An Expanded Spectrum for Congenital Disorders of Glycosylation

描述（由申请人提供）：该项目将基础科学与患者相关的转化研究相结合，因为它确定了损害蛋白质N-糖基化的罕见遗传疾病的原因。今天，已知有39个基因会导致先天性糖基化障碍（CDG），但我们知道许多导致CDG的基因仍未被发现。该提案使用一种新的、无偏见的、前瞻性的、功能性的方法来识别这些新基因。具体目标在基础科学和当今患者的遗传缺陷之间建立了一座双向桥梁。它将确定新的基因，指导和告知医生在未来的CDG患者的诊断在亚1000美元的基因组时代。CDG影响每一个器官系统，其令人叹为观止的可变临床表现跨越每一个医学专业。临床表现不确定的基因，然而，几乎所有目前的CDG患者有一个异常的血清转铁蛋白（Tf）糖基化生物标志物。没有Tf样细胞生物标志物可用于评估疑似糖基化缺陷患者的糖基化或补体候选基因。为了填补这一空白，我们构建了一个ER保留的GFP与N-糖基化位点（Glyc-ER-GFP），允许荧光只有当该网站是空的。这为糖基化受损的细胞提供了高度灵敏的N-聚糖生物传感器：它们发光。这种新的细胞生物标志物可以通过筛选成熟的人类全基因组siRNA敲低文库来识别新的N-糖基化基因。已知的CDG基因用作筛选的阳性对照。二级糖基化生物标志物和生化测定可以证实新基因参与N-糖基化。这种方法有即时和长期的回报。功能性糖基化分析可以鉴定基因，以查询今天的CDG患者的未知缺陷。将来，在整个外显子组（基因组）测序管道中显示这些基因突变的受试者将知道它影响N-糖基化途径。新发现的基因也将扩大我们目前对蛋白质糖基化的理解。

DESCRIPTION (provided by applicant): This project merges basic science with patient-relevant translational research because it identifies the causes of rare genetic diseases that impair protein N-glycosylation. Today, 39 genes are known to cause Congenital Disorders of Glycosylation (CDG), but we know that many CDG-causing genes remain undiscovered. This proposal uses a new, unbiased, forward-reaching, functional approach to identify these new genes. The Specific Aims build a bi-directional bridge between fundamental science and genetic defects in patients today. It will identify new genes to guide and inform physicians in their diagnosis of tomorrow's CDG patients in the sub-$1000-genome era. CDGs affect every organ system with a breath-taking array of variable clinical presentations that cross every medical specialty. Clinical presentations do not identify the gene, however, nearly all current CDG patients have an abnormal serum transferrin (Tf) glycosylation biomarker. There is no Tf-like cellular biomarker that can be used to assess glycosylation or complement candidate genes in suspected glycosylation-deficient patients. To fill this void, we constructed an ER-retained GFP with an N-glycosylation site (Glyc-ER-GFP) that allows fluorescence only when that site is unoccupied. This provides a highly sensitive N-glycan-biosensor for cells with impaired glycosylation: They glow. This novel cellular biomarker can identify new N-glycosylation genes by screening a well-established human genome-wide siRNA knockdown library for cells that glow. The known CDG genes serve as positive controls for the screening. Secondary glycosylation biomarkers and biochemical assays can confirm the novel gene's involvement in N-glycosylation. This approach has immediate and long-term payoffs. The functional glycosylation assay can identify genes to query today's CDG patients with unknown defects. In the future, subjects in the whole exome (genome) sequencing pipeline who show mutations in these genes will know it impacts the N-glycosylation pathway. The newly discovered genes will also expand the dimensions of our current understanding about protein glycosylation.