Establishment of a method to quantitatively detect FLT3 internal tandem duplication in acute myeloid leukemia with denaturing high-performance liquid chromatography

目的:建立一种应用变性高效液相色谱技术(dHPlC)相对定量检测急性髓细胞白血病(AMl)患者fMS样酪氨酸激酶3(flT3)基因的内部串联重复(ITd)突变的方法。方法:根据flT3-ITd突变基因多位于14外显子而设计引物,用聚合酶链反应(PCr)方法特异性扩增121例AMl患者flT3-ITd突变基因,再用dH-PlC技术相对定量检测flT3-ITd等位基因突变的情况;与毛细管电泳法(CE)检测突变的结果对比进行该方法的有效性检验;最后与121例样品PCr扩增产物的测序结果进行对比。结果:经dHPlC分析后均能得到特征性的洗脱峰。121例样本中检测到flT3-ITd突变阳性的样本13例,总阳性率为10.7%,阳性突变等位基因的比例不一,分布范围中位数为34.5%(11.4%-80.2%),为21-87 bP单个插入片段。阳性率和突变比例与CE方法检测结果相比较均无显著差异(P>0.05),并与121例样本flT3-ITd扩增PCr产物基因测序结果一致。结论:成功建立了一种应用dHPlC相对定量检测AMl患者flT3-ITd基因突变的方法。AIM: To establish a relatively-quantitative method to detect the internal tandem duplication(ITD) mutation of Fms-like tyrosine kinase 3(FLT3)gene in acute myeloid leukemia(AML) patients using denaturing high-performance liquid chromatography(DHPLC).METHODS: According to the fact that much more FLT3-ITD mutations are located in exon 14,we designed the primers,and use the method of polymerase chain reaction(PCR) to specifically amplify FLT3-ITD mutation gene in 121 cases of AML,and relatively quantified the situation of mutant allelic gene of FLT3-ITD by the method of DHPLC.The effectiveness of DHPLC was verified by the method of capillary electrophoresis(CE).The sequenced results from PCR amplified products of 121 samples were compared.RESULTS: A characteristic of elution peak was detected by DHPLC with 10.7% overall positive rate(13/121) and varied in the proportion of mutant alleles,with a single duplicated insert fragment from 21 bp to 87 bp.The median range of mutant alleles was 34.5%(11.4%-80.2%).No significant difference of the positive rates and mutation proportions between the results with DHPLC and the results with CE method was observed.The results of FLT3-ITD mutant gene of 121 samples were consistent with the results using sequencing method.CONCLUSION: A relatively-quantitative method to analyze AML patients with FLT3-ITD mutation by DHPLC is successfully established