The Investigation of disease causing genes in C. elegans

线虫致病基因的研究

• 批准号: 10706086
• 负责人: Andy Golden
• 金额: $ 115.85万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10706086
• 关键词: Academia; Address; Affect; Alleles; Anabolism; Animal Model; Animals; Arrhythmia; BSCL2 gene; Back; Biogenesis; Biological; Biological Assay; Biological Process; Biology; C. elegans genome; CRISPR/Cas technology; Caenorhabditis elegans; Calcium; Cells; Chemicals; Clinical; Collaborations; Complex; Coupled; Data; Defect; Diet; Disease; Drosophila genus; Embryo; Enhancers; Enzymes; Family; Fatty Acids; Functional disorder; Gene Deletion; Gene Expression; Genes; Genetic; Growth; Health; Heterozygote; Human; Investigation; Ion Channel; Ions; Iron; Lead; Learning; Ligase; Lipids; Lipodystrophy; Literature; Metabolic; Metabolic stress; Missense Mutation; Mitochondria; Molecular; Mothers; Movement; Mutate; Mutation; Natural History; Nonsense Mutation; Oocytes; Organism; Orthologous Gene; Ovulation; Oxygen Consumption; Pathway interactions; Patients; Permeability; Phenotype; Play; Process; Prostaglandins; Publishing; Regulation; Reporting; Reproducibility; Role; Series; Signal Transduction; Site; Spermatids; Sterility; Stress; Sulfur; Suppressor Mutations; Syndrome; Taiwan; Testing; Therapeutic; Thioctic Acid; Timothy syndrome; Translating; Work; Zebrafish; base; college; disease phenotype; disease-causing mutation; enzyme biosynthesis; gene function; gene replacement therapy; genetic analysis; genome sequencing; human disease; insight; lipid biosynthesis; loss of function mutation; mechanical force; meetings; migration; mitochondrial dysfunction; mutant; programs; response; sperm cell; standard of care; stem cell therapy; therapeutic target; tool; transcriptome sequencing; whole genome

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.

