Single molecule fluorescence detection of fragile X mutations

描述（申请人提供）：脆性X综合征（FXS），是遗传性精神发育迟滞的最常见形式，在所有种族中，每4000名男性中有1名患者，每6000名女性中有1名患者。此外，载波频率相当高。大约每250名女性中就有1名携带脆性X，每800名男性中就有1名携带脆性X。FXS是与三联核苷酸重复扩增有关的第一种人类疾病之一，三联核苷酸重复由FMR 1基因5'非翻译区中200-2000个CGG单位组成。然而，最近的研究表明，一系列认知、运动和生殖缺陷可归因于甚至更少的三联体重复，在约60-200的范围内。风险可以通过三联体重复数分层，如果早期发现，疾病表现对治疗反应良好。因此，脆性X基因是一个重要的诊断和筛查机会，建议对受影响的个体，携带者，携带者的后代和具有类似症状但没有疾病史的个体进行检测。我们的信念得到了医学界许多人的支持，即脆性X射线筛查将很快变得更加普遍。但是脆性X基因给目前的分子诊断程序带来了几个问题。使用Southern印迹和凝胶电泳对基因组DNA进行大小测定耗时且低通量。聚合酶通过CGG重复阅读有困难，因此大多数基因扩增方法（例如PCR）没有实现高保真和扩增FMR 1。此外，这些方法可能会误诊异质基因型的载体或嵌合体。我们的目标是开发一种具有足够保真度和通量的脆性X筛选方法。该提案描述了一种检测方法，该方法将我们的FMR 1 PCR程序与一种新型检测系统相结合，该方法针对三联体重复序列进行了广泛优化，该检测系统可以实现异质基因型的通量和分辨率。我们的PCR方法目前能够以非常高的灵敏度和保真度可靠地扩增超过900个CGG重复。然后，我们的检测方法能够单独调查所有PCR产物并确定分布重复数。本研究的重点是建立脆性X基因突变的检测方法，并将其与优化的PCR技术相结合，以证明高通量筛查脆性X基因突变的可行性。 公共卫生相关性：脆性X基因的突变是智力迟钝、记忆和运动缺陷以及生殖功能不全的重要原因。干预可以成功，但需要对疾病进行准确的分子诊断。我们的长期目标是开发脆性X基因突变筛查检测方法，以识别有发展或传递这些基因风险的个体。

DESCRIPTION (provided by applicant): Fragile X Syndrome (FXS), is the most common form of inherited mental retardation, affecting 1 in 4000 males and about 1 in 6000 females of all ethnicities. Additionally, carrier frequencies are quite high. About 1 in 250 females carry Fragile X and about 1 in 800 men carry Fragile X. FXS was one of the first human diseases to be linked to an expansion of triplet nucleotide repeats, consisting of anywhere from 200-2000 CGG units in the 5' untranslated region of the FMR1 gene. Recent research indicates, however, that a constellation of cognitive, motor and reproductive deficits can be attributed to even fewer triplet repeats, in the range of about 60-200. Risk can be stratified by triplet repeat number and the disease manifestations respond well to therapies If identified early. The Fragile X gene is, consequently, a substantial diagnostic and screening opportunity, and testing is recommended for affected individuals, carriers, offspring of carriers and individuals with similar symptoms but no history of the disease. Our belief, which is supported by many in the medical community, is that fragile X screening will soon become more prevalent. But the Fragile X gene poses several problems for current molecular diagnostic procedures. Sizing of genomic DNA using Southern blot and gel electrophoresis is time consuming and low-throughput. Polymerases have trouble reading through the CGG repeat, and so most gene amplification methods (e.g. PCR) have not achieved high fidelity and amplifying FMR1. Additionally, these methods may misdiagnose the heterogeneous genotypes of carriers or mosaics. Our goal is to develop a Fragile X screening method that has adequate fidelity and throughput. This proposal describes an assay that combines our FMR1 PCR procedures, extensively optimized for the triplet repeats, with a novel detection system that can achieve both throughput and resolution of heterogeneous genotypes. Our PCR methods are currently able to reliably amplify over 900 CGG repeats with very high sensitivity and fidelity. Our detection methods are then able to individually survey all PCR products and determine the distribution repeat numbers. This proposal focuses on developing the detection method and integrating it with optimized PCR in order to demonstrate feasibility of high throughput screening for Fragile X gene mutations. PUBLIC HEALTH RELEVANCE: Mutations of the Fragile X gene are a significant cause of mental retardation, memory and motor defects, and reproductive insufficiency. Interventions can be successful but require accurate molecular diagnosis of the disease. Our long-term goal is to develop Fragile X mutation screening assays that will identify individuals at-risk for developing or passing on these genes.