IgH不同区域基因重排引物的分析及在石蜡淋巴瘤诊断中的应用

Primers for detecting gene rearrangement in different regions of immunoglobulin heavy chain genes and their application in diagnosis of paraffin-embedded lymphoma tissues

目的通过生物信息学方法分析并优化免疫球蛋白重链(IgH)不同框架(FR)区域基因重排的引物,探索其对石蜡包埋的淋巴瘤组织基因重排检测中的应用。方法通过Clustal W软件比较44条有效的IgH可变区和6条J区的基因片段,选取3对(IgHFR1、FR2、FR3区各一对,分别为P1c,P2A,P31)IgH基因重排引物作为B细胞基因重排引物。另选一对TCRγ引物作为T细胞基因重排引物。通过PCR扩增,检测经形态学及免疫组织化学确诊的101例(80例B细胞淋巴瘤、14例T细胞淋巴瘤和7例淋巴结反应性增生组织)石蜡包埋组织标本的基因重排状况。以DG75、Jurkat淋巴瘤细胞系DNA作为B和T细胞淋巴瘤基因重排的阳性对照,以反应性增生组织DNA作为阴性对照。结果IgHFR1、FR2和FR3区域引物对80例B细胞淋巴瘤的阳性检出率分别为37.5%(30/80)、52.5%(42/80)和70.0%(56/80),在14例T细胞淋巴瘤中均为7.1%,三者的检出率两两之间差异有显著性;IgHFR3区和IgHFR2区引物的组合可将其检出率提高到83.9%。以上引物在7例反应性淋巴结中均未检出阳性。结论IgH不同区域引物比较,FR3区引物检出率明显高于FR1区和FR2区。FR3区和FR2区引物联合检测可明显提高石蜡组织中B细胞淋巴瘤基因重排的检出率。

Objective To analyze and optimize the gene rearrangement primers of different frame regions （FR） of immunoglobulin heavy chain （IgH） genes by bioinformatic methods and explore the application of these primers in the detection of paraffin-embedded lymphoma tissues. Methods Three pairs of primers from IgH FR1, FR2 and FR3 regions （Plc, P2A and P31, respectively） were selected as the B cell gene rearrangement primers after comparison of the gene fragments in 44 IgH variable and 6 joining regions. Using one pair of T cell receptor （TCR） γprimer as the T cell gene rearrangement primer, 101 histopathologically confirmed lymphoproliferative samples including 80 B cell lymphomas, 14 T cell lymphomas, and 7 reactive proliferative lymph nodes were examined by PCR for gene arrangement. The DNAs from DG75 and Jurkat cell lines were used as the positive controls for B and T cell lymphoma, respectively, with those from reactive proliferative lymph nodes as the negative control. Results The positivity rates of IgH primers （Plc, P2A and P31） in the 80 B cell lymphomas were 37.5% （30/80）, 52.5% （42/80） and 70.0% （56/80）, respectively, and only one of the 14 T cell lymphoma cases was positive for the primers, suggesting significant differences in the detection rates of B cell lymphomas by the 3 primers. The detection rate was increased to 83.9% by combining the results by P31 and P2A primers. No positivity was found in the proliferative reaction tissues. Conclusion Primers from IgH FR3 region genes are more sensitive than that from the FR1 and FR2 regions in the detection of gene rearrangement in paraffin-embedded lymphoma tissues. The detection rates can be increased by combining the results with the primers for IgH FR3 with that of FR2.