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

基本信息

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
项目类别：
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&apos 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

项目摘要

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性 X 综合征 (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，以满足人群筛查需求。由于脆性 X 细胞疾病的流行、出生时缺乏明确的表型、当前和新兴的早期干预选择以及相关综合征的影响，因此需要及时进行新生儿脆性 X 细胞筛查 (NBS)。获得廉价且有效的高通量筛选技术是 NBS 实施的最大障碍之一。为了弥补这一技术差距，Asuragen 配制了一套 PCR 试剂，可以重复检测临床样本纯化 DNA 中的全谱脆性 X 三联体重复扩增，包括具有 >1000 个 CGG 重复的扩增。在该提案中，描述了将利用这些 PCR 创新技术实现血点卡直接扩增并支持临床相关脆性 X 样本的准确检测的研究。脆性 X 染色体是最常见的遗传性精神发育迟滞，大约每 4,000 名男性和每 6,000 名女性中就有 1 人受到影响，女性携带率高达十分之一，男性携带率为 250 分之一。该疾病是由 FMR1 基因 5'-非翻译区中 CGG 三核苷酸序列的扩展引起的。疾病的严重程度与该基因区域的 CGG 重复次数密切相关。 CGG > 200 的患者通常表现为 FXS。更适度的扩张与生殖和认知/运动障碍有关。面对前景光明的 FXS 药物和行为疗法，改进诊断筛查尤其重要。已成功开发出商业化并已发表的 FMR1 PCR 技术验证，可检测所有类别的脆性 X 等位基因，包括以前只能通过 Southern 印迹分析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（例如女性接合性））这混淆了现有的基于 PCR 的诊断方法。在该项目中，这些 PCR 进展将得到扩展，以开发一种高通量 NBS 检测原型，该检测可以准确识别有脆性 X 病风险的新生儿。该提案的具体目标是： 1) 优化 Whatman #903 纸的血斑处理程序； 2) 优化FMR1 CGG RP PCR试剂和CE检测，用于处理过的血斑卡样本； 3) 自动优化血斑处理和 FMR1 PCR。这项研究的成果将证明建立一个准确、自动化且具有成本效益的新生儿脆性 X 染色体检测平台的可行性，并解决对可扩展技术长期未得到满足的需求，这些技术可以在更广泛的范围内识别男性和女性的脆性 X 染色体疾病。人口。公共健康相关性：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，用于筛查新生儿，以检测与脆性 X 综合征相关的特征性基因突变。脆性 X 染色体是最常见的遗传性精神发育迟滞之一，也可能导致注意力缺陷多动症和自闭症等疾病。诊断测试可从纸上滴下的一小滴血中检测 DNA，并将支持早期诊断，由于当前和新兴的早期干预选择，这可能是有益的。