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 are currently focusing on two type IV collagen genes, emb-9 and let-2, whose mutant phenotypes were previously characterized by Dr. James Kramer at Northwestern University. There are temperature-sensitive missense alleles of these genes, making them ideal for genetic suppression screens. Mutations in the human type IV collagens cause a number of diseases. We are also suppressing a ubiquitin activating enzyme, uba-1, which was originally identified and characterized by Dr. Harold Smith (Kulkarni and Smith, 2008), currently here in NIDDK. This allele is also temperature-sensitive. Mutations in the human UBA1 gene cause a specific form of Spinal muscular atrophy. We have also carried out suppressor screens with a temperature-sensitive allele of air-2, an Aurora ortholog. Mutations in the Aurora C kinase in humans causes Spermatogenic Failure 5. In addition to these genes, we have begun to characterize the germline defects of a deletion mutation in the lpd-8 ortholog of C. elegans;this is a gene that, when mutated, causes Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1). We are currently using the CRISPR technology to introduce the orthologous mutation into C. elegans to mimic the mutations found in humans. For each of these genes, mutations in them appear to reveal recessive, highly penetrant phenotypes. We are carrying out straightforward genetic suppression screens with these mutants. The phenotypes of the above genes range from embryonic and larval lethality to slow development and sterility. The uba-1 mutant has a number of distinct phenotypes and thus we will need to score our suppressors for their ability to suppress each of these phenotypes. So far, we have only isolated alleles that suppress the larval lethality of uba-1, but not the sperm defects. We are currently in the process of performing whole genome sequencing on our suppressed strains to molecularly identify our suppressor mutations. We will then characterize the phenotypes of these suppressor mutations in otherwise wildtype backgrounds to see if they perturb development. In the future, we hope to test drugs that mimic the effects of suppressor mutations to determine whether they might be worthy new therapies of diseases in which recessive mutations are known to be responsible for suppression. In the absence of effective gene therapy or stem cell therapy, drugs that target specific proteins may be beneficial to patients with such diseases.
