Suzanne de Bruijn

304 Chapter 6 to be collected for an individual case also requires a significant amount of time and expertise to be analyzed. How cost- and time-effective will the application of these technologies be? Currently, in a diagnostic setting, the sequencing and analysis costs of a single exome are around €1,000 and even higher when counseling is included. 126 The estimated turnaround time for a diagnostic report in the Netherlands is already three months. How much will these costs and turnaround time increase when multi-omics approaches will be adopted? Most realistically, the described technologies will remain in the research domains for many years to come, and a genomics-only approach (based onWES, or potentiallyWGS) will remain the first-line of screening in routine diagnostics. Besides time andmoney concerns, another important hurdle is the lack of interpretation guidelines. Currently, ACMG guidelines are not fitted to validate non-coding variation and there is an urgent need for an established framework. 127,128 For example, when aberrant gene expression levels are detected in RNA-seq experiment, which cut-off should be used to distinguish pathogenic from non-pathogenic differential expression changes? What is the minimum level of wildtype gene expression that is required to prevent disease? Exon-skipping studies have indicated that already ~20% of wildtype transcript can be enough for phenotypic rescue. 116,129 Additionally, when can an elevated expression level be considered toxic? Furthermore, there are tissue-related concerns. Most likely, routine RNA-seq would be performed in readily available cell types such as blood cells and fibroblasts. Although a significant number of HL- and RD-associated genes are expressed in these tissue types, the majority of these genes are not or at a too low extent and differentiation of patient-derived iPSCs will increase costs and turnaround times even more. Also, how translatable will the findings be? Long lists of candidate disease-associated genes are available for both HL and RD, for which putative pathogenic variants have only been reported in single cases or families. Evidently, the “low hanging fruit” has already been picked, and only the rare causes of disease remain.While collecting additional cases, which requiresworldwide data sharing efforts (e.g. ClinGen 130 or GeneMatcher 131 ) is still an option that should be explored, we should also consider the possibility that these mutational events are unique for these specific families. In these cases, functional and multi-omics analyses and potentially testing in animal models are required to provide conclusive evidence for causality of variants in these candidate disease genes. Is it feasible to perform all these analyses for all unique cases? Despite the fact that a wide implementation is unlikely, this thesis exemplifies that the use of the indicated technologies will provide important insights that will aid in the identification of the underlying genetic defects. Crucial lessons have been learned from