Chapter 7 220 Case-control vs. isogenic designs The 4H-iPSC cohort used throughout this thesis existed of 5 patients, with either POLR3A or POLR3B mutations. However, for monogenic diseases like leukodystrophies, gene editing to introduce disease variants into standard control lines or repair the disease mutation in patient-derived iPSCs is a very plausible option. In fact, the neuron and spheroid experiments (Chapter 3 and 4) included isogenic pairs for GLD and CD. However, looking at the PCA plots generated from both sequencing data sets, isogenic pairs were not more similar than non-isogenic lines. This raises the question: were isogenic lines valuable, and should we create such pairs for 4H in the future? As mentioned, isogenic controls in experiment design comes with certain advances such as increased power (Brunner et al., 2023). With a general failure of power in neuroscience this is very welcome (Button et al., 2013; Nord et al., 2017). However, their utility depends on the research question and the following considerations. Isogenic lines can elucidate subtle phenotypes that might be missed in heterogeneous case-control studies. For 4H, where neuronal and spheroid mono-cultures showed no striking phenotypes, isogenic lines might uncover additional pathologies. Introducing common mutations into standard control lines is advantageous for studying specific mechanisms. However, this approach lacks the link to the clinical context (patient-specificity), and information on the wide clinical heterogeneity seen in 4H patients, possibly caused by other (non)-genetic factors as well (Wolf et al., 2014). Therefore, patient-derived iPSC lines with diverse backgrounds remain crucial for translational research. Introducing mutations across multiple clones with varying genetic backgrounds could enhance understanding but would remain resource intensive. Considering all of the above, while 4H research could benefit from isogenic lines to discover more clear phenotypes addressing the variability and heterogeneity inherent to this disorder, future work should consider a balanced approach, integrating the strengths of both patient-derived and isogenic lines. As processes are time consuming, it is important to register and biobank those iPSC-lines so they can be shared with other 4H-leukodystrophy researchers. Collaboration to increase sample size One of the most elegant experimental designs used in this thesis has been the sample cohorts as presented in the spheroids chapter (Chapter 4). The collaboration between several institutes increased the available resources, respectively expertise, personnel and
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