A new diagnostic tool for rapid detection and characterization of REPEAT SEQUENCES in inherited diseases

一种新的诊断工具，用于快速检测和表征遗传性疾病中的重复序列

• 批准号: 10354657
• 负责人: Jason C Reed
• 金额: $ 25.57万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10354657
• 关键词: Affect; Age of Onset; Ataxia; Atomic Force Microscopy; Bar Codes; Benchmarking; Bioinformatics; CGG repeat; Capillary Electrophoresis; Child; Clinic; Clinical; Complex; Coupled; DNA; DNA Sequence Alteration; Decision Making; Detection; Development; Diagnosis; Diagnostic; Disease; Disease Management; Early Diagnosis; Elderly; Equilibrium; FMR1; FXTAS; Family; Family Planning; Family member; Fragile X Syndrome; Generations; Genes; Genetic; Genetic Anticipation; Genetic Counseling; Genetic Phenomena; Genetic Risk; Genome; Genomic DNA; Genomics; Goals; Healthcare; Hereditary Disease; Hospitals; Individual; Inherited; Insurance Carriers; Label; Lead; Length; Lesion; MJD1 protein; Measures; Medical; Medical Genetics; Meiosis; Memory Disorders; Methodology; Methods; Molecular; Monitor; Mood Disorders; Movement; Mutation; Nature; Patient Monitoring; Patients; Performance; Persons; Phenotype; Physicians; Polymerase; Polymerase Chain Reaction; Procedures; Process; Product Labeling; Prognosis; Relative Risks; Risk; Sampling; Severities; Skeletal Muscle; Southern Blotting; Speed; Spinocerebellar Ataxias; Stretching; Symptoms; System; Techniques; Technology; Testing; Time; Tremor; Trinucleotide Repeat Expansion; Trinucleotide Repeats; United States; Variant; Walking; base; biobank; clinically relevant; cost; design; diagnostic tool; genetic analysis; genetic disorder diagnosis; genetic testing; health care economics; improved; member; multiplex assay; multiplex detection; mutant; nanoparticle; nanopore; nervous system disorder; new therapeutic target; next generation sequencing; non-genetic; novel diagnostics; novel strategies; offspring; patient stratification; rapid detection; risk stratification; screening; sequencing platform; success; targeted treatment; tool; virtual

=. PROJECT SUMMARY …... Ataxia is a discoordination of voluntary muscle movement and can be seen as the primary symptom of multiple disorders including genetic and non-genetic etiologies. Several genes are implicated in causing Spinocerebellar ataxia through trinucleotide repeat expansions (TREs) and are thought to affect between 1.5 to 4 per 10,000 people globally. Similar trinucleotide repeat expansions in the FMR1 gene, including potentially symptomatic premutations, can be found in around 1 in 300 people in the United States. Detection of repeat expansion disorders in a family are important for understanding genetic risks and early detection in at risk members, especially since unstable repeat expansions lead to the phenomenon of genetic anticipation, where symptoms can present earlier or more severely across generations. Additionally, several recent studies have suggested development of targeted therapies specifically targeting expanded repeats as a novel therapeutic target. As such, earlier and rapid detection of these disorders is crucial for advancing their medical management. Traditionally, short TRE targets are assessed via repeat-primed PCR (PR-PCR) and capillary electrophoresis, while longer mutants are confirmed via Southern blotting. This is clinically important, especially where repeat lengths outside of the range quantifiable by PR-PCR are diagnostically relevant. Testing for most TRE genes remains difficult for short read sequencing platforms, where repeat tract lengths exceed average read length (~100 bp for Illumina systems). Long-read sequencing technologies like PacBio and Oxford Nanopore have shown success in measuring TREs, however a combination of high costs, large quantities of DNA input, complex bioinformatics, problems determining repeat region boundaries, and high error rates makes them an unlikely solution for widespread screening in their current state We developed a solution, PRECYSE, based on high-speed atomic force microscopy (HSAFM) paired with unique ‘nanoparticle barcoding’ that can potentially characterize complex structural variants for numerous ataxia conditions simultaneously at much lower cost than possible with next generation sequencing (NGS) sequencing and other emerging approaches. This extremely sensitive technique can be conducted without using polymerase chain reaction (PCR) and can span a very wide range of possible target sizes, thereby allowing extension to virtually unlimited molecular lengths. Our hypothesis is that this technology can be easily adapted to multiplexed detection of TRE targets of practically any length. If successfully developed, our new approach will change the way clinicians identify, understand, and monitor changes in the genome caused by trinucleotide repeat expansion diseases through multiplexed panels. At the end of this R21 project, we will have a platform for multiplexed genomic analysis that successfully purifies and detects trinucleotide repeat targets. However, a follow-on larger scale (50-60 samples) planned R01 project will provide statistical significance and further refine the methodology.

=。项目概要…… 共济失调是随意肌肉运动的不协调，可以被视为多发性运动障碍的主要症状。 疾病包括遗传性和非遗传性病因。几个基因与引起脊髓小脑损伤有关 通过三核苷酸重复扩增 (TRE) 导致共济失调，据认为每 10,000 人中有 1.5 至 4 人受影响 全球人民。 FMR1 基因中类似的三核苷酸重复扩增，包括潜在的症状 在美国，大约每 300 人中就有 1 人存在前突变。重复扩增检测 家庭疾病对于了解遗传风险和早期发现高危成员非常重要， 特别是因为不稳定的重复扩增会导致遗传预期现象，其中症状 可以在几代人之间出现更早或更严重。此外，最近的几项研究表明 开发专门针对扩展重复序列作为新治疗靶点的靶向疗法。作为 因此，更早、快速地发现这些疾病对于推进其医疗管理至关重要。 传统上，短 TRE 靶标通过重复引发 PCR (PR-PCR) 和毛细管进行评估 电泳，而更长的突变体则通过 Southern blotting 进行确认。这在临床上很重要，尤其是 其中超出 PR-PCR 可量化范围的重复长度具有诊断相关性。测试大多数 TRE 基因对于短读长测序平台来说仍然很困难，因为重复序列长度超过平均水平 读长（Illumina 系统约为 100 bp）。长读长测序技术，例如 PacBio 和 Oxford 纳米孔在测量 TRE 方面取得了成功，但成本高、数量大 DNA 输入、复杂的生物信息学、确定重复区域边界的问题以及高错误率使得 在目前的情况下，它们不太可能成为广泛筛查的解决方案 我们开发了一种基于高速原子力显微镜 (HSAFM) 的解决方案 PRECYSE 具有独特的“纳米粒子条形码”，可以潜在地表征许多复杂的结构变体 同时治疗共济失调，其成本比下一代测序 (NGS) 低得多 测序和其他新兴方法。这种极其敏感的技术可以在不使用 聚合酶链式反应 (PCR) 可以跨越非常广泛的可能目标尺寸，从而允许 延伸至几乎无限的分子长度。我们的假设是这项技术可以很容易地适应 对几乎任何长度的 TRE 目标进行多重检测。如果开发成功，我们的新方法 将改变临床医生识别、理解和监测三核苷酸引起的基因组变化的方式 通过多重面板重复扩增疾病。在这个 R21 项目结束时，我们将拥有一个平台 用于多重基因组分析，成功纯化和检测三核苷酸重复靶标。然而，一个 后续更大规模（50-60个样本）计划的R01项目将提供统计意义并进一步细化 方法论。