Cellular phenotype of mucopolysaccharidosis II for studies of genomic variants

用于基因组变异研究的粘多糖贮积症 II 的细胞表型

• 批准号: 10442244
• 负责人: William J Buchser
• 金额: $ 7.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10442244
• 关键词: A549; Address; Alleles; Attenuated; Benchmarking; Benign; Biochemical; Biological Assay; Birth; Blood; Blood Tests; CRISPR/Cas technology; Catabolism; Cataloging; Catalogs; Cell Line; Cells; Cellular Assay; Classification; ClinVar; Clinic; Clinical; Clinical Trials; Conflict (Psychology); DNA; Data; Databases; Detection; Development; Diagnosis; Diagnostic; Discrimination; Disease; Enzymes; Family; Filipin; Genes; Genotype; Glycosaminoglycans; Goals; Gold; Human; IGF Type 2 Receptor; Image; In Vitro; Incidence; Incubated; Individual; Infant; Laboratories; Left; Libraries; Link; Machine Learning; Measures; Methods; Missouri; Molecular; Molecular Diagnostic Testing; Morphology; Mucopolysaccharidosis II; Mutation; NPC1 gene; Neonatal Screening; Newborn Infant; Pathogenicity; Patients; Phenocopy; Phenotype; Population; Proteins; Publications; Publishing; Rosaniline Dyes; Running; Serum; Severities; Spottings; Stains; Sulfatases; Technology; Test Result; Testing; Urine; Variant; Visit; Work; X Chromosome; base; clinical phenotype; clinically relevant; diagnostic accuracy; disorder risk; experimental study; genetic testing; genetic variant; iduronate-2-sulfatase; improved; male; mannose 6 phosphate; protein function; response; restoration; risk prediction; screening; screening program; uptake; urinary; variant of unknown significance

ABSTRACT The diagnosis of mucopolysaccharidosis type II (MPS II, also known as Hunter syndrome) by newborn screening is principally made by dried blood spot assay for the iduronate-2-sulfatase enzyme, followed by assessment of glycosaminoglycans and molecular testing for the IDS gene located on the X chromosome. However, diagnosis is complicated by the difficulty in interpreting molecular diagnostic testing results due to the large number of variants of uncertain significance in the IDS gene. Furthermore, we do not have a thorough understanding of the pseudodeficiency alleles, which are defined by an apparent deficiency of a protein that does not cause disease. While the pseudodeficiency of an enzyme like iduronate-2-sulfatase shows low activity against an artificial substrate, it results in normal catabolism of the natural substrate. Here, we propose to develop high-throughput, cell-based assays and analysis methods to support the comprehensive functional assessment of IDS gene variants. The core hypothesis outlined in this proposal is that experimental data measuring the direct functional effects of variants will inform accurate disease risk prediction. In addition, we hypothesize that an in vitro, cell-based assay will more accurately detect abnormal processing of the natural substrate predictive of disease than assays using artificial substrates. Here, we will develop a functional assay in human A549 cells with known pathogenic and benign variants generated using CRISPR/Cas9. These will be studied on the CellRaft Technology platform in the Buchser laboratory, which uses machine learning to determine the combination of morphological phenotypes that define pathogenicity. A cellular phenotype will be established and then tested using 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-sulfatase activity on newborn screening.

抽象的 新生儿诊断II型粘二糖（MPS II，也称为Hunter综合征）的诊断 筛查主要是由iduronate-2-硫酸酶的酶干燥的血点测定进行的，其次是 评估位于X染色体上的IDS基因的糖胺聚糖和分子测试。 但是，由于难以解释分子诊断测试结果的困难，诊断变得复杂 IDS基因中具有不确定意义的大量变体。此外，我们没有彻底的 理解伪缺陷等位基因，这些等位基因由蛋白质的明显缺乏来定义 不会引起疾病。虽然像iduronate-2-硫酸酶这样的酶的伪缺陷显示低 对人工底物的活性，会导致天然底物的正常分解代谢。在这里，我们建议 开发高通量，基于细胞的测定和分析方法以支持全面的功能 评估IDS基因变体。该提案中概述的核心假设是实验数据 测量变体的直接功能效应将为准确的疾病风险预测提供信息。另外，我们 假设基于细胞的基于细胞的测定将更准确地检测到天然的异常处理 比使用人工底物测定的底物预测疾病。在这里，我们将开发功能分析 在使用CRISPR/CAS9产生的已知病原和良性变体的人类A549细胞中。这些将是 在Buchser实验室的CellRaft技术平台上进行了研究，该平台使用机器学习来 确定定义致病性的形态表型的组合。细胞表型将是 建立并使用第二组变体与救援实验结合进行了测试。结果将 在IDS分子测试中告知变体分类并改善个人的诊断 在新生儿筛查中通过低iduronate-2-硫酸酶活性鉴定。