应用YAC探针进行荧光原位杂交检测慢性粒细胞白血病BCR基因重排

YAC PROBE DETECTION OF REARRANGEMENT OF BCR GENE IN CHRONIC MYELOID LEUKEMIA BY FLUORESCENCE IN SITU HYBRIDIZATION

为检测慢性粒细胞白血病（ＣＭＬ）中ＢＣＲ基因重排，采用酵母人工染色体（ＹＡＣ）ＤＮＡ的Ｉｎ－ｔｅｒ－Ａｌｕ－ＰＣＲ产物为探针进行荧光原位杂交（ＦＩＳＨ）研究了１０例ＣＭＬ，其中包括初诊患者２例，ＣＭＬ急变并接受化疗２例，α干扰素治疗２例，自体骨髓移植术（ＡＢＭＴ）后３例和Ｐｈ染色体阴性ＣＭＬ１例。同时进行细胞遗传学和ＲＴ－ＰＣＲ检测。结果：９例ＣＭＬ的４６％～１００％的可分析核型显示ｔ（９；２２）易位，其中携带ｔ（９；２２）细胞最少者为１例自体骨髓移植术后８个月的患者，其６４％的分裂相存在ｔ（９；２２），３６％为正常核型；１例Ｐｈ（－）ＣＭＬ未见ＢＣＲ基因易位，而ＲＴ－ＰＣＲ（＋），提示ＡＢＬ基因片段插入２２ｑ１１，造成隐匿性Ｐｈ染色体。结果表明：应用ＹＡＣ探针进行原位杂交的定位明确。ＦＩＳＨ检测微小残留病（ＭＲＤ）比常规细胞遗传学方法更敏感，而且可以完成ＰＣＲ方法不易进行的定量分析。

To detect the rearrangement of BCR gene, 10 patients with chronic myeloid leukemia(CML) including 2 cases at diagnosis, 2 cases in blastic crisis with chemotherapy, 2 cases treated with IFN α, 3 cases after autologous bone marrow transplantation (ABMT) and one case with Ph negative CML were analysed by fluorescence in situ hybridization (FISH) using a DNA probe made by Inter Alu PCR from a yeast artificial chromosome correlation BCR gene. Cytogenetics and RT PCR were simultaneously performed. 9 out of 10 demonstrated t(9;22) in 64%-100% of evaluated metaphases. The least for carrying cells with t(9;22), 8 months after ABMT, showed the presence of chimerism of 64% with t(9;22) while 36% had no rearrangement of BCR gene; one case, Ph negative CML, lacked translocation of BCR genes, but was identified with masked Ph chromosome after RT PCR(+). This BCR ABL fusion is the result of a submicroscopic insertion translocation of ABL into 22q11. The results indicate that rearrangement of BCR genes can be specifically detected with YAC probe through metaphase FISH, which is more sensitive and quantifiable than conventional cytogenetic and PCR methods respectively for MRD examination.