FXTAS: Mechanisms and Modifiers

总结 脆性X相关震颤/共济失调综合征（FXTAS）是一种成人发病的神经退行性疾病，影响 脆性X染色体智力低下1（FMR 1）前突变等位基因（55-~200个CGG重复）。共同 FXTAS的特征包括进行性意向性震颤、步态共济失调、帕金森综合征和认知衰退。 外显率与年龄有关，男性携带者在80岁时达到~75%。高达15%的女性 前突变也显示FXTAS的症状。FXTAS的神经病理学标志包括泛素- 全脑核内包涵体阳性，小脑浦肯野神经元明显脱落。 在分子水平上，FMR 1前突变等位基因表现出FMR 1 mRNA和表达增加2至8倍。 含有长（~100）CGG三联体的突变体mRNA在细胞和动物模型中显示出毒性。 目前的数据支持FXTAS的两种非相互排斥的分子发病机制：1）RNA获得- 功能，其中RNA中扩增的CGG（rCGG）的表达干扰RNA结合的子集。 蛋白质（RBP），通过螯合功能限制其可用性，和2）重复相关的非AUG (RAN)翻译，由此通过rCGG（和/或反义rCCG）重复的翻译导致 产生毒性同源多肽，其中最丰富的是FMRpolyGlycine（FMRpolyG）， 干扰细胞功能。我们和其他人已经开发并使用了多种小鼠模型来研究 这些机制。纳尔逊、托德、艾伦和金小组先前使用模式生物（苍蝇、小鼠）开展的工作 并且细胞模型已经鉴定了几种受rCGG表达影响的RBP。其中包括Pur α、hnRNP A2/B1、DROSHA/DGCR 8和TDP 43。增加这些蛋白的表达可以调节rCGG介导的 模型系统中的毒性，支持FXTAS的RNA介导的螯合模型。此外，RAN 翻译产物存在于患者夹杂物和小鼠模型中，并且似乎也赋予毒性， 许多研究。在确定RAN翻译和RBP螯合的贡献的研究中， FXTAS发病机制，我们已经产生了表达hnRNPA 2/B1的转基因小鼠系， 和抑制rCGG重复介导的毒性而不改变FMRpolyG阳性包涵体。平行 埃默里脆性X染色体研究中心的研究工作使用全基因组序列（WGS）分析前突变携带者， FXTAS的早期或晚期发作，结合果蝇遗传筛选，以确定其他遗传修饰剂， 影响FXTAS发病年龄。了解这些基因中变异的作用可以提出候选基因。 治疗目标在这个应用中，我们建议确认和扩展遗传修饰剂的识别 通过序列分析和分析在埃默里大学使用人类遗传学鉴定的FXTAS的潜在修饰剂， 在贝勒、埃默里和密歇根进行的模型系统研究，使用了额外的苍蝇、细胞和小鼠模型。在 与项目1和3的协调，我们希望提高对FXTAS机制的理解， 其他脆性X相关疾病，如脆性X相关的原发性卵巢功能不全（FXPOI）。

SUMMARY Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects individuals with premutation alleles (55–~200 CGG repeats) in fragile X mental retardation 1 (FMR1). Common features of FXTAS include progressive intention tremor, gait ataxia, Parkinsonism, and cognitive decline. Penetrance is age-dependent and reaches ~75% in male carriers by age 80. Up to ~15% of women with premutations also show symptoms of FXTAS. The neuropathological hallmarks of FXTAS include ubiquitin- positive intranuclear inclusions throughout the brain and marked dropout of Purkinje neurons in the cerebellum. At the molecular level, FMR1 premutation alleles exhibit a 2 to 8-fold increase in FMR1 mRNA and expression of mutant mRNAs containing long (~100) CGG triplets has been shown to be toxic in cell and animal models. Current data support two non-mutually exclusive molecular pathogenesis mechanisms for FXTAS: 1) RNA gain- of-function, in which the expression of expanded CGGs in RNA (rCGG) interferes with a subset of RNA-binding proteins (RBPs), functionally limiting their availability through sequestration, and 2) Repeat-associated non-AUG (RAN) translation, whereby translation through the rCGG (and/or antisense rCCG) repeats leads to the production of toxic homo-polypeptides, the most abundant of which is FMRpolyGlycine (FMRpolyG), that in turn interfere with cellular functions. Multiple mouse models have been developed and used by us and others to study these mechanisms. Previous work by the Nelson, Todd, Allen and Jin groups using model organisms (flies, mice) and cell models has identified several RBPs affected by expression of rCGGs. Among these are Pur α, hnRNP A2/B1, DROSHA/DGCR8, and TDP43. Increasing expression of these proteins can modulate rCGG-mediated toxicity in model systems, supporting the RNA-mediated sequestration model of FXTAS. In addition, RAN translation products are found in patient inclusions and mouse models and appear to also confer toxicity in numerous studies. In studies to determine the contributions of both the RAN translation and RBP sequestration mechanisms to FXTAS pathogenesis, we have generated transgenic lines of mice that express hnRNP A2/B1 and suppression of rCGG repeat-mediated toxicity without alteration of FMRpolyG positive inclusions. Parallel efforts at the Emory Fragile X Center used whole genome sequence (WGS) analysis of premutation carriers with early or late onset of FXTAS, combined with fly genetic screens to identify additional genetic modifiers that influence age of onset of FXTAS. Understanding the role of variation in these genes could suggest candidate therapeutic targets. In this application, we propose to confirm and extend identification of genetic modifiers through sequence analysis and analyze potential modifiers of FXTAS identified at Emory using human genetics and model system studies at Baylor, Emory and Michigan using additional fly, cell and mouse models. In coordination with Projects 1 and 3, we expect to improve understanding of mechanisms that lead to FXTAS and other Fragile X-associated Disorders, such as Fragile X associated Primary Ovarian Insufficiency (FXPOI).