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针对脆弱的X综合征（FXS）（FXS）和相关疾病开发准确，快速，高通量和具有成本效益的诊断测试，以期待人口筛查需求。由于疾病的患病率，出生时缺乏明显的表型，早期干预措施的当前和新兴选择以及相关综合症的影响，易碎X的新生儿筛查（NB）是及时的。获得廉价且有效的高通量筛查技术是NBS实施最重要的障碍之一。为了解决这一技术差距，Asuragen制定了一个PCR试剂集，可以在临床样品中纯化的DNA中可重复地检测到脆弱的X三重膨胀范围，包括具有> 1000 CGG重复序列的膨胀。在此提案中，描述了将利用这些PCR创新的调查，以从血点卡中直接放大，并支持对临床上相关的脆弱X样品的准确检测。脆弱的X是遗传性智力低下的最常见形式，大约影响4,000名男性中有1个，而女性中有1个，女性的载率在130名中，男性为130，而男性为250名。该疾病源于FMR1基因的5'-非翻译区域中CGG三核苷酸序列的扩展。疾病的严重程度与该基因区域中的CGG重复次数密切相关。 > 200个CGG的患者通常表现出FXS。更适度的扩展与生殖和认知/运动障碍有关。面对FXS的有前途的药物和行为疗法，改进的诊断筛查尤其重要。已成功开发了一种商业化并发布了FMR1 PCR技术的验证，可以检测所有类别的脆弱X等位基因，包括仅通过Southern印迹分析可检测到的非常大的完整突变扩展，并解决其他长期存在的技术问题（例如女性zygosity），以混淆基于PCR的现有诊断方法。对于该项目，将扩展这些PCR的进步，以开发一种原型高通量NBS分析，该测定方法可以准确地确定有脆弱X疾病风险的新生儿。该提案的具体目的是：1）使用Whatman＃903纸优化血点处理程序； 2）优化FMR1 CGG RP PCR试剂和CE检测，可与加工的血点卡样品一起使用； 3）自动化优化的血点处理和FMR1 PCR。这项研究的结果将证明可行性，可为新生儿中脆弱的X检测提供准确，自动化和具有成本效益的平台，并解决了对可扩展技术的长期未满足的需求，这些技术可以鉴定出更广泛人群中男性和女性的脆弱X疾病。 公共卫生相关性：我们正在开发一种自动化，快速且具有成本效益的分子测试，用于筛选新生儿，以检测与脆弱X综合征相关的特征遗传突变。脆弱的X是智力低下的最常见的形式之一，也可能导致ADHD和自闭症等条件。诊断测试可检测到纸上发现的一小滴血液中的DNA，并将支持早期诊断，这可能是由于早期干预措施的当前和新兴选择而有益的。