Functional Assessment of Variants in Organisms of Research (FAVOR) - Profiling Canonical Human Genes and their Variants through Disease Model Phenotyping.

项目摘要/摘要 全基因组DNA测序现在正被用于临床实践，并且越来越多的变种 在癫痫相关基因中被发现，然而对新变体的临床解释是具有挑战性的。 已知一些变异是病态的或良性的，然而大多数基因变异仍然存在。 它们的功能后果未知。大量不确定意义变量(VU)是 在人类疾病的基因中变得司空见惯，为诊断和治疗提供了一个重大障碍 实施治疗。生物信息学方法可以为VUS的致病概率提供一些洞察力 等位基因，但通常需要在动物模型系统中进行功能研究来确定致病性 任务。生产老鼠模型的费用和时间较长，使得替代方案的使用很少 动物模型很吸引人。在这项建议中，​Celegans​线虫被用作替代模型，能够 快速高通量生产和筛选。人类基因被安装为基因互换的替代物 原生疾病--基因同源。在前期工作中，​UNC-18​基因座STXBP1STXBP1的基因交换人源化 挽救了基因敲除动物中存在的严重运动和行为缺陷。致病变异体 进入STXBP1STXBP1DNA基因互换位点会导致活性的显著中断。在这项提议中，意义重大而新颖 对我们现有的用于体内变异功能分析的管道进行了改进。在目标1中， 通过提高线虫模型系统对人类疾病研究的相关性和可扩展性 多个相关基因座的同时人性化。在目标2中，发展了新的分子检测方法 表型，提高输入致病性确定算法的分辨率，并产生 不同操作的作用机制水平读数。在目标3中，对管道的改进是 进行测试，以量化在一组测试变种的致病性确定中的收益。

Project Summary / Abstract Genome wide DNA sequencing is now being adopted in clinical practice and an increasing number of variants are identified in epilepsy-associated genes, yet the clinical interpretation of the new variants is challenging. Some of the variants are known to be either pathological or benign, yet a majority of the gene variations remain unknown for their functional consequence. A large number of Variant of Uncertain Significance (VUS) are becoming commonplace in genes for human diseases, providing a significant barrier in making diagnoses and implementing therapies. Bioinformatic approaches can provide some insight into pathogenic probability of VUS alleles, but functional studies in animal model systems are often needed to make definitive of pathogenicity assignments. The expense and long timelines of mouse model production make the use of alternative small animal models attractive. In this proposal, the ​C. elegans​nematode is used as an alternative model capable of fast high-throughput production and screening. Human genes are installed as gene-swap replacements of the native disease-gene homologs. In preliminary work, gene-swap humanization of STXBP1 in the ​unc-18​locus rescued severe locomotion and behavior defects present in the gene knock-out animals. Pathogenic variants into the STXBP1​​gene-swap loci leads to significant disruption of activity. In this proposal, significant and novel improvements are made to our existing pipeline for the functional analysis of variants in vivo. In Aim 1, the relevance and extensibility of the C. elegans model system for studying human disease is improved through simultaneous humanization of multiple related loci. In Aim 2, new methods are developed for molecular phenotyping, improving the resolution of inputs pathogenicity determination algorithms, and yielding mechanism-of-action level readouts to variant manipulations. In Aim 3, the improvements to the pipeline are tested to quantify gains in pathogenicity determination on a test set of variants.