The role of non-coding variants in Usher disease

非编码变异在亚瑟病中的作用

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要初级纤毛或非运动纤毛几乎存在于每个哺乳动物细胞中。纤毛病会导致多个器官的疾病谱，包括脑、肾和肝。最让人衰弱的，然而，感觉神经病会导致耳聋和失明。视网膜纤毛病占占所有遗传性视网膜疾病 (IRD) 的三分之一，是视力障碍和视力障碍的主要原因儿童失明。 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听力逐渐下降，带来了巨大的健康负担想象。 Usher 2 型 (USH2) 是最常见的亚型，USH2A 基因负责大多数情况。 USH2 以常染色体隐性方式遗传；然而，大约 20% 在具有 USH2 典型临床特征的患者中，仅发现了一种 USH2A 突变外显子组测序，从而排除了明确的诊断。第二种疾病变异可能存在于 USH2A 基因的非编码区。鉴于 IRD 的蛋白质编码变体已被在人类和其他模型系统中研究发现，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，尤其是在基因产物与 USH2A 一样大且复杂（跨度 800 KB，包含 72 个外显子），因此仍处于诊断不足状态。 IRD 患者的基因组测序可识别致病性非编码监管区域的变化来解释原因变化，并且越来越多地被用来对患有疑似单基因疾病的患者进行基因诊断。定相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 基因座及其周围调控区域的定相将提供更准确的检测单等位基因患者的致病变异。这将通过结合 Illumina 短读长测序的基本精度，其中较长读长来自基于纳米孔的免扩增靶向测序。非编码变体的识别及其阶段信息是了解其对患者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异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) 分化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异的致病性和突变性负担。该提议具有巨大的治疗潜力，因为非编码深内含子变异已一直是各个IRD基因打靶和基因编辑技术的焦点。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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。

DOI：
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

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.)
影响因子：
0
作者：
Mustafi,Debarshi;Huang,Johnson;Ting,MichelleA;Waligorski,Natalie;Stacey,AndrewW;Huang,LauraC
通讯作者：
Huang,LauraC

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