title: SMA screening system using dried blood spots on filter paper: application of COP-PCR to the SMN1 deletion test.
abstract: BACKGROUND
  Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletions. Thus, the SMN1 deletion test should be performed initially as part of the diagnostic process. However, SMN2, a highly homologous gene, hampers detection of SMN1 deletion. To differentiate between SMN1 and SMN2, many analysis methods have been developed yet they are not all available worldwide.
  
  
  OBJECTIVE
  To establish a simple but accurate SMN1-deletion detection system that can be used worldwide.
  
  
  METHODS
  Fifty DNA samples (29 SMA patients and 21 controls) from dried blood spots (DBS) on filter paper were assayed. All participants had previously been screened for SMA by PCR-restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from freshly collected blood. DNA was extracted from DBS that had been stored at room temperature (20-25℃) for between 1 and 8 years. Competitive oligonucleotide priming-PCR (COP-PCR) was performed to distinguish SMN1 and SMN2 exon7.
  
  
  RESULTS
  DNA yield from an 11-mm diameter DBS circle was 21,171 ± 7,485 ng (mean ± SD), with an 260/280 OD ratio from 1.49 to 2.1(mean ± SD; 1.67 ±0.13). Nucleotide sequencing confirmed gene-specific amplification of SMN1 and SMN2 by COP-PCR. SMN1 and SMN2 COP-PCR results are completely consistent with those obtained by PCR-RFLP.
  
  
  CONCLUSIONS
  We have combined DNA extraction from DBS on filter paper with COP-PCR that specifically detects SMN1 and SMN2, establishing a new SMN1-deletion detection system with practical application worldwide.
authors: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.|Kobe Pharmaceutical University, 4-19-1 Motoyamakitamachi, Higasinada-ku, Kobe 658-8558 Japan.
authors: Nozomu
authors: N
authors: Kato
authors: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.|Basic Medical and Biomedical Sciences, Graduate School of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.
authors: Nihayatus
authors: N
authors: Sa'Adah
authors: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Mawaddah
authors: M
authors: Ar Rochmah
authors: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Nur Imma Fatimah
authors: NI
authors: Harahap
authors: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Dian Kesumapramudya
authors: DK
authors: Nurputra
authors: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Hideyuki
authors: H
authors: Sato
authors: Basic Medical and Biomedical Sciences, Graduate School of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.|Department of Biochemistry, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.
authors: Ahmad Hamim
authors: AH
authors: Sadewa
authors: Basic Medical and Biomedical Sciences, Graduate School of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.|Department of Pharmacology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.
authors: Indwiani
authors: I
authors: Astuti
authors: Basic Medical and Biomedical Sciences, Graduate School of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.|Department of Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.
authors: Sofia Mubarika
authors: SM
authors: Haryana
authors: Division of Child Neurology, Department of Neurology, National Hospital Organization Toneyama National Hospital, Toneyama, Japan.
authors: Toshio
authors: T
authors: Saito
authors: Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan.
authors: Kayoko
authors: K
authors: Saito
authors: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Noriyuki
authors: N
authors: Nishimura
authors: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Hisahide
authors: H
authors: Nishio
authors: Kobe Pharmaceutical University, 4-19-1 Motoyamakitamachi, Higasinada-ku, Kobe 658-8558 Japan.
authors: Atsuko
authors: A
authors: Takeuchi
mesh_terms: D017931:DNA Primers; D059788:Dried Blood Spot Testing; D017353:Gene Deletion; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics*; D055540:Survival of Motor Neuron 2 Protein / Q000235:genetics
chemical_list: D017931:DNA Primers; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein
keywords: COP-PCR; SMN1; SMN2; dried blood spot; spinal muscular atrophy