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：
10682387
负责人：
Jason C Reed
金额：
$ 20.33万
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/10682387
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 Etiology 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 Managed Care 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.

=。项目摘要…... 共济失调是自愿运动的不和谐，可以看作是多重症状的主要症状包括遗传和非遗传病因的疾病。暗示引起脊椎小脑的几种基因通过三核苷酸重复扩展（TRE）的共济失调，被认为影响到每10,000次1.5至4 全球人。 FMR1基因中类似的三核苷酸重复膨胀，包括潜在的症状在美国，大约有300人中有大约1人可以找到预言。检测重复扩展家庭中的疾病对于理解遗传风险和AT风险成员的早期发现很重要，特别是因为不稳定的重复扩张导致遗传预期的现象，症状可以在几代人的早期或更严重的情况下提出。此外，最近的一些研究表明靶向疗法的开发专门针对扩展的重复序列，作为一种新的治疗靶点。作为这些疾病的早期和快速检测对于推进其医疗管理至关重要。传统上，通过重复提交的PCR（PR-PCR）和毛细管评估短TRE目标电泳，而通过Southern印迹证实了更长的突变体。这在临床上很重要，特别是在PR-PCR可量化的范围之外重复长度的情况下，在诊断上是相关的。大多数测试对于简短读取测序平台而言，TRE基因仍然很难，其中重复道超过平均值读取长度（Illumina系统的100 bp）。 PACBIO和牛津等长阅读测序技术纳米孔在测量TRE方面表现出成功，但是高成本的组合，大量 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他们不太可能在当前状态下进行宽度筛查我们基于配对的高速原子力显微镜（HSAFM）开发了一种解决方案，具有独特的“纳米颗粒条形码”，可以潜在地表征许多复杂的结构变体共济失调条件的成本仅比下一代测序（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这种非常敏感的技术聚合酶链反应（PCR）可以跨越非常广泛的可能目标大小，从而允许扩展到几乎无限的分子长度。我们的假设是，该技术很容易适应多重检测的TRE靶标几乎任何长度。如果成功发展，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展疾病。在此R21项目的结尾，我们将有一个平台为了成功净化和检测三核苷酸重复靶标的多路复用基因组分析。但是，随后的大规模（50-60个样本）计划的R01项目将提供统计意义，并进一步完善方法。