Amplification Strategies for Detection of Fragile X Trinucleotide Repeats

描述（由申请人提供）：本项目的总体目标是开发和实施一套全面的技术，以改善与脆性X综合征（FXS）相关的疾病的筛查和诊断。FXS是由FMR 1基因5 '非翻译区的胞嘧啶-鸟嘌呤-鸟嘌呤（CGG）三联体重复扩增引起的。FXS影响1/4000男性和1/6000女性。完全扩增至大于200个重复与FMR 1基因的超甲基化和FMR 1蛋白质产生的完全丧失相关。CGG重复序列的适度扩增与老年男性脆性X相关震颤/共济失调综合征（FX-TAS）和女性原发性卵巢功能不全（FX-POI）相关。最近发表的指南建议对自闭症谱系障碍（ASD）的诊断进行后续染色体和脆性X检测，ASD影响1/150的个体-大约是脆性X人群发病率的33倍。此外，有希望的药物目前正在临床试验中，需要准确和早期识别脆性X患者。脆性X染色体检测的改进将对与这种疾病相关的多种精神和健康状况的所有年龄段的广泛个体产生重要影响。目前脆性X染色体检测的诊断方法是进行PCR，然后根据需要进行Southern印迹，以确定CGG重复数和FMR 1甲基化状态。然而，PCR的一个主要限制是不能扩增完整的突变，甚至许多突变前的等位基因。目前所有的PCR方法都缺乏分辨纯合性的能力。因此，近50%的临床实验室目前将所有样品反射到更昂贵、费力且低通量的Southern印迹分析。因此，尽管对基于人群的FXS筛查（新生儿和/或携带者筛查）有强有力的论据，但无法可靠地扩增全突变重复扩增使得广泛的筛查对于实施来说过于昂贵和不准确。Asuragen已经开始努力改善长GC富集重复序列的扩增，以便更好地服务于脆性X突变的稳健诊断和筛查的重要临床目标。在我们的初步数据中，我们现在证明了高达至少~1000个CGG重复的PCR扩增，这是商业上可获得或已发表的最长重复长度。这种能力是基于PCR的检测的基础，该检测可以报告前突变和完全突变等位基因，并显著减少或消除了被反射到Southern印迹的病例数量。在第一阶段的SBIR项目中，我们将建立有效的，自动化的测试，开发一个基于PCR的测试来解决女性样本的接合性，并开发一个基于PCR的方法来确定FMR 1基因的甲基化状态。这将导致一个诊断工作流程，可以支持常规筛查，从而使早期干预和改善患者的治疗选择。 公共卫生相关性：该项目的长期目标是改善脆性X综合征和相关疾病的筛查和诊断。在这个项目中，我们将利用我们在FMR 1基因PCR方面的突破，开发一套具有成本效益和效率的强大而准确的测试。这将使广泛的筛查，以确定携带者，并允许早期诊断和干预脆性X综合征患者。

DESCRIPTION (provided by applicant): The overall objective for this project is to develop and implement a comprehensive set of technologies to improve screening and diagnosis of conditions associated with Fragile X Syndrome (FXS). FXS is caused by an expansion of a cytosine-guanine-guanine (CGG) triplet repeat in the 5'-untranslated region of the FMR1 gene. FXS affects 1/4000 men and 1/6000 women. Full expansion to greater than 200 repeats is associated with hypermethylation of the FMR1 gene and complete loss of FMR1 protein production. A more modest expansion of the CGG repeats is associated with Fragile X-associated Tremor/Ataxia Syndrome (FX-TAS) in older men and primary ovarian insufficiency (FX-POI) in women. Recently published guidelines suggest follow-up chromosome and Fragile X testing for a diagnosis of autism spectrum disorder (ASD), which impacts 1/150 individuals-roughly 33 times the population incidence of Fragile X. Furthermore, promising drugs are currently in clinical trials and will require accurate and early identification of Fragile X patients. Improvements in testing for Fragile X will have important implications for a broad range of individuals of all ages across multiple mental and health conditions associated with this disorder. The current diagnostic approach for Fragile X testing is to perform PCR, followed by Southern blot as necessary, to determine the CGG repeat number and FMR1 methylation state. However, a major limitation of PCR is the inability to amplify full mutations and even many pre-mutation alleles. All current PCR methods lack the ability to resolve homozygosity. As a result, nearly 50% of clinical laboratories currently reflex all samples to the more expensive, laborious, and low-throughput Southern blot assay. Therefore, despite strong arguments for population-based screening for FXS (either newborn and/or carrier screening), the inability to reliably amplify full mutation repeat expansions makes wide-spread screening too expensive and inaccurate for implementation. Asuragen has undertaken an effort to improve the amplification of long GC-rich repeats in order to better serve the important clinical goals of robust diagnosis and screening for Fragile X mutations. In our preliminary data, we now demonstrate PCR amplification of up to at least ~1000 CGG repeats, the longest repeat length that is commercially available or published. This capability is the foundation for a PCR-based assay that can report both premutation and full mutation alleles and dramatically reduce or eliminate the number of cases reflexed to Southern blots. In this phase I SBIR project, we will establish efficient, automatable testing, develop a PCR_based test to resolve zygosity in female samples, and develop a PCR-based method for determining methylation status of the FMR1 gene. This will result in a diagnostic workflow that can support routine screening, and thus enable earlier interventions and improved treatment options for patients. PUBLIC HEALTH RELEVANCE: The long-term goal for this project is to improve screening and diagnosis of Fragile X Syndrome and related conditions. In this project, we will leverage our breakthrough in PCR of the FMR1 gene to develop a set of robust and accurate tests that are cost-effective and efficient. This will enable widespread screening to identify carriers and permit earlier diagnosis and intervention for Fragile X Syndrome patients.