One of the key problems in clinical genetics and in the use of genomic sequencing is the problem of gene function and difficulty in intrepreting human genomic variation and assigning pathogenicity in the clinic.
We began developing an approach to use Drosophila. Initially our efforts involved
accelerating cross-species studies for novel genes by working simultaneously with a large forward genetic screen in Drosophila and a genomic database from 2,000 individuals ascertained for Mendelian disease. This rich resource was published in Cell (Yamamoto et al., 2014), and this approach was outlined in an article in Genetics which has been selected for Faculty of 1000 (Wangler et al., 2015). We have recently developed an efficient pipeline using Drosophila melanogaster to screen nearly ANY conserved gene and variant of interest from genomic sequencing studies. We have successfully applied these tools to solve cases in the Centers for Mendelian Genomics (www.mendelian.org), and we are currently applying this pipeline to de novo missense variants from the Simons Simplex Collection as well as unsolved cases from the Undiagnosed Diseases Network (UDN).
The key features of our approach are shown in this figure (from Yamamoto and Bellen, Cell 2015).
- Variants of interest are screened with bioinformatic prioritization involving prediction of the Drosophila ortholog, and cross-referencing with independent genomic databases.
- A cassette is introduced into an intron of the gene of interest using CRISPR this cassette relies on latest fly technology to produce both a strong loss-of-function allele, and the expression of a GAL4 from endogenous enhancers. Strong loss of function alleles allowing studies of gene function
- Human cDNA is introduced into a transgenic strain into a docking site in the fly genome. GAL4 from the endogenous enhancer drives expression of human or variant cDNA from UAS. With phenotypic characterization and then with an integration with the clinical data, to address variant pathogenicity
This process allows for Humanization of the fly for the gene of interest. This pipeline establishes a large screen in human and fly genomes to understand gene function.