The role of non-coding variants in Usher disease

项目总结/摘要 初级纤毛，或非运动纤毛，存在于几乎每一个哺乳动物细胞。睫状体病导致 多器官疾病谱，包括脑、肾和肝。最让人虚弱的， 然而，感觉神经病变导致耳聋和失明。视网膜睫状体病 三分之一的遗传性视网膜疾病（IRD），是视力障碍的主要原因， 儿童群体的失明。Usher综合征是最常见的视网膜睫状体病， 由于先天性听力障碍和听力的逐渐下降， 视野Usher 2型（USH2）是最常见的亚型，USH2A基因占 大多数案件。USH2以常染色体隐性方式遗传;然而，在大约20%的 在具有USH2典型临床特征的患者中，仅鉴定出USH2A的一种突变， 外显子组测序，从而排除了明确的诊断。第二种疾病变体可能存在于 USH2A基因的非编码区。然而，IRD的蛋白质编码变体已经被 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 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 基因产物大而复杂，如USH2A（跨越800个内切酶，含有72个外显子），因此 仍然诊断不足。IRD患者的基因组测序可以识别致病性非编码 调节区域的变化来解释因果变化，并越来越多地用于 对疑似单基因疾病的患者进行基因诊断。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体型 USH2A基因座及其周围调控区域的定相将提供更准确的 检测单等位基因患者中的致病性变体。这将通过将 Illumina短读段测序的碱基水平准确度，其中较长的读段长度从 基于纳米孔的无扩增靶向测序。非编码变体的鉴定及其应用 相位信息是了解其在患者中致病作用的重要第一步， 衍生干细胞将使用荧光素酶敲入来探索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（iPSC）在 体外形成视网膜类器官（RO）。RO的使用将提供来自患者的临床相关组织， 可以使用CRISPR-Cas9技术进行编辑，以确定变体致病性和突变性。 负担这一提议具有巨大的治疗潜力，因为非编码深层内含子变体具有 一直是各种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.