Mochizuki Hiroyuki, Nakamura Kenji, Sato Hirofumi, Goto-Koshino Yuko, Sato Masahiko, Takahashi Masashi, Fujino Yasuhito, Ohno Koichi, Uchida Kazuyuki, Nakayama Hiroyuki, Tsujimoto Hajime
Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Vet Immunol Immunopathol. 2011 Sep 15;143(1-2):38-45. doi: 10.1016/j.vetimm.2011.05.030. Epub 2011 Jun 23.
Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. PCR amplification of complementarity-determining region 3 (CDR3) of the immunoglobulin heavy-chain variable region (IGHV) gene can be used to assess clonality of B-cell populations as a supportive diagnostic tool for B-cell neoplasms. Because of the sequence variation and possible somatic hypermutation of the IGHV gene, sensitivity of the PCR-based assay to detect clonal IGHV gene rearrangement largely depends on the sequences and numbers of primer sets. Prior to the development of an efficient assay, we cloned and sequenced 97 IGHV complementary DNAs (48 IGHV-1 and 49 IGHV-3 clones) from normal cat spleens. On the basis of these sequences, we designed 6 forward primers at the variable region and 5 reverse primers at the joining region. Using each of 6 forward primers and a mixture of 5 reverse primers, we amplified CDR3 of IGHV genes and analyzed the PCR products by conventional PAGE and Genescan analyses using fluorescence-labeled primers. Twenty-six feline B-cell neoplasms diagnosed by histopathological and immunohistochemical examinations were subjected to the newly developed analysis of IGHV gene rearrangement. Clonal IGHV gene rearrangement was detected in 22 of 26 (84%) samples by both PAGE and Genescan analyses. To reduce the number of PCR reactions, we constructed a multiplex PCR analysis system using a mixture of IGHV-1- and IGHV-3-specific primers as forward primers and a mixture of 5 joining region reverse primers. Results of the multiplex PCR were 100% concordant with those obtained by each of the singleplex PCRs. The multiplex PCR-based assay and Genescan analysis developed in the present study would be useful and practical tools to detect clonal IGHV gene rearrangement in feline B-cell neoplasms.
淋巴样肿瘤通常根据细胞学和组织病理学检查结果进行诊断。然而，在某些情况下，区分淋巴样肿瘤与反应性淋巴样增生是困难的。免疫球蛋白重链可变区（IGHV）基因互补决定区3（CDR3）的PCR扩增可用于评估B细胞群体的克隆性，作为B细胞肿瘤的辅助诊断工具。由于IGHV基因的序列变异和可能的体细胞超突变，基于PCR的检测方法检测克隆性IGHV基因重排的灵敏度很大程度上取决于引物组的序列和数量。在开发高效检测方法之前，我们从正常猫脾脏中克隆并测序了97个IGHV互补DNA（48个IGHV-1和49个IGHV-3克隆）。基于这些序列，我们在可变区设计了6条正向引物，在连接区设计了5条反向引物。使用6条正向引物中的每一条和5条反向引物的混合物，我们扩增了IGHV基因的CDR3，并通过传统的聚丙烯酰胺凝胶电泳（PAGE）和使用荧光标记引物的基因扫描分析来分析PCR产物。对26例经组织病理学和免疫组织化学检查诊断的猫B细胞肿瘤进行了新开发的IGHV基因重排分析。通过PAGE和基因扫描分析，在26个样本中的22个（84%）中检测到克隆性IGHV基因重排。为了减少PCR反应的数量，我们构建了一个多重PCR分析系统，使用IGHV-1和IGHV-3特异性引物的混合物作为正向引物，以及5条连接区反向引物的混合物。多重PCR的结果与每个单重PCR的结果100%一致。本研究中开发的基于多重PCR的检测方法和基因扫描分析将是检测猫B细胞肿瘤中克隆性IGHV基因重排的有用且实用的工具。
