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.

PROJECT NARRATIVE Diagnostic DNA testing for Hunter syndrome is often inconclusive, as we cannot always determine whether a given DNA change in the Hunter syndrome gene will cause disease. We will use cells to develop a method to determine whether specific DNA changes cause Hunter syndrome or are benign DNA changes. The results can help better predict whether someone will have Hunter syndrome from DNA testing.