BIOMED-2引物系统检测急性淋巴细胞白血病患者免疫球蛋白基因重排

摘要

Objective To investigate the sensitivity of BIOMED-2 primer system in adult acute lymphoblastic leukemia (AIJ,) patients Ig gene rearrangement, and to analyze their frequency, corearrangement pattern, utilization of V, D and J genes and composition of junctional regions. Methods Amplification of rearranged IgH (complete and incomplete), IgK, IgK-Kde and IgL was performed in standard PCR in 29 adult ALL patients. Monoclonal PCR products were subjected directly to DNA sequencing. Sequences were identified by comparison with all known human Ig germline sequences to analyze the recombination patterns, somatic mutations and germline gene segments usage. Results IgH, incomplete IgH, IgK, igK-Kde and Igl, rearrangements were found with positive rate of 70.8%, 12.5% , 29.2% , 25.0% and 0 of B-ALL patients, respectively. All B-ALL patients displayed at least one pattern of Ig gene rearrangements. In TALL, one of five patients was found with incomplete IgH rearrangement, two patients were found with IgK rearrangements and two patients were PCR-negative. The sequence analysis showed that the most frequently used V, D, J segments in adult B-ALL patients were from VH3/VH4 families, DH3 family and JH6 family, respectively. Four of five IgK rearrangement used VκI family. 23.5% B-ALL IgH contained scattered replacement mutations with replacement to silent substitution ratio < 1 in complementarity determining regions. Conclusion BIOMED-2 multiplex PCR analysis strategy is a reliable and useful technique in the adult BALL patients.%目的 探讨BIOMED-2引物系统检测成人急性淋巴细胞白血病(ALL)患者Ig基因重排的敏感性,分析Ig基因重排方式、各胚系基因的利用频率等.方法 采用BIOMED-2引物系统扩增29例成人ALL患者Ig重链(IgH)和Ig轻链(IgL)重排基因.将PCR产物直接测序,使用IMGT/V-QUEST等生物信息资源分析B细胞-ALL(B-ALL)患者IgH和IgL基因重排类型、胚系基因片段利用及体细胞突变情况.结果 24例B-ALL患者中IgH完全重排阳性率为70.8%,不完全重排为12.5%,IgK VH-JH重排为29.2%,IgK删失元件(IgK-Kde)重排为25.0%,未检测出IgL阳性重排.所有B-ALL患者Ig基因重排检出率可达100%.5例T细胞-ALL(T-ALL)患者1例检测到IgH不完全重排阳性,2例IgK VH-JH重排阳性,2例未检测出Ig基因重排.B-ALL中IgH重排优先利用的V、D、J家族分别为VH3和VH4、DH3和JH6.5例IgK重排中4例利用了 Vκ1家族.重排基因中发生替代突变的基因占23.5%.替代突变零星散布于Ig基因全长,互补决定区中替代突变与静寂突变的比例<1.结论 BIOMED-2引物系统可以检测出绝大多数B-ALL患者的Ig基因重排,国内的临床检测资料亦然.这是一种有效的检测工具,为定量分析微小残留病(MRD)水平提供了基础资料.