Detection of the MYD88 mutation by the combination of the allele‐specific PCR and quenching probe methods

• Published in: International journal of laboratory hematology 2017-04, Vol.39 (2), p.163-168
• Main Authors: Nogami, S., Kawaguchi‐Ihara, N., Shiratori, E., Ohtaka, M., Itoh, M., Tohda, S.
• Format: Article
• Language: English
• Subjects: Alleles; allele‐specific PCR; Cell Line, Tumor; DNA Mutational Analysis - methods; Freezing; high‐resolution melting analysis; Humans; Lymphoma; Lymphoma - diagnosis; Lymphoma - genetics; Macroglobulinemia; Missense mutation; Mutation; Mutation, Missense; MYD88; MyD88 protein; Myeloid Differentiation Factor 88 - genetics; Polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; quenching probe; Restriction fragment length polymorphism; Sensitivity and Specificity; Time Factors; Waldenstrom Macroglobulinemia - diagnosis; Waldenstrom Macroglobulinemia - genetics; Waldenström's macroglobulinemia
• ISSN: 1751-5521; 1751-553X
• DOI: 10.1111/ijlh.12598

Summary Introduction 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. Method 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. Conclusion The AS‐QP analysis is rapid and minimally improves detection sensitivity compared to the AS‐PCR.