Amplification Strategies for Detection of Fragile X Gene Trinucleotide Repeats

用于检测脆弱 X 基因三核苷酸重复的扩增策略

• 批准号: 8126187
• 负责人: ANDREW G HADD
• 金额: $ 48.59万
• 依托单位: AMBION DIAGNOSTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8126187
• 关键词: 5' Untranslated Regions; Address; Affect; Age; Alleles; Archives; Ataxia; Automation; Biological Assay; Chromosomes; Clinical; Clinical Trials; Computer Assisted; Computer Interface; Computer software; Cytosine; DNA; Data Analyses; Data Storage and Retrieval; Databases; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Disease; Early Diagnosis; Early identification; Early treatment; Electrophoresis; Elements; Employee Strikes; Ensure; FMR1; FMR1 Gene; FXTAS; Female; Fragile X Gene; Fragile X Mental Retardation Protein; Fragile X Syndrome; GC Rich Sequence; Genes; Genetic; Goals; Grant; Guanine; Guidelines; Hypermethylation; Incidence; Individual; Laboratories; Length; Mental Health; Methods; Methylation; Metric; Modeling; Outcome; Patients; Pharmaceutical Preparations; Phase; Population; Premature Menopause; Procedures; Production; Publishing; Quality Control; Reaction; Reagent; Reflex action; Reporting; Reproducibility; Research Project Grants; Risk Assessment; Running; Sampling; Screening procedure; Southern Blotting; Specimen; Syndrome; Technology; Testing; Time; Tremor; Trinucleotide Repeats; Triplet Multiple Birth; Tube; Turner' s Syndrome; Woman; autism spectrum disorder; base; clinical practice; cost; cost effective; flexibility; follow-up; improved; instrumentation; meetings; men; novel; older men; phase 1 study; public health relevance; research clinical testing; tool

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.

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 FMR1 基因 5'-非翻译区中胞嘧啶-鸟嘌呤-鸟嘌呤 (CGG) 三联体重复序列的扩展引起的。 FXS 影响 1/4000 的男性和 1/6000 的女性。完全扩增至超过 200 个重复序列与 FMR1 基因的超甲基化和 FMR1 蛋白产生的完全丧失有关。 CGG 重复序列的适度扩张与老年男性的脆性 X 相关震颤/共济失调综合征 (FX-TAS) 和女性的原发性卵巢功能不全 (FX-POI) 相关。最近发布的指南建议通过后续染色体和脆性 X 细胞检测来诊断自闭症谱系障碍 (ASD)，该疾病影响 1/150 的个体，大约是脆性 X 细胞人群发病率的 33 倍。此外，有前景的药物目前正在进行临床试验，需要准确、早期识别脆性 X 细胞患者。脆性 X 染色体检测的改进将对与这种疾病相关的多种心理和健康状况的各个年龄段的广大个体产生重要影响。 我们的第一阶段资助重点是开发扩增技术，能够快速、高通量和灵敏地检测 CGG 重复数以及与 FXS 相关的甲基化。此外，这些技术将能够解决女性中混杂的接合性问题，这是基于商业 PCR 的 FXS 测试所不具备的功能。第一阶段的所有主要里程碑均已达到并超越。我们开发了三种 PCR 技术，其中一些具有重叠功能，可以解决与 FXS 测试相关的所有主要问题。这些技术有望完全取代繁琐且缓慢的 Southern 印迹反射测试。在本次资助的第二阶段部分，我们提出研究将 PCR 技术整合到全面和简化的测试中。这包括开发一套完整的控制、质量控制程序以及用于数据分析和存储的计算机接口。综合测试工作流程将在存档的临床标本中进行评估。该第二阶段拨款将产生一个全面的诊断工作流程，可以支持常规测试和筛查，从而为患者提供早期干预和改进的治疗选择。 公共卫生相关性： 该项目的长期目标是改善脆性 X 综合征及相关病症的筛查和诊断。在这个项目中，我们将利用我们在 FMR1 基因 PCR 方面的突破来开发一套稳健、准确、经济高效的测试。这将使广泛的筛查能够识别携带者，并允许对脆性 X 综合征患者进行早期诊断和干预。