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

开发可靠且标准化的脆性 x 蛋白分子测定方法

• 批准号: 8904904
• 负责人: GARY J LATHAM
• 金额: $ 22.5万
• 依托单位: ASURAGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-18 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8904904
• 关键词: 5' Untranslated Regions; Address; Antibodies; Attention deficit hyperactivity disorder; Autistic Disorder; Basic Science; Behavioral; Biological; Biological Assay; Biology; Blood; Blood Cells; Brain; CGG repeat; CGG repeat expansion; Case Study; Cells; Clinical; Clinical Management; Clinical Research; Clinical Trials; Cognitive; Collection; Communities; Data Set; Development; Diagnosis; Diagnostic; Diagnostic tests; Disease; Drops; Emotional; Epigenetic Process; FMR1; FMR1 Gene; FMRP; Female; Fragile X Gene; Fragile X Mental Retardation Protein; Fragile X Syndrome; Functional disorder; Gene Deletion; Gene Silencing; Genetic; Genotype; Goals; Guidelines; Heterogeneity; Human; Hypermethylation; Indium; Individual; Inherited; Institutes; Intellectual functioning disability; Link; Lymphocyte; Malignant Neoplasms; Measures; Mental Retardation; Methylation; Molecular; Mood Disorders; Mosaicism; Mutation; Nervous System Physiology; Neurologic; New York; Paper; Patients; Performance; Proteins; Protocols documentation; Publishing; Reagent; Reproducibility; Research; Research Personnel; Sampling; Schizophrenia; Site; Source; Specificity; Specimen; Spottings; Technology; Testing; Tissues; Validation; Variant; Whole Blood; X Inactivation; brain cell; brain tissue; clinical application; clinical phenotype; companion diagnostics; diagnostic assay; improved; male; novel; outcome forecast; patient oriented; prevent; prognostic; promoter; protein expression; public health relevance; reagent standard; reproductive; reproductive function; screening; targeted treatment; theranostics; validation studies

 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.

 描述(由申请人提供)：该项目的目标是开发一种有效的、定量的脆性X智力低下蛋白(FMRP)检测方法，以帮助脆性X综合征(FXS)、自闭症和其他疾病患者的临床治疗，并促进FXS的研究。FXS是遗传性智力残疾的主要形式，从轻微到严重不等，是自闭症中最常见的已知突变。它是由X连锁的FMR1基因中CGG重复扩增引起的。在未受影响的个体中，FMR1 5‘非编码区出现6到44个重复，而在FXS患者中，观察到&gt；200重复，导致FMRP和智力残疾水平显著降低。扩展到55-200次重复会导致FMRP正常或小得多的下降。这些人没有受到影响，或者表现出不同的、不太严重的认知、行为和情绪功能障碍。重复次数少于55次的个体几乎从来没有表现出FMR1相关的临床特征。FMRP诊断测试已在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受到生物学和技术问题的破坏。迫切需要一种有效和标准化的FMRP定量分析，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。对可靠解释FMR1分子测试的生物学挑战包括体细胞嵌合体，或在不同组织中存在不同的基因类型和/或甲基化状态。重要的是，案例研究揭示了脆性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口腔细胞可能比血液更能提供临床信息。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限制， 抗体不合格，缺乏定量FMRP标准。最近发布了一种FMRP检测方法，解决了这些问题，包括与干血斑(DBS)的兼容性，以及Luminex平台上可修改为常规测试的工作流程。我们的总体目标是通过减少变异的来源来改进这项测试，用带注释的临床样本验证测试，然后推出用于分析稳定在“903”试纸上的血液和口腔细胞的测试。这项技术将能够从单个样本中准确地量化FMRP和FMR1基因分型，从而简化全面的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：使用常用试剂和样品完成对已发表的化验结果的多点验证。具体目标2：开发用于使DBS样本中产生FMRP的血细胞正常化的方案和试剂。具体目标3：证明使用储存在903篇论文上的口腔细胞来检测FMRP的可行性，并进行初步的临床研究。

项目成果

A Genotype-Phenotype Study of High-Resolution FMR1 Nucleic Acid and Protein Analyses in Fragile X Patients with Neurobehavioral Assessments.

• DOI: 10.3390/brainsci10100694
• 发表时间: 2020-09-30
• 期刊: Brain sciences
• 影响因子: 3.3
• 作者: Budimirovic DB;Schlageter A;Filipovic-Sadic S;Protic DD;Bram E;Mahone EM;Nicholson K;Culp K;Javanmardi K;Kemppainen J;Hadd A;Sharp K;Adayev T;LaFauci G;Dobkin C;Zhou L;Brown WT;Berry-Kravis E;Kaufmann WE;Latham GJ
• 通讯作者: Latham GJ