Molecular mechanisms underlying strabismus risk

斜视可以是视觉和社会衰弱和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恢复完整的视觉功能，并且不能解决视觉障碍。 潜在的病理学斜视有一个明确的遗传成分，但精确的遗传机制还没有 已定义。我们最近发现了三种罕见的，经常性的基因重复，增加了内斜视的风险。 这些重复中的每一个都包括一个长的非编码RNA（lncRNA），它通常参与染色质 重塑和基因表达的调节。重复也可以通过插入 调节元件在新的位置或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。这一点得到了 研究发现，已知的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节。本建议旨在（1）界定 这些复制对基因表达、染色质结构和神经元形态的影响， 功能，（2）评估单核苷酸变异（SNV）或小插入的内斜视和外斜视患者 在复制中包括的或受复制影响的基因和调控区中的插入或缺失（indel）。 重复，和（3）通过全基因组测序确定斜视的其他遗传原因， 大斜视家庭。将确定复制品的精确断点和插入点 通过长读全基因组测序，然后将每个重复引入诱导多能干细胞 通过CRISMERE（CRISPR/cas9的变体）培养iPSC细胞。基因表达，增强子活性，和 将比较干细胞、神经祖细胞和分化的神经元之间的染色质构象 无论是否重复。每一个复制对神经元形态和功能的影响将是 评估。Fluidigm多路复用和下一代测序将允许具有成本效益的筛选我们的大型 斜视组群中包括的基因的编码和调控区中的SNV和插入缺失， 复制，以及其表达被复制改变的基因。在多个国家中发现的变异体 将用体外功能研究来评估个体和预测的生物信息学损害。 将入组具有多名斜视成员的额外家庭，并对编码、非编码和 将通过全基因组测序鉴定结构变异。将根据以下因素对变体进行优先排序： 联系，生物信息学预测和人口频率。此外，表观遗传和3D相互作用组 来自神经祖细胞和神经元的图谱将用于区分变体的优先级。功能研究将在 高优先级的已识别变体。这项工作，通过识别基因和信号通路，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 新的斜视治疗或基于潜在病理生理学的预防性干预。

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.