title: Detection of MYD88 L265P mutation by real-time allele-specific oligonucleotide polymerase chain reaction.
abstract: MYD88 L265P mutation has been reported in ∼90% of Waldenström's Macroglobulinemia (WM) patients and immunoglobulin M (IgM) monoclonal gammopathies of uncertain significance (MGUS), as well as in some cases of lymphoma and chronic lymphocytic leukemia. The present study aimed to develop a real-time allele-specific oligonucleotide PCR (ASO-RQ-PCR) to detect the MYD88 L265P mutation. We first evaluated the reproducibility and sensitivity of the technique with a diluting experiment of a previously known positive sample. Then, we evaluated the applicability of the methodology by analyzing 30 selected patients (10 asymptomatic WM, 10 symptomatic WM, and 10 IgM MGUS) as well as 10 healthy donors. The quantitative ASO-PCR assay could detect the MYD88 L265P mutation at a dilution of 0.25%, showing an inverse correlation between the tumor cell percentage and the cycle threshold (CT) value, thus allowing for tumor burden quantitation. In addition, mutated cases were distinguished from the unmutated by >10 cycles of difference between CTs. To sum up, ASO-RQ-PCR is an inexpensive, robust, and optimized method for the detection of MYD88 L265P mutation, which could be considered as a useful molecular tool during the diagnostic work-up of B-cell lymphoproliferative disorders.
authors.affiliation: *Department of Hematology, University Hospital of Salamanca †Center of Investigation in Cancer (CIC), Instituto Biosanitario de Salamanca (IBSAL), Salamanca, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Jiménez
authors.forename: María Del Carmen
authors.initials: Mdel C
authors.lastname: Chillón
authors.forename: Ana
authors.initials: A
authors.lastname: Balanzategui
authors.forename: Noemí
authors.initials: N
authors.lastname: Puig
authors.forename: Elena
authors.initials: E
authors.lastname: Sebastián
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcoceba
authors.forename: María E
authors.initials: ME
authors.lastname: Sarasquete
authors.forename: Isabel P
authors.initials: IP
authors.lastname: Conde
authors.forename: Rocío
authors.initials: R
authors.lastname: Corral
authors.forename: Luis A
authors.initials: LA
authors.lastname: Marín
authors.forename: Bruno
authors.initials: B
authors.lastname: Paiva
authors.forename: Montserrat
authors.initials: M
authors.lastname: Ruano
authors.forename: Alicia
authors.initials: A
authors.lastname: Antón
authors.forename: Rebeca
authors.initials: R
authors.lastname: Maldonado
authors.forename: Jesús F
authors.initials: JF
authors.lastname: San Miguel
authors.forename: Marcos
authors.initials: M
authors.lastname: González
authors.forename: Ramón
authors.initials: R
authors.lastname: García-Sanz
mesh_terms: D000483:Alleles; D004252:DNA Mutational Analysis / Q000379:methods*; D003937:Diagnosis, Differential; D006801:Humans; D007075:Immunoglobulin M / Q000378:metabolism; D008998:Monoclonal Gammopathy of Undetermined Significance / Q000175:diagnosis* / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D047368:Tumor Burden; D008258:Waldenstrom Macroglobulinemia / Q000175:diagnosis* / Q000235:genetics
chemical_list: D007075:Immunoglobulin M; D053594:Myeloid Differentiation Factor 88