A single-tube allele specific-polymerase chain reaction to detect T315I resistant mutation in chronic myeloid leukemia patients

Wanwisa Wongboonma,Chirayu U Auewarakul,Wanna Thongnoppakhun

doi:10.1186/1756-8722-4-7

Wanwisa Wongboonma, Chirayu U Auewarakul + Show 1 more

Open Access

https://doi.org/10.1186/1756-8722-4-7

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Abstract

BackgroundBCR-ABL kinase domain (KD) mutation is the major mechanism contributing to suboptimal response to tyrosine kinase inhibitors (TKI) in BCR-ABL-positive chronic myeloid leukemia (CML) patients. T315I mutation, as one of the most frequent KD mutations, has been shown to be strongly associated with TKI resistance and subsequent therapeutic failure. A simple and sensitive method is thus required to detect T315I mutation at the earliest stage.MethodsA single-tube allele specific-polymerase chain reaction (AS-PCR) method was developed to detect T315I mutation in a mixture of normal and mutant alleles of varying dilutions. Denaturing high performance liquid chromatography (DHPLC) and direct sequencing were performed as a comparison to AS-PCR.ResultsT315I mutant bands were observed in the mixtures containing as low as 0.5-1% of mutant alleles by AS-PCR. The detection sensitivity of DHPLC was around 1.5-3% dilution whereas sequencing analysis was unable to detect below 6.25% dilution.ConclusionA single-tube AS-PCR is a rapid and sensitive screening method for T315I mutation. Detection of the most resistant leukemic clone in CML patients undergoing TKI therapy should be feasible with this simple and inexpensive method.

Highlights

BCR-ABL kinase domain (KD) mutation is the major mechanism contributing to suboptimal response to tyrosine kinase inhibitors (TKI) in BCR-ABL-positive chronic myeloid leukemia (CML) patients
Failure to respond to imatinib developed in some CML patients as a result of resistant mutations arising in the BCR-ABL kinase domain (KD), leading to shortened survivals of CML patients with these mutations as contrasted to those without [6,7,8,9,10,11]
Detection of T315I mutation in dilution mixtures by allele specific-polymerase chain reaction (AS-PCR) AS-PCR was designed to detect T315I mutations using the Complementary DNA (cDNA) templates synthesized from RNA with known percentages of the mutant allele

Summary

Introduction

BCR-ABL kinase domain (KD) mutation is the major mechanism contributing to suboptimal response to tyrosine kinase inhibitors (TKI) in BCR-ABL-positive chronic myeloid leukemia (CML) patients. Failure to respond to imatinib developed in some CML patients as a result of resistant mutations arising in the BCR-ABL kinase domain (KD), leading to shortened survivals of CML patients with these mutations as contrasted to those without [6,7,8,9,10,11]. The majority of mutations in imatinib-resistant patients usually occurred within the nine amino acid positions of KD including G250E, Y253H/F, E255K/V, T315I, M351T, F359V, and H396 with varying sensitivities to TKI [17,18,19,20,21]. Screening for T315I mutations is recommended for all CML patients undergoing TKI treatment and should be performed as early as possible to detect the lowest levels of the mutant clone [24,25]

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