Unbiased Genetic Modifier Screen for CMT

We chose to screen for EP lines on the Drosophila X-chromosome as a strategy to identify YARS1^CMT-relevant genetic interactions in an unbiased manner, and yet avoid a lengthy whole-genome screening, or screening for EP lines of the much larger chromosomes 2 or 3. The screening was performed as described in ref. ^{39 (link)}. Briefly, GMR-Gal4; UAS-YARS1^E196K/TM6B virgins were crossed with EP males. In case of EP male sterility or lethal EP-element insertion, crosses were repeated vice versa, using EP virgins and GMR-Gal4; UAS-YARS1^E196K/TM6B males. In F1, at least 20 female flies heterozygous for GMR-Gal>YARS1^E196K>EP and GMR-Gal4>TM6B>EP genotypes were compared with each other. Initial positive hits were selected when rough eye phenotype was present in flies with the first genotype and was absent in flies with the second genotype, respectively. The crosses for the positive hits were repeated at least three independent times. In a next step, the initially positive hit went through a rigorous validation process, where the major criterium to be claimed as a genuine CMT modifier was to induce a rough-eye phenotype only upon co-expression with YARS1^E196K and not with YARS1^WT. Additionally, the genetic interaction had to be evolutionary conserved (i.e., present both with human and Drosophila versions of YARS1^E196K) (see Fig. 1a for a schematic representation of the screening strategy).