Development of a reliable and standardized molecular assay for fragile x protein

 描述（由申请人提供）：该项目的目标是开发一种有效的脆性X智力低下蛋白（FMRP）定量检测方法，以帮助脆性X综合征（FXS），自闭症和其他疾病的临床管理，并推进FXS研究。FXS是遗传性智力残疾的主要形式，从轻度到重度不等，是自闭症中最常见的已知突变。它是由X连锁FMR 1基因中的CGG重复扩增引起的。在未受影响的个体中，FMR 1 5' UTR中出现6至44个重复，而在患有FXS的个体中，观察到>200个重复，导致FMRP水平大幅降低和智力障碍。扩增至55-200个重复导致FMRP的正常或小得多的降低。这些人不受影响或呈现不同的，不太严重的认知，行为和情感功能障碍。少于55个重复的个体几乎从不表现FMR 1相关的临床特征。 FMRP诊断测试已经在2013年ACMG脆性X指南中得到认可。然而，FMRP水平与患者诊断和预后之间的可靠相关性已被生物学和技术问题破坏。迫切需要一种经过充分验证和标准化的定量FMRP测定，以支持研究人员和临床医生努力了解FXS生物学并帮助患者管理。 FMR 1分子检测可靠解释的生物学挑战包括体细胞嵌合现象，或不同组织中不同基因型和/或甲基化状态的存在。重要的是，病例研究揭示了脆性X突变的组织特异性差异，其中与脑组织共享外胚层发育谱系的颊细胞可能比血液更具临床信息。我们将测试使用BCs检测FMRP的可行性，因为它们可能更好地代表大脑中的脆性X生物学。 技术挑战包括分析前的局限性， 不合格的抗体，以及缺乏定量的FMRP标准。最近发表的FMRP检测解决了这些问题，包括与干血斑（DBS）的兼容性以及Luminex平台上可用于常规检测的工作流程。我们的总体目标是通过减少变异来源，用注释的临床样品验证测试，然后启动用于分析稳定在“903”纸上的血液和口腔细胞的测试来改进该测试。该技术将能够从单个样本中准确定量FMRP和FMR 1基因分型，从而简化全面FXS数据集的收集，实现诊断和预后应用，并提高我们对FXS生物学的整体理解。具体目标1：使用常用试剂和样本完成已发布检测试剂盒的多中心验证。具体目标2：开发方案和试剂，以标准化DBS样本中产生FMRP的血细胞。具体目标3：证明使用903张纸上储存的颊细胞测定FMRP的可行性，并进行初步临床研究。

 DESCRIPTION (provided by applicant): The goal of this project is to develop a validated, quantitative assay for the fragile X mental retardation protein (FMRP) to aid in the clinical management of those with fragile X syndrome (FXS), autism and other disorders and to advance FXS research. FXS is the leading form of inheritable intellectual disability, ranging from mild to severe and is the most common known mutation in autism. It is caused by CGG repeat expansion in the X-linked FMR1 gene. In non-affected individuals, 6 to 44 repeats occur in the FMR1 5' UTR, whereas in those with FXS, >200 repeats are observed, causing substantially reduced levels of FMRP and intellectual disability. Expansion to 55-200 repeats results in normal or much smaller reductions in FMRP. These individuals are unaffected or present a varying, less serious degree of cognitive, behavioral and emotional dysfunctions. Individuals with fewer than 55 repeats almost never manifest FMR1-related clinical features. FMRP diagnostic testing is already recognized in the 2013 ACMG fragile X guidelines. However, reliable correlation between FMRP levels and patient diagnosis and prognosis has been undermined by both biological and technical issues. There is a critical need for a well validated and standardized quantitative FMRP assay to support researchers and clinicians in their efforts to understand FXS biology and to aid in patient management. Biological challenges to the reliable interpretation of FMR1 molecular tests include somatic mosaicism, or the presence of varying genotypes and/or methylation states in different tissues. Importantly, case studies have revealed tissue-specific differences for fragile X mutations, wherein buccal cells that share an ectodermal developmental lineage with brain tissue may be more clinically informative than blood. We will test the feasibility of using BCs to assay FMRP because they may better represent fragile X biology in the brain. Technical challenges include pre-analytical limitations, substandard antibodies, and the lack of quantitative FMRP standards. Recently an FMRP assay was published that addresses these issues, including compatibility with dried blood spots (DBS) and a workflow on the Luminex platform that is amendable to routine testing. Our overall objective is to make improvements to this assay by reducing sources of variation, validating the test with annotated clinical samples, and then launching the assay for the analysis of blood and buccal cells stabilized on "903" paper. This technology will enable the accurate quantification of FMRP and FMR1 genotyping from a single sample, thereby simplifying the collection of a comprehensive FXS dataset, enabling diagnostic and prognostic applications, and improving our overall understanding of FXS biology. Specific Aim 1: Complete a multi-site validation of the published assay using common reagents and samples. Specific Aim 2: Develop the protocols and reagents to normalize for the FMRP-producing blood cells in a DBS sample. Specific Aim 3: Demonstrate the feasibility of using buccal cells stored on 903 papers for assaying FMRP, and perform a pilot clinical study.