Spinal Muscular Atrophy: New Screening System with Real-Time mCOP-PCR and PCR-RFLP for SMN1 Deletion

• Published in: Kobe journal of the medical sciences Vol. 65; no. 2; pp. E44 - E48
• Main Authors: Niba, Emma Tabe Eko, Rochmah, Mawaddah Ar, Harahap, Nur Imma Fatimah, Awano, Hiroyuki, Morioka, Ichiro, Iijima, Kazumoto, Takeshima, Yasuhiro, Saito, Toshio, Saito, Kayoko, Takeuchi, Atsuko, Lai, Poh San, Bouike, Yoshihiro, Matsuo, Masafumi, Nishio, Hisahide, Shinohara, Masakazu
• Format: Journal Article
• Language: English
• Published: Japan Kobe University School Of Medicine 2019
• Subjects: SMN2; targeted pre-amplification; spinal muscular atrophy; PCR-RFLP; SMN1; mCOP-PCR
• ISSN: 1883-0498; 0023-2513; 1883-0498

Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration or loss 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, it is necessary to differentiate SMN1 from its highly homologous gene, SMN2. We developed a modified competitive oligonucleotide priming-PCR (mCOP-PCR) method using dried blood spot (DBS)-DNA, in which SMN1 and SMN2-specific PCR products are detected with gel-electrophoresis. Next, we added a targeted pre-amplification step prior to the mCOP-PCR step, to avoid unexpected, non-specific amplification. The pre-amplification step enabled us to combine mCOP-PCR and real-time PCR. In this study, we combined real-time mCOP-PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) to develop a new screening system for detection of SMN1 deletion. 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), the products of which were then used as a template in the real-time PCR with mCOP-primer sets. To confirm the results, the pre-amplified products were subject to the second PCR-RFLP. The real-time mCOP-PCR separately amplified SMN1 and SMN2 exon7, and clearly demonstrated SMN1 deletion in an SMA patient. The results of the real-time mCOP-PCR using DBS-DNA were completely consistent with those of the first and second PCR-RFLP analysis. In our new system for detection of SMN1 deletion, real-time mCOP-PCR rapidly proved the presence or absence of SMN1 and SMN2, and the results were easily tested by PCR-RFLP. This solid genotyping system will be useful for SMA screening.