Abstract
BackgroundSpinal muscular atrophy (SMA) is an autosomal recessive hereditary disorder caused by mutations of the survival motor neuron 1 (SMN1) gene. Recently, high-resolution DNA melting analysis (HRMA) with saturation LC Green dyes has become a powerful post-PCR technique for genotyping or mutation scanning. So far, no studies have applied HRMA to the molecular analysis of SMA.MethodsThe exon 7 and the flanking area of the SMN1 and SMN2 genes of 55 SMA patients and 46 unrelated normal individuals were amplified with asymmetric PCR with unlabeled probe and symmetric PCR without probe, respectively. The saturation LC Green dyes were added to the PCR system. The PCR products were loaded onto the LightScanner system and were melted from 60°C to 95°C slowly. The melting curves were acquired and analyzed by the LightScanner software.ResultsThree types of melting curves that correlated with the presumed genotype of SMA patients and controls were clearly separated on the HRMA chromatogram with the unlabeled probe. The 55 SMA patients and 46 non-SMA controls were identified with HRMA with a 100% clinical sensitivity.ConclusionThe HRMA with saturation LC Green dyes and unlabeled probe appears to be a suitable, alternative method for the diagnosis of SMA, with high sensitivity and specificity.
Highlights
Spinal muscular atrophy (SMA) is an autosomal recessive hereditary disorder caused by mutations of the survival motor neuron 1 (SMN1) gene
SMN1 can be distinguished from SMN2 by two nucleotide changes in exon 7 and 8, which can be used to detect the deletion of SMN1 and establish the diagnosis of SMA
Four assays have been described for detecting the absence of SMN1: singlestranded conformation polymorphism (SSCP), restriction enzyme digestion analysis, denaturing high-performance liquid chromatography (DHPLC) analysis and liquid microbead arrays
Summary
Spinal muscular atrophy (SMA) is an autosomal recessive hereditary disorder caused by mutations of the survival motor neuron 1 (SMN1) gene. SMN1 is the critical gene involved in SMA, as more than 90% of SMA patients have SMN1 exon 7 homozygous deletions [1,2,3]. Four assays have been described for detecting the absence of SMN1: singlestranded conformation polymorphism (SSCP), restriction enzyme digestion analysis, denaturing high-performance liquid chromatography (DHPLC) analysis and liquid microbead arrays. While the liquid microbead arrays is a sensitive, high-throughput approach that can be used to detect SMN1 exon 7 deletions from blood spots, the cost may prohibit its application in many laboratories [10]
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