IDENTIFICATION AND CLARIFICATION OF THE UNDERLYING MECHANISM OF NOVEL DEANESS GENE BY MUTAGENESIS PROGRAM

近5年来，国外发现了20个与遗传性耳聋有关的新基因，但日本从未发现过新的遗传性耳聋基因。这主要是由于日本家系规模小、耳聋表型分类困难、定位克隆、候选基因定位等标准遗传学方法难以识别新基因所致。因此，为了发现新的耳聋基因，我们采用了随机诱变计划，包括对小鼠基因的诱变和耳聋的筛选，以及对致病基因的鉴定。对小鼠耳聋模型的听觉分析提供了耳聋的潜在机制。此外，这一动物研究还可以应用于人类研究，为临床应用提供了可能性。我们利用EUN技术在胚胎干细胞中创造了耳聋基因点突变，并对听性脑干反应产生的小鼠进行了筛选。共检出耳聋小鼠13个品系56只。对纯音诱发的听觉反应进行详细分析，得到轻度听力损失动物12只，中度听力损失动物22只，重度听力损失动物32只。病理组织学检查发现Corti器13只，螺旋神经节细胞9只，血管纹5只，螺旋韧带4只，覆盖膜2只，2只动物未见异常。我们发现，编码Ca-ATPase的基因至少导致了这些小鼠模型中的一条线。

In recent 5 years, twenty novel genes responsible for hereditary deafness have been discovered in foreign countries, but no new identification of deafness gene has never appeared in Japan. It will be explained by small size of Japanese families, difficulty in classification of phenotype of deafness, difficulty in standard genetic approach to identify novel genes such as positioning cloning and candidate gene approach. Therefore, we use random mutagenesis project, which includes mutagenesis of genes in mouse and screening of deafness, and identification of the responsible gene in order to discover novel deafness genes. Auditory analysis of the mouse model for deafness provides the underlying mechanism of deafness. Furthermore, this animal research can be applied to human study, leading to possibilities of clinical application.We created point mutation of deafness gene in embryonic stem cell by EUN and screened the mice created by auditory brainstem response. Totally 56 mice of 13 lines could be detected deafness. The detailed analysis of auditory responses by tone burst resulted in 12 animals of slight hearing loss, 22 animals of moderate hearing loss, and 32 animals of severe hearing loss. Based upon histopathological analysis, pathological findings were observed in 13 animal in the organ of Corti, 9 in spiral ganglion cells, 5 in stria vascularis, 4 spiral ligament and 2 in tectorial membrane, and no abnormal finding was observed in 2 animals. We discovered that the gene encoding Ca-ATPase was responsible for de at least one line of these mouse models.