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，我们使用C。研究该基因的功能，并从遗传学上鉴定在同一途径中起作用的其他因子。为了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和明确的C。秀丽隐杆线虫直向同源物第二，这个基因中必须有一个已经存在的突变或缺失。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。表型越明显越好。如果满足这些标准，可以进行遗传抑制和增强筛选，以确定与任何给定基因及其功能的生物过程起作用的相互作用因子。在过去的一年里，我们已经确定了一些C。秀丽隐杆线虫是人类致病基因的直系同源基因。 我们已经确定，这些候选人中的许多人满足上述所有标准-这些基因中存在突变，并且它们揭示了非常明显和可评分的表型。 我们已经启动了一个项目，以模拟人类颅面综合征在C。优雅我们的同事来找我们，以确定是否在唯一的C。Twist碱性螺旋-环-螺旋（bHLH）转录因子的elegans直向同源物在C.优雅人类有两个Twist基因，Twist 1和Twist 2。扭转突变已经与其他颅面疾病如Saethre-Chotzen综合征有关。有趣的是，我们的临床同事最近发现，Twist 1和Twist 2的保守DNA结合基本结构域中的保守谷氨酸残基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，并假设导致Twist的显性阴性变体。在每种情况下，这种保守的谷氨酸被改变为五个其他氨基酸残基之一。 利用CRISPR/Cas9基因组编辑技术，我们对C. elegans hlh-8基因，人类Twist基因的唯一直系同源物。我们能够通过PCR、限制性内切酶和测序来筛选我们的突变，并且能够产生所有期望的突变。 我们的每一个突变都导致了一个非常明显的表型;纯合子动物是产卵缺陷型（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，以确定这些表型中的每一种的渗透性如何。我们还将这些菌株与GFP报告菌株杂交，以观察已知HLH-8靶标的表达。我们还研究了hlh-8：：gfp报告基因的表达，以了解更多关于发育中幼虫的M谱系的信息。M谱系负责产生肌肉，这些肌肉构成C中的产卵和排便系统。优雅使用这个标记，我们可以确定M后代何时分裂和迁移。这个标记还允许我们分析性肌肉（SM）的产生。有了这些标记，我们能够在细胞和分子水平上描述这些hlh-8等位基因的突变表型，并更好地将我们的等位基因分成不同的表型类别。有趣的是，这些突变体还显示出不同的雄性尾部表型，进一步使我们能够将这些突变体分为不同的类别。到目前为止，我们的数据表明，外阴肌肉对这些突变等位基因中的每一个都最敏感，而负责排便的肠肌的敏感性更易变。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。我们怀疑它们也以显性负性方式起作用，干扰靶基因的正确表达。我们渴望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？我们还计划在遗传抑制筛选中测试最弱的等位基因，以确定我们是否可以分离出逆转突变表型的抑制突变体，并将其恢复到更像野生型的状态。

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.