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℃) 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.
Keywords: COP-PCR; SMN1; SMN2; dried blood spot; spinal muscular atrophy.