Mutational analysis to understand the role of CHML in developmental regression

 描述（由申请人提供）：对罕见疾病的研究很重要，因为它们有助于揭示疾病的潜在机制，可能为患者带来新的治疗方法。这些努力也为发现在生物医学研究中具有重大意义的新生物过程提供了难得的机会。该应用程序旨在探索一个独特的基因型-表型关系的基础上，谁是在美国国立卫生研究院未诊断疾病计划（UDP）登记的患者。患者在CHML基因中携带两个复合杂合错义突变，其中一个突变等位基因遗传自父母双方。患者表现出发育退化和神经变性，包括进行性皮质和白色物质丢失。异常表型在患者的兄弟姐妹和他们的父母中不存在，他们不携带任何突变。在人类和其他哺乳动物中，旁系同源基因CHM和CHML分别编码Rab护送蛋白REP-1和REP-2。Rab家族由大量GTP酶组成，其调节复杂的膜运输事件。翻译后异戊烯化是将Rabs靶向特定亚细胞膜所必需的。REP对于Rab的适当异戊烯化是必不可少的。在斑马鱼中，只有一个REP编码基因chm，该基因的无义突变导致多个器官的退化。已经对哺乳动物CHM直系同源物进行了广泛的功能研究。CHM的错义突变导致无脉络膜患者眼组织变性。已经提出，这些患者的眼外组织，如脑组织，可能受到CHML的保护。然而，令人惊讶的是，几乎没有CHML直系同源物的功能研究，尽管他们认为的重要性，直到第一个证据的影响，CHML从这个UDP的情况。在本申请中，我们建议成为第一组来测试相关的主要假设，即CHML是一种不可或缺的保护脑组织的变性使用小鼠突变的CHML基于其高度保守的人类直系同源物。在目标1中，我们将在小鼠中工程化Chml无效等位基因和UDP患者特异性错义突变。在目标2中，我们将分析错义突变和无效等位基因作为功能丧失对照的复合杂合性的表型后果。表型表征将包括脑结构的磁共振成像（MRI）和体视学分析以及运动和认知功能的行为分析。我们还将分析突变小鼠中的Rab异戊烯化，以探索基因型和表型之间的机制联系。该项目的成功将首次揭示CHML在维持中枢神经系统完整性方面的重要作用，从而为UDP患者的未来研究奠定机制基础，包括治疗研究。该项目的成功还将开辟CHML突变和CHML相关生物过程可能在其他神经退行性疾病以及自闭症谱系障碍中发挥关键作用的可能性，其中发育退化是重要的临床特征。

 DESCRIPTION (provided by applicant): Studies on rare diseases are important because they help to unravel the underlying mechanisms of diseases which may lead to novel therapies for patients. These efforts also offer rare opportunities to discover novel biological processes with major significance in biomedical research. This application is designed to explore a unique genotype-phenotype relationship based on a patient who is enrolled in the NIH Undiagnosed Disease Program (UDP). The patient harbors two compound heterozygous missense mutations in the CHML gene, with one mutant allele inherited from each parent. The patient exhibits developmental regression and neurodegeneration, including progressive cortical and white matter loss. The abnormal phenotypes are absent in the patient's siblings, who do not carry either mutation, and in their parents. In humans and other mammals, the paralogous genes CHM and CHML code for the Rab escort proteins REP-1 and REP-2, respectively. The Rab family consists of a large number of GTPases, which regulate complex membrane trafficking events. Posttranslational prenylation is required for targeting Rabs to specific subcellular membranes. REPs are essential for appropriate prenylation of Rabs. In zebrafish, there is only a single REP-coding gene, chm, and a nonsense mutation of the gene leads to degeneration of multiple organs. Extensive functional studies have been carried out on mammalian CHM orthologs. Null and missense mutations of CHM lead to degeneration of tissues in the eyes of patients with choroideremia. It has been proposed that tissues outside of the eyes in these patients, such as brain tissues, are likely protected by CHML. However, surprisingly, there are almost no functional studies of CHML orthologs despite their perceived importance until the first evidence of the impact of CHML from this UDP case. In this application, we propose to be the first group to test the related major hypothesis that CHML is an indispensable protector of brain tissues from degeneration using mouse mutations of Chml based on its high conservation with the human ortholog. In Aim 1, we will engineer Chml null alleles and the UDP patient-specific missense mutations in mice. In Aim 2, we will analyze the phenotypic consequences of compound heterozygosity of the missense mutations and the null alleles as loss-of-function controls. Phenotypic characterizations will include magnetic resonance imaging (MRI) and stereology analysis of brain structures as well as behavioral analysis of motor and cognitive functions. We will also analyze Rab prenylation in the mutant mice to explore the mechanistic link between genotypes and phenotypes. The success of this project will reveal for the first time the essential role of CHML in maintaining the integrity of the central nervous system, thereby laying the mechanistic foundation for future studies on the UDP patient, including therapeutic studies. The success of this project will also open up possibilities that CHML mutations and CHML-associated biological processes may play critical roles in other neurodegenerative disorders as well as autism spectrum disorder in which developmental regression is an important clinical feature.