Mawaddah Ar Rochmah1, Nur Imma Fatimah Harahap1, Emma Tabe Eko Niba1, Kenta Nakanishi1, Hiroyuki Awano2, Ichiro Morioka2, Kazumoto Iijima2, Toshio Saito3, Kayoko Saito4, Poh San Lai5, Yasuhiro Takeshima6, Atsuko Takeuchi7, Yoshihiro Bouike8, Maya Okamoto9, Hisahide Nishio10, Masakazu Shinohara1. 1. Department of Community Medicine and Social Health Care, Kobe University Graduate School of Medicine, Kobe, Japan. 2. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan. 3. Division of Child Neurology, Department of Neurology, National Hospital Organization Toneyama National Hospital, Toyonaka, Japan. 4. Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan. 5. Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 6. Department of Pediatrics, Hyogo College of Medicine, Nishinomiya, Japan. 7. Analytical Laboratory, Kobe Pharmaceutical University, Kobe, Japan. 8. Faculty of Nutrition, Kobe Gakuin University, Kobe, Japan. 9. Center for Public Health, Pharmacological Evaluation Institute of Japan, Kawasaki, Japan. 10. Department of Community Medicine and Social Health Care, Kobe University Graduate School of Medicine, Kobe, Japan; Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan. Electronic address: nishio@med.kobe-u.ac.jp.
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 deletion. SMA is the leading genetic cause of infant death, and has been considered an incurable disease. However, a recent clinical trial with an antisense oligonucleotide drug has shown encouraging clinical efficacy. Thus, early and accurate detection of SMN1 deletion may improve prognosis of many infantile SMA patients. METHODS: A total of 88 DNA samples (37 SMA patients, 12 carriers and 39 controls) from dried blood spots (DBS) on filter paper were analyzed. All participants had previously been screened for SMN genes 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°C) for 1week to 5years. To ensure sufficient quality and quantity of DNA samples, target sequences were pre-amplified by conventional PCR. Real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) with the pre-amplified PCR products was performed for the gene-specific amplification of SMN1 and SMN2 exon 7. RESULTS: Compared with PCR-RFLP using DNA from freshly collected blood, results from real-time mCOP-PCR using DBS-DNA for detection of SMN1 exon 7 deletion showed a sensitivity of 1.00 (CI [0.87, 1.00])] and specificity of 1.00 (CI [0.90, 1.00]), respectively. CONCLUSION: We combined DNA extraction from DBS on filter paper, pre-amplification of target DNA, and real-time mCOP-PCR to specifically detect SMN1 and SMN2 genes, thereby establishing a rapid, accurate, and high-throughput system for detecting SMN1-deletion with practical applications for newborn screening.
BACKGROUND:Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletion. SMA is the leading genetic cause of infantdeath, and has been considered an incurable disease. However, a recent clinical trial with an antisense oligonucleotide drug has shown encouraging clinical efficacy. Thus, early and accurate detection of SMN1 deletion may improve prognosis of many infantile SMA patients. METHODS: A total of 88 DNA samples (37 SMA patients, 12 carriers and 39 controls) from dried blood spots (DBS) on filter paper were analyzed. All participants had previously been screened for SMN genes 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°C) for 1week to 5years. To ensure sufficient quality and quantity of DNA samples, target sequences were pre-amplified by conventional PCR. Real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) with the pre-amplified PCR products was performed for the gene-specific amplification of SMN1 and SMN2 exon 7. RESULTS: Compared with PCR-RFLP using DNA from freshly collected blood, results from real-time mCOP-PCR using DBS-DNA for detection of SMN1 exon 7 deletion showed a sensitivity of 1.00 (CI [0.87, 1.00])] and specificity of 1.00 (CI [0.90, 1.00]), respectively. CONCLUSION: We combined DNA extraction from DBS on filter paper, pre-amplification of target DNA, and real-time mCOP-PCR to specifically detect SMN1 and SMN2 genes, thereby establishing a rapid, accurate, and high-throughput system for detecting SMN1-deletion with practical applications for newborn screening.