A context-based approach to unravel Variants of (Uncertain) Significance

The Netherlands boasts one of the best healthcare systems globally, yet a subset of severely ill children remains overlooked. These children lack an official diagnosis despite extensive follow-up. Often, these children exhibit genetic variation of unknown clinical significance, termed Variants of Uncertain Significance (VUS). A VUS may be benign or hint towards underlying genetic disease. Currently, there are no clear guidelines on how to structure diagnostic follow-up for these patients. This thesis was aimed to develop new tools and frameworks for interpreting VUSes. The initial chapters examine three cases of patients with VUSes, highlighting the challenges in solving these cases. Our efforts led to the discovery of three novel metabolic diseases linked to VUSes in NAA80, NAE1, and LIMK1, providing diagnoses for these eight patients. We then explored functional cellular screening using a novel technique called Imaging Flow Cytometry to expedite VUS elucidation. This method identified specific cellular phenotypes in patients with VUS, resulting in diagnoses for all patients with VUS in known genes and one patient with a VUS in a novel disease gene. The success of this approach led to its application in well-known genetic metabolic diseases, including mitochondrial diseases and mitochondrial aminoacyl tRNA deficiencies (ARS2 deficiencies). Imaging Flow Cytometry in mitochondrial disease revealed distinct functional cellular profiles, correlating with patients' clinical characteristics. In ARS2 deficiencies, it uncovered the potential of amino acid treatment, tested in a small cohort of eight patients, resulting in improvements for most. Together, this thesis underscores the transformative power of context in unraveling the intricacies of genetic diseases.