Nozomu Kato1,2, Nihayatus Sa'Adah1,3, Mawaddah Ar Rochmah1, Nur Imma Fatimah Harahap1, Dian Kesumapramudya Nurputra1, Hideyuki Sato1, Ahmad Hamim Sadewa3,4, Indwiani Astuti3,5, Sofia Mubarika Haryana3,6, Toshio Saito7, Kayoko Saito8, Noriyuki Nishimura1, Hisahide Nishio1, Atsuko Takeuchi2. 1. 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. 2. Kobe Pharmaceutical University, 4-19-1 Motoyamakitamachi, Higasinada-ku, Kobe 658-8558 Japan. 3. Basic Medical and Biomedical Sciences, Graduate School of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. 4. Department of Biochemistry, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. 5. Department of Pharmacology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. 6. Department of Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. 7. Division of Child Neurology, Department of Neurology, National Hospital Organization Toneyama National Hospital, Toneyama, Japan. 8. Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan.
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. AIM: 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. CONCLUSION: 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.
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. AIM: 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 SMN2COP-PCR results are completely consistent with those obtained by PCR-RFLP. CONCLUSION: 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: K Vill; A Blaschek; U Schara; H Kölbel; K Hohenfellner; E Harms; B Olgemöller; Maggie C Walter; W Müller-Felber Journal: Nervenarzt Date: 2017-12 Impact factor: 1.214