title: Diagnosis of spinal muscular atrophy via high-resolution melting analysis symmetric polymerase chain reaction without probe: a screening evaluation for SMN1 deletions and intragenic mutations.
abstract: OBJECTIVE
  Spinal muscular atrophy (SMA) is a well-defined autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron 1 (SMN1) gene. The most frequently observed mutation is a deletion of exon 7, which has been documented in >95% of SMA patients. A novel technique for detecting mutations known as high-resolution melting analysis (HRMA) has rapidly become the tool of choice for screening pathogenic genetic variants. In the present study, we attempt to validate the applicability of HRMA to the detection of exon 7 deletions and other intragenic mutations in SMN1.
  
  
  RESULTS
  Three primer sets were adopted in our HRMA screening for deletion of SMN1 exon 7. In screening attempts utilizing two primer sets, the results of HRMA were not compatible with those obtained by polymerase chain reaction-restriction fragment length polymorphism. Therefore, we applied a modified protocol using revised primer sets, which resulted in an absolute compatibility of results between HRMA and polymerase chain reaction-restriction fragment length polymorphism. With regard to screenings for intragenic mutations in SMN1 exon 3, two primer sets were adopted for use in HRMA. In the initial HRMA screening using the first primer set, we failed to identify any intragenic mutations; however, when using a revised primer set, HRMA successfully detected the presence of a c.275G>C mutation.
  
  
  CONCLUSIONS
  HRMA is a simple but versatile tool to add to the existing arsenal of diagnostic techniques that could aid clinicians/researchers in diagnosing SMA. However, as we demonstrate in the present study, the design and selection of primers is of monumental importance in ensuring the successful application of HRMA to screening for pathogenic variants.
authors.affiliation: Department of Epidemiology, Kobe University Graduate School of Medicine, Kobe, Japan. msatoru@med.kobe-u.ac.jp
authors.forename: Satoru
authors.initials: S
authors.lastname: Morikawa
authors.forename: Indra Sari Kusuma
authors.initials: IS
authors.lastname: Harahap
authors.forename: Richard Hideki
authors.initials: RH
authors.lastname: Kaszynski
authors.forename: Tomoto
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Dian Kesuma
authors.initials: DK
authors.lastname: Pramudya
authors.forename: Huyen Thi Van
authors.initials: HT
authors.lastname: Pham
authors.forename: Tri Budi
authors.initials: TB
authors.lastname: Hartomo
authors.forename: Myeong Jin
authors.initials: MJ
authors.lastname: Lee
authors.forename: Ichiro
authors.initials: I
authors.lastname: Morioka
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Nishimura
authors.forename: Naoki
authors.initials: N
authors.lastname: Yokoyama
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ueno
authors.forename: Masafumi
authors.initials: M
authors.lastname: Matsuo
authors.forename: Hisahide
authors.initials: H
authors.lastname: Nishio
mesh_terms: D016022:Case-Control Studies; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes; D017353:Gene Deletion; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007438:Introns / Q000235:genetics; D009134:Muscular Atrophy, Spinal / Q000175:diagnosis* / Q000235:genetics; D009154:Mutation / Q000502:physiology; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics*
chemical_list: D015342:DNA Probes; C526471:SMN1 protein, human; D055533:Survival of Motor Neuron 1 Protein