Cellular Phenotypes of Genetic Variants in Mucopolysaccharidosis

粘多糖贮积症遗传变异的细胞表型

• 批准号: 10638709
• 负责人: William J Buchser
• 金额: $ 45.15万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638709
• 关键词: A549; Affect; Attenuated; Benign; Biochemical; Biological Assay; Biopsy; Birth; Cell Line; Cells; Cellular Morphology; Chimeric Proteins; Classification; Clinical; Complementary DNA; Consent; DNA; Data; Dermatan Sulfate; Diagnosis; Diagnostic; Diagnostic tests; Discrimination; Disease; Enzymes; Generations; Genes; Genetic Diseases; Genetic Variation; Genome engineering; Genomics; Glycosaminoglycans; Goals; Heparitin Sulfate; Human; Image; Impairment; In Vitro; Individual; Inherited; Intellectual functioning disability; Knock-out; Laboratories; Libraries; Link; Lysosomes; Machine Learning; Measurement; Measures; Medical; Medical Records; Membrane; Methods; Molecular; Morphology; Mucopolysaccharidoses; Mucopolysaccharidosis II; Neonatal Screening; Neurons; Pathogenicity; Patients; Phenotype; Population; Reporting; Research; Resolution; Sampling; Severities; Skin; System; Technology; Test Result; Testing; Transmembrane Domain; Universities; Variant; Washington; Whole Blood; cell type; diagnostic accuracy; disorder risk; experimental study; genetic testing; genetic variant; iduronate-2-sulfatase; improved; induced pluripotent stem cell; molecular diagnostics; novel; phenotypic data; polysulfated glycosaminoglycan; prevent; risk prediction; single cell sequencing; stem cells; variant of unknown significance

ABSTRACT There is presently an urgent need to develop methodical approaches to evaluate the function of genomic vari- ants. Clinical genomic testing is growing rapidly, and with it a larger-than-ever number of genetic variants that cannot be defined as either disease-causing or benign. The problem affects clinicians and their patients, who struggle to understand and interpret molecular diagnostic reports, the implications of the results, and how to manage their patients in the absence of definitive information. Systems that involve single cells to generate high-content, high-resolution functional data are of paramount importance to solving the problem posed by var- iants of uncertain significance. Here, we propose to utilize a novel cell-based platform that uses machine learn- ing to determine the combination of morphological phenotypes that define pathogenicity. We will apply the technology to the comprehensive functional assessment of variants in IDS, the gene responsible for Hunter syndrome. The core hypothesis outlined in this proposal is that experimental data measuring the direct func- tional effects of variants will inform accurate disease risk prediction. In addition, we hypothesize that an in vitro, cell-based assay based on morphological features will more accurately detect disease compared to existing biochemical testing using artificial substrates. We will perform a functional assay in a variant library using the RaftSeq pipeline in the Buchser laboratory, where a cellular phenotype will be established and then tested us- ing a second set of variants combined with rescue experiments. The results will inform variant classification in IDS molecular testing and improve diagnosis of individuals including those identified by low iduronate-2-sulfa- tase activity on newborn screening.

抽象的 目前迫切需要开发系统方法来评估基因组变异的功能 蚂蚁。临床基因组测试正在迅速发展，随之而来的遗传变异数量也比以往任何时候都多。 不能被定义为致病或良性。这个问题影响到临床医生和他们的患者，他们 难以理解和解释分子诊断报告、结果的含义以及如何 在缺乏明确信息的情况下管理患者。涉及单细胞生成的系统 高内容、高分辨率的功能数据对于解决变量带来的问题至关重要 意义不确定的事物。在这里，我们建议利用一种新颖的基于细胞的平台，该平台使用机器学习- 旨在确定定义致病性的形态学表型的组合。我们将应用 技术对 IDS 变异的综合功能评估，该基因负责 Hunter 综合症。该提案中概述的核心假设是测量直接函数的实验数据 变异的影响将为准确的疾病风险预测提供信息。此外，我们假设在体外， 与现有技术相比，基于形态学特征的细胞检测将更准确地检测疾病 使用人造基质进行生化测试。我们将使用以下方法在变体库中进行功能测定 Buchser 实验室的 RaftSeq 流程将建立细胞表型，然后进行测试- 结合救援实验的第二组变体。结果将告知变异分类 IDS 分子检测并改善个体的诊断，包括那些艾杜糖醛酸-2-磺胺含量低的个体 新生儿筛查中的 Tase 活性。