Figure 4 – Patient variants behave as strong or mild loss-of-function alleles in flies.

A mutant form of myo that corresponds to 3 of the proband’s variants (p.R295P, p.E306K, and p.Y336*) along with a wild type myo construct (WT) and an empty UAS-vector (negative control) were expressed with various GAL4 drivers to determine their effect when overexpressed. (A) Ubiquitous overexpression of myo-WT and overexpression with myo-T2A-GAL4 allele is lethal except at low temperatures (18°C) when GAL4 is less abundant. Ubiquitous overexpression of myo-E500K mirrors the lethality of myo-WT, myo-R498P is viable at higher temperatures and no lethality is observed when myo-F530* is expressed at any temperature. When overexpressed specifically in muscles, myo-WT and myo-E500K are only lethal at 29°C while myo-R498P and myo-F530X are viable. When overexpressed specifically in glial cells, the toxicity mirrors that seen with ubiquitous overexpression. The numbers of viable animals were quantified for ubiquitous expression (B), glial expression (C), and with myo-T2A-GAL4 expression (D). These data indicate a decreasing scale of toxicity of myo-WT>myo-E500K>myo-R489P>myo-F530X. This trend is also seen with repo-GAL4 and myo-T2A-GAL4 at 18°C. (B-D) Lower case letters represent groups significantly different (χ², p <0.05) from each other. (E) When myo-E500K and myo-R489P variants are expressed ubiquitously at 18°C a rough eye phenotype is observed indicating a developmental issue. All eye pictures are taken under the same magnification and were processed identically. Scale bar = 200μm. Error bars = SD.