FUNCTIONAL CHARACTERIZATION OF NOVEL DETERMINANTS OF HOLOPROSENCEPHALY (HPE)

总结 先天性缺陷是全世界发病的主要原因，造成33万新生儿死亡， 每年出生。脑畸形似乎是最常见的先天性异常， 死亡和终身残疾的原因。在大多数情况下，由于复杂性，原因仍然不确定。 以及这些异常的多基因起源。编码转录因子（TF）和表观遗传的基因 鉴于这些蛋白质在整合信号传导中的核心作用， 级联并协调多个生物过程。其功能的缺陷可能会干扰整个 转录程序，涉及几个基因和分子途径。 在这里，我们将联合收割机小鼠遗传学和表观基因组学方法相结合，以揭示PRDM15的作用，PRDM15是一种以前被认为是一种基因突变。 在先天性脑畸形中，未被怀疑的疾病相关的表观遗传调节因子。此外，由 我们发现了PRDM15下游效应子（如NOTCH和WNT/PCP途径）的功能特征 迄今为止，在患有脑畸形（即HPE和小头畸形）的患者中未被充分认识的基因突变。 初步数据：我们已经表征了PRDM15在调节小鼠幼稚ESC状态中的功能。 然后，我们扩大了我们的发现，以证明在体内，PRDM15缺失导致：1）胚胎 E12.5-E14.5时的致死性; 2）影响前/后图案形成和前脑发育的图案形成缺陷。 特别是，我们观察到未能正确形成轴向中内胚层（AME），胚胎结构 3）最后，与M. Muenke（NIH）和 Hildebrandt（哈佛），我们已经鉴定了PRDM 15中的杂合和纯合突变， 全原脑畸形（HPE）和小头畸形，分别在超过100个PRDM15调节基因的突变 （~20%可能是损害性的）在一个大的HPE患者队列（132个三胞胎和188个单胞胎）中。 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被激活的。 在染色质水平调节驱动前脑发育的转录程序。接下来，在AIM 2中， 将在人类PRDM15突变、HPE相关遗传变异之间建立因果联系 作为PRDM15转录靶点，以及相关的脑畸形谱（从HPE到 小头畸形）。 这些研究的意义在于PRDM15是迄今为止尚未表征的胚胎发育的关键调节因子。 下游调控途径的开发和知识将有助于再生领域的研究。 这将对前脑无裂畸形和小头畸形患者的诊断和临床意义。 这些研究的临床影响是，鉴于HPE的多基因起源， PRDM15及其关键下游靶点可能被添加到有风险家庭的常规基因检测中 携带其他导致HPE的突变（例如SHH，ZIC 2，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).