A Saliva Based Kit for the Epigenetic Assessment of Heavy Alcohol Intake

大量饮酒是常见的，但可以避免的死亡，残疾和人类痛苦的原因。这些 如果及早发现大量饮酒，不良后果是可以避免的。然而，目前的筛查 慢性酒精消耗的方法仅在测试前几小时内捕获酒精使用，或者依赖于 不敏感的非特异性蛋白质测定。在最近一个非常成功的第二阶段项目中， 全血，我们证明了仅仅两个甲基化敏感液滴数字PCR的组合， （MSddPCR）测定可以相当准确地识别最近大量饮酒的受试者。以来 然后，我们继续推进这些发现，并开发了更多更强大的 MSddPCR分析和改进我们的过程，只需要在一滴血中发现的DNA。在 与我们的道德和营销团队合作，我们现在正在全国范围内推出这种产品。 然而，更普遍地实施这种颠覆性技术的一个重大障碍是， 血的要求。可以想象，一种可以使用唾液中DNA的测试可以绕过患者 担心针头或刺血针刺伤的不适。此外，它还将使非医疗 面向酒精治疗计划和面向远程医疗的治疗计划， 病人对治疗的反应这对于治疗老年人的酒精中毒尤其重要， 通常不能或不愿意前往酒精治疗机构。 在第一阶段的建议中，我们打算采取下一步措施，建立唾液DNA筛查测试 因为酗酒最近，我们已经收集并制备了150名受试者的唾液DNA， 接受酒精治疗和150名年龄、性别和种族匹配的对照组。我们亦 开发了一种MSddPCR检测方法，可以调整唾液DNA中的细胞异质性。 在本申请中，我们将使用这些资源，以确定唾液DNA的MSddPCR评估是否也可以 用于检测重度酒精消耗。具体而言，在目标1中，我们将确定在4 CpG基因座对检测饮酒具有高预测能力， 含量可用于校正唾液中的细胞混合物。然后，在目标2中，我们将进行一系列 唾液DNA的质量评估。最后，在目标3中，我们将分析来自我们的4个高度预测的数据， 位点，必要时对细胞混合物进行校正，以确定能够预测重 酒精使用状况此外，我们将分析质量控制措施，以确定那些唾液DNA 提供不可靠的样品。作为该项目的结果，我们将生产一套MSddPCR检测试剂盒和一个 这是一个严格的多地点第二阶段测试的质量控制指标，这个“不流血”的NextGen产品。

Heavy alcohol use is common, yet avoidable cause of death, disability and human suffering. These adverse outcomes are potentially avoidable if heavy alcohol use is spotted early. However, current screening methods for chronic alcohol consumption capture alcohol usage only in the hours prior to testing or rely on insensitive, non-specific protein assays. In a recent highly successful Phase II project, using DNA from whole blood, we demonstrated that a combination of just two methylation sensitive droplet digital PCR (MSddPCR) assays could quite accurately identify subjects with recent heavy alcohol consumption. Since then, we have continued to advance those findings and have developed additional, even more powerful MSddPCR assays and refined our processes to only require the DNA found in a single drop of blood. In conjunction with our ethics and marketing team, we are now in the process of rolling this product nationwide. However, a significant barrier to a more universal implementation of this disruptive technology is the requirement for blood. Conceivably, a test that could use the DNA from saliva could circumvent patient concerns about the discomfort of a needle or lancet stick. In addition, it would enable both non-medically oriented alcohol treatment programs and telemedicine oriented treatment programs to more easily assess client response to treatment. This will be particularly critical for treatment of alcoholism in the elderly, who are often unable or unwilling to travel to alcohol treatment facilities. In this Phase I proposal, we intend to take the next steps to the creation of a saliva DNA screening test for heavy alcohol consumption. Already, we have collected and prepared saliva DNA on 150 subjects recently admitted to alcohol treatment and 150 age, gender and ethnicity matched controls. In addition, we have also developed an MSddPCR assay that can adjust for cellular heterogeneity in the saliva DNA. In this application, we will use these resources to whether MSddPCR assessments of saliva DNA can also be used to detect heavy alcohol consumption. Specifically, in Aim 1, we will determine methylation status at 4 CpG loci with high predictive power for detecting alcohol consumption and one locus whose information content can be used to correct for cellular admixture in saliva. Then, in Aim 2, we will conduct a series of quality assessment of the saliva DNA. Finally, in Aim 3, we will analyze the data from our 4 highly predictive loci, correcting when necessary for cellular admixture, to determine an algorithm capable of predicting heavy alcohol use status. In addition, we will analyze the quality control measures to identify those saliva DNA samples which give unreliable. As a result of this project, we will produce a set of MSddPCR assays and a quality control metric for a rigorous multisite Phase II test of this “bloodless” NextGen product.