EB病毒即刻早期BRLF1基因在淋巴瘤组织中多态性研究（英文）

Polymorphism Research on Epstein-Barr virus Immediate-early Gene BRLF1 in Lymphomas

目的:EB病毒(Epstein-Barr virus,EBV)感染宿主细胞包括潜伏期和裂解期,它的即刻早期基因BRLF1编码产物Rta蛋白是EBV从潜伏期到裂解期的关键性调节因子。目前,Rta蛋白已成为治疗EBV相关肿瘤的靶蛋白,但有关BRLF1基因在EBV阳性淋巴瘤中的研究甚少,本研究旨在明确EB病毒即刻早期基因BRLF1在EB病毒阳性淋巴瘤组织中的变异规律,探讨其变异意义。方法:原位杂交筛选EB病毒阳性淋巴瘤(BL),提取EB病毒DNA。PCR结合DNA测序检测EBV阳性淋巴瘤中BRLF1基因多态性,应用DNAStar软件对序列进行对比分析。结果:共筛选出27例EBV阳性淋巴瘤标本,在27例BL阳性标本中成功扩增BRLF1基因,与B95-8标准序列比对分析,共发现20处无义突变和19处有义突变。8例属于BR1-A亚型,18例属于BR1-C亚型,1例属于BR1-E亚型,其中以BR1-A亚型和BR1-C亚型最为常见,分别为29.6%(8/27)、66.7%(18/27)。与EBVaGC、NPC和TW相比,4种人群BRLF1亚型的分布不同,其中BR1-A亚型更多出现在健康人群中,BR1-C亚型更多出现在NPC和BL中。在Rta蛋白功能区中,二聚体区域呈高度保守,在DNA结合区域和反式激活区域均检测出序列突变,16种CTL抗原表位中,只有NAA、QKE和ERP表位发生改变,且NAA和QKE在健康人群中突变率较高。结论:山东地区EB病毒阳性淋巴瘤BRLF1基因变异类型主要为BR1-A亚型和BR1-C亚型。BR1-C亚型可能与BL相关,发生在Rta蛋白DNA结合区域和反式激活区域的突变可能对其功能产生影响,多数CTL表位序列保守提示BRLF1可以用作EBV相关淋巴瘤免疫治疗的靶基因。

Objective： Epstein-Barr virus （EBV） infected the host cells eluding the latency and lytic phase. The Rta protein coded by the immediate early gene BRLF1 of EBV plays a key role in the swtich from latency to lytic. At present, the Rta protein has become a target for treating EBV associated tumors. However, little is know about the study of BRLFlgene in EBV-positive lymphomas. This study aimed to clear variation of BRLF 1 gene in EBV-positive lymphoma tissues, and to explore the significance of variation. Methods： In situ hybridization with EBV-encoded small RNA 1 （EBER1） was used to screen EBV positive lymphomas, and then extract the DNA of EBV, and then amplify DNA fragments by PCR and sequence it. Finally, the DNAStar software was used to analyze and compare the sequencing results. Results： A total of 27 EBV positive lymphoma specimens were screened. The sequence of BRLF1 gene was successfully amplified in 27 BLs, compared with that of prototype B95-8, 19 loci were found to be affected by amino acid changes, 20 loci by silent nucleotide changes. 8 specimens belonged to BR1-A subtype, 18 specimens belonged to BR1-C subtype, only 1 specimn belonged to BR1-E subtype. The subtype BR1-A and BR1-C were the most universal, being detected in 29.6 %（8/27）, 66.7 %（18/27） of samples, respectively. Compared with EBVaGCs, NPCs and TWs, the distributions of these two subtypes among four types of specimens were significantly different. The subtype BR1-A preferentially existed in healthy donors, while BR1-C was seen more in biopsies of nasopharyngeal carcinomas and BLs. Among three functional domains of Rta, the dimerization domain of Rta showed a stably conserved sequence, while DNA binding and transactivation domains were detected to have multiple mutations. Three of sixteen CTL epitopes, NAA, QKE and ERP, were affected by amino acid changes and NAA, QKE changes were observed more in healthy donors. Conclusion： The BR1-A and BR1-C are the dominant subtypes of BRLF1 in EBV positive lymphomas from Shandong region, and BR1-C may associate with BLs. Mutations in the DNA binding and transactivation domains of Rta may affect its functions. Conservative nature of most CTL epitopes of Rta suggested that BRLF1 gene could be used as the potential target gene in the immunotherapy of EBV-associated lymphomas.