Amplification Strategies for Detection of Fragile X Gene Trinucleotide Repeats

描述（由申请人提供）： 该项目的总体目标是开发和实施一套全面的技术，以改善脆性X综合征（FXS）相关疾病的筛查和诊断。FXS是由FMR 1基因5 '非翻译区的胞嘧啶-鸟嘌呤-鸟嘌呤（CGG）三联体重复扩增引起的。FXS影响1/4000男性和1/6000女性。完全扩增至大于200个重复与FMR 1基因的超甲基化和FMR 1蛋白质产生的完全丧失相关。CGG重复序列的适度扩增与老年男性脆性X相关震颤/共济失调综合征（FX-TAS）和女性原发性卵巢功能不全（FX-POI）相关。最近发表的指南建议对自闭症谱系障碍（ASD）的诊断进行后续染色体和脆性X染色体检测，ASD的发病率为1/150，大约是脆性X染色体发病率的33倍。此外，有希望的药物目前正在临床试验中，需要准确和早期识别脆性X患者。脆性X染色体检测的改进将对与这种疾病相关的多种精神和健康状况的所有年龄段的广泛个体产生重要影响。 我们的第一阶段资助重点是开发扩增技术，这些技术可以快速，高通量和灵敏地检测CGG重复数和与FXS相关的甲基化。此外，这些技术将能够解决女性中的混淆接合性，这是基于商业PCR的FXS测试中不可用的特征。第一阶段的所有主要里程碑都已达到和超过。我们已经开发了三种PCR技术，其中一些具有重叠的功能，可以解决与FXS测试相关的所有主要问题。这些技术显示出完全取代需要用Southern印迹进行反射测试的希望，Southern印迹繁琐且缓慢。在第二阶段，我们建议研究将PCR技术整合到综合和简化的测试中。这包括开发一套完整的控制、质量控制程序和用于数据分析和存储的计算机接口。将在存档的临床标本中评价综合检测工作流程。第二阶段赠款将产生一个全面的诊断工作流程，可以支持常规检测和筛查，从而使早期干预和改善患者的治疗选择。 公共卫生相关性： 该项目的长期目标是改善脆性X综合征及相关疾病的筛查和诊断。在这个项目中，我们将利用我们在FMR 1基因PCR方面的突破，开发一套具有成本效益和效率的强大而准确的测试。这将使广泛的筛查，以确定携带者，并允许早期诊断和干预脆性X综合征患者。

DESCRIPTION (provided by applicant): The overall objective for this project is to develop and implement a comprehensive set of technologies to improve screening and diagnosis of conditions associated with Fragile X Syndrome (FXS). FXS is caused by an expansion of a cytosine-guanine-guanine (CGG) triplet repeat in the 5'-untranslated region of the FMR1 gene. FXS affects 1/4000 men and 1/6000 women. Full expansion to greater than 200 repeats is associated with hypermethylation of the FMR1 gene and complete loss of FMR1 protein production. A more modest expansion of the CGG repeats is associated with Fragile X-associated Tremor/Ataxia Syndrome (FX-TAS) in older men and primary ovarian insufficiency (FX-POI) in women. Recently published guidelines suggest follow- up chromosome and Fragile X testing for a diagnosis of autism spectrum disorder (ASD), which impacts 1/150 individuals-roughly 33 times the population incidence of Fragile X. Furthermore, promising drugs are currently in clinical trials and will require accurate and early identification of Fragile X patients. Improvements in testing for Fragile X will have important implications for a broad range of individuals of all ages across multiple mental and health conditions associated with this disorder. Our phase I grant was focused on developing amplification technologies that can enable rapid, high throughput and sensitive detection of CGG repeat numbers, and methylation associated with FXS. Furthermore, these technologies would be able to address confounding zygosity in females, a feature that is not available on commercial PCR based FXS tests. All of the major milestones of phase I have been met and exceeded. We have developed three PCR technologies, some of them with overlapping functionality that can address all major issues associated with FXS testing. These technologies show promise for completely replacing the need for reflex testing with Southern blots, which are cumbersome and slow. In the phase II portion of this grant we propose studies to integrate the PCR technologies into comprehensive and streamlined test. This includes the development of a complete set of controls, quality control procedures and a computer interface for data analysis and storage. The comprehensive test workflows will be evaluated in archived clinical specimens. This phase II grant will result in a comprehensive diagnostic workflow that can support routine testing and screening, and thus enable earlier interventions and improved treatment options for patients. PUBLIC HEALTH RELEVANCE: The long-term goal for this project is to improve screening and diagnosis of Fragile X Syndrome and related conditions. In this project, we will leverage our breakthrough in PCR of the FMR1 gene to develop a set of robust and accurate tests that are cost-effective and efficient. This will enable widespread screening to identify carriers and permit earlier diagnosis and intervention for Fragile X Syndrome patients.