Figure 3 .

Drosophila lethality and homeotic transformation phenotype rescue experiments demonstrate that mutations in Sce corresponding to patient variants in RNF2 are LoF alleles. (A) A schematic diagram of the Drosophila Sce locus [third chromosome right arm (3R)] and the genomic rescue (GR) constructs inserted on chromosome 2 (VK37) of the fly. The relative locations of the mutations/variants used/studied in this work are shown using the Sce gene or GR constructs, respectively. (B) Lethality rescue experiments in Drosophila of recessive lethal Sce alleles using GR-WT or variant (GR-R65H, GR-S77R, GR-S77N) Sce protein. GR-WT and GR-S77N (gnomAD variant) are able to rescue the lethality caused by loss of Sce function, whereas GR-R65H and GR-S77R variants fail to do so. (C) A diagram and photographs of Drosophila legs from WT and grh^MI05134/+; Sce¹/+ male flies. Endogenous or ectopic sex combs are shown using blue or red arrows, respectively. A WT fly always has two sex combs (one on each L1), whereas grh^MI05134/+; Sce¹/+ male flies develop ectopic sex combs in L2 and L3 legs at high frequency. (D) Average number of legs with sex combs per animal in grh^MI05134/+; Sce^1/+ male flies with or without WT or variant genomic transgenes. We scored all six legs per fly for the presence of sex combs. GR-WT and GR-S77N (gnomAD variant) are able to rescue the ectopic sex comb phenotype in grh^MI05134/+; Sce^1/+ double heterozygous flies, whereas GR-R65H fail to do so. GR-S77R has weak activity in this assay. Note that while the antimorphic Sce¹ genetically interacts with grh^MI05134 to induce ectopic sex combs in most legs, LoF Sce alleles (Sce^KO and Sce^33M2) do not show this defect. Further introduction of genomic rescue transgenes that express WT or variant Sce also do not modify this phenotype in grh^MI05134/+; Sce^KO/+ and grh^MI05134/+; Sce^33M2/+ animals.