Subtle cellular phenotypes inform pathological and benign genetic mutants in the Iduronate-2 sulfatase gene.

Molecular genetic testing is increasingly used in clinical care to identify genetic variants and their impact on disease burden. However, variants of uncertain significance (VUS) hamper the utility of molecular diagnostic testing. In patients presenting with Hunter Syndrome and VUS in the IDS gene, clinical testing for iduronate-2-sulfatase enzyme activity has been the mainstay to determine whether a variant is likely damaging. However, enzyme assays alone fail to predict disease severity. In this study, we developed an image-based cellular assay using genome-engineered cells with IDS variants to determine whether a specific variant causes morphological changes that are associated with disease. Specifically, we generated twelve mutant cell lines and documented both IDS biochemical activity and reproducible phenotypic differences therein. Next, we examined patient-derived cell lines and found the same phenotypic differences compared to parental controls. The morphological changes were complex, but measured on a single scale, which we termed PathScoreLC. To determine whether the observed changes are specific to IDS, we reintroduced a recombinant human IDS enzyme (rhIDS) to rescue both the biochemical and phenotypic changes of these cells. We found a partial rescue in the presence of corrective levels of IDS enzyme. Finally, we examined the differences in gene expression and found that a recombinant enzyme was not sufficient to fully restore transcriptional changes in the mutant lines at the time points studied. This proof-of-concept study establishes preliminary validation of the method and sets the stage for future functional studies and broader IDS variant testing.