This protocol allows us to determine whether missense variants in human genes that are linked to diseases affect protein function. This is difficult to accomplish computationally even with state-of-the-art algorithms. This technique allows a rapid analysis of missense variants in human proteins using an in vivo system Drosophila melanogaster which is faster and more cost efficient than vertebrate model organisms.
Demonstrating that a specific variant affects protein function directly contributes to the diagnosis of rare diseases and serves as a starting point for disease mechanism analysis and potential therapies. This protocol is particularly useful when studying variants linked to rare diseases, but it can be applied to more common diseases like autism and cancer. Identifying reproducible phenotypes in flies that reflect protein function can be challenging.
The phenotypes may vary from gene to gene and can be subtle. Functional studies of variants in human proteins using Drosophila can be performed based on rescue experiments or overexpression studies. Before beginning the procedure, gather information about the human gene of interest and its model organism orthologs.
To test variant function by overexpressing reference and variant human proteins in the fly visual system, generate transgenic flies that express reference or variant human cDNAs of interest under the control of the Gal4 upstream activating sequence or UAS system. To select a specific Gal4 line to express the human proteins in a specific fly tissue, cross three to five virgin females from the Gal4 line with three to five males from each of the UAS human cDNA transgenic lines in single vials or in duplicates. Transfer the crosses every two to three days to obtain as many animals eclosing from a single cross as possible and examine the eclosed animal under a dissection microscope to identify any differences between the reference and variant strains.
If there is a visible defect, image the flies to document the phenotypes. To generate flies to test for functional defects in the visual system, cross virgin females from the rhodopsin 1 Gal4 line to males with reference or variant UAS human cDNA transgenes to express the human proteins of interest in the photoreceptors R1 to R6 in the fly retina. Once the flies begin to eclose, gather the progeny into fresh vials and return the vials to an incubator set to the appropriate experimental temperature for an additional three days to allow the visual system to mature.
At the end of the incubation, immobilize the flies with an appropriate anesthesia method and gently glue one side of each fly onto a glass microscope slide. After setting up an electroretinogram rig, place a 1.2 millimeter glass capillary into a needle puller and break the capillary tube to obtain two sharp hollow tapered electrodes with less than 0.5 millimeter diameters. Fill the capillaries with 100 millimolar saline solution taking care to avoid air bubbles and slide the glass capillaries over the silver wire electrodes.
Clamp the capillaries in place and acclimate the flies to complete darkness for at least 10 minutes at room temperature. At the end of the habituation, place the slide containing the flies onto the recording apparatus and move the micromanipulators carrying the reference and recording electrodes close to the fly of interest. Watching the tip of the electrode, carefully place the reference electrode into the thorax of the fly penetrating the cuticle and place the recording electrode on the surface of the eye.
For successful electroretinogram recordings, take care to apply an appropriate amount of pressure to the recording electrode so that it causes a small dimple without penetrating the eye. When the electrodes have been placed, turn off the primary light for three minutes to re-acclimate the flies to the dark environment. At the end of the re-acclimation period, open and close the shutter once a second for 20 seconds recording the electroretinograms from at least 15 flies per slide per genotype per condition using the same electrodes.
When the recordings from the reference and variant flies are complete, compare the electroretinogram recordings from the reference, variant and controls to assess for differences. Then evaluate the electroretinogram data for changes in on transients, depolarization, off transients and repolarization. In this representative experiment, overexpression-based studies were performed for TBX2 since the human reference TBX2 was unable to functionally replace the orthologous fly gene making rescue-based strategies impossible.
When reference human TBX2 was overexpressed in the developing eye and parts of the brain using eyeless Gal4, the overexpression results in an approximately 85%lethality and a significant reduction in the eye size. In contrast, the variant transgene is less potent in causing lethality and induces a small eye phenotype using the same driver under identical experimental conditions suggesting that the variant affects protein function. In addition, when a reference human TBX2 is overexpressed in the photoreceptors using rhodopsin Gal4, the overexpression causes a significant alteration in the electroretinogram trace.
This phenotype is milder when the variant protein is expressed providing further evidence that the variant of interest affects protein function in vivo. It is important to note that every gene is unique so there is no single experiment that can assess the impact of all disease associated variants. In addition to working with human cDNAs, one can also determine if the variant has functional consequences by introducing analogous variants into evolutionarily conserved residues of the orthologous fly gene.
Using this method, we have contributed to a number of human disease gene discovery studies in collaboration with the Undiagnosed Disease Network and other groups.
