title: High-resolution melting (HRM) analysis as a feasible method for detecting spinal muscular atrophy via dried blood spots.
abstract: BACKGROUND
  Spinal muscular atrophy (SMA) is a neurodegenerative disease with the leading genetic cause of infant mortality. More than 95% of patients with SMA have a homozygous disruption in the survival motor neuron1 (SMN1) gene, caused by mutation, deletion, or rearrangement. Recently, treatment in humans in the immediate postnatal period, prior to the development of weakness or very early in the course of the disease, may be effective. Therefore, our objective was to establish a feasible method for SMA screening. High-resolution melting (HRM) analysis is rapidly becoming the most important mutation-scanning methodology that allows mutation scanning and genotyping without the need for costly labeled oligonucleotides. In the current study, we aim to develop a method for identifying the substitution of single nucleotide in SMN1 exon 7 (c.840C>T) by HRM analysis.
  
  
  METHODS
  Genomic DNA was extracted from peripheral blood samples and dried blood spots obtained from 30 patients with SMA and 30 normal individuals. All results were previously confirmed by denaturing high-performance liquid chromatography (DHPLC).
  
  
  RESULTS
  In order to identify the substitution of single nucleotide in SMN1 exon 7 (c.840C>T) by HRM analysis, a primer set was used in HRM analysis. At first, we failed to identify the substitution of single nucleotide in SMN1 exon 7 (c.840C>T) by HRM analysis because the homozygous CC and homozygous TT cannot be distinguished by HRM analysis. Therefore, all samples were mixed with a known SMN1/SMN2 copy number (SMN1/SMN2=0:3), which we may call driver. This strategy is used to differentiate between homozygous CC and homozygous TT. After mixing with driver, the melting profile of homozygous CC becomes heteroduplex; however, the homozygous TT remains the same in the normalized and temperature-shifted difference plots.
  
  
  CONCLUSIONS
  HRM analysis can be successfully applied to screen SMA via DNA obtained from whole blood and dried blood spots. We strongly believe that HRM analysis, a high-throughput method, could be used for identifying affected infants prior to the presentation of clinical symptoms in future.
authors: Division of Molecular Diagnostics, Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
authors: Tze-Kiong
authors: TK
authors: Er
authors: Tzu-Min
authors: TM
authors: Kan
authors: Yu-Fa
authors: YF
authors: Su
authors: Ta-Chih
authors: TC
authors: Liu
authors: Jan-Gowth
authors: JG
authors: Chang
authors: Shih-Ya
authors: SY
authors: Hung
authors: Yuh-Jyh
authors: YJ
authors: Jong
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods; D059788:Dried Blood Spot Testing / Q000379:methods*; D005091:Exons; D005615:Freezing; D006720:Homozygote; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000097:blood / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics*
chemical_list: C526471:SMN1 protein, human; D055533:Survival of Motor Neuron 1 Protein