Development of Novel Diagnostics for Fragile X Syndrome

脆性 X 综合征新型诊断方法的开发

• 批准号: 8066422
• 负责人: SEIYU HOSONO
• 金额: $ 53.71万
• 依托单位: JS GENETICS, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-25 至 2012-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8066422
• 关键词: 5' Untranslated Regions; Adult; Autistic Disorder; Award; Base Pairing; Binding; Biological Assay; Blood; Blood capillaries; Boston; CGG repeat; CGG repeat expansion; Child; Chromosome Band; Clinical; DNA; Development; Diagnosis; Diagnostic; Diagnostic tests; Electrophoresis; Etiology; FMR1; FMR1 Gene; FXTAS; Family history of; Female; Fetus; Fragile X Gene; Fragile X Mental Retardation Protein; Fragile X Syndrome; Future Generations; Gel; Gene Mutation; Genes; Genetic; Genetic screening method; Genome; Guanosine; Guanosine Triphosphate; Incidence; Individual; Infertility; Laboratories; Language; Lead; Learning Disabilities; Mental Retardation; Methods; Methylation; Molecular; Molecular Weight; Motor; Neonatal Screening; Neurons; Parkinsonian Disorders; Pediatric Hospitals; Pervasive Development Disorder; Phase; Primary Health Care; Professional counselor; Promoter Regions; Proteins; Provider; Reaction; Risk; Sampling; Screening procedure; Site; Small Business Innovation Research Grant; Southern Blotting; Speech; Spottings; Stretching; Syndrome; Taq Polymerase; Testing; Time; Translating; Translations; Tremor; Trinucleotide Repeats; Woman; X Chromosome; autism spectrum disorder; base; capillary; commercial application; cost; design; equilibration disorder; gel electrophoresis; high throughput screening; male; medical schools; meetings; neurodevelopment; novel; novel diagnostics; nucleotide analog; pregnant; prenatal testing; programs; public health relevance; rapid technique; tool

DESCRIPTION (provided by applicant): Fragile X Syndrome (FRAX) is the most common genetic cause of mental retardation in males. FRAX is caused by the expansion of a CGG trinucleotide repeat of the 5' untranslated region (UTR) of the FMR1 gene. In normal individuals, the 5' UTR of the FMR1 gene contains 5 to 45 CGG repeats; however, individuals with FRAX have over 200 repeats. Presently, Southern Blot analysis is used to determine the size of the repeat segment and methylation status of the FRAX gene. This test only detects the gross size of CGG repeats and is labor intensive and expensive. PCR and gel electrophoresis is typically used to determine the size of the CGG expansion. This approach is limited, as PCR reactions typically fail to amplify long stretches of CGG expansions (>25 repeats) and molecular weight determination by electrophoresis via capillary or slab gels is labor intensive. In our Phase I SBIR award, we developed a novel, highly efficient and accurate screening test for diagnosing FRAX. In Step 1, Whole Genome Multiple Displacement Amplification using 7-deaza-2-Guanosine (7-deaza GTP) nucleotide analog is incorporated into multiple copies of the CGG FMR1 expansion. In Step 2, Site Specific Multiple Displacement Amplification (SSMDA) using 7-deaza GTP is performed to weaken the GC base pairings, making the GCC expansion more accessible to Taq DNA Polymerase in real-time PCR. In Step 3, SSMDA is followed by quantitative assessment of the numbers of CGG triplet repeats using TaqMan real-time PCR without the need for sizing by gel electrophoresis or Southern blotting. We hypothesize that using this new molecular-based method, we can develop an effective, rapid and low-cost screening test for FRAX with broad commercial application. In this Phase II application we propose to: SA1: Optimize our assay for CGG repeat copy number determination using a large number of DNA samples with varying degrees of CGG repeats. SA2: Test our assay in a clinical FRAX diagnostic laboratory. SA3: Test our assay in blood spots for potential newborn screening. We anticipate that this Phase II application will lead to the optimization and implementation of a test that is suitable for low cost high-throughput screening for FRAX. As such, this test has the potential to markedly change how we currently screen for FRAX. These studies will involve the combined expertise of JS Genetics, which has been developing novel DNA-based diagnostic tests, and Dr. Bai-Lin Wu of Boston Children's Hospital and Harvard Medical School, who an extensive track record of developing clinical DNA diagnostic studies and in clinical laboratory testing of FRAX. PUBLIC HEALTH RELEVANCE: Fragile X Syndrome (FRAX) is the most common genetic cause of mental retardation in males. We propose the development of a new molecular-based method, for rapid and low-cost screening of FRAX with broad commercial application.

描述(由申请人提供)： 脆性X综合征(FRAX)是男性智力低下最常见的遗传原因。FRAX是由FMR1基因5‘非翻译区的CGG三核苷酸重复序列扩大引起的。在正常人中，FMR1基因的5‘非编码区包含5到45个CGG重复；然而，FRAX患者有200多个重复。目前，Southern Blot分析用于确定重复片段的大小和FRAX基因的甲基化状态。这项测试只检测CGG重复的粗大小，而且是劳动密集型和昂贵的。聚合酶链式反应和凝胶电泳法通常用于确定CGG扩增的大小。这种方法是有限的，因为聚合酶链式反应通常不能扩增较长的CGG扩增片段(重复25次)，而且通过毛细管或平板凝胶进行电泳法测定分子量是劳动密集型的。在我们的第一阶段SBIR奖中，我们开发了一种用于诊断FRAX的新颖、高效和准确的筛查测试。在步骤1中，使用7-去氮-2-鸟苷(7-去氮GTP)核苷酸类似物的全基因组多重置换扩增被整合到CGG FMR1扩增的多个副本中。在步骤2中，使用7-deaza GTP进行位点特异性多重置换扩增，以弱化GC碱基配对，使Taq DNA聚合酶更容易获得GCC扩增片段。在步骤3中，SSMDA之后使用TaqMan实时聚合酶链式反应对CGG三联体重复序列的数量进行定量评估，而不需要通过凝胶电泳法或Southern blotting来确定大小。我们推测，利用这种新的基于分子的方法，我们可以开发出一种有效、快速、低成本的FRAX筛查方法，具有广泛的商业应用前景。在这个第二阶段的应用中，我们建议：SA1：使用大量具有不同程度CGG重复的DNA样本来优化我们的CGG重复拷贝数测定方法。SA2：在临床FRAX诊断实验室测试我们的分析。SA3：在血点中测试我们的检测方法，以进行潜在的新生儿筛查。我们预计，这一第二阶段的应用将导致一种适用于FRAX低成本高通量筛选的测试的优化和实施。因此，这项测试有可能显著改变我们目前筛选FRAX的方式。这些研究将涉及JS Genetics和波士顿儿童医院和哈佛医学院的吴柏林博士的专业知识，前者一直在开发新型的基于DNA的诊断测试，后者在开发临床DNA诊断研究和FRAX的临床实验室测试方面有着广泛的记录。 公共卫生相关性： 脆性X综合征(FRAX)是男性智力低下最常见的遗传原因。我们提出了一种新的基于分子的方法，用于快速、低成本地筛选FRAX，具有广泛的商业应用前景。