New Congenital Disorders of Glycosylation: Therapy and Models

项目总结 我们发现并有时用单糖治疗罕见的先天性糖基化紊乱(CDG) (单糖)。但为什么它们的工作效率如此之高仍是个谜。一条线索来自我们最近的发现 表明一种活性糖，GDP-岩藻糖，不是存在于单一的均质池中，而是发生在 在多个不同的、非均一的池中，由岩藻糖是否来自从头途径或 抢救/节食。这些池选择性地为不同的多糖的合成提供原料。使用稳定的同位素标记的糖， 我们想了解岩藻糖进入细胞的机制，以及它是如何被利用的 选定的多聚糖。同样的技术也可以应用于目前正在使用的半乳糖的研究 来治疗其他CDG。在半乳糖血症患者中，半乳糖是有毒的，必须从他们的饮食中去除。还没有 依从性患者也有糖基化缺陷和一些持续的症状。少量 半乳糖促进糖基化，但没有数据表明不同的糖基化途径如何利用外源 半乳糖。我们假设半乳糖也存在于高度复杂的、非均一的池中 不同的糖基化途径。 我们发现了两个新的CDG，每个都是由基因的反复从头突变引起的，这些基因具有良好的- 已知但非常不同的常染色体隐性遗传。我们想要了解 并提出研究新表型的模型。 索尔·威尔逊综合征(SWS)患者患有进展性侏儒症，智力正常，原因是 CDG基因COG4的重复性新突变。具有双等位基因COG4突变的患者有非常严重的 通常致命的表型伴随改变的N-糖基化。SWS不会改变N-糖基化，但有选择性地影响 硫酸软骨素蛋白多糖修饰和胶原堆积/分泌。这表明，在 生长板上的细胞外基质。我们提出了一系列的细胞模型来评估分子细节 SWS中COG4的异常表达可以解释这些表型。 我们发现了一种独特的凝血障碍和血清N-糖基化异常。这些病人 在SLC37A4中有单一从头突变。该基因编码内质网定位的葡萄糖-6-P转运蛋白 来维持葡萄糖的稳态。当常染色体隐性遗传时，它会导致糖原储存障碍，GSD-Ib。在De中 Novo紊乱，它只改变肝脏衍生蛋白的N-糖基化。患者iPS细胞显示部分移位 SLC37A4从急诊室到高尔基。我们假设，该突变消除了一个关键的内质网滞留信号，错配- 将全功能传送器的一半本地化。在新的位置，Glc-6-P聚集在高尔基体腔内。如果 其内质网定位的葡萄糖-6-磷酸酶伴侣G6PC的出现，PI也可能积聚和改变 糖基化，通过限制多种酶的关键阳离子的溶解度/可用性。我们将创建CRISPR- 派生的模型系统来探索和验证这一假设。潜在的小分子疗法已经存在。

PROJECT SUMMARY We discover and sometimes treat rare Congenital Disorders of Glycosylation (CDG) with simple sugars (monosaccharides). But why they work so efficiently is a puzzle. One clue comes from our recent discoveries showing that one activated sugar, GDP-fucose, does not reside in a single homogenous pool, but instead occurs in multiple, distinct, non-homogenous pools determined by whether fucose comes from a de novo pathway or salvage/diet. These pools selectively feed the synthesis of different glycans. Using stable isotope-labeled sugars, we want to understand the mechanisms that underlie the import of fucose into the cell and how it is utilized for selected glycans. The same technology can be applied to the study of galactose, which is currently being used to treat other CDGs. In galactosemia patients, galactose is toxic and must be eliminated from their diet. Yet compliant patients also have glycosylation deficiencies and some persistent symptoms. Small amounts of galactose improve glycosylation, but there is no data on how different glycosylation pathways use exogenous galactose. We hypothesize that galactose also resides in highly complex, non-homogenous pools that feed different glycosylation pathways. We discovered two new CDGs, each caused by recurrent de novo mutations in genes that have well- known, but very different, autosomal recessive presentations. We want to understand the underlying basis of the new disorders and propose models to study the novel phenotypes. Saul Wilson Syndrome (SWS) patients have a progeroid dwarfism and normal intelligence due to a recurrent de novo mutation in the CDG gene, COG4. Patients with bi-allelic COG4 mutations have a very severe, often lethal phenotype with altered N-glycosylation. SWS does not alter N-glycosylation, but selectively effects chondroitin sulfate proteoglycan modification and collagen accumulation/secretion. This suggests defects in the extracellular matrix at the growth plate. We propose series of cellular models to assess the molecular details of COG4 dysregulation in SWS that can explain the phenotypes. We discovered patients with a unique coagulopathy and abnormal serum N-glycosylation. These patients have a single de novo mutation in SLC37A4. The gene encodes the ER-localized glucose-6-P transporter used for glucose homeostasis. When autosomal recessive, it causes glycogen storage disorder, GSD-Ib. In the de novo disorder, it alters only liver-derived protein N-glycosylation. Patient iPS cells show relocation of a portion of SLC37A4 from ER to the Golgi. We hypothesize that the mutation eliminates a critical ER-retention signal, mis- localizing half of the fully functional transporter. In the new location Glc-6-P accumulates in the Golgi lumen. If G6PC, its ER-localized glucose-6-phosphatase partner, comes along, Pi might also accumulate and alter glycosylation, by limiting solubility/availability of critical cations for multiple enzymes. We will create CRISPR- derived model systems to explore and validate this hypothesis. Potential small molecule therapies already exist.