Comprehensive Deep Phenotyping and Multi-omics to Develop Clinical and Molecular Biomarkers for MeCP2-related Diseases

摘要 X连锁基因MECP2(甲基CpG结合蛋白2)与两种主要的 神经发育障碍：雷特综合征(RTT)，由MeCP2功能丧失突变引起，以及 MeCP2重复综合征(MDS)，由过量的MeCP2引起。RTT是最常见的遗传性疾病之一 女性智力残疾的原因，而(MDS)是最常见的基因组重排之一 男性。因为MeCP2蛋白调节多个大脑中数千个基因的表达 在不同地区，每种疾病的表型远远超出智力障碍的范围，影响情绪、运动控制、 和自主功能1。大脑对MeCP2的数量非常敏感：只有16%的下降 MeCP2水平足以产生Rett样症状。这一事实对大多数人来说是一个突出的挑战 这些疾病正在开发有希望的治疗方法：通过增加剂量来治疗RTT MeCP2水平过高会导致MDS；在MDS中过多抑制MeCP2水平会导致RTT。为了避免 简单地将一组令人衰弱的症状换成另一组，我们需要可靠的方法来衡量治疗 并评估我们是否使用了正确的剂量。 对人源化小鼠的临床前研究令人信服地证明，反义寡核苷酸 (ASO)可以降低MeCP2水平并逆转MDS表型；最近的工作发现了激酶和 调节MeCP2稳定性的磷酸酶，同样在小鼠身上取得了良好的效果。这两个选项都是非常 很有希望，但我们如何测量患者的MeCP2水平？MeCP2是一种核染色质结合蛋白 这在大脑中的表达水平非常高，在那里它无法直接测量。我们有 因此一直在寻找其他与MeCP2水平相关但在血液中可以测量到的分子 样本或其他相对非侵入性的手段。我建议，对于RTT和MDS这样的复杂疾病， 在判断治疗反应方面，复合生物标记物面板将优于任何单一的方法。我们的 初步研究已经确定了两个跟踪MeCP2水平的重要分子生物标志物 在老鼠身上；我们也有几个额外的候选者。因此，这项研究的目的是开发一组临床 以及分子生物标记物，将指导治疗努力，以防止这些患者治疗过度或治疗不足 疾病。德克萨斯儿童医院拥有全国最多的RTT和MDS患者， 因此，我们处于有利地位，可以实现1)开发MDS的结果衡量标准；2)将表型与 Xq28基因座的基因组结构；以及3)在人类中验证跟踪变化的分子生物标志物 在小鼠体内的MeCP2水平。完成这三个目标将为ASOS的临床试验奠定基础 并为涉及其他等位基因疾病的研究铺平了道路，涉及太多或太少的 同样的基因产物。

Abstract The X-linked gene MECP2 (methyl CpG-binding protein 2) is associated with two major neurodevelopmental disorders: Rett Syndrome (RTT), caused by loss-of-function mutations in MeCP2, and MECP2 duplication syndrome (MDS), caused by too much MeCP2. RTT is one of the most common genetic causes of intellectual disability in females, while (MDS) is one of the most common genomic rearrangements in males. Because the MeCP2 protein regulates the expression of thousands of genes across multiple brain regions, the phenotype of each disease extends well beyond intellectual disability to affect mood, motor control, and autonomic functions1. The brain is exquisitely sensitive to the quantity of MeCP2: a drop of just 16% in MeCP2 levels is enough to produce Rett-like symptoms. This single fact is a salient challenge to the most promising therapies being developed for these diseases: slightly over-shooting treatment for RTT by increasing MeCP2 levels too much will cause MDS; suppressing MeCP2 levels too much in MDS will cause RTT. To avoid simply exchanging one set of debilitating symptoms for another, we need reliable ways to measure treatment responses and to assess whether we are administering the correct dose. Preclinical studies in humanized mice have convincingly demonstrated that antisense oligonucleotides (ASO) can reduce MeCP2 levels and reverse the MDS phenotype; more recent work identified kinases and phosphatases that regulate MeCP2 stability, again with good results in mice. These options are both extremely promising, but how do we measure MeCP2 levels in patients? MeCP2 is a nuclear, chromatin-bound protein that is expressed at very high levels in the brain, where it is not accessible to direct measurement. We have therefore been searching for other molecules that correlate with MeCP2 levels but are measurable in blood samples or other relatively noninvasive means. I propose that, for such complex diseases as RTT and MDS, a composite biomarker panel will be superior to any single-modality measure to judge treatment response. Our preliminary studies have already identified two important molecular biomarkers that track with MeCP2 levels in mice; we have several additional candidates as well. This study therefore aims to develop a panel of clinical and molecular biomarkers that will guide therapeutic efforts to prevent over- or under-treatment in these diseases. Texas Children's Hospital has the largest patient populations in the country for both RTT and MDS, so we are well-positioned to accomplish 1) Develop outcome measures for MDS; 2) Correlate phenotypes with the genomic structure at Xq28 locus; and 3) Validate in humans molecular biomarkers that track with changes in MeCP2 levels in mice. Completing these three aims will lay the groundwork for clinical trials of ASOs in MDS and pave the path forward for studies involving other allelic disorders involving too much or too little of the same gene product.