Screen and functional validation of Pediatric Cardiomyopathy genetic variants in Drosophila

项目摘要 小儿心肌病（PCM）是一种罕见但严重的心肌疾病， 发病，治疗选择有限，由于有效治疗有限，往往导致儿童死亡。一 最近发表的对528名患有各种类型PCM的患者的外显子组测序研究，是迄今为止最大的， 揭示了PCM的遗传结构，并在125个基因中鉴定了343个变体。这些发现 证实儿童CM与成人发病CM具有不同的遗传基础。它进一步表明， PCM的遗传复杂性;患者携带多个基因的变异，表明多基因性质，和 同一基因的变异与不同的PCM类型相关。此外，大多数候选基因没有 以前在心里研究过。为了弥合候选变异和疾病病因之间的差距，我们的团队 已经开发了一种计算机管道来识别适合在果蝇心脏中建模的候选人， 根据可能导致蛋白质功能缺陷的可能性对它们进行优先排序，并指定最佳建模方法 在果蝇中的变异类型然后，我们将通过我们的“基因替代”方法， 高效的“基因水平”和“变体水平”的功能验证，以快速筛选343 PCM 候选变体。具体而言，我们将：1）使用针对果蝇的生物信息学工具，筛选候选人 针对PCM基因及其变异体在果蝇心脏中的可行性进行建模，确定最佳建模方案 方法，并提供种间（苍蝇-人）心脏表型相关性，以与 临床和研究社区。2)使用我们的果蝇心脏特异性RNAi基因沉默系统，提供“基因- 所有候选PCM变体的“水平”验证，可能导致功能丧失，并指定为可行 在果蝇的心脏中建模。我们将扩大苍蝇的心脏表型测定，以开发一种新的方法。 可以区分不同PCM类型的全面评估。我们将把这些发现用于 PCM飞行器建模以产生心脏表型文库。3)使用我们的果蝇“基因替换”方法 为功能增益或不确定影响的候选PCM变体提供“变体级”验证， 以及为携带多种遗传变异的患者生成精确的多基因模型。成功 完成拟议目标后，将建立一个高效率的“计算机-体内”管道， 从功能上验证从PCM患者中鉴定出的遗传变异，为数百种 PCM候选变异，并建立从果蝇到人类的种间心脏表型相关性。 在这个项目中产生的许多苍蝇PCM模型将是“精确的疾病模型”，因为它们携带了 在苍蝇心脏中发现了特定的人类患者变体，因此可以在未来用于研究疾病机制 并测试靶向治疗。

PROJECT SUMMARY Pediatric cardiomyopathies (PCM) are rare but serious disorders of the cardiac muscle with early childhood onset, limited treatment options, that often culminate in childhood death due to limited effective treatments. A recently published exome sequencing study of 528 patients with various types of PCM, the largest to date, revealed insights into the genetic architecture of PCM and identified 343 variants in 125 genes. The findings affirmed that pediatric CM has different genetic underpinnings than adult-onset CM. It further demonstrated the genetic complexity of PCM; with patients carrying variant in multiple genes, suggesting a polygenic nature, and variants in the same gene associated with different PCM types. Moreover, most of the candidate genes have not been studied in the heart before. To bridge the gap between candidate variant and disease causation, our team has developed an in silico pipeline to identify the candidate suited for modeling in the Drosophila heart, to prioritize them by likelihood to cause a functional defect in the protein, and assign the best modeling approach in fly based on the mutation type. We will then follow this through with our “gene replacement” approach for efficient “gene-level” and “variant-level” functional validations in the fly heart, to rapidly screen the 343 PCM candidate variants. Specifically, we will: 1) Use bioinformatic tools tailored to Drosophila, to screen the candidate PCM genes and variants for their feasibility to be modeled in the fly heart, to determine the best modeling approach for each, and to provide interspecies (fly-human) cardiac phenotypic correlations to be shared with the clinical and research communities. 2) Use our fly heart-specific RNAi gene silencing system to provide “gene- level” validation for all candidate PCM variants, likely to cause loss-of-function, and designated feasible for modeling in the Drosophila heart. We will expand the cardiac phenotype assays for fly to develop a comprehensive assessment that can distinguish between the different PCM types. We will use the findings in the PCM fly models to generate a cardiac phenotype library. 3) Use our Drosophila “gene replacement” approach to provide “variant-level” validation for candidate PCM variants that are gain-of-function or of uncertain effect, as well as to generate precision polygenic models for patients carrying multiple genetic variants. Successful completion of the proposed aims will establish a highly efficient “in silico - in vivo” pipeline to screen and functionally validate genetic variants identified from patients with PCM, provide causal association for hundreds of PCM candidate variants, and establish interspecies cardiac phenotypic correlation from Drosophila to humans. Many of the fly PCM models generated in this project will be “precision disease models” since they carry the specific human patient variants in the fly heart and thus could be used in the future to study disease mechanisms and to test targeted therapies.