儿童急性淋巴细胞白血病IgH基因重排的定量检测研究  

Quantitative determination of immunoglobulin heavy chain gene rearrangements in childhood acute lymphoblastic leukemia

作　　者：崔蕾[1] 李志刚[1] 高超[1] 吴敏媛[1] 

机构地区：[1]首都医科大学附属北京儿童医院血液病中心,100045

出　　处：《中华检验医学杂志》2007年第10期1119-1122,共4页Chinese Journal of Laboratory Medicine

基　　金：北京市科技计划基金(D0905001040431);北京市科技新星计划基金(2005806)

摘　　要：目的建立免疫球蛋白重链(IgH)的实时定量(RQ)-PCR 检测体系,以用于儿童急性淋巴细胞白血病(ALL)微小残留病(MRD)检测。方法采用定性 PCR 筛选109例儿童 B 细胞 ALL(B-ALL)IgH 基因单克隆重排,根据连接区序列设计等位基因特异性引物,应用 RQ-PCR 技术,采用胚系 Taqman 探针与引物,在41例患儿中建立 RQ-PCR 定量检测方法,并分析其敏感度、特异性等。结果109例儿童 B-ALL 初诊标本中 IgH 单克隆重排有48例,经测序分析发现,V、D、J 片段频率最高的分别为 V3家族、D3家族和 J4家族。RQ-PCR 方法扩增48例 IgH 单克隆重排的初诊 DNA,其中41例可重复敏感度和最大敏感度均≤10^(-4),非特异性扩增的循环阈值(Ct 值)均>40,与特异性扩增 Ct值的差距均>3。标准曲线斜率均值为-3.31±0.19,截距均值为37.66±1.23,相关系数均为0.97以上。结论以 IgH 基因重排为靶分子,采用 RQ-PCR 方法和胚系探针策略检测儿童 B-ALL,敏感性≤10^(-4),并具有较高的特异性,适于 MRD 的定量研究。Objective To establish the methodology for quantitative detection of immunoglobulin heavy chain （ IgH ） gene rearrangements in childhood acute lymphoblastic leukemia （ALL） by real-time quantitative polymerase chain reaction（RQ-PCR）. Methods Clonal IgH gene rearrangements in 109 B cell acute lymphoblastic leukemia （B-ALL） patients at diagnosis were identified by multiplex PCR assay followed by sequencing. Allele specific oligonucleotide （ASO） primers were designed complementary to the junctional region. Germline JH TaqMan probe and primer was used to establish RQ-PCR for determination of IgH gene rearrangements in 41 childhood B-ALL The sensitivity and specificity were assessed as well. Results IgH rearrangements was identified in 48 cases out of 109 patients with B-ALL The sequence analysis showed that the most frequently used V, D, J segments were from V3 family, D3 family and J4 family, respectively. 41 cases were repeatable among 48 cases. Both sensitivity and specificity was less than 10^-4. The Ct values are more than 40 in all nonspecific amplification. The difference is more than 3 compared with that in specific amplification. The mean slope of the standard curves was -3.31 ±0. 19 and the mean intercept was 37.66 ± 1.23. The correlation coefficients of all standard curves were above 0. 97. Conclusions RQ-PCR analysis of IgH gene rearrangements is highly sensitive （ ≤ 10^-4 ） and specific. It is applicable to detect MRD in childhood ALL.

关 键 词：白血病 淋巴细胞 急性 肿瘤 残余 免疫球蛋白类 重链 基因重排 

分 类 号：R733.7[医药卫生—肿瘤]