81008730

open access

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SMA Screening System Using Dried Blood Spots on Filter Paper: Application of COP-PCR to the SMN1 Deletion Test

English (英語)

The Kobe journal of the medical sciences

60(4)

78-85

Kobe University School of Medicine

2014

2015-02-02

BACKGROUND: Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletions. Thus, the SMN1 deletion test should be performed initially as part of the diagnostic process. However, SMN2, a highly homologous gene, hampers detection of SMN1 deletion. To differentiate between SMN1 and SMN2, many analysis methods have been developed yet they are not all available worldwide. AIM: To establish a simple but accurate SMN1-deletion detection system that can be used worldwide. METHODS: Fifty DNA samples (29 SMA patients and 21 controls) from dried blood spots (DBS) on filter paper were assayed. All participants had previously been screened for SMA 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 between 1 and 8 years. Competitive oligonucleotide priming-PCR (COP-PCR) was performed to distinguish SMN1 and SMN2 exon7. RESULTS: DNA yield from an 11-mm diameter DBS circle was 21,171 ± 7,485 ng (mean ± SD), with an 260/280 OD ratio from 1.49 to 2.1(mean ± SD; 1.67 ±0.13). Nucleotide sequencing confirmed gene-specific amplification of SMN1 and SMN2 by COP-PCR. SMN1 and SMN2 COP-PCR results are completely consistent with those obtained by PCR-RFLP. CONCLUSION: We have combined DNA extraction from DBS on filter paper with COP-PCR that specifically detects SMN1 and SMN2, establishing a new SMN1-deletion detection system with practical application worldwide.

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