Molecular biological studies on the characterization of novel human endogenous retrovirus associated with autoimmune diseases.

人内源性逆转录病毒（HERV）序列分布于整个人类基因组中，可作为人类基因组研究的遗传标记。此外，HERV可能通过其启动子活性激活周围基因或产生病毒蛋白从而产生免疫复合物而参与自身免疫性疾病的发病机制。由于CpG岛的去甲基化与转录活性相关，因此去甲基化的HERV可能在体内具有转录活性。本研究采用抑制PCR方法对HERV周围基因的甲基化模式进行了研究，旨在获得HERV与自身免疫性疾病相关的信息。简而言之，用甲基化敏感性限制性内切酶HpaII消化基因组DNA，并将其连接到接头引物上。随后使用衔接子和HERV特异性引物进行PCR。作为对照，使用甲基化非依赖性酶Mspl。通过自动测序仪分析该片段。当我们比较从中性粒细胞和淋巴结中分离的DNA的结果时，MspI片段的模式是相同的，表明在HpaII/MspI RFLP方面，样品之间不存在遗传差异。相反，HpaII片段模式彼此不同，可能反映了它们之间的表观遗传差异（甲基化）。RA患者外周血和滑膜液细胞Hpa Ⅱ片段型不同。抗风湿治疗前后中性粒细胞的HpaII型也有差异。因此，抑制PCR可能是一种方便的方法，用于研究各种自身免疫性疾病中HERV周围甲基化模式的差异，

Human endogenous retroviruses (HERV) sequences are dispersed throughout the human genome, which may be useful as a genetic marker for human genome research. In addition, it is possible that HERV may contribute to the pathogenesis of autoimmune diereses, either by activating the surrounding genes via its promoter activiites or generating virus proteins and thereby yielding immune complexes. Since demethylation of CpG islands is Associated with transcriptional activities, demethylated HERV may be transcriptionally active in vivo. In the present study, we investigated the methylation pattern of genes surrounding HERV, using suppression PCR method, for the purpose of obtaining the information as to the HERV associated with autoimmune diseases. In brief, genomic DNA was digested with methylation-sensitive restriction enzyme, HpaII, and was ligated to adapter primer. PCR was subsequently performed using adapter and HERV-specific primer. As control, methylation-independent enzyme, Mspl, was used. The fragment was ananalyzed by an automated sequencer. When we compare the results between DNA isolated from neutrophils and that of lymph nodes, the pattern of MspI fragment was the same, indicating that there exists no genetic difference, in terms of HpaII/MspI RFLP, between the samples. In contrast, HpaII fragment Pattern was different from each other, presumably reflecting the epigenetic difference (methylation) between them. In RA, HpaII fragment pattern of peripheral blood was different from that of synovila fluid cells. There was also a difference of HpaII pattern of neutrophils, before and after anti-rheumatic treatments. Suppression PCR may thus be a convenient way, to study the difference of methylation pattern around HERV in various autoimmune diseases,