Cellular phenotype of mucopolysaccharidosis II for studies of genomic variants

摘要 新生儿粘多糖病II(MPS II，又称Hunter综合征)的诊断 筛选主要通过艾杜酸-2-硫酸酯酶的干血斑点试验进行，其次是 糖胺多聚糖的鉴定和位于X染色体上的入侵检测基因的分子检测。 然而，由于以下原因，解释分子诊断测试结果的难度使诊断变得复杂 在入侵检测系统基因中存在大量意义不确定的变异。此外，我们还没有彻底的 对假性缺乏等位基因的理解，假性缺乏等位基因是由一种蛋白质的明显缺乏来定义的 不会导致疾病。而像艾杜酸-2-硫酸酯酶这样的酶的假缺陷率很低 对人造底物的活性，它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、基于细胞的分析和分析方法，以支持全面的功能 对入侵检测系统基因变异的评估。这项提案中概述的核心假设是实验数据 测量变异体的直接功能效应将有助于准确预测疾病风险。此外，我们 假设体外、基于细胞的化验将更准确地检测到天然的 底物对疾病的预测比使用人工底物的检测更准确。在这里，我们将开发一种功能分析 在人类A549细胞中，使用CRISPR/Cas9产生了已知的致病和良性变异。这些将是 研究了Buchser实验室的CellRaft技术平台，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。一种细胞表型将是 建立，然后使用第二套变种结合救援实验进行测试。结果将会是 告知入侵检测系统分子检测中的变体分类，并改进对包括 在新生儿筛查中通过低艾杜酸-2-硫酸酯酶活性鉴定。

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.