Dataset Information

High-resolution melting analysis for rapid and sensitive MYD88 screening in chronic lymphocytic leukemia.

ABSTRACT: High resolution melting (HRM) assay is a novel technology for the fast, high-throughput, sensitive, post-PCR analysis of genetic mutations. Myeloid differentiation primary response 88 (MYD88) mutations are frequently reported in chronic lymphocytic leukemia (CLL) and confer a worse prognosis. The objective of the present study was to assess the value of HRM analysis for the rapid screening of MYD88 mutations in patients with CLL. Genomic DNA samples were extracted from the bone marrow of 129 newly diagnosed patients with CLL. A plasmid with an MYD88-L265P mutation was constructed, and the p.L265P substitution, which is the predominant MYD88 mutation in CLL, was detected using HRM analysis and direct sequencing. The plasmid pCMV-MYD88-L265P-Mu was successfully constructed as a positive control, and was verified by direct sequencing. The normalized and shifted melting curves of 6/129 (4.65%) samples were clearly different from those of other patients by HRM analysis. In addition, the 794T>C mutation in MYD88 was identified in 6 (4.65%) patients by direct sequencing. Sensitivity evaluation revealed that the HRM assay had a higher sensitivity (to 1% dilution) than direct sequencing, in addition to being convenient and time-saving. The MYD88 p.L256P mutation has been implicated to be associated with adverse prognosis in CLL. HRM analysis has the potential to be a routine prescreening technique to identify the MYD88 p.L256P mutation and may facilitate the clinical treatment of CLL.

SUBMITTER: Jiang M

PROVIDER: S-EPMC6540357 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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High-resolution melting analysis for rapid and sensitive MYD88 screening in chronic lymphocytic leukemia.

Jiang Min M Li Jie J Zhou Jun J Xing Chao C Xu Jing-Jing JJ Guo Feng F

Oncology letters 20190513 1

High resolution melting (HRM) assay is a novel technology for the fast, high-throughput, sensitive, post-PCR analysis of genetic mutations. Myeloid differentiation primary response 88 (MYD88) mutations are frequently reported in chronic lymphocytic leukemia (CLL) and confer a worse prognosis. The objective of the present study was to assess the value of HRM analysis for the rapid screening of MYD88 mutations in patients with CLL. Genomic DNA samples were extracted from the bone mar ...[more]

PMID: 31289558

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Project description:Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5.Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (Tm). A standardized software algorithm was used to assign amplicons as 'reference' or 'variant' as compared to duplicate reference sequence DNA controls for each SNP.Intra-assay (n=5) precision of Tms for all SNPs was ?0.19%, while inter-assay (n=20) precision ranged from 0.04% to 0.21%. When compared to a reference method of Sanger sequencing, the HRM assay produced no false negative results, and overcall frequency ranged from 0% to 26%, depending on the SNP. Furthermore, HRM genotyping displayed accuracy over input DNA concentrations ranging from 10 to 200 ng/?L.The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of 'reference' or 'variant' available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.

Dataset Information

High-resolution melting analysis for rapid and sensitive MYD88 screening in chronic lymphocytic leukemia.

Publications

High-resolution melting analysis for rapid and sensitive <i>MYD88</i> screening in chronic lymphocytic leukemia.

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