The role of non-coding variants in Usher disease

项目摘要/摘要 初级纤毛，或非运动纤毛，几乎存在于每一个哺乳动物细胞中。纤毛病导致 包括大脑、肾脏和肝脏在内的多个器官的疾病谱。最令人衰弱的， 然而，感觉神经病会导致耳聋和失明。视网膜纤毛疾病占了 占所有遗传性视网膜疾病(IRD)的三分之一，是视力障碍和 儿科人群中的失明。Usher综合征是最常见的视网膜纤毛病变 由于先天性听力障碍和进行性下降 幻象。Usher2型(USH2)是最常见的亚型，USH2a基因是导致 大多数情况下。USH2以常染色体隐性遗传方式遗传；然而，在大约20% 在具有典型USH2临床特征的患者中，只有一种USH2A突变被确定为 外显子组测序，从而排除了明确的诊断。第二种疾病变种可能存在于 USH2A基因的非编码区。鉴于IRD的蛋白质编码变体一直是 在人类和其他模型系统中研究，非蛋白质编码变体的作用有助于 人们对单等位基因疾病的了解要少得多。非编码变体很难分类，特别是在 像USH2A一样大而复杂的基因产物(跨度800千碱基，包含72个外显子)，因此 仍然被低估了。IRD患者的基因组测序可以识别致病的非编码 监管区域的变化来解释引起的变化，并越来越多地被用来 对疑似单基因疾病患者进行基因诊断。阶段性遗传变异是 对人类疾病研究也至关重要。我们假设基因组测序和单倍型 USH2A基因座及其周围监管区域的阶段化将提供更准确的 单等位基因患者致病变异的检测。这将通过将 获得较长读取长度的Illumina短读测序的基本精度 基于纳米孔的无扩增靶向测序。非编码变体及其变异体的鉴定 时相信息是了解其对患者的致病影响的重要第一步- 衍生干细胞。将使用荧光素酶敲入技术来研究非编码变体的致病性 细胞培养系统以及患者来源的诱导多能干细胞(IPSCs)在 在体外形成视网膜类器官(ROS)。ROS的使用将提供来自患者的临床相关组织 可以使用CRISPR-Cas9技术编辑以确定变异的致病性和突变 负担。这一提议具有巨大的治疗潜力，正如非编码深层内含子变体所具有的那样 成为各种IRD中基因打靶和基因编辑技术的焦点。

PROJECT SUMMARY/ABSTRACT Primary cilia, or non-motile cilia, are present in almost every mammalian cell. Ciliopathies cause a spectrum of diseases in multiple organs, including the brain, kidney and liver. The most debilitating, however, are sensory neuropathies leading to deafness and blindness. Retinal ciliopathies account for one third of all inherited retinal diseases (IRDs), and are a major cause of visual impairment and blindness in the pediatric population. Usher syndrome is the most common retinal ciliopathy and presents a tremendous health burden due to congenital hearing impairment and progressive decline in vision. Usher type 2 (USH2) is the most common subtype with the USH2A gene accounting for the majority of cases. USH2 is inherited in an autosomal recessive manner; however, in approximately 20% of patients with clinical features typical for USH2, only one mutation of USH2A has been identified by exome sequencing, thereby precluding a definitive diagnosis. The second disease variant may reside in non-coding regions of the USH2A gene. Whereas the protein coding variants of IRDs have been studied in human and other model systems, the role of non-protein coding variants contributing to monoallelic disease is much less understood. Non-coding variants are difficult to classify, especially in gene products as large and complex as USH2A (spans 800 kilobases and contains 72 exons), and thus remain under-diagnosed. Genome sequencing of IRD patients can identify pathogenic non-coding variations in regulatory regions to explain causative changes and is increasingly being used to genetically diagnose patients suffering from suspected monogenic disease. Phasing genetic variation is also critical for human disease studies. We hypothesize that genomic sequencing and haplotype phasing of the USH2A locus and its surrounding regulatory regions will provide a more accurate detection of pathogenic variants in monoallelic patients. This will be accomplished by combining the base-level accuracy of Illumina short-read sequencing with the longer read lengths obtained from Nanopore based amplification-free targeted sequencing. Identification of non-coding variants and their phase information are essential first steps towards understanding their pathogenic effects in patient- derived stem cells. The pathogenicity of non-coding variants will be explored using luciferase knock-in cell culture systems as well as patient-derived induced pluripotent stem cells (iPSCs) differentiated in vitro to form retinal organoids (ROs). Use of ROs will provide clinically relevant tissue from patients that can be edited using CRISPR-Cas9 technology to determine variant pathogenicity and mutational burden. This proposal has tremendous therapeutic potential as non-coding deep-intronic variants have been the focus of gene targeting and gene editing technologies in various IRDs.