The role of non-coding variants in Usher disease

非编码变异在亚瑟病中的作用

• 批准号: 10588508
• 负责人: Debarshi Mustafi
• 金额: $ 23.03万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10588508
• 关键词: Accounting; Adult; Alleles; Biological Assay; Biological Models; Blindness; Brain; CRISPR/Cas technology; Catalogs; Cell Culture System; Cell Culture Techniques; Cells; Childhood; Cilia; Classification; Clinical; Code; Complex; Data Set; Detection; Diagnosis; Disease; Electrophysiology (science); Engineering; Exons; Family member; Gene Targeting; Genes; Genetic Variation; Genomics; Genotype; Haplotypes; Health; Human; In Vitro; Inherited; Integral Membrane Protein; Kidney; Knock-in; Length; Light; Liver; Luciferases; Mammalian Cell; Maps; Mendelian disorder; Microscopy; Modeling; Mutation; Nucleic Acid Regulatory Sequences; Organ; Organoids; Pathogenicity; Patients; Pattern; Phase; Phenotype; Photoreceptors; Population; Proteins; Reporter; Retina; Retinal Degeneration; Retinal Diseases; Role; Sensorineural Hearing Loss; Technology; Therapeutic; Tissues; Transport Process; USH2A gene; Untranslated RNA; Usher Syndrome; Usher Syndrome Type 2; Variant; Vision; Visual impairment; autosome; base; ciliopathy; clinically relevant; cohort; congenital hearing loss; deafness; disease phenotype; epigenomics; exome sequencing; gene product; genome sequencing; human disease; induced pluripotent stem cell; nanopore; novel; rare variant; reconstruction; sensory neuropathy; stem cell differentiation; stem cells; targeted sequencing; variant detection

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.

项目摘要/摘要 几乎每个哺乳动物细胞中都存在原发性纤毛或非运动纤毛。纤毛疾病会导致a 多个器官的疾病范围，包括大脑，肾脏和肝脏。最令人沮丧的， 但是，有感觉神经病导致耳聋和失明。视网膜纤毛病 所有遗传性视网膜疾病（IRD）的三分之一，是视觉障碍的主要原因 小儿人群的失明。 Usher综合征是最常见的视网膜纤毛病， 由于先天性听力障碍和逐步下降，给予巨大的健康负担 想象。 USHER 2型（USH2）是USH2A基因的最常见亚型 大多数案件。 USH2以常染色体隐性方式继承；但是，大约20％ 在USH2典型临床特征的患者中，仅确定了一个USH2A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基因的非编码区域。 IRD的蛋白质编码变体是 在人类和其他模型系统中进行了研究，非蛋白质编码变体的作用有助于 单一疾病的了解程度要少得多。非编码变体很难分类，尤其是在 与USH2A一样大的基因产物（跨度为800千酶，包含72个外显子），因此 保持未诊断。 IRD患者的基因组测序可以鉴定致病性非编码 监管区域的变化以解释原因变化，并越来越多地用于 遗传诊断患有可疑单基因疾病的患者。相位遗传变异是 对于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基因座及其周围的监管区域的分阶段将提供更准确的 单位型患者的致病变异检测。这将通过结合 Illumina简短读取测序的基础水平精度，从较长的读取长度获得 基于纳米孔的无扩增靶向测序。识别非编码变体及其 阶段信息是了解其在患者中的致病作用的重要第一步。 衍生的干细胞。使用荧光素酶敲入将探索非编码变体的致病性 细胞培养系统以及患者衍生的诱导多能干细胞（IPSC）在 体外形成视网膜器官（ROS）。 ROS的使用将为患者提供临床相关的组织 可以使用CRISPR-CAS9技术进行编辑以确定变异的致病性和突变 负担。该建议具有巨大的治疗潜力，因为非编码的深入性变体具有 是各种IRD中基因靶向和基因编辑技术的重点。

项目成果

Contributed Session I: Correlating cone structure and function in retinitis pigmentosa using coarse-scale optoretinography (CoORG).

贡献会议 I：使用粗尺度视视网膜成像 (CoORG) 关联色素性视网膜炎中的视锥细胞结构和功能。

• 发表时间: 2023
• 期刊: Journal of vision
• 影响因子: 1.8
• 作者: Liu,Teng;Pandiyan,VimalPrabhu;Wendel,Benjamin;Slezak,Emily;Mustafi,Debarshi;Chao,Jennifer;Sabesan,Ramkumar
• 通讯作者: Sabesan,Ramkumar

Familial co-segregation and the emerging role of long-read sequencing to re-classify variants of uncertain significance in inherited retinal diseases.

• DOI: 10.1038/s41525-023-00366-9
• 发表时间: 2023-08-10
• 期刊: NPJ GENOMIC MEDICINE
• 影响因子: 5.3
• 作者: Gupta, Pankhuri;Nakamichi, Kenji;Bonnell, Alyssa C. C.;Yanagihara, Ryan;Radulovich, Nick;Hisama, Fuki M. M.;Chao, Jennifer R. R.;Mustafi, Debarshi
• 通讯作者: Mustafi, Debarshi

Successful Treatment Response of a Juxtapapillary Retinal Capillary Hemangioblastoma Due to von Hippel-Lindau Syndrome with Belzutifan in a Pediatric Patient.

使用贝尔祖替芬对一名儿科患者因冯·希佩尔-林道综合征引起的近视乳头状视网膜毛细血管母细胞瘤的成功治疗反应。

• DOI: 10.1097/iae.0000000000003973
• 发表时间: 2024
• 期刊: Retina (Philadelphia, Pa.)
• 作者: Mustafi,Debarshi;Huang,Johnson;Ting,MichelleA;Waligorski,Natalie;Stacey,AndrewW;Huang,LauraC
• 通讯作者: Huang,LauraC