Cellular phenotype of mucopolysaccharidosis II for studies of genomic variants

摘要 新生儿诊断粘多糖病II型（MPS II，也称为亨特综合征） 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 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，诊断由于难以解释分子诊断测试结果而变得复杂， IDS基因中存在大量意义不明的变异体。此外，我们没有一个彻底的 假缺陷等位基因的理解，这是由蛋白质的明显缺陷， 不会引起疾病。虽然假缺乏酶如艾杜糖醛酸-2-硫酸酯酶显示低的 活性对人造底物，它导致天然底物的正常catalysis。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 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效应将为准确的疾病风险预测提供信息。另外我们 假设体外的、基于细胞的测定将更准确地检测天然细胞的异常加工， 与使用人工底物的测定相比，使用人工底物的测定更能预测疾病。在这里，我们将开发一种功能性测定方法， 在具有使用CRISPR/Cas9产生的已知致病性和良性变体的人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 建立并然后使用第二组变体与拯救实验相结合进行测试。结果将 在IDS分子测试中提供变异体分类信息并改进对包括那些 通过新生儿筛查中艾杜糖醛酸-2-硫酸酯酶活性低鉴定。

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.