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- we have made mutations in these genes and they reveal very penetrant and scorable phenotypes. Our very first attempt at this strategy was 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 had 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. We were able to characterize these mutants with a variety of molecular and cellular assays and essentially generated an allelic series. This first proof-of-principle study was published in June of 2017. My colleague, Dr. Ann Corsi, is currently screening for suppressors of one of the phenotypes. We initiated a few other projects in the past two years based on diseases we learned about at meetings, the Undiagnosed Disease Program's Clinical Rounds, or from the literature. Using CRISPR/Cas9 to edit the C. elegans genome, we have made deletion alleles to determine the null phenotype of each gene and have also made patient-specific alleles to mimic the specific mutation that is associated with disease. We are currently investigating Long QT Syndrome (LQTS, using the kqt-3 gene) to model arrhythmias in C. elegans. Our assay involves electropharyngeograms where we can quantify the rhythmic pumping of the pharynx. We are also trying the same assay with a mutation that causes the very rare Timothy Syndrome, an arrhythmia syndrome that is coupled with numerous other health problems. The mutation in C. elegans causes embryonic lethality when homozygous and so we are looking at the effects of this disease allele in heterozygotes. In humans, Timothy Syndrome is dominant and so our analysis of heterozygotes may prove informative. We have also initiated a study of the genes involved in Multiple Mitochondrial Dysfunctions Syndromes (MMDS, using the gene lpd-8). The genes that cause these syndromes are all involved in the biogensis of Fe-S clusters, key co-factors for a number of mitochondrial enzymes as well as many non-mitochondrial enzymes. Homozygous mutations in lpd-8 result in sterility. We are currently testing a number of other genes that function in Fe-S cluster biogenesis to determine if they also yield similar phenotypes when mutated. Another project focuses on the first Congenital Disorder of Deglycosylation. This disease, called NGLY1 deficiency, affects about 50 children worldwide. We are generating patient-specific alleles of the C. elegans ortholog, png-1, to learn more about the processes in which this gene functions. 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. We will continue seeking genes implicated in human disease that have C. elegans orthologs and for which we can mutate them and study their phenotypic consequences. This strategy should help in understanding the cellular and molecular role that these genes play in both C. elegans and humans. Suppressor screens should also prove informative in identifying interacting and regulatory factors that influence the function of that gene. Hopefully, our findings will lead to investigations in other model organisms and potentially to genes that might prove useful as therapeutic targets.
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