Circulating Organ-enriched microRNAs as biomarkers of Rett Syndrome

摘要 Rett综合征(RTT)是一种罕见的X连锁神经发育障碍，由MECP2基因突变引起。 这种疾病几乎只影响女性，因为携带MECP2突变的男性胎儿通常会早死 出生。受影响的女性在出生时看起来是正常的；疾病通常在6-18个月大时明显出现， 以神经退化、运动刻板印象、呼吸不规律和其他障碍为特征的。临床 严重程度似乎取决于多种因素，包括MECP2突变的类型，X- MECP2的失活和可能的遗传修饰物。尽管诊断性MECP2基因检测可用于 RTT，RTT的外周生物标志物，包括微创的、基于血液的疾病严重程度指标和 进步，都是缺乏的。分子诊断公司Diamir开发了专有平台 基于循环分析的病理生理过程早期检测与监测技术 器官丰富，包括大脑丰富，血浆中的微小RNA(MiRNAs)。到目前为止在 该公司发现了神经退行性变和其他疾病的有前景的miRNA生物标记物候选 在此，我们建议扩大使用我们的技术来建立RTT的miRNA生物标记物。 MECP2的缺失会导致突触功能障碍和脑内miRNAs的显著失调。我们 假设循环中的miRNAs丰富在不同的大脑区域，存在于突触/神经突起中，并且 在血浆中可以检测到与RTT发展相关的病理生理过程。此外, 分析肝脏、肺和肌肉中丰富的miRNAs水平可以反映RTT相关的变化 这些纸巾。数据归一化采用“miRNA对”方法，包括一个生物标记物候选者 两个miRNAs的比例。为了更高的精度，2-3个miRNA对被组合到miRNA分类器中。 对四种小鼠模型和人类患者进行的初步研究确定了miRNA对和分类器 区分野生型RTT小鼠模型和年龄匹配对照组(AMC)RTT患者模型； 某些miRNA对在小鼠和人类中是常见的，这表明潜在的miRNA之间的相似性 病理过程。目前的研究将使用在Rett中心收集的血浆样本进行 在蒙特菲奥里医疗中心。具体目标包括：(1)评估区分RTT和AMC的可行性 通过在受RTT(脑、肝、 肺、肌肉)(60RTT/60AMC)；以及(2)评估循环miRNAs预测疾病的潜力 通过分析先前研究的19个miRNAs的血浆水平来监测疾病的严重性和进展 在3年后从同一研究参与者(30RTT/30AMC)收集的纵向样本集中。 SBIR第二阶段将涉及更大规模的临床研究，以验证miRNA生物标记物候选。化验结果将是 最初是作为临床试验分析而开发的，在积累临床数据后，作为体外诊断。

SUMMARY Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder caused by mutations in the MECP2 gene. The disease affects females almost exclusively, since male fetuses with a MECP2 mutation usually die before birth. Affected females appear normal at birth; disease onset is typically evident by 6-18 months and is characterized by neurological regression, motor stereotypies, irregular breathing, and other handicaps. Clinical severity appears to depend on multiple factors, including the type of MECP2 mutation, skewing of the X- inactivation and likely genetic modifiers of MECP2. Although diagnostic MECP2 genetic testing is available for RTT, peripheral biomarkers of RTT, including minimally invasive, blood-based indicators of disease severity and progression, are lacking. DiamiR, a molecular diagnostics company, has developed proprietary platform technology for early detection and monitoring of pathophysiological processes based on analysis of circulating organ-enriched, including brain-enriched, microRNAs (miRNAs) in plasma. Studies conducted to date at the company resulted in identification of promising miRNA biomarker candidates for neurodegenerative and other diseases, and here we propose extending the use of our technology to establish miRNA biomarkers of RTT. Loss of MECP2 results in synaptic dysfunction and marked dysregulation of miRNAs in the brain. We hypothesize that circulating miRNAs enriched in different brain regions, present in synapses/neurites, and detectable in plasma can detect pathophysiological processes associated with RTT development. In addition, analysis of levels of miRNAs enriched in liver, lung and muscle can be reflective of RTT associated changes in these tissues. An “miRNA pair” approach is used for data normalization, and a biomarker candidate is comprised of a ratio of two miRNAs. Two-three miRNA pairs are combined into a miRNA classifier for higher accuracy. Preliminary studies performed with four murine models and human patients identified miRNA pairs and classifiers differentiating both RTT mouse models from wild-type mice and RTT patients from age-matched control (AMC); certain miRNA pairs were common to mice and humans, indicating the similarity between the underlying pathological processes. The current study will be performed using plasma samples collected at the Rett Center at Montefiore Medical Center. Specific aims include: (1) assessing the feasibility of differentiating RTT from AMC by plasma levels of 38 pre-selected circulating miRNAs enriched in organs/tissues affected by RTT (brain, liver, lung, muscle) (60 RTT/60 AMC); and (2) evaluating the potential of circulating miRNAs for predicting disease severity and monitoring disease progression by analysis of plasma levels of the previously studied 19 miRNAs in the longitudinal set of samples collected 3 years later from the same study participants (30 RTT/30 AMC). SBIR Phase II will involve larger clinical studies to validate miRNA biomarker candidates. The assay will be initially developed as a clinical trial assay, and, upon accumulation of clinical data, as in vitro diagnostics.