儿童急性T淋巴细胞性白血病T细胞受体β链基因重排的特点及其在微小残留病定量检测中的意义

Characteristics of T cell receptor beta gene rearrangements and its role in minimal residual disease detection in childhood T-cell acute lymphoblastic leukemia

目的建立以T细胞受体β链（TCRβ）基因重排为分子标志定量检测儿童急性T淋巴细胞性白血病（T—ALL）微小残留病（MRD）的实时定量聚合酶链反应（RQ—PCR）检测体系。方法应用BIOMED-2欧洲协作组设计的PCR检测体系，检测26例初发T—ALL患儿的TCRβ基因重排，并进行序列分析与比对。对其中6例患儿根据基因重排后连接区序列设计等位基因特异性寡核苷酸（ASO）引物，结合胚系TaqMan探针与引物，建立RQ—PCR检测方法，行缓解期标本MRD定量追踪检测。结果92．3％的T—ALL患儿可检测到克隆性TCRβ基因重排[84．6％具有Vβ-（Dβ）-Jβ完全重排，50％具有Dβ-Jβ不完全重排]。通过序列分析，V片段使用频率最高的家族为BV5、BV6，J区使用频率最高的家族为Jβ2．7，J1．3、J2．4、J2．6未被使用。6例患儿ASO标准曲线的斜率为-3．54-3．37，截距为19．35-20．51，相关系数〉0．98。定量范围均达到10^-4。对随访期标本MRD检测发现，复发患儿的MRD水平在复发前显著升高。结论以TCRβ基因重排为分子标志对T—ALL患儿行MRD定量检测，具有较高的敏感度和特异性，动态追踪检测随访期标本MRD水平具有重要的临床价值。

Objective To explore the characteristics of T cell receptor beta （TCRβ） gene rearrangements in children with T-cell acute lymphoblastic leukemia （T-ALL） and establish a system of quantitative detection of MRD with real-time quantitative （RQ-PCR） targeted at TCRβ gene rearrangement. Methods Multiplex polymerase chain reaction （PCR） designed by BIOMED-2 was used to detect TCRβ gene rearrangements in the bone marrow samples of 26 children with T-ALL. Sequence of junction region were then compared and analyzed in IMGT database. Allele specific oligonucleotide （ASO） upstream primers were designed complementary to the V-D-J or D-J junctional region of TCRβ gene rearrangements. Samples at diagnosis were serially diluted in DNA obtained from mononuclear cells （MNC） from a pool of 20 healthy donors to generate the patient specific standard curves. Subsequently, a TCRβ RQ- PCR assay to quantify MRD with germline Jβ primer/probe combinations was applied in six patients. To check the quantity and quality of DNA, the investigators used RQ-PCR analysis for the N-ras gene. Results Clonal rearrangements were identified in 92. 3% childhood T-ALL （ Vβ-Dβ-Jβ rearrangements in 84. 6% , Dβ-Jβ rearrangements in 50% ）. Comparative sequence analysis of 42 TCRβ recombinations revealed two downstream Vβ families （BV5, BV6） were preferentially used. The segment Jβ2. 7 in childhood T-ALL was preferentially used. Jβ1. 3, Jβ2.4, and Jβ2. 6 were not found to be used. The slope of the standard curves was from -3.54 to -3.37 and the intercepts were from 19. 35 to 20. 51. The correlation coefficients of all 6 standard curves were excellent （ 1〉 0. 98 ） . All the RQ-PCR quantitative range reached 10^-4. MRD analysis of follow up samples showed that MRD increased before relapse. Conclusion RQ-PCR analysis of TCRβ gene rearrangements was highly sensitive and specific, it will be of high value for future T-ALL MRD studies. And quantitative and serial study of MRD may be of prognostic importance.