对于已知相关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，并从基因上鉴定在同一途径中起作用的其他因素。为了使该策略发挥作用，必须满足许多标准。首先，必须有一个令人信服且明确的线虫直系同源物。其次，该基因中必须已经存在突变或缺失。为此，我们正在使用 CRISPR 技术来生成类似于人类疾病中发现的总基因缺失和突变等位基因。第三，必须有可评分、可重复的表型。表型的渗透性越强越好。如果满足这些标准，则可以进行遗传抑制子和增强子筛选，以识别与任何给定基因及其发挥作用的生物过程起作用的相互作用因子。在过去的一年里，我们继续对一些人类致病基因的线虫直系同源物进行研究。 我们已经确定，这些候选者中有许多满足上述所有标准——我们对这些基因进行了突变，它们揭示了非常渗透和可评分的表型。去年我们发布了其中两个项目的报告。 今年我们的重点是五种不同的疾病，其中之一是了解压电直系同源物在秀丽隐杆线虫中的功能作用。这两种人类直向同源物的突变会导致多种不同的疾病。压电是一系列机械敏感离子通道，可将机械力转化为生物反应，其中钙离子是重要的离子。完整的基因缺失和许多患者特异性等位基因是通过 CRISPR/Cas9 在秀丽隐杆线虫 pezo-1 直向同源物中生成的，并且所有这些都渗透到不寻常的表型；它们会导致排卵缺陷，导致卵母细胞在通过受精囊时被压碎。这会导致育雏规模减小，并且可以轻松观察和量化。信号传递似乎也存在缺陷，每次排卵后从受精囊中冲出的精子细胞无法迁移回受精囊，从而耗尽受精囊中的精子。这也导致我们的 pezo-1 突变体的巢体尺寸较小。这个最初的故事发表在 eLife 上。为了进一步了解精子迁移缺陷，我们现在专注于前列腺素（PG）生物合成和脂质生物学。 PG 是已知的精子迁移到受精囊的引诱剂。 PEZO-1 如何影响 PG 生物合成是一个谜，我们希望更好地理解这个过程。来自我们的 pezo-1 突变体的 RNA-seq 数据确实显示了许多脂质生物合成酶的有趣调节，我们现在正在测试这些基因如何参与精子表型以及它们如何与 PEZO-1 连接。 在几年前启动的另一个项目中，我们描述了秀丽隐杆线虫的另一种不寻常的表型。人类 seipin 基因突变会导致脂肪营养不良。我们制作了无效突变和患者特异性错义突变，并观察到这些突变体是亚存活的；大量纯合母亲的后代在胚胎时死亡。我们已经证明，这种致死率是由蛋壳形成缺陷引起的，导致渗透性胚胎受到渗透压。与博士合作。奥尔森（波莫纳学院）和王（台湾中央研究院），我们已经证明饮食中的某些脂肪酸可以挽救这种致命的表型。这部作品最近出版了。我们正在继续表征这种表型，并开始进行抑制筛选，以确定可能恢复这些突变体活力的其他因素。我们目前正在绘制许多抑制因子并进行全基因组测序，以识别相关基因。我们认为脂滴的形成受到这些突变的影响，并且这些脂滴必定有助于蛋壳渗透性屏障的形成，蛋壳是蛋壳的富含脂质的层。然而，这种联系可能并不那么直接，因为我们的一些抑制剂抑制胚胎致死率，但不抑制脂滴大小的变化。 过去两年，我们根据在会议、未确诊疾病项目的临床查房或文献中了解到的疾病启动了一些其他项目。使用 CRISPR/Cas9 编辑线虫基因组，我们制作了删除等位基因以确定每个基因的无效表型，并制作了患者特异性等位基因来模拟与疾病相关的特定无义或错义突变。我们目前正在研究蒂莫西综合症（TS，使用egl-19基因），这是一种非常罕见的心律失常综合症，伴有许多其他健康问题。当纯合子时，线虫中的突变会导致胚胎致死，因此我们正在研究这种疾病等位基因对杂合子的影响。在人类中，蒂莫西综合征占主导地位，因此我们对杂合子的分析可能会提供丰富的信息。 我们还在研究最近文献中报道的一些 TS 等位基因，看看它们是否也具有致命性。除了我们对秀丽隐杆线虫的研究之外，我们还即将与凯瑟琳·蒂莫西（Katherine Timothy）合作完成一项自然历史研究，她是第一个认识到这种综合征的人。除了这些线虫研究之外，我们最近还培育了一条 TS 斑马鱼来检查其表型。 我们在涉及多发性线粒体功能障碍综合征（MMDS，使用基因 nfu-1，正式名称为 lpd-8）的基因研究方面也取得了良好进展。引起这些综合征的基因都与 Fe-S 簇的生物发生有关，Fe-S 簇是许多线粒体酶以及许多非线粒体酶的关键辅助因子。 nfu-1 中纯合的患者特异性错义突变会导致生长缓慢、运动不良和不育。我们现在正在进行代谢测定，以解决这些突变体中线粒体的功能障碍。除了耗氧量缺陷外，我们还发现这些突变动物存在严重的代谢应激，触发至少三种应激途径的基因表达。我们发表了关于缺失突变体和 5 个患者特异性等位基因的初步研究结果。我们的患者特异性等位基因代表了一个等位基因系列，并且大多数具有与我们的缺失等位基因相似的表型。我们目前正在测试许多在 Fe-S 簇生物发生中发挥作用的其他基因，以确定它们在突变时是否也产生类似的表型。最近，我们启动了一项对另一种线粒体基因 mecr-1 基因的研究。该基因参与硫辛酸的生物发生。这与我们的 nfu-1 项目密切相关，因为 NFU-1 的功能是将铁硫复合物传递给硫辛酸合成酶。我们预计这两种疾病在表型上可能有一些重叠。 我们将继续寻找与人类疾病有关的具有秀丽隐杆线虫直向同源物的基因，我们可以对其进行突变并研究其表型后果。这种策略应该有助于理解这些基因在秀丽隐杆线虫和人类中发挥的细胞和分子作用。抑制筛选还应该在识别影响该基因功能的相互作用和调节因素方面提供信息。希望我们的发现能够促进对其他模式生物的研究，并有可能导致对可能被证明可用作治疗靶点的基因的研究。