Yogik Onky Silvana Wijaya1, Emma Tabe Eko Niba1, Mawaddah Ar Rochmah1, Nur Imma Fatimah Harahap1, Hiroyuki Awano2, Yasuhiro Takeshima3, Toshio Saito4, Kayoko Saito5, Atsuko Takeuchi6, Poh San Lai7, Yoshihiro Bouike8, Hisahide Nishio1,9, Masakazu Shinohara1. 1. Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan. 2. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan. 3. Department of Pediatrics, Hyogo College of Medicine, Nishinomiya, Japan. 4. Division of Child Neurology, Department of Neurology, National Hospital Organization Toneyama National Hospital, Toneyama, Japan. 5. Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan. 6. Kobe Pharmaceutical University, Kobe, Japan. 7. Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 8. Faculty of Nutrition, Kobe Gakuin University, Kobe, Japan. 9. Faculty of Rehabilitation, Kobe Gakuin University, Kobe, Japan.
Abstract
BACKGROUND: Spinal Muscular Atrophy (SMA) is a common autosomal recessive disorder caused by SMN1 gene deletion. SMA has been considered an incurable disease. However, a newly-developed antisense oligonucleotide drug, nusinersen, brings about a good outcome to SMA patients in the clinical trials. Now, a screening for SMA is required for early diagnosis and early treatment so as to give a better clinical outcome to the patients. We have invented a new technology, mCOP-PCR, for SMA screening using dried blood spot (DBS) on the filter paper. One of the problems encountered in SMA screening is poor quality and quantity of DNA extracted from DBS. METHODS: DNA was extracted from DBS of six individuals. Fresh blood DNA of each individual had already been genotyped using PCR/RFLP. The fragments including the sequence of SMN1/SMN2 exon 7 were pre-amplified with conventional PCR. To determine which pre-amplified product is a better template for the real-time mCOP-PCR, we did pre-amplification with a single PCR or pre-amplification with a nested PCR. RESULTS: The real-time mCOP-PCR using pre-amplified products with a single PCR brought about ambiguous results in some SMN1-carrying individuals. However, the results of real-time mCOP-PCR following pre-amplification with a nested PCR were completely matched with those of PCR-RFLP. CONCLUSION: In our study on the real-time mCOP-PCR screening system for SMA, a nested PCR secured the DNA template quality and quantity, leading to unambiguous results of SMA screening.
BACKGROUND:Spinal Muscular Atrophy (SMA) is a common autosomal recessive disorder caused by SMN1 gene deletion. SMA has been considered an incurable disease. However, a newly-developed antisense oligonucleotide drug, nusinersen, brings about a good outcome to SMApatients in the clinical trials. Now, a screening for SMA is required for early diagnosis and early treatment so as to give a better clinical outcome to the patients. We have invented a new technology, mCOP-PCR, for SMA screening using dried blood spot (DBS) on the filter paper. One of the problems encountered in SMA screening is poor quality and quantity of DNA extracted from DBS. METHODS: DNA was extracted from DBS of six individuals. Fresh blood DNA of each individual had already been genotyped using PCR/RFLP. The fragments including the sequence of SMN1/SMN2 exon 7 were pre-amplified with conventional PCR. To determine which pre-amplified product is a better template for the real-time mCOP-PCR, we did pre-amplification with a single PCR or pre-amplification with a nested PCR. RESULTS: The real-time mCOP-PCR using pre-amplified products with a single PCR brought about ambiguous results in some SMN1-carrying individuals. However, the results of real-time mCOP-PCR following pre-amplification with a nested PCR were completely matched with those of PCR-RFLP. CONCLUSION: In our study on the real-time mCOP-PCR screening system for SMA, a nested PCR secured the DNA template quality and quantity, leading to unambiguous results of SMA screening.