title: Detection of the MYD88 mutation by the combination of the allele-specific PCR and quenching probe methods.
abstract: BACKGROUND
  The MYD88 missense mutation c.794T>C, p.Leu265Pro, is found in patients with Waldenstörm's macroglobulinemia and lymphoma. Direct sequencing, allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and high-resolution melting analysis (HRM) are currently used to detect the mutation; however, they are either time-consuming or have low detection sensitivity. Here, we developed a novel highly sensitive and rapid detection method based on the quenching probe (QP) technique and AS-PCR.
  
  
  METHODS
  A lymphoma cell line heterozygous for the MYD88 mutation, two wild-type cell lines, and two samples from Waldenstörm's macroglobulinemia patients were analyzed by AS-PCR, PCR-RFLP, HRM, and QP, and their detection sensitivity was examined using the mixtures of the mutant and wild-type DNA.
  
  
  RESULTS
  For mutation-carrying heterozygous samples, the QP method produced W-shaped melting profiles presenting curves derived from the wild-type and mutant alleles. The QP analysis was performed in 2 h and demonstrated the detection limit of 5%, which was similar to that of the other methods. However, the combination of AS-PCR and QP (AS-QP) improved the sensitivity to 0.62% of the mutant allele.
  
  
  CONCLUSIONS
  The AS-QP analysis is rapid and minimally improves detection sensitivity compared to the AS-PCR.
authors.affiliation: Department of Laboratory Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Nogami
authors.affiliation: Department of Health Sciences, Saitama Prefectural University, Koshigaya-city, Saitama, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Kawaguchi-Ihara
authors.affiliation: Department of Laboratory Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: E
authors.initials: E
authors.lastname: Shiratori
authors.affiliation: Department of Laboratory Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Ohtaka
authors.affiliation: Department of Laboratory Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Itoh
authors.affiliation: Department of Laboratory Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Tohda
mesh_terms: D000483:Alleles; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods; D005615:Freezing; D006801:Humans; D008223:Lymphoma / Q000175:diagnosis / Q000235:genetics; D020125:Mutation, Missense; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013997:Time Factors; D008258:Waldenstrom Macroglobulinemia / Q000175:diagnosis / Q000235:genetics
chemical_list: C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88
keywords: MYD88; Waldenström's macroglobulinemia; allele-specific PCR; high-resolution melting analysis; quenching probe