Enabling use of blood spot cards for accurate high-throughput Fragile X screening

能够使用血点卡进行准确的高通量脆性 X 筛查

• 批准号: 8124769
• 负责人: GARY J LATHAM
• 金额: $ 34.63万
• 依托单位: ASURAGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8124769
• 关键词: 5' Untranslated Regions; Address; Affect; Alleles; Attention deficit hyperactivity disorder; Autistic Disorder; Automation; Behavioral; Biological Assay; Birth; Blinded; Blood; CGG repeat; Categories; Characteristics; Clinical; Cognitive; Collection; DNA; Data; Data Analyses; Detection; Development; Diagnostic; Diagnostic Procedure; Diagnostic tests; Disease; Drops; Early Diagnosis; Early treatment; FMR1; FMR1 Gene; Female; Fragile X Syndrome; Gene Mutation; Genes; Goals; Hereditary Disease; Human; Inherited; Investigation; Length; Link; Measures; Mental Retardation; Methods; Molecular; Mutation; Neonatal Screening; Newborn Infant; Outcomes Research; Paper; Patients; Performance; Pharmacologic Substance; Phase; Phenotype; Pilot Projects; Population; Preparation; Prevalence; Procedures; Process; Protein Analysis; Publishing; Reagent; Risk; Sampling; Screening procedure; Severity of illness; Southern Blotting; Spottings; Syndrome; Technology; Testing; Therapeutic; Time; Trinucleotide Repeats; Triplet Multiple Birth; Validation; Whole Blood; base; clinically relevant; cost effective; design; high throughput screening; improved; innovation; male; motor disorder; programs; prototype; reproductive; research clinical testing

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop accurate, rapid, high-throughput and cost-effective diagnostic tests for Fragile X syndrome (FXS) and related disorders in anticipation of population screening needs. Newborn screening (NBS) for Fragile X is timely due to the disease prevalence, lack of a clear phenotype at birth, current and emerging options for early interventions, and the impact of associated syndromes. Access to an inexpensive and effective high-throughput screening technology is one of the most significant hurdles to NBS implementation. To address this technology gap, Asuragen has formulated a PCR reagent set that can reproducibly detect the full spectrum of Fragile X triplet repeat expansions, including those with >1000 CGG repeats, in purified DNA from clinical samples. In this proposal, investigations are described that will leverage these PCR innovations to enable direct amplification from blood spot cards and support the accurate detection of clinically relevant Fragile X samples. Fragile X is the most common form of inherited mental retardation, affecting roughly 1 in 4,000 males and 1 in 6,000 females, with carrier rates as high at 1 in 130 in females and 1 in 250 in males. The disease arises from expansion of a CGG trinucleotide sequence in the 5'-untranslated region of the FMR1 gene. Disease severity is strongly linked to the number of CGG repeats in this region of the gene. Patients with >200 CGG often manifest FXS. More modest expansions are associated with reproductive, and cognitive/motor disorders. Improved diagnostic screening is particularly relevant in the face of promising pharmaceutical and behavioral therapeutics for FXS. A commercialized, and published the validation of FMR1 PCR technologies have been successfully developed that can detect all categories of Fragile X alleles, including very large full mutation expansions previously detectable only by Southern blot analysis, and resolve other longstanding technical issues (such as female zygosity) that confound existing PCR-based diagnostic methods. For this project, these PCR advances will be extended to develop a prototype high-throughput NBS assay that can accurately identify newborns at risk for fragile X disorders. The specific aims of the proposal are to: 1) Optimize procedures for blood spot processing with Whatman #903 paper; 2) Optimize FMR1 CGG RP PCR reagents and CE detection for use with processed blood spot card samples; and 3) Automate optimized blood spot processing and FMR1 PCR. The outcome of this research will be demonstrated feasibility for an accurate, automated, and cost- effective platform for fragile X detection in newborns, and address a longstanding unmet need for scalable technologies that can identify fragile X disorders in both males and females in the broader population. PUBLIC HEALTH RELEVANCE: We are developing an automated, rapid, and cost-effective molecular test for screening newborns to detect the characteristic genetic mutation associated with Fragile X Syndrome. Fragile X is one of the most commonly inherited forms of mental retardation and can also cause conditions such as ADHD and autism. The diagnostic tests detects DNA from a small drop of blood spotted on paper and will support early diagnosis which may be beneficial because of current and emerging options for early interventions.

描述(由申请人提供)：该项目的长期目标是开发准确、快速、高通量和成本效益高的脆性X综合征(FXS)和相关疾病的诊断测试，以满足人口筛查需求。由于疾病流行、出生时缺乏明确的表型、当前和正在出现的早期干预选择以及相关综合征的影响，新生儿脆性X筛查(NBS)是及时的。获得廉价有效的高通量筛查技术是国家统计局实施的最大障碍之一。为了解决这一技术差距，Asuragen开发了一套聚合酶链式反应试剂组，可以重复检测临床样本中纯化的DNA中脆性X三联体重复序列的全谱扩展，包括那些具有&gt；1000 CGG重复序列的DNA。在这项提案中，描述了利用这些PCR创新技术实现血斑卡直接扩增并支持对临床相关的脆性X样本进行准确检测的研究。脆性X基因是最常见的遗传性智力低下，大约每4000名男性和每6000名女性中就有一人患病，女性携带率高达每130人中有1人，男性为每250人中有1人携带。这种疾病是由于FMR1基因5‘-非翻译区CGG三核苷酸序列的扩张引起的。疾病的严重程度与该基因这一区域的CGG重复数密切相关。患有&gt；200 CGG的患者通常表现为FXS。较温和的扩张与生殖、认知/运动障碍有关。面对前景看好的FXS药物和行为疗法，改进的诊断筛查尤其相关。一种商业化并公开发表的FMR1聚合酶链式反应技术已经成功开发，它可以检测所有类别的脆性X等位基因，包括以前只能通过Southern杂交分析才能检测到的非常大的完全突变扩展，并解决了困扰现有基于聚合酶链式反应的诊断方法的其他长期存在的技术问题(如女性合子)。在这个项目中，这些聚合酶链式反应的进展将被扩展到开发一个原型的高通量NBS分析，可以准确地识别有脆性X疾病风险的新生儿。该建议的具体目的是：1)优化Whatman#903试纸的血点处理程序；2)优化FMR1 CGG RP PCR试剂和CE检测，以用于已处理的血斑卡样本；以及3)自动化优化的血点处理和FMR1 PCR。这项研究的结果将证明建立一个准确、自动化和具有成本效益的新生儿脆性X检测平台的可行性，并解决长期未得到满足的可扩展技术的需求，这种技术可以在更广泛的人群中识别男性和女性的脆性X疾病。 公共卫生相关性：我们正在开发一种自动化、快速和经济高效的分子检测方法，用于筛查新生儿，以检测与脆性X综合征相关的特征基因突变。脆性X是最常见的遗传性智力低下形式之一，也可能导致ADHD和自闭症等疾病。诊断测试从纸上斑点的一小滴血中检测DNA，并将支持早期诊断，这可能是有益的，因为目前和正在出现的早期干预选择。