外套细胞淋巴瘤bcl-1/IgH基因重排的检测与序列分析

Detection and analysis of bcl-1/IgH rearrangement in mantle cell lymphoma

目的 :寻找一种敏感、特异而又快捷的方法检测外套细胞淋巴瘤中 bcl- 1/ Ig H 基因重排 ,并分析 bcl- 1/Ig H 基因重排产物的序列 ,以了解 bcl- 1/ Ig H 基因重排发生的确切机理。方法 :对 2 8例经临床确诊的外套细胞淋巴瘤的新鲜冰冻组织 ,通过半巢式 PCR检测 bcl- 1/ Ig H基因重排 ,并对 bcl- 1/ Ig H基因重排产物进行克隆和序列分析。结果 :通过半巢式 PCR测到有 bcl- 1/ Ig H基因重排者计 17/ 2 8例。而采用一步法 PCR仅 9例有此基因重排 ,两者相比 ,差异显著 (χ2 =4 .59,P<0 .0 5)。对 bcl- 1/ Ig H基因重排产物进行序列分析后发现 ,bcl- 1/ Ig H基因重排产物为 74～ 16 2 bp大小 ,融合基因连接区为 6～ 2 4 bp大小。在断裂点集中的 6 5bp范围内 ,找到 10个不同的断裂点 ,其中 5个未见文献报道。对 JH 基因序列的分析 ,发现 bcl- 1/ Ig H 基因重排时 ,JH1,JH3,JH4 ,JH5和 JH6都可能参与重排 ,其中 ,以 JH4多见 ,而 JH2则没有涉及。结论 :该半巢式 PCR方法是检测外套细胞淋巴瘤中 bcl- 1/ Ig H基因重排的一种敏感而特异的方法 ,对外套淋巴细胞淋巴瘤的明确诊断和治疗都有一定的指导意义 ,而对 bcl- 1/Ig H基因重排的序列分析 ,则将为外套细胞淋巴瘤的的发病机理研究提供理论依据。

Objective: To investigate the specificity and sensitivity of the PCR technique in the identification of bcl 1/IgH gene rearrangement in mantle cell lymphoma(MCL) and to characterize the bcl 1/IgH junction DNA sequences. Methods: A semi nested PCR method to amplify bcl 1/IgH gene rearrangement in DNA from fresh frozen lymphonode specimen was established. Twenty eight cases of mantle cell lymphoma were analyzed for the presence of bcl 1/IgH gene rearrangement. The rearrangement products was cloned and sequenced to recognize the junction sequences, the breakpoints in the bcl 1 region, and J H gene involved in the rearrangement. Results: A bcl 1/IgH gene rearrangement was detected in 17 out of 28 cases of MCL, while only 9 cases was detected with single step PCR method ( χ 2 =4.59, P <0.05). The rearrangement product varied in size between 74 to 162 base pairs, and the length of the junction sequences ranged from 6 to 24 base pairs. Ten different bcl 1 breakpoints were clustered within 65 base pair spans, among which, 5 breakpoints (located at 430, 440, 451, 486 and 492) were never reported by other authors. The most common J H gene segments utilized in the translocation were J H 4(8/18), then J H 5(3/18), J H 6(2/18), J H 4/5(2/18), J H 1(2/18), and J H 3(1/18). Conclusion: These results indicate that the semi nested PCR is a specific and sensitive method for the detection of bcl 1/IgH gene rearrangement in mantle cell lymphoma, which has implications for both the diagnosis and clinical management of mantle ccell lymohoma. The recognization the potential mechanism of bcl 1/IgH gene rearrangement will help us to know the exact pathogenic mechanisms of MCL.