Elucidation of molecular pathophysiology and development of the methods for molecular diagnosis of the disease associated with WFS1 gene mutations.

Wolfram(DIDMOAD)综合征是一种常染色体隐性遗传病，伴有幼年型胰岛素依赖型糖尿病和进行性视神经萎缩。我们最近发现了WFS1(Wolfram综合征1)基因，这是一个新的功能未知的基因。在这项研究中，我们产生了一种针对WFS1蛋白COOH末端的特异性抗体，并研究了它在培养细胞中的亚细胞定位。并对其在大鼠脑内的分布进行了研究。生化研究表明，WFS1蛋白是一种完整的、内糖苷酶H敏感的膜糖蛋白，主要定位于内质网。与此一致的是，WFS1的免疫荧光细胞染色在细胞质和核膜上显示出特征的网状图案。没有获得WFS1与线粒体共定位的证据，这与早期的临床假说--Wolfram综合征是线粒体介导的疾病--相矛盾。在大鼠脑内，WFS1被发现存在于前…更不祥的是，在海马CA1区、杏仁体区、嗅结节和皮质浅层的选定神经元中。这些表达部位，即边缘系统的组成部分或与该系统密切相关的结构，可能参与了该综合征特有的精神、行为和情绪异常。WFS1的内质网定位表明，该蛋白在膜运输、蛋白质加工和/或调节内质网钙稳态中发挥的作用尚不明确。作为阐明WFS1功能的一步，我们创建了WFS1-/-小鼠。此外，我们从这些小鼠身上建立了WFS1-/-小鼠胚胎成纤维细胞。为了解WFS1的作用，正在进行全身(小鼠)和细胞水平的研究。我们还研究了WFS1基因突变在1型糖尿病发病中的可能参与。我们对21例日本非免疫性1型糖尿病患者进行了基因突变筛查。几个核苷酸替换(单核苷酸多态)被发现与这种形式的糖尿病没有明显的关联。较少

Wolfram (DIDMOAD) syndrome is an autosomal recessive disorder accompanied by juvenile-onset insulin-dependent diabetes mellitus and progressive optic atrophy. We recently identified the WFS1 (Wolfram syndrome 1) gene, a novel gene of unknown function. In this study, we generated a specific antibody against the COOH terminus of the WFS1 protein and investigated its subcellular localization in cultured cells. We also studied its distributions in rat brain. Biochemical studies indicated the WFS1 protein to be an integral, endoglycosidase H-sensitive membrane glycoprotein that localizes primarily in the endoplasmic reticulum. Consistent with this, immunofluorescence cell staining of WFS1 showed a characteristic reticular pattern over the cytoplasm and nuclear envelope. No evidence of co-localization of WFS1 with mitochondria was obtained, arguing against an earlier clinical hypothesis that Wolfram syndrome is a mitochondria-mediated disorder. In rat brain, WFS1 was found to be present pred … More ominantly in selected neurons in the hippocampus CA1, amygdaloid areas, olfactory tubercle and superficial layer of the allocortex. These expression sites, that is, components of the limbic system or structures closely associated with this system, may be involved in the psychiatric, behavioral and emotional abnormalities characteristic of this syndrome. ER localization of WFS1 suggests this protein to play as yet undefined roles in membrane trafficking, protein processing, and/or regulation of ER calcium homeostasis.As a step to elucidate the function of WFS1, we created a WFS1 -/- mice. In addition, we established WFS1 -/- mouse embryonic fibroblast from these mice. The studies for understanding the roles of WFS1 are being undertaken at whole body (mice) and cellular levels.We also studied the possible involvement of WFS1 gene mutations in the development of type 1 diabetes mellitus. We screened for the mutations in 21 Japanese patients with non-immune type 1 diabetes mellitus. Several nucleotide substitutions (single nucleotide polymorphisms) were identified without obvious association with this form of diabetes. Less