Identification of the genes for human diseases by the nucleotide analyses in wide range and developing the model mouse for them

基于我们已有的结果，我们认为人类白细胞抗原区域(跨度为1.8Mb)位于6p21的短臂。3在人类6号染色体已经被高精度测序的基础上，我们试图找出两种人类疾病(Bechet病[BA]和银屑病[PA])的候选基因，并建立这两种疾病的动物模型。以下是我们在过去三年中取得的成就。遗传分析显示，PA的致病区域位于人类白细胞抗原C基因座端粒两侧89~143kb的范围内。在该区域，有1个已知基因和4个新基因在皮肤中表达。测序结果表明，这4个新基因中有1个表现出明显的多态性。该基因被命名为“SEEK1”，并被发现在患有PA2的皮肤中过度表达。对于BD的候选基因，人类白细胞抗原B基因本身或其邻近的MICA或MICB基因被认为是最好的候选基因。然而，最近的遗传分析表明，人类白细胞抗原-B51基因是BD.3的致病基因。我们获得了几个携带SEEK1基因30kb片段的转基因(TG)株系。目前正在对这些线路进行进一步分析。我们已经建立了携带MICA或MICB基因的TG株，就在决定人类白细胞抗原B51基因是BD的致病基因之前。这些MICA或MICB TG系表现出几种异常，如白细胞和体重减少，皮肤角化过度，提示这些基因可能参与了BD.5的发病机制。我们还建立了携带人类白细胞抗原-B51和人类b2-微球蛋白基因的转基因小鼠，发现在这些小鼠中，细胞表面的人类白细胞抗原-B51分子的表达大大增加。利用B2M基因敲除小鼠成功培育出携带人B2M和人类白细胞抗原B51基因，但缺乏小鼠B2M基因的小鼠。

Based on our previous achievement that HLA region (spanning 1.8 Mb) positioned at the short arm of 6p21. 3 in the human chromosome 6 has already been sequenced with high accuracy, we attempted to identify candidate genes responsible for two human diseases (Bechet's disease [BA] and Psoriasis-[PA]) and create animal models for these two diseases. The following is the achievement we have performed in the past three years.1. A region responsible for PA was confined to a region spanning 89 to 143 kb from the telomere side of HLA-C locus by genetic analyses. In this region, there were one known and 4 novel genes expressed in a skin. Sequencing of this region revealed that one of the 4 novel gene exhibited a significant polymorphism. This gene was termed "SEEK1" and found to overexpress in the skin suffered with PA.2. For the candidate gene for BD, HLA-B gene itself or its neiboring MICA or MICB gene has been considered for the best candidate. However, recent genetic analysis revealed that HLA-B51 gene is the responsible gene for BD.3. We produced several transgenic (Tg) lines carrying a 30-kb fragment containing SEEK1 gene. Further analysis of these lines is now underway.4. We have already produced Tg lines carrying MICA or MICB gene, just prior to decision that HLA-B51 gene is the responsible gene for BD. These MICA or MICB Tg lines exhibited several abnormalities such as reduction in the nubmer of leukocytes and body weight, and hyperkeratosis in the skin, suggesting possible involvement of these genes in the pathogenesis of BD.5. We also produced Tg lines carrying HLA-B51 and human b2-microglobulin genes and found that in these mice expression of HLA-B51 molecules on the cell surface was greatly elevated. Mice carrying human b2-microglobulin (b2m) and HLA-B51 genes, but lacking mouse b2m gene are now successfully produced using b2m-knock out mice.