Utilization of Drosophila in rare disease gene discovery and functional classification of VUS. To compile a list of papers published using Drosophila melanogaster as a model organism to study rare disease genes and variants between January 2020 and April 2024, we first performed manual inspections of all abstracts published in the following journals where many new human disease gene discovery and phenotypic expansion papers are published: American Journal of Human Genetics, European Journal of Human Genetics, Genetics in Medicine, Genome Research, Human Molecular Genetics, Nature Genetics. We also utilized the advanced search option on PubMed by performing the search with the keywords [“Drosophila” + “Variant”, “Human Mutation”, “de novo”, “biallelic”, or “missense allele”] in the title or abstract and manually filtered the results to identify relevant papers that were published in other journals. Note that this search strategy may have omitted some relevant papers that were not captured by the selected keywords. (A) The number of the papers that studied GUS and VUS utilizing Drosophila models has continuously increased between 2020 and 2023 based on our search methods. The majority of the studies explored the function of specific variants in addition to assessing the function of the orthologous fly gene. (B) The DIOPT scores of fly-human ortholog candidate pairs used in each study. A great majority of papers studied ortholog candidate pairs with a DIOPT score of 10 or more, but some papers conducted functional studies of genes with lower DIOPT scores. (C) The percentages of papers that used each Drosophila-based functional approach in human genetic studies. Approximately 70% of papers assessed the loss-of-function of the orthologous genes as part of the functional study of the gene of interest, which are often GUS. Approximately 30% of manuscripts performed humanization of orthologous fly gene, and ∼35% of papers performed functional assessment of the VUS based on overexpression and/or fly analogous variant studies. (D) Breakdown of functional studies of rare variants by the number of Drosophila approaches employed. Whereas approximately half of the papers used a single method (Loss-of-function, Overexpression, Humanization, or Fly analogous variant investigation), the other half integrated multiple strategies. (E) Out of 98 papers that assessed the function of variants, 54 papers classified the variants of interest as LOF and 34 papers designated the variants as GOF alleles. Some studies found both LOF and GOF variants that contribute to clinical phenotypes and one study reported variants that have properties of both GOF and LOF (referred to here as “mixed” due to their mixed properties). (F) Of the 43 papers that identified GOF variants, ∼75% provided data to further classify them as specific Muller's morphs. See Supplemental Table 1 for details.
