Genetic modifiers of Cep290-mediated retinal degeneration

摘要 Leber先天性黑色素是一组遗传性视网膜退行性疾病，其特征是 眼球震颤和失明，通常在生命的第一年表现出来。就像几个视网膜退行性变 疾病，许多形式的LCA涉及光感受器纤毛功能障碍。CEP290基因的突变是 LCA最常见的原因，暗示CEP290是疾病的主要贡献者。CEP290编码一个 大蛋白质被认为通过连接纤毛的光感受器调节蛋白质的运输 内段和外段。使用CEP290介导的LCA的小鼠模型，RD16小鼠，我们发现 小鼠CEP290表型的相对严重程度对遗传背景高度敏感。在这里，我们 建议使用小鼠进行实验，利用这种背景敏感性来识别遗传修饰物 CEP290介导的视网膜变性。这些修饰物的鉴定既有基础的，也有临床的， 意义。从基础生物学的角度来看，对遗传修饰物的研究可以揭示基础生物学 CEP290、光感受器纤毛的功能及其在视网膜疾病中的作用 背景。从临床的角度来看，基因修饰因子的识别提供了一个识别 治疗性代用品。我们目前提案的前提、其可行性以及我们有能力 进行视网膜变性的定量修饰物研究都源于一个相对较大的新近 工作。我们已经与小鼠进行了大规模的遗传杂交，并确定了修改视网膜的数量性状基因座 Cep290rd16突变纯合子小鼠的疾病严重程度。在基因组中被鉴定为 尤其重要的是，我们目前的建议集中在视网膜退行性变数量性状的修饰上 位于小鼠12号染色体上的基因座1(Mrdq1)。这个修饰符的一个独特特征有助于我们的能力 要确定它的分子基础是它表现出印记--它的影响因原产地而异。 使用物理图谱结合对视网膜表达基因的研究，我们有 在以前没有研究过的微小RNA中发现了一种明显的突变，极有可能是病因 突变。这一提议的实验描述了严格确认我们已经识别出 Mrdq1修饰物的精确突变(特定目标1)，并开始研究其作用机制 通过鉴定光感受器中存在的microRNA的下游靶标。完成后，我们将 预计这项工作将对CEP290介导的LCA产生影响，并促进对如何 两种研究不足的现象--通过microRNAs进行的基因调控和印记--影响了视网膜疾病。

Abstract Leber congenital amaurosis (LCA) is a group of inherited retinal degenerative diseases characterized by nystagmus and blindness that typically manifest in the first year of life. As with several retinal degenerative diseases, many forms of LCA involve dysfunction of photoreceptor cilia. Mutations in the CEP290 gene are the most common cause of LCA, implicating CEP290 as a major contributor to the disease. CEP290 encodes a large protein proposed to regulate protein transport through the photoreceptor connecting cilium spanning the inner and outer segments. Using a mouse model of CEP290-mediated LCA, the rd16 mouse, we have found that the relative severity of Cep290 phenotypes in mice is highly sensitive to genetic background. Here, we propose experiments using mice that take advantage of this background sensitivity to identify genetic modifiers of Cep290-mediated retinal degeneration. Identification of these modifiers has both basic, and clinical, significance. From a basic biology perspective, studies of genetic modifiers can uncover basic biological functions of CEP290, photoreceptor cilia, and their gestalt contributions to retinal disease in a physiologic context. From a clinical perspective, identification of genetic modifiers offers an opportunity to identify therapeutic surrogates. The premise for our current proposal, its feasibility, and evidence of our ability to conduct quantitative modifier studies of retinal degeneration all stem from a relatively large body of recent work. We have performed large genetic crosses with mice and identified quantitative trait loci modifying retinal disease severity of mice homozygous for the Cep290rd16 mutation. Among regions of the genome identified as particularly important, our current proposal focuses on the Modifier of retinal degeneration quantitative trait locus 1 (Mrdq1) located on mouse chromosome 12. A unique feature of this modifier that has aided our ability to identify its molecular basis is that it shows imprinting—its influence varies according to parent-of-origin. Using physical mapping in combination with a study of retinal expressed genes that are imprinted, we have identified an overt mutation within a previously unstudied microRNA that is highly likely to be the causative mutation. Experiments of this proposal describe the work to stringently confirm that we have identified the precise mutation underlying the Mrdq1 modifier (Specific Aim 1), and begin to study its mechanisms of action through identification of downstream targets of the microRNA present in photoreceptors. At completion, we expect this work to have impact on CEP290-mediated LCA, as well as promote a better understanding of how two understudied phenomena, gene regulation via microRNAs and imprinting, influence retinal disease.