Development of Novel Diagnostics for Fragile X Syndrome

描述（由申请人提供）： 脆性X综合征（FRAX）是男性智力低下的最常见遗传原因。FRAX由FMR 1基因5'非翻译区（UTR）的CGG三核苷酸重复扩增引起。在正常个体中，FMR 1基因的5' UTR含有5至45个CGG重复;然而，患有FRAX的个体具有超过200个重复。目前，Southern印迹分析用于确定FRAX基因的重复片段的大小和甲基化状态。该测试仅检测CGG重复序列的总大小，并且劳动密集且昂贵。PCR和凝胶电泳通常用于确定CGG扩增的大小。这种方法是有限的，因为PCR反应通常不能扩增CGG扩增的长片段（>25个重复），并且通过毛细管或平板凝胶电泳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的新型，高效和准确的筛查测试。在步骤1中，将使用7-脱氮-2-鸟苷（7-脱氮GTP）核苷酸类似物的全基因组多重置换扩增掺入CGG FMR 1扩增的多个拷贝中。在步骤2中，使用7-脱氮GTP进行位点特异性多重置换扩增（SSMDA）以减弱GC碱基配对，使得GCC扩增在实时PCR中更容易被Taq DNA聚合酶获得。在步骤3中，SSMDA之后使用TaqMan实时PCR定量评估CGG三联体重复的数量，而不需要通过凝胶电泳或Southern印迹进行大小测定。 我们推测，使用这种新的分子基础的方法，我们可以开发一种有效的，快速的和低成本的FRAX筛选测试与广泛的商业应用。在该II期申请中，我们提出：SA 1：使用大量具有不同程度CGG重复的DNA样品优化我们的CGG重复拷贝数测定测定方法。 SA 2：在临床FRAX诊断实验室中测试我们的检测试剂盒。 SA 3：在血斑中测试我们的检测试剂盒，以进行潜在的新生儿筛查。 我们预计，这第二阶段的应用将导致优化和实施的测试，是适合于低成本的高通量筛选FRAX。因此，这项测试有可能显著改变我们目前筛查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Bai-Lin Wu博士的综合专业知识，JS Genetics一直在开发新的基于DNA的诊断测试，而Bai-Lin Wu博士在开发临床DNA诊断研究和FRAX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）是男性智力低下的最常见遗传原因。我们建议开发一种新的基于分子的方法，用于快速、低成本地筛查具有广泛商业应用的FRAX。

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.