The Investigation of disease causing genes in C. elegans

对于已知负责基因的人类常染色体隐性和显性疾病，我们正在使用秀丽隐杆线虫来研究该基因的功能，并从遗传学上确定在同一途径中起作用的其他因素。要使这一战略发挥作用，必须满足若干标准。首先，必须有一个令人信服的、清晰的秀丽隐杆线虫同源物。其次，这个已经存在的基因必须发生突变或缺失。为此，我们正在使用CRISPR技术来产生类似于人类疾病中发现的基因缺失和突变等位基因。第三，必须有一个可评分的、可复制的表型。表型渗透性越强越好。如果满足这些标准，就可以进行基因抑制和增强筛选，以确定与任何给定基因起作用的相互作用因素及其起作用的生物过程。在过去的一年里，我们继续研究了一些秀丽隐杆线虫与人类致病基因的同源物。我们已经确定，许多候选基因满足上述所有标准——我们对这些基因进行了突变，它们显示出非常具有渗透性和可评分的表型。在过去的一年里，我们已经发表了其中两个项目的报告。

For human autosomal recessive and dominant 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 total gene deletions and mutant alleles analogous to those found in human diseases. Third, there would have to be a scorable, reproducible 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 continued our investigations of 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. We have published reports for two of these projects in the past year. Our focus this year has been on five different diseases, one being to understand the functional roles of a Piezo ortholog in C. elegans. Mutations in the two human orthologs cause a multitude of distinct diseases. Piezo is a family of mechanosensitive ion channels that translate mechanical force into a biological response with calcium being the ion of importance. The full gene deletion and a number of patient-specific alleles were generated by CRISPR/Cas9 in the C. elegans pezo-1 ortholog and all are penetrant for an unusual phenotype; they cause defects in ovulation such that oocytes are crushed as they pass through the spermatheca. This results in reduced brood sizes and can be easily observed and quantified. There also seem to be defects in signaling such that the spermatids that are washed out of the spermatheca after each ovulation fail to migrate back to the spermatheca, thus depleting the spermatheca of sperm. This also contributes to the low brood size of our pezo-1 mutants. This initial story was published in eLife. In further pursuit of understanding the sperm migration defect, we are now focusing on prostaglandin (PG) biosynthesis and lipid biology. PGs are known attractants for sperm to migrate to the spermatheca. How PEZO-1 influences PG biosynthesis is a mystery and we hope to better undersatnd this process. RNA-seq data from our pezo-1 mutants does show interesting regulation of numerous lipid biosynthesis enzymes and we are now testing how these genes are involved in sperm phenotypes and how they connect to PEZO-1. In another project initiated a few years ago, we have described another unusual phenotype in C. elegans. Mutations in the human seipin gene cause lipodystrophy. We have made null and patient-specific missense mutations and observed that these mutants are sub-viable; a significant number of progeny of homozygous mothers die as embryos. We have shown that this lethality is caused by defects in eggshell formation, resulting in permeable embryos that are osmotically stressed. In collaboration with Drs. Olson (Pomona College) and Wang (Academia Sinica, Taiwan), we have shown that certain fatty acids in the diet can rescue this lethal phenotype. This work was recently published. We are continuing to characterize this phenotype and have started a suppressor screen to identify other factors that may restore viability to these mutants. We have a number of suppressors that we are currently mapping and performing whole genome sequencing with in order to identify the responsible gene. We believe the formation of lipid droplets is affected by these mutations and that these lipid droplets must contribute to the formation of the permeability barrier of the eggshell, which is a lipid-rich layer of the eggshell. However, this connection may not be so straight forward as some of our suppressors suppress the embryonic lethality but not the alterations in size of the lipid droplets. 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 nonsense or missense mutation that is associated with disease. We are currently investigating Timothy Syndrome (TS, using the egl-19 gene), a very rare 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 are also investigating some TS alleles that have been recently reported in the literature to see if they are lethal as well. In addition to our studies in C. elegans, we are close to completing a Natural History Study in collaboration with Katherine Timothy, who first recognized this syndrome. In addition to these C. elegans studies, we have recently generated a TS zebrafish to examine its phenotypes. We have also made good progress in our investigation of the genes involved in Multiple Mitochondrial Dysfunctions Syndromes (MMDS, using the gene nfu-1, formally known as lpd-8). The genes that cause these syndromes are all involved in the biogenesis of Fe-S clusters, key co-factors for a number of mitochondrial enzymes as well as many non-mitochondrial enzymes. Homozygous patient-specific missense mutations in nfu-1 result in slow growth, poor movement, and sterility. We are performing metabolic assays now to address the dysfunction in mitochondria in these mutants. In addition to defects in oxygen consumption, we have shown that there is severe metabolic stress in these mutant animals, triggering the gene expression of at least three stress pathways. We published our initial findings with a deletion mutant and 5 patient-specific alleles. Our patient-specific alleles represent an allelic series and most have a phenotype similar to that of our deletion allele. 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. Most recently, we initiated a study of the mecr-1 gene, another mitochondrial gene. This gene is involved in the biogenesis of lipoic acid. This ties in nicely with our nfu-1 project, since NFU-1 functions to deliver iron-sulfur complexes to lipoic acid synthetase. We anticipate that these two diseases may have some overlap in phenotype. 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.