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.

斜视在视觉和社会上可以使人衰弱及其潜在的病理生理机制保持不当理解。当前的治疗通常不会恢复完整的视觉功能，也不会解决潜在的病理。 Strabismus具有明显的遗传成分，但精确的遗传机制尚未已定义。我们最近确定了三个罕见的，复发性的遗传重复，以增加息肉的风险。这些重复中的每一个都包括长的非编码RNA（LNCRNA），这些RNA通常参与染色质基因表达的重塑和调节。复制也会通过插入来影响基因表达新位置的调节元素或3D染色质结构的破坏。因此，我们假设基因表达的调节是斜视的重要机制。这是由发现斜视的已知环境风险因素，包括早产，孕妇吸烟和低出生体重，通过甲基化的变化影响表观遗传调节。该建议旨在（1）定义这些重复对基因表达，染色质结构和神经元形态的后果以及功能，（2）评估单核苷酸变体（SNV）或少量插入的息肉和外部患者或在重复中包括的基因和调节区域中的缺失（indels）或受重复，（3）通过整个基因组测序确定斜视的其他遗传原因大型斜视家庭。将确定重复的确切断点和插入点通过长阅读的整个基因组测序，然后将每个重复引入诱导的多能茎细胞（IPSC）通过CRISMERE（CRISPR/CAS9的变体）。基因表达，增强子活性和将比较干细胞，神经元素和分化神经元之间的染色质构象有或没有每个重复。每个重复对神经元形态和功能的影响将是评估。流体多路复用和下一代测序将允许对我们的大型筛选 SNV和INIDELS的斜视队列在所包含的基因的编码和调节区域中复制以及表达因重复改变的基因。多个鉴定的变体通过体外功能研究，将评估个体并被预测会损害生物信息。将会招募有多个具有斜视的成员的其他家庭，编码，非编码以及结构变体将通过整个基因组测序识别。将根据联系，生物信息学预测和人口频率。另外，表观遗传和3D相互作用组来自神经生殖器和神经元的地图将用于优先考虑变体。功能研究将进行高优先级确定了变体。这项工作，通过识别有助于的基因和信号通路斜视的发展将提供对斜视发病机理的见解，这将允许发展基于潜在的病理生理学的新斜视治疗或预防性干预措施。