Development of Novel Diagnostics for Fragile X Syndrome

描述（由申请人提供）：脆性X综合征（FRAX），是男性智力迟钝的最常见原因。FRAX的发病率在男性中为1 / 4000，在女性中为1 / 8000。FRAX是由FMR1基因5'非翻译区（UTR）的CGG三核苷酸重复扩增引起的。该基因位于染色体Xq27.3上。在正常人中，FMR1基因的5' UTR包含5到45个CGG重复序列。然而，在患有FRAX的个体中发现了200多个重复序列。目前，Southern Blot分析用于检测FRAX基因重复片段的大小和甲基化状态。然而，该测试仅检测CGG重复序列的总大小，并且是高度劳动密集型和昂贵的。PCR分析可用于检测CGG重复序列的大小。这种方法是有限的，因为PCR反应不能扩增长片段的CGG扩增。最近，我们开发了一种新型、高效、准确的FRAX诊断方法。该测试依赖于通过位点特异性多重位移扩增（SSMDA）扩增CGG重复扩增，该扩增能够扩增基因组中非常长的富含gc的区域。SSMDA之后使用实时聚合酶链反应定量评估CGG三联体重复序列的数量。我们假设利用这种新的基于分子的筛选方法，我们可以开发出一种有效、快速、低成本的FRAX筛选方法，具有广泛的商业应用前景。我们预计，第一阶段的应用将导致FRAX的新筛选程序的开发，该程序灵敏、准确、廉价，并且适用于高通量使用。如果成功，我们预计这些1期研究将导致2期研究。如果得到广泛应用，该策略将适用于FRAX携带者和FRAX患者的诊断。

DESCRIPTION (provided by applicant): Fragile X Syndrome (FRAX), is the most common cause of mental retardation in males. The incidence of FRAX is 1 per 4000 in males and 1 per 8000 in females. FRAX is caused by the expansion of a CGG trinucleotide repeat of the 5' untranslated region (UTR) of FMR1 gene. This gene is located at chromosome Xq27.3. In normal individuals, the 5' UTR of the FMR1 gene contains 5 to 45 CGG repeats. However, over 200 repeats are found in individuals with FRAX. Presently, Southern Blot analysis is used to examine the size of the repeat segment and methylation status of the FRAX gene. Yet, this test only detects the gross size of CGG repeats and is highly labor intensive and expensive. PCR analysis can be used to examine the size of CGG repeats. This approach is limited, as the PCR reaction fails to amplify long stretches of CGG expansions. Recently, we developed a novel, highly efficient, accurate, test for diagnosing FRAX. This test relies on amplifying CGG repeat expansions by Site Specific Multiple Displacement Amplification (SSMDA), which capably amplifies very long stretches of GC-rich regions in the genome. SSMDA is followed by quantitative assessment of the numbers of CGG triplet repeats using real-time Polymerase Chain Reaction. We hypothesize that using this new molecular-based screening method, we can develop an effective, rapid and low-cost screening test for FRAX with broad commercial application. We anticipate that this Phase 1 application will lead to the development of a new screening procedure for FRAX that is sensitive, accurate, inexpensive, and adaptable for high-throughput use. If successful, we anticipate these Phase 1 studies will lead to Phase 2 studies. If broadly applied, this strategy will be applicable for diagnosing carriers of FRAX and individuals with FRAX.