Molecular mechanisms underlying strabismus risk

斜视风险的分子机制

基本信息

批准号：
10365004
负责人：
Mary Catherine Whitman
金额：
$ 66.16万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-01 至 2026-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10365004
关键词：
3-Dimensional Address Affect Area Binding Binocular Vision Bioinformatics Biological Assay Blindness CRISPR/Cas technology Cells Cephalic Child Chromatin Chromatin Structure Chromosome 10 Chromosome 2 Chromosome 4 Code Copy Number Polymorphism DNA Defect Development Enhancers Enrollment Environmental Risk Factor Epigenetic Process Esotropia Exotropia Eye Family Family Study First Degree Relative Frequencies Functional disorder Gene Cluster Gene Dosage Gene Expression Gene Expression Profile Gene Expression Regulation Genes Genetic Genetic Transcription Genetic study Goals Heritability Human In Vitro Individual Inherited Introns Knowledge Location Low Birth Weight Infant Luciferases Maps Methylation MicroRNAs Molecular Molecular Conformation Morphology Motor Neurons Muscle Neurons Nucleic Acid Regulatory Sequences Occupational Operative Surgical Procedures Paralysed Pathogenesis Pathology Patients Penetrance Population Population Study Premature Birth Proteins Recurrence Refractive Errors Regulation Regulatory Element Relative Risks Reporting Risk Risk Factors Running Signal Pathway Single Nucleotide Polymorphism Strabismus Syndrome Testing Twin Studies Untranslated RNA Variant Vision Work base chromatin remodeling cohort cost effective epigenetic regulation gene function genetic pedigree genome sequencing genome wide association study genomic locus imprint induced pluripotent stem cell insertion/deletion mutation insight maternal cigarette smoking member nerve stem cell neuron development next generation sequencing premature preventive intervention proband screening social stem cells therapy design whole genome

项目摘要

Strabismus can be both visually and socially debilitating and its underlying pathophysiological mechanisms remain poorly understood. Current treatments often do not restore full visual function and do not address the underlying pathology. Strabismus has a clear hereditary component, but precise genetic mechanisms have not been defined. We recently identified three rare, recurrent genetic duplications that increase risk of esotropia. Each of these duplications includes a long non-coding RNA (lncRNA), which are often involved in chromatin remodeling and regulation of gene expression. Duplications can also affect gene expression by insertion of regulatory elements in new locations or disruption of the 3D chromatin structure. We therefore hypothesize that regulation of gene expression is an important mechanism underlying strabismus. This is bolstered by the findings that known environmental risk factors for strabismus, including prematurity, maternal smoking, and low birth weight, affect epigenetic regulation through changes in methylation. This proposal aims to (1) define the consequences of these duplications on gene expression, chromatin structure, and neuronal morphology and function, (2) evaluate esotropic and exotropic patients for single nucleotide variants (SNVs) or small insertions or deletions (indels) in the genes and regulatory regions included in the duplications or affected by the duplications, and (3) identify additional genetic causes of strabismus through whole genome sequencing of large strabismus families. The precise breakpoints and insertion points of the duplications will be determined by long-read whole genome sequencing, then each duplication will be introduced into induced pluripotent stem cells (iPSCs) through CRISMERE (a variant of CRISPR/cas9). Gene expression, enhancer activity, and chromatin conformation will be compared between stem cells, neuroprogenitors, and differentiated neurons with and without each duplication. The effects of each duplication on neuronal morphology and function will be assessed. Fluidigm multiplexing and next-generation sequencing will allow cost-effective screening of our large strabismus cohort for SNVs and indels in the coding and regulatory regions of the genes included in the duplications, as well as genes whose expression is altered by the duplications. Variants identified in multiple individuals and predicted to be damaging bioinformatically will be evaluated with in vitro functional studies. Additional families with multiple members with strabismus will be enrolled, and coding, non-coding, and structural variants will be identified through whole genome sequencing. Variants will be prioritized based on linkage, bioinformatic predictions, and population frequency. In addition, the epigenetic and 3D interactome maps from neuroprogenitors and neurons will be used to prioritize variants. Functional studies will be done on high priority identified variants. This work, by identifying genes and signaling pathways that contribute to development of strabismus, will provide insights into strabismus pathogenesis, which will allow development of new strabismus treatments or preventative interventions based on the underlying pathophysiology.

斜视可能会导致视力和社交能力下降及其潜在的病理生理机制仍然知之甚少。目前的治疗通常不能完全恢复视觉功能，也不能解决潜在的病理学。斜视有明显的遗传成分，但尚无确切的遗传机制被定义。我们最近发现了三种罕见的、反复出现的基因重复，它们会增加内斜视的风险。每个重复都包含一个长非编码 RNA (lncRNA)，它通常参与染色质基因表达的重塑和调控。重复也可以通过插入来影响基因表达新位置的调控元件或 3D 染色质结构的破坏。因此我们假设基因表达的调控是斜视的重要机制。这得到了支持研究结果表明，已知斜视的环境危险因素包括早产、母亲吸烟和低出生体重，通过甲基化的变化影响表观遗传调控。该提案旨在 (1) 定义这些重复对基因表达、染色质结构和神经元形态的影响功能，(2) 评估内向性和外向性患者的单核苷酸变异 (SNV) 或小插入或重复或受重复影响的基因和调控区域中的缺失（插入缺失）重复，以及（3）通过全基因组测序确定斜视的其他遗传原因斜视大家族。将确定重复的精确断点和插入点通过长读长全基因组测序，然后将每个重复引入诱导多能干细胞（iPSC）通过 CRISMERE（CRISPR/cas9 的一种变体）。基因表达、增强子活性和将比较干细胞、神经祖细胞和分化神经元之间的染色质构象有和没有每次重复。每次重复对神经元形态和功能的影响将是评估。 Fluidigm 多重分析和下一代测序将能够对我们的大样本进行经济有效的筛选斜视队列中包含的基因的编码和调控区域中的 SNV 和插入缺失重复，以及其表达因重复而改变的基因。多处发现的变种生物信息学上预计会造成损害的个体将通过体外功能研究进行评估。将招募多名成员患有斜视的其他家庭，并进行编码、非编码和结构变异将通过全基因组测序来鉴定。变体将根据以下条件确定优先级：连锁、生物信息学预测和群体频率。此外，表观遗传学和 3D 相互作用组神经祖细胞和神经元的图谱将用于确定变异的优先顺序。功能研究将进行高优先级已识别变体。这项工作通过识别有助于斜视的发展，将提供对斜视发病机制的见解，这将允许发展基于潜在病理生理学的新斜视治疗或预防干预措施。