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.

摘要