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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的新生儿粘多糖贮积症 II 型（MPS II，又称亨特综合征）的诊断筛选主要通过干血斑测定艾杜糖醛酸-2-硫酸酯酶进行，然后进行糖胺聚糖的评估和位于 X 染色体上的 IDS 基因的分子检测。然而，由于难以解释分子诊断测试结果，诊断变得复杂。 IDS 基因中存在大量意义不确定的变异。此外，我们还没有一个彻底的了解假缺陷等位基因，其定义为蛋白质的明显缺陷不会引起疾病。虽然艾杜糖醛酸-2-硫酸酯酶等酶的假性缺陷显示出较低的对人工底物的活性，它会导致天然底物的正常分解代谢。在此，我们建议开发高通量、基于细胞的测定和分析方法，以支持综合功能 IDS 基因变异的评估。该提案中概述的核心假设是实验数据测量变异的直接功能影响将为准确的疾病风险预测提供信息。此外，我们假设体外、基于细胞的测定将更准确地检测天然产物的异常加工与使用人工底物的测定相比，底物预测疾病。在这里，我们将开发一种功能测定使用 CRISPR/Cas9 生成具有已知致病性和良性变异的人类 A549 细胞。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确定定义致病性的形态表型组合。细胞表型将是建立并使用第二组变体结合救援实验进行测试。结果将为 IDS 分子检测中的变异分类提供信息，并改善对个体（包括以下人群）的诊断在新生儿筛查中通过低艾杜糖醛酸-2-硫酸酯酶活性来鉴定。