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

基本信息

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
项目类别：
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

项目摘要

=. 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。包括遗传和非遗传病因在内的疾病有几个基因与脊髓小脑的病因有关。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 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 人发现重复扩增。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焦虑现象，其中症状最近的几项研究表明，这种疾病可能会在几代人之间出现得更早或更严重。靶向治疗的发展特别扩大了靶向重复作为新的治疗靶点。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，而较长的突变体通过Southern印迹证实，这在临床上很重要，尤其是。对于大多数情况，重复长度超出 PR-PCR 可量化范围具有诊断相关性。 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），例如 PacBio 和 Oxford 等长读长测序技术。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可以在不使用的情况下进行。聚合酶链式反应 (PCR) 可以跨越非常广泛的可能目标尺寸，从而允许我们的假设是，这项技术可以很容易地适应。如果开发成功，我们的新方法将能够对几乎任何长度的 TRE 目标进行多重检测。将改变人们青睐的识别、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。用于成功纯化和检测三核苷酸重复靶标的多重基因组分析。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。