Technologies for Mapping Interspersed AGG Sequences in the FMR1 Gene

描述(由申请人提供)：该项目的总体目标是验证、商业化并进一步建立一套全面的基因分型分析的临床实用性，以改进与脆性X综合征(FXS)相关的疾病的筛查和诊断。脆性X智力低下(FMR1)基因扩增的群体研究提出了CGG重复序列中散布的AGG序列检测的相关性。已经提出了重复CGG元件内的AGG重复的数量和序列背景，以赋予世代扩展的稳定性。尽管AGG错位与三联体重复扩张的风险相关，但以前在大多数女性样本中无法确定这些错位。我们建议使用我们开发的决定性的AGG图谱分析方法，通过将AGG散布信息纳入对存档DNA样本的FMR1等位基因传播的分析中，来完善中间和前突变等位基因的风险估计。我们的检测方法利用高通量聚合酶链式反应，并承诺改进诊断和风险评估，以实现准确的AGG基因分型，并最终确定每个样本等位基因的连续CGG重复数。我们达到或超过了第一阶段授予的所有拟议目标。这一成功导致了FMR1的补充分子遗传分析，具有全面的agg基因分型能力。我们的学术合作者参与了大型临床试验，这些试验将检查对FMR1重复扩增重要的遗传因素，我们的技术将成为评估散布的AGG图谱对一系列FMR1疾病的临床应用的核心。第二阶段的目标是：目标1：将第一阶段开发的检测技术应用于回溯性临床样本，以更准确地确定AGG基因分型在CGG重复扩增中的临床应用。目的2：建立一套检测AGG重复序列的对照和标准，并将试剂、对照和质量控制指标整合到一个支持基于聚合酶链式反应的AGG基因检测的综合工作流程中。目标3：开发和测试用户友好的软件组件，以绘制最不发达国家的位置图。目标4：整合和评估目标2和目标3中开发的综合测试功能。

DESCRIPTION (provided by applicant): The overall objective for this project is to validate, commercialize, and further establish the clinical utility of a comprehensive set of genotyping assays to improve screening and diagnosis of conditions associated with fragile X syndrome (FXS). The relevance of testing for interspersed AGG sequences in CGG repeats has been proposed from population studies of fragile X mental retardation (FMR1) gene expansion. Both the number and sequence context of AGG repeats within the repeat CGG element have been proposed to confer stability to generational expansion. Although AGG interspersions are associated with the risk of triplet repeat expansion, these interspersions previously could not be determined in most female samples. We propose to refine the risk estimates for intermediate and premutation alleles by incorporating AGG interspersion information into the analysis of FMR1 allele transmissions from archived DNA samples using the definitive AGG mapping assays that we have developed. Our assay approach, leverages high throughput PCR and promises improved diagnostic and risk assessments for enabling accurate AGG genotyping, and definitive determination of the number of consecutive CGG repeats for each sample allele. We met or exceeded all proposed aims in the phase I grant. This success has resulted in complementary molecular genetic assays for FMR1 with comprehensive AGG genotyping capabilities. Our academic collaborators are involved in large clinical trials that will examine the genetic factors important for FMR1 repeat expansions, and our technology will be the centerpiece to evaluate the clinical utility of the interspersed AGG mapping for a range of FMR1 disorders. The aims for phase II are: Aim 1: Apply the assay technologies developed in Phase I to retrospective clinical samples to more precisely define the clinical utility of AGG genotyping in CGG repeat expansions. Aim 2: Develop a set of controls and standards for detection of AGG repeats and integrate reagents, controls and QC metrics into a comprehensive workflow that supports the PCR-based detection of AGG genotypes. Aim 3: Develop and test user-friendly software components for mapping AGG positions. Aim 4: Integrate and evaluate comprehensive test features developed in Aims 2 and 3.