SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA

Emma Tabe Eko Niba; Mawaddah Ar Rochmah; Nur Imma Fatimah Harahap; Hiroyuki Awano; Ichiro Morioka; Kazumoto Iijima; Toshio Saito; Kayoko Saito; Atsuko Takeuchi; Poh San Lai; Yoshihiro Bouike; Hisahide Nishio; Masakazu Shinohara

SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA

Kobe J Med Sci. 2017 Dec 18;63(3):E80-E83.

Affiliations

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 Division of Child Neurology, Department of Neurology, National Hospital Organization Toneyama National Hospital, Toneyama, Japan.
⁴ Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan.
⁵ Kobe Pharmaceutical University, Kobe, Japan.
⁶ Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
⁷ Faculty of Nutrition, Kobe Gakuin University, Kobe, Japan.

PMID: 29434179
PMCID: PMC5826024

Abstract

Background: Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA.

Aim: To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA.

Methods: DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus.

Results: The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP.

Conclusion: Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first-tier diagnostic method of SMA.

Keywords: spinal muscular atrophy; mCOP-PCR; SMN1; SMN2; real-time PCR.

Publication types

Evaluation Study

MeSH terms

Case-Control Studies
DNA / blood
DNA / genetics
Exons
Gene Deletion
Humans
Infant
Infant, Newborn
Mass Screening
Real-Time Polymerase Chain Reaction / methods
Spinal Muscular Atrophies of Childhood / diagnosis*
Spinal Muscular Atrophies of Childhood / genetics*
Survival of Motor Neuron 1 Protein / genetics*
Survival of Motor Neuron 2 Protein / genetics

Substances

SMN1 protein, human
SMN2 protein, human
Survival of Motor Neuron 1 Protein
Survival of Motor Neuron 2 Protein
DNA