Molecular mechanisms underlying strabismus risk

斜视可损害视力和社交能力，其潜在的病理生理机制 人们对此仍然知之甚少。目前的治疗方法往往不能完全恢复视觉功能，也不能解决 潜在的病理学。斜视有明确的遗传成分，但确切的遗传机制没有 已经被定义了。我们最近发现了三种罕见的、反复发生的基因重复，它们会增加内斜视的风险。 这些复制中的每一个都包括一个长的非编码RNA(LncRNA)，它通常与染色质有关 基因表达的重塑和调控。复制也可以通过插入 新位置的调控元件或3D染色质结构的破坏。因此，我们假设 基因表达调控是斜视发生的重要机制。这一点得到了 研究发现，导致斜视的已知环境风险因素，包括早产、母亲吸烟和低血压 出生体重，通过甲基化的变化影响表观遗传调节。本提案旨在(1)界定 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)评估内斜视和外斜视患者的单核苷酸变异(SNV)或小插入 或基因和调控区域的缺失(Indels)，这些基因和调控区域包括在复制中或受 重复，以及(3)通过对斜视的全基因组测序来确定斜视的其他遗传原因 斜视大家庭。将确定复制的精确断点和插入点 通过长时间的全基因组测序，将每个复制引入诱导多能干细胞 细胞(IPSCs)通过CRISMERE(CRISPR/Cas9的变体)。基因表达、增强子活性和 将比较干细胞、神经前体细胞和分化的神经元之间的染色质构象。 使用和不使用每个复制。每次复制对神经元形态和功能的影响将是 评估过了。Fluidigm多路传输和下一代测序将使我们能够经济高效地筛选我们的大型 斜视队列的SNV和INDELs在编码和调节区的基因包括 复制，以及其表达被复制改变的基因。在多个版本中确定的变体 将通过体外功能研究对被预测为生物信息损害的个体进行评估。 将招收更多有多名斜视成员的家庭，并进行编码、非编码和 结构变异将通过全基因组测序来识别。变体将根据以下因素确定优先级 连锁、生物信息学预测和种群频率。此外，表观遗传学和3D互作组 来自神经前体细胞和神经元的地图将被用来对变体进行优先排序。功能研究将在以下方面进行 高度优先确定的变种。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，这将使斜视的发展 基于潜在病理生理学的斜视新疗法或预防性干预。

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.