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

=。项目摘要…..。 共济失调是一种随意肌肉运动的失调，可被视为多发性 疾病包括遗传性和非遗传性病因。有几个基因与脊髓小脑有关 通过三核苷酸重复扩增(TRE)造成的共济失调，据认为每10,000人中有1.5到4人患有共济失调 全球各地的人。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人存在预突变。重复扩增的检测 家庭中的疾病对于了解遗传风险和早期发现高危成员很重要， 特别是因为不稳定的重复扩增会导致基因预期现象，症状 可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， 开发专门针对扩大重复序列的靶向治疗作为新的治疗靶点。AS 这种更早、更快地发现这些疾病对于推进他们的医疗管理至关重要。 传统上，短的tre靶点是通过重复启动的聚合酶链式反应(PR-PCR)和毛细管进行评估的。 而较长的突变体则通过Southern blotting得到确认。这在临床上很重要，尤其是 在PR-PCR可量化的范围之外的重复长度在诊断上具有相关性。针对MOST的测试 TRE基因对于短读测序平台来说仍然是困难的，那里的重复序列长度超过了平均长度 读取长度(对于Illumina系统约为100个bp)。像PacBio和Oxford这样的长时间测序技术 纳米孔在测量TRE方面已经显示出成功，然而，高昂的成本、大量的 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使 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复杂的结构变体 与下一代测序(NGS)相比，共济失调的成本要低得多 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(聚合酶链式反应)，可以跨越非常广泛的可能的靶尺寸，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 涉及对几乎任何长度的TrE目标的多路检测。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 通过多路面板重复扩张性疾病。在这个R21项目结束时，我们将拥有一个平台 用于成功提纯和检测三核苷酸重复靶标的多重基因组分析。然而，a 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。

=. 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.