慢性髓系白血病STI571耐药与Bcr-Abl基因点突变的关系

Analysis of mutation of ATP binding domain of BCR-Abl gene in chronic myeloid leukemia resistant to STI571

目的通过Bcr-Abl激酶ATP结合区测序分析,探讨慢性髓系白血病(CML)STI571耐药与Bcr-Abl基因点突变的关系。方法将CML患者分为对STI571耐药和非耐药两组,细胞系分为STI571耐药细胞系K562/G01、野生型K562/W细胞,进行骨髓单个核细胞或细胞系细胞Abl基因ATP结合区双向测序分析,了解Abl酪氨酸激酶ATP结合区点突变情况。PCR产物的序列长度为344bp,其中的180bp(98～277bp)为ABL激酶的第906～1085bpDNA序列片段,涵盖了Abl激酶的ATP结合区序列。结果K562/G01耐药细胞ABL激酶ATP结合区DNA测序与K562/W相同,与ABL激酶原序列相比,有两个同义点突变。在STI571耐药急变的CML患者骨髓单个核细胞Abl激酶ATP结合区测序结果发现:在8例耐药急变的患者中2例患者第944位核苷酸C→T单个碱基出现点突变,使苏氨酸(Thr)突变为异亮氨酸(Ile)。1例CML-CP的患者第1070碱基A→G替换,导致357位赖氨酸(Lys)→精氨酸(Arg)。结论Bcr/Abl融合基因ATP结合区的点突变是CML中STI571耐药的重要原因之一,对指导临床用药具有重要价值。

Objective To analyze point mutation of BCR-Abl gene with chronic myeloid leukemia （CML） resistant to ST1571.Methods CML patients were divided into resistant to ST1571 and non- resistant to ST1571; cell lines were divided into resistant cell line K562/G01 and non-resistant eell line K562/W. The point mutations of ATP binding domain of ABL protein tyrosine kinase （PTK） were taken by bidirection detection of sequence of ATP binding domain of BCR-ABL gene in CML patient bone marrow mononucleareell and cells of cell lines. Length of PCR production sequencing was 344bp, 180bp （98bp-277bp）of the total was order 906bp-1085bp DNA tract, sequence of ATP binding domain of ABL kinase was included.Results Sequence of ATP binding domain of Abl kinase had two synonymous point mutations in K562/G01 and K562/W. This single nucleotide C→T change results in a substitution of threonine to isoleucine at position 315 （Thr→lle） of c-ABL kinase in two acquired ST1571-resistant patients. This single nucleotide A→G change results in a lysine to Arg substitution at position 1070 （Lys→Argl） of c-Abl kinase in a CML-CP patient. Conclusion ATP binding domain mutants of Abl kinase is one of the major mechanisms of resistance to imatinib.