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...... 共济失调是一种随意肌肉运动的不协调，可以被视为多发性硬化症的主要症状。 包括遗传和非遗传病因的疾病。有几个基因与脊髓小脑症有关 通过三核苷酸重复扩增（TREs）导致共济失调，据认为影响1.5至4/10，000 全球的人。FMR 1基因中类似的三核苷酸重复扩增，包括潜在的症状性 在美国，大约每300人中就有1人有这种突变。重复扩增检测 家族中的疾病对于了解遗传风险和早期发现处于风险中的成员是重要的， 特别是因为不稳定的重复扩增导致遗传预期的现象，其中症状 可以在几代人中更早或更严重地出现。此外，最近的几项研究表明， 开发特异性靶向扩展重复序列作为新的治疗靶点的靶向治疗。作为 这种早期和快速检测这些疾病对于促进其医疗管理至关重要。 传统上，通过重复引物PCR（PR-PCR）和毛细管PCR（PCR）来评估短TRE靶标。 电泳，而更长的突变体通过Southern印迹确认。这在临床上很重要，尤其是 其中在可通过PR-PCR定量的范围之外的重复长度是诊断相关的。测试大多数 TRE基因仍然难以用于短读段测序平台，其中重复序列长度超过平均值 读取长度（对于Illumina系统为~100 bp）。PacBio和Oxford等长读段测序技术 纳米孔已经在测量TREs方面显示出成功，然而，高成本、大量的纳米孔和纳米孔的组合是不可能的。 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， 在他们目前的状态下，他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）配对的解决方案PRECYSE。 具有独特的“纳米颗粒条形码”，可以潜在地表征许多 以比下一代测序（NGS）低得多的成本同时治疗共济失调 排序和其他新兴方法。这种极其敏感的技术可以在不使用 聚合酶链式反应（PCR）可以是一种非常有效的方法，并且可以跨越非常宽范围的可能的靶大小，从而允许 延伸到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 涉及实际上任何长度的TRE靶的多重检测。如果研发成功，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。 重复扩增疾病。在这个R21项目结束时，我们将有一个平台， 用于成功纯化和检测三核苷酸重复目标的多重基因组分析。但 后续更大规模（50-60份样本）计划的R 01项目将提供统计学意义并进一步完善 方法论。