence; D001773:Blood Cells / Q000378:metabolism; D016022:Case-Control Studies; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005091:Exons; D018628:Gene Dosage; D006801:Humans; D018482:Muscle, Skeletal / Q000378:metabolism; D009134:Muscular Atrophy, Spinal / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000097:blood / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D055532:SMN Complex Proteins; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein