白血病患者FLT3基因第二酪氨酸激酶结构域点突变分析

目的研究FLT3基因酪氨酸激酶结构域(TKD)点突变与急性白血病的关系及临床意义。方法采用PCR结合限制性内切酶酶切及序列测定,检测143例急性髓系白血病(AML)、25例急性淋巴细胞白血病(ALL)、2例急性杂合细胞白血病(AHL)、17例骨髓增生异常综合征(MDS)和7例慢性粒细胞白血病急变期(CMLBC)患者骨髓单个核细胞中FLT3基因外显子20中的TKD点突变,分析其临床相关性。结果143例AML患者中9例(6.3%)存在FLT3TKD点突变(FLT3TKD+),阳性率显著低于FLT3基因内部串联重复(ITD)突变(25.9%,P<0.01)。FLT3TKD+存在于AMLM2(3/53)、M3(3/40)、M5(2/23)、M6(1/2)亚型中。2例FLT3-TKD+患者同时存在FLT3-ITD突变,M2、M3各1例。在25例ALL、2例AHL、17例MDS和7例CMLBC患者中未检测到FLT3TKD+。测序分析显示TKD点突变累及密码子D835,未改变FLT3的开放式阅读框架,为错义突变。D835的第1个核苷酸G被T替换,即D835Y。FLT3-TKD+组和FLT3-TKD组在性别、年龄组成、白细胞计数、骨髓原始细胞比例及化疗缓解率方面差异无统计学意义。结论AML患者中FLT3-TKD点突变的阳性率显著低于FLT3ITD。TKD点突变累及密码子D835,为错义突变。TKD点突变可单独存在,也可与ITD同时存在。与FLT3-ITD相比,TKD点突变与高白细胞无关。

Detection of point mutation at second tyrosine kinase domain of FLT3 gene in acute myeloid leukemia

OBJECTIVE: To evaluate the prevalence of a novel FLT3 activating mutation in tyrosine kinase domain (TDK) in acute leukemia patients and its clinical implication. METHODS: Genomic DNA from bone marrow mononuclear cells of 143 cases of acute myeloid leukemia (AML), 25 acute lymphocytic leukemia (ALL), 2 acute hybrid leukemia (AHL), 17 myelodysplastic syndromes (MDS) and 7 chronic myelogenous leukemia in blast crisis (CML-BC) was screened by polymerase chain reaction (PCR) and gel electrophoresis for FLT3-TKD point mutations. RESULTS: Among AML patients, FLT3-TKD point mutation (FLT3-TKD(+)) rate was 6.3% (9/143), an incidence significantly lower than that of internal tandem duplication (ITD) mutation (37/143, 25.9%, P < 0.01). According to the FAB classification, FLT3-TKD point mutation was found in 3/53 of M(2), 3/40 of M(3), 2/23 of M(5) and 1/2 of M(6) subtypes. Two patients showed both ITD and TKD mutations. No FLT3-TDK mutation was found in 25 ALL, 2 AHL, 17 MDS and 7 CML-BC patients. Sequence analysis showed that one of the mutations occurred at codon 835 of FLT3. The substitution of the first nucleotide T for G of D835, resulted in D835Y, a missense mutation. The presence of TKD mutations was related neither to age, sex, nor to WBC counts, the marrow blast percentages, and CR rates for induction therapy. CONCLUSION The incidence of FLT3-TKD(+) is significantly lower than that of ITD mutation. FLT3-TKD mutations may occur alone or together with ITD mutation. In contrast to FLT3-ITD mutation, TKD mutations were not associated with leukocytosis or low complete remission rate.