Emma Tabe Eko Niba1, Mawaddah Ar Rochmah1, Nur Imma Fatimah Harahap1, Hiroyuki Awano2, Ichiro Morioka2, Kazumoto Iijima2, Yasuhiro Takeshima3, Toshio Saito4, Kayoko Saito5, Atsuko Takeuchi6, Poh San Lai7, Yoshihiro Bouike8, Masafumi Matsuo9, 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 neuromuscular disease characterized by defects of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion using dried blood spot (DBS), we developed a new combined system with real-time "modified competitive oligonucleotide priming"-polymerase chain reaction (mCOP-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). Although our real-time mCOP-PCR method is secured enough to be gene-specific, its amplification efficiency is not as good because the reverse primers carry a nucleotide mismatched with the sequence of the pre-amplified product. The mismatch has consequently been generated in the process of introducing a restriction enzyme site in the pre-amplified products for PCR-RFLP. METHOD: DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification). Pre-amplified products were used as template in the real-time mCOP-PCR, and, on the other hand, were digested with DraI enzyme (PCR-RFLP). To improve the amplification efficiency of mCOP-PCR, one nucleotide change was introduced in the original reverse primers (SMN1-COP and SMN2-COP) to eliminate the mismatched nucleotide. RESULTS: The real-time mCOP-PCR with a new primer (SMN1-COP-DRA or SMN2-COP-DRA) more rapidly and specifically amplified SMN1 and SMN2, and clearly demonstrated SMN1 deletion in an SMA patient. With the new primers, the amplification efficiencies of real-time mCOP-PCR were improved and the Cq values of SMN1 (+) and SMN2 (+) samples were significantly lowered. CONCLUSION: In the advanced version of our screening system for homozygous SMN1 deletion using DBS, the real-time mCOP-PCR with newly-designed reverse primers demonstrated the presence or absence of SMN1 and SMN2 within a shorter time, and the results were easily tested by PCR-RFLP. This rapid and accurate screening system will be useful for detection of newborn infants with SMA.
BACKGROUND:Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. More than 95% of SMApatients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion using dried blood spot (DBS), we developed a new combined system with real-time "modified competitive oligonucleotide priming"-polymerase chain reaction (mCOP-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). Although our real-time mCOP-PCR method is secured enough to be gene-specific, its amplification efficiency is not as good because the reverse primers carry a nucleotide mismatched with the sequence of the pre-amplified product. The mismatch has consequently been generated in the process of introducing a restriction enzyme site in the pre-amplified products for PCR-RFLP. METHOD: DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification). Pre-amplified products were used as template in the real-time mCOP-PCR, and, on the other hand, were digested with DraI enzyme (PCR-RFLP). To improve the amplification efficiency of mCOP-PCR, one nucleotide change was introduced in the original reverse primers (SMN1-COP and SMN2-COP) to eliminate the mismatched nucleotide. RESULTS: The real-time mCOP-PCR with a new primer (SMN1-COP-DRA or SMN2-COP-DRA) more rapidly and specifically amplified SMN1 and SMN2, and clearly demonstrated SMN1 deletion in an SMApatient. With the new primers, the amplification efficiencies of real-time mCOP-PCR were improved and the Cq values of SMN1 (+) and SMN2 (+) samples were significantly lowered. CONCLUSION: In the advanced version of our screening system for homozygous SMN1 deletion using DBS, the real-time mCOP-PCR with newly-designed reverse primers demonstrated the presence or absence of SMN1 and SMN2 within a shorter time, and the results were easily tested by PCR-RFLP. This rapid and accurate screening system will be useful for detection of newborn infants with SMA.
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Authors: S Lefebvre; L Bürglen; S Reboullet; O Clermont; P Burlet; L Viollet; B Benichou; C Cruaud; P Millasseau; M Zeviani Journal: Cell Date: 1995-01-13 Impact factor: 41.582