title: A Rapid, Accurate and Simple Screening Method for Spinal Muscular Atrophy: High-Resolution Melting Analysis Using Dried Blood Spots on Filter Paper.
abstract: BACKGROUND
  Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutation of the survival of the motor neuron 1 (SMN1) gene. More than 95% of SMA patients carry a homozygous deletion of SMN1. SMA can be screened for by polymerase chain reaction and high-resolution melting analysis (PCR-HRMA) using DNA extracted from dried blood spots (DBSs) stored on filter paper. However, there are two major problems with this approach. One is the frequent poor quality/quantity of DNA extracted from DBSs on filter paper, and the other is the difficulty in designing primer sets or probes to separate allele-specific melting curves. In this study, we addressed these problems and established a rapid, accurate and simple screening system for SMA with PCR-HRMA using DNA extracted from DBSs on filter paper.
  
  
  METHODS
  Seventy individuals were assayed in this study, 42 SMA patients and 28 controls, all of whom had been previously been screened for SMA by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) using DNA extracted from freshly collected blood. In this study, the DNA of each individual was extracted from dried blood that had been spotted onto cards and stored at room temperature (20 - 25 degrees C) for between 1 and 8 years. PCR amplification of 30 or 45 cycles was performed using 50 ng of DNA and was immediately followed by HRMA. SMN1 and SMN2 products were co-amplified using a previously designed primer set (R111 and 541C770) containing two single nucleotide differences.
  
  
  RESULTS
  The absorbance ratio at 260/280 of DNA extracted from DBSs ranged from 1.49 to 2.1 (mean ± SD; 1.66 ± 0.12), suggesting high-purity DNA. Thirty cycles of PCR amplification were insufficient to amplify the target alleles; PCR with 45 cycles was, however, successful in 69 out of 70 samples. PCR-HRMA using the R111/541C770 primer set enabled separation of the normalized melting curves of the samples with no SMN1 from those with SMN1 and SMN2.
  
  
  CONCLUSIONS
  DBSs on filter paper can be a good source of DNA for the diagnosis of diseases and PCR-HRMA using DNA extracted from DBSs is an alternative method to detect the SMN1 deletion. These findings suggest that the SMA screening system using PCR-HRMA with DBSs on filter paper is practicable in a large population study over a long time period.
authors.forename: Nihayatus
authors.initials: N
authors.lastname: Sa'adah
authors.forename: Nur Imma Fatimah
authors.initials: NI
authors.lastname: Harahap
authors.forename: Dian Kesumapramudya
authors.initials: DK
authors.lastname: Nurputra
authors.forename: Mawaddah Ar
authors.initials: MA
authors.lastname: Rochmah
authors.forename: Satoru
authors.initials: S
authors.lastname: Morikawa
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Nishimura
authors.forename: Ahmad Hamim
authors.initials: AH
authors.lastname: Sadewa
authors.forename: Indwiani
authors.initials: I
authors.lastname: Astuti
authors.forename: Sofia Mubarika
authors.initials: SM
authors.lastname: Haryana
authors.forename: Toshio
authors.initials: T
authors.lastname: Saito
authors.forename: Kayoko
authors.initials: K
authors.lastname: Saito
authors.forename: Hisahide
authors.initials: H
authors.lastname: Nishio
mesh_terms: D016022:Case-Control Studies; D004247:DNA / Q000097:blood / Q000737:chemistry; D008403:Mass Screening; D009134:Muscular Atrophy, Spinal / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics
chemical_list: C526471:SMN1 protein, human; D055533:Survival of Motor Neuron 1 Protein; D004247:DNA