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

描述（由申请人提供）：本项目的长期目标是开发准确，快速，高通量和成本效益的诊断测试脆性X综合征（FXS）和相关疾病的预期人群筛查需求。新生儿脆性X染色体筛查（NBS）是及时的，因为疾病的流行，出生时缺乏明确的表型，目前和新兴的早期干预选择，以及相关综合征的影响。获得廉价有效的高通量筛选技术是NBS实施的最重要障碍之一。为了解决这一技术差距，Asuragen已经制定了一套PCR试剂，可以重复检测来自临床样本的纯化DNA中的脆性X三联体重复扩增的全谱，包括具有>1000个CGG重复的那些。在本提案中，描述了将利用这些PCR创新的研究，以实现从血斑卡直接扩增，并支持临床相关的脆性X样本的准确检测。脆性X染色体是最常见的遗传性智力低下形式，大约每4,000名男性中有1名，每6,000名女性中有1名，女性中的携带率高达130分之1，男性中为250分之1。该疾病由FMR 1基因5 '非翻译区中CGG三核苷酸序列的扩增引起。疾病的严重程度与该基因区域中CGG重复的数量密切相关。CGG>200的患者通常表现为FXS。更温和的扩张与生殖和认知/运动障碍有关。面对FXS有前途的药物和行为疗法，改进的诊断筛查特别重要。已经成功开发了一种商业化的并已发表的FMR 1 PCR技术验证，该技术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印迹分析检测到的非常大的全突变扩增，并解决了其他长期存在的技术问题（如女性接合性），这些问题混淆了现有的基于PCR的诊断方法。对于这个项目，这些PCR的进展将被扩展到开发一个原型高通量NBS检测，可以准确地识别新生儿脆性X染色体疾病的风险。该提案的具体目的是：1）使用Whatman #903纸优化血斑处理程序; 2）优化FMR 1 CGRP PCR试剂和CE检测，以用于处理过的血斑卡样本; 3）自动化优化的血斑处理和FMR 1 PCR。这项研究的结果将证明一个准确，自动化和成本效益的新生儿脆性X检测平台的可行性，并解决长期未满足的可扩展技术需求，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形式之一，也可能导致多动症和自闭症等疾病。诊断测试可以检测纸上斑点的一小滴血中的DNA，并将支持早期诊断，这可能是有益的，因为当前和新兴的早期干预选择。

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.