FUNCTIONAL CHARACTERIZATION OF NOVEL DETERMINANTS OF HOLOPROSENCEPHALY (HPE)

摘要 先天性缺陷是全球发病率的主要原因，占33万新生婴儿的死亡。 每年都会出生。脑畸形似乎是最常见的先天性畸形，是一种主要的 死因和终生残疾。在大多数情况下，由于原因的复杂性，原因仍然不确定 以及这些异常的多成因来源。编码转录因子和表观遗传学的基因 考虑到这些蛋白质在整合信号方面的核心作用，调节器已经成为相关的候选对象 级联和协调多个生物过程。它们的功能缺陷可能会扰乱整个 转录程序，涉及多个基因和分子途径。 在这里，我们结合小鼠遗传学和表观基因组学方法来揭示PRDM15的作用，这是一个以前的 在先天性脑畸形中，意外的疾病相关表观遗传调节。此外，通过 我们发现的PRDM15下游效应器(例如，Noch和WNT/PCP通路)的功能特征 到目前为止，在患有脑畸形(即HPE和小头畸形)的患者中，基因突变被低估了。 初步数据：我们已经确定了PRDM15在调节小鼠幼稚ESC状态中的功能。 然后，我们扩展了我们的发现，证明在体内，PRDM15缺失会导致：1)胚胎 E12.5-E14.5的致命性；2)图案缺陷影响前/后图案和前脑发育。 特别是，我们观察到未能正确形成轴中胚层(AME)，这是一种胚胎结构 对于适当的前路规范是必要的；3)最后，与M.Muenke(NIH)和 F.Hildebrandt(哈佛大学)，我们已经发现PRDM15的杂合和纯合子突变与 全脑发育异常(HPE)和小头畸形，以及100多个PRDM15调节基因的突变 (约20%可能是破坏性的)在一个大的HPE患者队列中(132个三联体和188个单胞胎)。 在AIM1中，我们建议定义PRDM15功能的分子基础，特别是了解它是如何 在染色质水平上调节驱动前脑发育的转录程序。接下来，在AIM2中，我们 将在人类PRDM15突变和已发现的HPE相关遗传变异之间建立致病联系 作为PRDM15-转录靶点，以及相关的脑畸形谱(从HPE，到 小头畸形症)。 这些研究的意义在于，PRDM15是一种迄今未被描述的胚胎发育的关键调节因子 下游调控通路的开发和认识将对再生领域有所帮助 并将对无前脑畸形和小头畸形的患者具有诊断和临床意义。 这些研究的临床影响是，鉴于HPE的多基因起源，靶向测序 PRDM15及其关键的下游靶点可能会被添加到高危家庭的常规基因检测中 携带其他导致HPE的突变(如SHH、ZIC2、TGIF)。

SUMMARY Congenital defects are a leading cause of morbidity worldwide, accounting for the deaths of 330,000 new- born every year. Brain malformations appear to be the most common congenital anomalies and are a major cause of death and lifelong disability. In the majority of cases the cause remains uncertain, due to the complexity and the multi-genic origin of these anomalies. Genes encoding Transcription Factors (TFs) and epigenetic regulators have become relevant candidates given the central role of these proteins in integrating signalling cascades and orchestrating multiple biological processes. Deficiency in their function may disturb entire transcriptional programs, involving several genes and molecular pathways. Here we combine mouse genetics and epigenomic approaches to uncover the role of PRDM15, a previously unsuspected disease-associated epigenetic regulator, in congenital brain malformations. Moreover, by functionally characterizing PRDM15 downstream effectors (e.g. NOTCH and WNT/PCP pathways) we uncover hitherto underappreciated genes mutated in patients with brain malformations (i.e. HPE and microcephaly). Preliminary data: We have characterized the function of PRDM15 in regulating the mouse naïve ESC state. We have then expanded our findings to demonstrate that, in vivo, PRDM15 depletion leads to: 1) embryonic lethality at E12.5-E14.5; 2) patterning defects affecting Anterior/Posterior patterning and forebrain development. In particular we have observed a failure to properly form the Axial Mesendoderm (AME), an embryonic structure necessary for proper anterior specification; 3) Finally, in collaboration with the groups of M.Muenke (NIH) and F.Hildebrandt (Harvard), we have identified heterozygous and homozygous mutations in PRDM15 linked to Holoprocensephaly (HPE) and microcephaly, respectively and mutations in over 100 PRDM15-regulated genes (~20% likely to be damaging) in a large cohort of HPE patients (132 trios and 188 singletons). In AIM1 we propose to define the molecular basis of PRDM15 function, specifically to understand how it regulates, at the level of chromatin, the transcriptional program driving forebrain development. Next, in AIM2 we will establish a causative link between human PRDM15 mutations, HPE-associated genetic variants identified as PRDM15-transcriptional targets, and the associated spectrum of brain malformations (ranging from HPE, to Microcephaly). The significance of these studies is that PRDM15 is a so far uncharacterized critical regulator of embryonic development and knowledge of the downstream regulated pathways will be useful to the field of regenerative medicine and will have diagnostic and clinical implications for patients with holoprosencephaly and microcephaly. The clinical Impact of these studies is that given the multigenic origin of HPE, targeted sequencing of PRDM15, and its key downstream targets, can potentially be added to routine genetic testing in families at risk of carrying other HPE-causing mutations (e.g. SHH, ZIC2, TGIF).