Screen and functional validation of Pediatric Cardiomyopathy genetic variants in Drosophila

果蝇小儿心肌病遗传变异的筛选和功能验证

• 批准号: 10634898
• 负责人: ZHE HAN
• 金额: $ 65.04万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-04 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10634898
• 关键词: 1 year old; Address; Adult; Affect; Alleles; Antibodies; Biological Assay; Candidate Disease Gene; Cardiac; Cardiomyopathies; Cessation of life; Child; Childhood; Classification; Clinical; Communities; Complex; Data; Defect; Development; Diagnosis; Dilated Cardiomyopathy; Disease; Disease Progression; Disease model; Drosophila genus; Dyes; Etiology; Family; Feedback; Future; Gene Combinations; Gene Silencing; Genes; Genetic; Genetic Heterogeneity; Genotype; Goals; Heart; Heart Diseases; Heart Transplantation; Homologous Gene; Human; Hypertrophic Cardiomyopathy; Infant; Injections; Libraries; Modeling; Mutation; Myopathy; Nature; Newborn Infant; Pathologic; Patients; Pediatric Cardiomyopathy; Phenotype; Proteins; Publishing; RNA Interference; Rapid screening; Research; Research Personnel; Sample Size; Series; Stains; System; Testing; Transgenic Organisms; Uncertainty; Validation; Variant; bioinformatics tool; candidate identification; candidate validation; disease mechanisms study; early childhood; effective therapy; exome sequencing; fly; gain of function; gene function; gene replacement; genetic architecture; genetic variant; improved; in silico; in vivo; insight; loss of function; mortality; mutant; new technology; novel; screening; targeted treatment; tool

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.

项目概要 小儿心肌病 (PCM) 是一种罕见但严重的儿童早期心肌疾病 发病时，治疗选择有限，由于有效治疗有限，通常最终导致儿童死亡 A。 最近发表的外显子组测序研究涉及 528 名不同类型 PCM 患者，这是迄今为止规模最大的研究， 揭示了对 PCM 遗传结构的见解，并鉴定了 125 个基因中的 343 个变异。 确认儿童 CM 与成人发病 CM 具有不同的遗传基础，这进一步证明了这一点。 PCM 的遗传复杂性；患者携带多个基因变异，表明具有多基因性质； 此外，大多数候选基因没有与不同 PCM 类型相关的同一基因的变异。 为了弥合候选变异和疾病因果关系之间的差距，我们的团队之前已经在心脏中进行过研究。 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者，以 根据导致蛋白质功能缺陷的可能性对它们进行优先级排序，并指定最佳建模方法 然后，我们将根据突变类型，通过我们的“基因替换”方法来遵循这一点。 第343章 PCM 具体来说，我们将： 1）使用针对果蝇的生物信息学工具来筛选候选变体。 PCM 基因和变体在果蝇心脏中建模的可行性，以确定最佳建模 方法，并提供种间（果蝇-人类）心脏表型相关性以与 2) 使用我们的果蝇心脏特异性 RNAi 基因沉默系统来提供“基因- 对所有候选 PCM 变体进行“级别”验证，可能会导致功能丧失，并指定为可行的 我们将扩展果蝇心脏表型以开发一种分析方法。 我们将使用可以区分不同 PCM 类型的综合评估。 PCM 果蝇模型生成心脏表型库 3) 使用我们的果蝇“基因替换”方法。 为功能增益或效果不确定的候选 PCM 变体提供“变体级别”验证，如 以及为携带多种遗传变异的患者生成精确的多基因模型。 完成拟议目标将建立一个高效的“计算机-体内”管道来筛选和 从功能上验证从 PCM 患者中识别出的遗传变异，为数百个患者提供因果关系 PCM 候选变体，并建立从果蝇到人类的种间心脏表型相关性。 该项目中生成的许多飞行 PCM 模型将是“精准疾病模型”，因为它们携带 果蝇心脏中存在特定的人类患者变异，因此可以在未来用于研究疾病机制 并测试靶向治疗。