王欣, 张旭东, 陈清江, 王冠男, 胡俊霞, 吴少璇, 马咪静, 尹美凤, 杨万秋, 董萌, 丁梦杰, 张明智, 朱利楠. NK/T细胞淋巴瘤基因组中EBV DNA整合检测及分析[J]. 中国肿瘤临床, 2018, 45(23): 1194-1200. DOI: 10.3969/j.issn.1000-8179.2018.23.175
引用本文: 王欣, 张旭东, 陈清江, 王冠男, 胡俊霞, 吴少璇, 马咪静, 尹美凤, 杨万秋, 董萌, 丁梦杰, 张明智, 朱利楠. NK/T细胞淋巴瘤基因组中EBV DNA整合检测及分析[J]. 中国肿瘤临床, 2018, 45(23): 1194-1200. DOI: 10.3969/j.issn.1000-8179.2018.23.175
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Wang Xin, Zhang Xudong, Chen Qingjiang, Wang Guannan, Hu Junxia, Wu Shaoxuan, Ma Mijing, Yin Meifeng, Yang Wanqiu, Dong Meng, Ding Mengjie, Zhang Mingzhi, Zhu Linan. Detection and analysis of EBV DNA integration in NK/T cell lymphoma genome[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2018, 45(23): 1194-1200. DOI: 10.3969/j.issn.1000-8179.2018.23.175
Citation: Wang Xin, Zhang Xudong, Chen Qingjiang, Wang Guannan, Hu Junxia, Wu Shaoxuan, Ma Mijing, Yin Meifeng, Yang Wanqiu, Dong Meng, Ding Mengjie, Zhang Mingzhi, Zhu Linan. Detection and analysis of EBV DNA integration in NK/T cell lymphoma genome[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2018, 45(23): 1194-1200. DOI: 10.3969/j.issn.1000-8179.2018.23.175
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NK/T细胞淋巴瘤基因组中EBV DNA整合检测及分析

Detection and analysis of EBV DNA integration in NK/T cell lymphoma genome

    摘要
  • 摘要:
      目的  明确NK/T细胞淋巴瘤（NK/T cell lymphoma，NKTCL）基因组中是否存在EB病毒（Epstein-Barr virus，EBV）DNA整合，初步分析NKTCL细胞系基因组中EBV DNA整合信息。
      方法  利用PCR法扩增EBV DNA和原位杂交法检测EBER表达，验证由郑州大学第一附属医院生物样本库提供的5例EBV（+）及4例EBV（-）NK/T样本EBV感染情况。测序全基因组DNA样本并进行生物信息学分析。利用全基因组序列比对捕获EBV整合序列；使用Blast比对样本EBV fasta文件与EBV fasta库。采用CREST软件提取softclip reads，过滤paired reads并对过滤后的reads进行染色体分布的统计。使用IGV比对染色体部分区域reads分布情况，采用PCR法扩增EBV DNA高频整合区域并行sanger测序。
      结果  5例EBV（+）NK/T样本中均检测出EBV DNA和EBER表达，4例EBV（-）NK/T样本则未能检出。样本的测序深度、覆盖深度、覆盖率和比对率均满足后续研究要求。比对结果显示捕获的序列为病毒序列。EBV（+）NKTCL细胞系SNK、YTS和EBV（+）鼻腔NTKCL组织的reads数目最多，且在2号染色体上呈非随机性富集。chr2：30234084-30234483 400 bp区域存在EBV DNA整合，并导致chr2p23.1位点的插入缺失。
      结论  EBV（+）NKTCL细胞在chr2p23.1位点存在EBV DNA的高频整合，提示可能影响相关基因表达。

     

    Abstract:
      Objective  To investigate the presence of integrated Epstein-Barr virus (EBV) DNA in the NK/T cell lymphoma (NKTCL) genome and analyze the integration information in the genome of NKTCL cell lines.
      Methods  PCR and in situ hybridization were used to detect EBV infection in five EBV (+) NK/T samples and four EBV (-) NK/T samples provided by the biobanks of the First Affiliated Hospital of Zhengzhou University. Whole-genome DNA of the samples was sequenced and subjected to bioinformatics analysis. Whole-genome sequence alignment was used to identify the EBV integration sequence. BLAST analysis was used to compare EBV fasta files of the samples and EBV fasta library. CREST software was used to extract softclip reads, filter all paired reads, and enumerate their distribution on chromosomes. The integrated genomics viewer (IGV) was used to compare the distribution of reads in partial regions of chromosome. PCR was used to amplify the high-frequency integration region of the EBV DNA. The amplified fragments were sanger sequenced.
      Results  EBV DNA and EBER expression were detected in five EBV (+) NK/T samples but not in the four EBV (-) NK/T samples. Sequencing depth, coverage depth, proportion of coverage, and proportion of alignment all met the requirements for subsequent research. Sequence alignment revealed that the captured sequences were viral sequences. Filtered reads were most numerous in EBV (+) NKTCL cell line SNK, YTS, and EBV (+) nasal NKTCL tissue. The reads were non-randomly enriched in chromosome 2. EBV DNA integration in the 400 bp region of chr2:30234084-30234483 caused insertion or deletion in the chr2p23.1 site.
      Conclusions  EBV DNA is highly integrated in the chr2p23.1 site of EBV (+) NKTCL cells and may affect the expression of related genes.

     

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