To the homozygous WT peak (0 MT). T315I peak had a
To the homozygous WT peak (0 MT). T315I peak had a different peak pattern from other common mutations (Y253F, Y253H, E255K, M351T, and F359V) (data no shown). For direct sequencing, a “T” peak indicated the AUY922 solubility presence of T315I which could be clearly seen in 100 , 50 , 25 , 12.5 , and 6.25 . A “C” peak which represented the WT BCR-ABL KD allele could be seen in 50 , 25 , 12.5 , 6.25 , 3.13 , and 0 dilution (100 WT) (Figure 2B).3.3 Detection for T315I in DHPLC and sequencing positive patientsNine CML patients were tested for T315 mutation using DHPLC followed by sequencing and AS-PCR. Abnormal DHPLC patterns strongly supportive of T315I mutation were observed and all were confirmed by sequencing as shown in Figure 3A. Mutant bands were comparably seen by AS-PCR. Representative AS-PCR results of four CML cases with abnormal DHPLC and sequencing results (Patients no.360, no.461, no.504, and no. 509) are shown in Figure 3B.4. Discussion Several methods have been utilized to detect the existence of BCR-ABL KD mutations such as direct sequencing, DHPLC, restriction fragment length polymorphism (RFLP), pyrosequencing, double-gradient denaturing electrophoresis, AS-PCR, AS real-time PCR, array based assays, and high-resolution melt curve analysis (HRM), with varying sensitivity and specificity [25,26,29-35]. In this study, we established an AS-PCR-based method to detect T315I which is the most resistant genotype associated with the highest impact on clinical outcome of CML patients. The sensitivity of our AS-PCR method was better than the sensitivities reported from most previously reported detection techniques and was slightly better than DHPLC and direct sequencing analysis in our hands. By AS-PCR, T315I mutant bands were observed in the mixtures containing as low as 0.5 of mutant alleles whereas DHPLC was unable to detect the mutants below 1.56 dilution and sequencing was unable to detect below 6.25 . The detection sensitivity of DHPLC in our study was in the range of previously published articles (1-5 ) [30,31]. Although DHPLC is considered a useful tool toFigure 2 Sensitivity of T315I mutation detection by DHPLC and sequencing analysis . DHPLC chromatogram patterns generated by each mutant allele concentration are shown in Figure 2A and sequencing results corresponding to each DHPLC-generated chromatogram are shown in Figure 2B; Red arrow indicates c.947 C > T mutation; C, Wild-type; T, Mutant.screen for the presence of either known or unknown mutations, chromatograms generated from DHPLC were sometimes difficult to interpret and sequencing analysis is always needed to confirm their results. In our study, sequencing analysis was not able to detect mutant allelesWongboonma et al. Journal of Hematology Oncology 2011, 4:7 http://www.jhoonline.org/content/4/1/Page 5 ofFigure 3 Detection of T315I in CML patient samples by DHPLC, sequencing and AS-PCR; Figure 3A shows DHPLC patterns followed by sequencing analysis if a suspicious peak was observed; T315I cell lines and wild types are also shown; Figure 3B demonstrates representative AS-PCR results of four CML cases with abnormal PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 DHPLC and sequencing results (patients no.360, no.461, no.504, and no. 509) .below the 6.25 dilution. Therefore, it is the least sensitive method in our hands. Direct sequencing is recognized as a confirmation method for any screening tests because of its high specificity [25]. However, its disadvantage is its high costs and low sensitivity (15-25 ) [26], rendering.
