A Novel Platform for Maternal Alcohol Consumption Screening

描述(申请人提供)：孕妇在怀孕期间酗酒会导致胎儿酒精谱障碍(FASD)，这是一种终生残疾，其特征是一系列神经解剖学和神经行为缺陷。目前估计FASD在美国学龄儿童中的患病率约为2%-5%。因此，及早发现孕妇饮酒对预防FASD至关重要。母亲的自我报告在很大程度上是不可靠的，目前的生物标志物在怀孕期间的用途也是有限的。在这些标记物中，蛋白质转铁蛋白的糖基化变体(碳水化合物缺陷型转铁蛋白，CDT)被报道为慢性酒精滥用最具诊断特异性的生物标记物。然而，平均灵敏度约为65.4%，范围很大，取决于所采用的分析方法。此外，利用传统的电泳法、层析法和免疫计量学方法，到目前为止，很难表征所有糖类的组成和丰度。这一点很关键，因为酒精对多糖产生复杂的影响，包括唾液酸的损失、糖的部分损失和/或低聚糖链的完全损失。在广泛使用的FASD大鼠模型中，我们首次利用最先进的质谱仪，获得了表征母体血浆转铁蛋白N-连接寡糖的序列和丰度的初步数据，该序列和丰度保持了N-糖位的位置。基于我们的新数据，我们提出了以下具体目标：利用传统的生物标记物(转铁蛋白的变体)开发和验证一种新的、灵敏的高通量平台，用于在FASD大鼠模型中筛查母亲的酒精消费。提出了两个子目标来表征和验证该蛋白质在母体血浆中的翻译后糖基化特征丰度分布，并将其与酒精暴露的剂量和持续时间联系起来。我们将利用一种孕鼠模型，在妊娠期间给予不同时间的分级酒精剂量。将进行血浆蛋白(转铁蛋白)鉴定、序列作图、具有诊断碎裂光谱的糖肽鉴定以及高分辨率/准确质量定量。验证研究将包括N-连锁糖位确认和基于凝集素的探测。基于我们的初步数据，我们预计所提出的平台将提供高度敏感的蛋白质翻译后修饰特征图谱，这将极大地增强检测窗口 而且还能清楚地区分酒精剂量。最后，我们计划将糖基化特征与FASD神经行为测量相关联。我们还相信，这些新的方法未来可以扩展到孕妇，并形成高通量筛查工具的基础，这将有助于建立标准化的诊断标准，不仅在诊断方面，而且在治疗和预防方面也具有真正的临床影响。

DESCRIPTION (provided by applicant): Maternal alcohol abuse during pregnancy can result in Fetal Alcohol Spectrum Disorders (FASD), a lifelong disability characterized by a range of neuroanatomical and neurobehavioral deficits. Current estimates of FASD prevalence is about 2-5% among young school children in the United States. Therefore, early detection of alcohol use among pregnant women is critical to prevent FASD. Maternal self-reporting is largely unreliable and the utility of current biomarkers is also limited during pregnancy. Among these markers, glycosylated variants of the protein transferrin (carbohydrate deficient transferrin, CDT), is reported to be the most diagnostically specific biomarker for chronic alcohol abuse. However, the mean sensitivity is about ~65.4% with a wide range depending upon the analytical method employed. Moreover, utilizing the conventional electrophoretic, chromatographic, and immunometric methods, it has so far been difficult to characterize the composition and abundance of all glycoforms. This is critical because alcohol produces complex effects on the glycans including loss of sialic acids, partial loss of sugars, and/or complete loss of oligosaccharide chains. In a widely utilized FASD rat model, we have for the first time utilized state of the art mass spectrometry and obtained preliminary data characterizing the sequences and abundances of maternal plasma transferrin N- linked oligosaccharides preserving the location of the N-glycosite. Based on our novel data, we propose the following Specific Aim: Develop and validate a novel and sensitive high throughput platform utilizing a traditional biomarker (variants of transferrin) for maternal alcohol consumption screening in a FASD rat model. Two sub aims are proposed to characterize and validate the protein's post-translational glycosylation signature abundance profile in the maternal plasma and relating it to the dose and duration of alcohol exposure. We will utilize a pregnant rat model exposed to graded alcohol doses administered for different durations of exposure during gestation. Plasma protein (transferrin) identification, sequence mapping, glycopeptide identification with diagnostic fragmentation spectra, and High Resolution/Accurate Mass quantitation will be performed. Validation studies will include N-linked Glycosite confirmation and lectin-based probing. Based on our preliminary data, we expect the proposed platform to provide a highly sensitive protein post-translational modification signature profile that will greatly enhance the window of detection and also clearly distinguish between the alcohol doses. At the end, we plan to correlate the glycosylation signature profile with FASD neurobehavioral measures. We also believe that these novel methodologies can be in the future extended to pregnant women and form the basis for a high throughput screening tool that will help establish a standardized diagnostic criteria with a real clinical impact not only in diagnosis but also in treatment and prevention.