胞苷脱氨酶基因多态性与阿糖胞苷敏感性关系的研究

目的 探讨人类胞苷脱氨酶(CDA)基因编码区单核苷酸多态性(cSNP)与儿童急性白血病(AL)患者阿糖胞苷(Ara-C)敏感性的关系.方法 采集87例AL患儿骨髓及199例对照儿童的外周血,制备cDNA,采用变性梯度凝胶电泳及DNA测序进行CDA基因cSNP分析.将人类CDA基因分别转入大肠杆菌和酵母细胞,利用高效液相色谱分析确定不同等位型CDA的催化活性;应用生长抑制实验检测酵母转化体对Ara-C处理的敏感性.结果 在对286例中国儿童进行CDA基因cSNP检测中发现了3个已知的cSNP,即79A/C(K27Q)、208G/A(A70T)和435T/C,其等位基因频率分别为12.1%、0.5%和76.2%.这些多态性与白血病的易感性无相关性.携带人类CDA70T的大肠杆菌转化体的胞苷及Ara-C脱氨活性分别较CDA70A降低53%和63%(P值均＜0.01),Ara-C对携带人类CDA70T的酵母转化体作用15 h的细胞半数抑制浓度(IC50)值[(735±31)μmol/L]也显著低于Wt转化体[(973±61)μmol/L](P＜0.05),降低25%.结论 确定了中国人CDA基因中3种cSNP以及各自的基因型分布和等位基因频率.证实了一个人类CDA208A基因型,其对Ara-C的敏感性高于CDA208G基因型,提供了一个对脱氧胞苷同系物潜在敏感的亚群.

Study on the relationship between human cytidine deaminase gene polymorphisms and Ara-C sensitivity

Objective To study the relationship between coding single-nucleotide polymorphisms(cSNPs)in the human cytidine deaminase(CDA)gene and cytosine arabinoside(Ara-C)sensitivity in childhood acute leukemia(AL).Methods cDNAs from 87 leukemia and 199 control blood samples were analyzed for the cSNPs in CDA by PCR-denaturing gradient gel electrophoresis(DGGE)and sequencing.Human CDA genes were transformed into E.coli and yeast,respectively.Catalytic activities of the allele CDA and variant CDAs were determined by HPLC assay.The Ara-C sensitivity of the yeast transformants Was messured by growth inhibition assays.Results Three known different polymorphisms,namely,79A/C(K27Q),208G/A(ATOT)and 435T/C(silent)were identified in the coding msion of CDA from an investigated Chinese population and displayed allelie frequencies of 12.1%,0.5%and 76.2%,respectively.No association with susceptibility to disease Was observed.Compared with that of CDA70A,the deamination activities for cytidine and Am-C substrates of the E.coli transformants carrying human CDA70T were decreased by 53%and 63%,respectively(P＜0.01),and the Ara-C IC50 value of the yeast transformants was also significantly decreased by 25%[(973±61)μmol/L to(735±31)μmol/L,P＜0.05].Conclusions The 3 known cSNPs and their allelic frequencies of CDA are identified in a Chinese childhood AL.The 208A genotype is shown to be more sensitive to Ara-C than 208G genotype.