The Investigation of disease causing genes in C. elegans

对于已知致病基因的人类常染色体隐性遗传病，我们正在使用线虫来研究该基因的功能，并从基因上识别在相同途径中起作用的其他因素。要使这一战略奏效，必须满足一些标准。首先，必须有令人信服和清晰的线虫直系同源物。其次，这个基因必须存在突变或缺失。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突变等位基因。第三，必须有一个可评分的表型。表型越有穿透性越好。如果满足这些标准，就可以进行基因抑制和增强子筛选，以确定与任何给定基因起作用的相互作用因素及其发挥作用的生物过程。在过去的一年里，我们已经鉴定了一些与人类致病基因同源的线虫。我们已经确定，这些候选基因中的许多都满足上述所有标准--这些基因存在突变，它们显示出非常渗透性和可评分的表型。 我们已经启动了一个在线虫中模拟人类头面部综合征的项目。我们的同事与我们联系，以确定Twist碱性螺旋-环-螺旋(BHLH)转录因子的唯一同源突变是否导致线虫不同的表型。人类有两个Twist基因，Twist1和Twist2。扭曲突变已经与其他颅面疾病有关，如Saethre-Chotzen综合征。有趣的是，我们的临床同事最近表明，Twist1和Twist2保守的DNA结合基本域中保守的谷氨酸残基的突变与其他三种不同的颅面综合征有关，所有这些都是常染色体显性遗传的，并假设导致Twist的显性-阴性变异。在每种情况下，这种保守的谷氨酸被改变为其他五个氨基酸残基之一。 利用CRISPR/Cas9基因组编辑技术，我们对线虫hlh-8基因中的这种保守的谷氨酸进行了同源改变，这是人类Twist基因的唯一同源基因。我们能够通过聚合酶链式反应、限制性内切酶和测序来筛选我们的突变，并能够产生所有想要的突变。我们的每个突变都导致了一种非常明显的表型；纯合子动物是产卵缺陷(Egl)。有趣的是，只有一些突变导致便秘(CON)表型，并显示出非常畸形的尾巴。我们对这些菌株进行了定量表征，以确定每种表型的渗透性。我们还将这些菌株与GFP报告菌株杂交，以观察已知的HLH-8靶标的表达。我们还检测了hlh-8：：GFP记者的表达，以了解更多关于发育中的幼虫M谱系的信息。M世系负责产生肌肉，这些肌肉构成线虫的产卵和排泄系统。使用这个标记，我们可以确定M个后代何时分裂和迁徙。这个标记物还可以用来分析性肌(SMS)的生成。有了这些标记，我们已经能够在细胞和分子水平上表征这些HLH-8等位基因的突变表型，并更好地将我们的等位基因划分为不同的表型类别。有趣的是，这些突变体还表现出不同的雄性尾巴表型，进一步允许我们将这些突变体划分为不同的类别。到目前为止，我们的数据表明，外阴肌对这些突变的等位基因中的每一个都最敏感，而负责排便的肠肌的敏感度变化更大。这些等位基因中的每一个，就像人类的疾病一样，在自然界中也是半显性的。我们怀疑它们也在以显性-负向的方式起作用，干扰目标基因的正常表达。我们急于确定这些表型的分子机制：这些突变体是否与启动子结合，并不适当地打开或关闭导致这些表型的基因？我们还计划测试遗传抑制基因筛选中最弱的等位基因，以确定是否可以分离出逆转突变表型的抑制突变，并将它们恢复到更接近野生型的条件。

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 have 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.