For human autosomal recessive diseases in which the responsible gene is known, we are using C. elegans to study the function of that gene and to genetically identify other factors that act in the same pathway. There are a number of criteria that must be met in order for this strategy to work. First, there must be a convincing and clear C. elegans ortholog. Second, there would have to be a mutation or deletion in this gene that already exists. Towards this end, we are using CRISPR technology to generate mutant alleles analogous to those found in human diseases. Third, there would have to be a scorable phenotype. The more penetrant the phenotype, the better. If these criteria are met, genetic suppressor and enhancer screens could be performed to identify interacting factors that function with any given gene and the biological process in which it functions. In the past year, we have identified a number of C. elegans orthologs of human disease-causing genes. We have determined that many of these candidates satisfy all of the above criteria- there are mutations in these genes and they reveal very penetrant and scorable phenotypes. We have initiated a project to model human craniofacial syndromes in C. elegans. We were approached by colleagues to determine whether mutations in the sole C. elegans orthologs of the Twist basic helix-loop-helix (bHLH) transcription factor results in distinct phenotypes in C. elegans. There are two Twist genes in humans, Twist1 and Twist2. Twist mutations have already been implicated in other craniofacial disorders such as Saethre-Chotzen Syndrome. Interesting, our clinical colleagues have recently shown that mutations in a conserved glutamic acid residue in the conserved DNA-binding basic domain of Twist1 and Twist2 are implicated in three other distinct craniofacial syndromes, all of which are autosomal dominant and hypothesized to result in dominant-negative variants of Twist. In each case, this conserved glutamic acid is altered to one of five other amino acid residues. Using CRISPR/Cas9 genome-editing technology, we made the orthologous changes in this conserved glutamic acid in the C. elegans hlh-8 gene, the sole ortholog of the Twist genes in humans. We were able to screen for our mutations by PCR, restriction digests, and sequencing and were able to generate all of the desired mutations. Each of our mutations resulted in a very visible phenotype; homozygous animals were egg-laying defective (Egl). Interestingly, only some of the mutations resulted in a constipated (Con) phenotype and displayed a very deformed tail. We have characterized these strains quantitatively to determine how penetrant each of these phenotypes are. We have also crossed these strains with GFP reporter strains to look at the expression of known HLH-8 targets. We have also examined the expression of an hlh-8::gfp reporter to learn more about the M lineage in the developing larvae. The M lineage is responsible for the generation of the muscles that make up the egg-laying and defecation systems in C. elegans. Using this marker, we can determine when the M descendants divide and migrate. This marker also allows us to assay the generation of the sex muscles (SMs). With all these markers, we have been able to characterize the mutant phenotypes of these hlh-8 alleles at the cellular and molecular level and better group our alleles into distinct phenotypic classes. Interestingly, these mutants also display diverse male tail phenotypes, further allowing us to put these mutants into distinct classes. To date, our data suggest that the vulval muscles are most sensitive to each of these mutant alleles, while the enteric muscles responsible for defecation are more variable in their sensitivity. And each of these alleles, like the human diseases, is also semi-dominant in nature. We suspect that they are also acting in a dominant-negative fashion, interfering with the proper expression of target genes. We are eager to determine the molecular mechanism of these phenotypes: do these mutants bind promoters and inappropriately turn on or turn off genes that lead to these phenotypes? We also plan to test the weakest alleles in genetic suppressor screens to determine whether we can isolate suppressor mutants that reverse the mutant phenotypes and restore them to a more wild-type-like condition. In other projects, we have used CRISPR/Cas9 to edit the C. elegans genome to make patient alleles of genes involved in Long QT Syndrome (using the kqt-3 gene), genes involved in Mitochondrial disorders (using the gene lpd-8), and Congenital Disorders of Deglycosylation, such as NGLY1 deficiency (using the png-1 gene). We are currently developing assays to characterize the mutant phenotypes of these genes. We hope to initiate suppressor screens with each of these disease alleles in order to identify other genes that act in the same genetic pathways.
