Human genetics and molecular mechanisms of Vein of Galen aneurysmal malformation

项目总结 对人类严重先天性脑血管畸形的遗传学研究有助于深入了解其发病机制。 正常的血管发育，并确定治疗干预的目标。Galen静脉动脉瘤 脑动静脉畸形是儿童脑动静脉畸形中最常见、最严重的一种 (Avms)。我们对VOGMS分子发病机制的理解存在显著差距，阻碍了VOGMS的 制定改进的诊断和治疗措施。基因座异质性与散发性 许多VOGM病例构成了VOGM基因发现的根本障碍。我们最近应用了全套 外显子组测序(WES)克服这些障碍并鉴定从头基因和遗传性基因突变 这约占散发性VOGM病例的30%(Duran等人，Neuron，2019年)。其中包括全基因组的 动静脉关键调节因子EphB4(EphB4)罕见的破坏性突变带来的重大负担 在家族性AVM综合征、毛细血管畸形(CM)-AVM II型(CM-AVM2)中，规范也发生了突变。 我们还在其他基因中发现了新的突变，这些基因在相同的EPhin信号相互作用组中发挥作用， 包括RASA1(在CM-AVM1中也发生突变)。我们进一步证明了EphB4存在于一种 目前已经解决了RASA1的第一个多域晶体结构。尽管如此， 大多数VOGM病例在遗传上仍未解决，VOGM相关的分子机制 人们对突变知之甚少。为了解决这些知识差距，我们提出了功能基因组学 以原子级分辨率发现和机械解释VOGM相关突变的方法。 我们假设WES将发现新的VOGM基因和突变，包括花叶和体细胞 破坏EphB4-RASA1信号复合体调节活性的“二次打击”突变 对于动静脉发育来说是必不可少的。基于我们在识别结构脑方面的成功经验 在过去的几年里，Aim 1将确定更多的VOGM病例-父母三人组和 在我们不断增长的队列(已经是世界上最大的队列)上执行WES，以发现新奇和 皮损组织中传播性生殖系VOGM基因突变、马赛克变异和体细胞突变。在AIM 2，我们将利用生化、生物物理、 结构生物学和细胞生物学技术，通过尸检的VOGM组织和在 伴发皮肤血管畸形的VOGM患者的皮肤活检。成功完成 这些目标将增加我们对人类脑血管发育和VOGM的了解 病理生理学。这些进展将改善疾病管理和遗传咨询，并将 刺激VOGM靶向治疗的发展，这可能与其他血管广泛相关 病变，包括动静脉动静脉畸形和颅内动脉瘤。

PROJECT SUMMARY The genetic study of severe human congenital cerebrovascular anomalies can shed insight into mechanisms of normal vascular development and identify targets for therapeutic intervention. Vein of Galen aneurysmal malformations (VOGMs) are the most common and severe of pediatric brain arterio-venous malformations (AVMs). Significant gaps in our understanding of the molecular pathogenesis of VOGMs impede the development of improved diagnostic and therapeutic measures. Locus heterogeneity and the sporadic nature of VOGM cases have constituted fundamental obstacles to VOGM gene discovery. We recently applied whole exome sequencing (WES) to overcome these obstacles and identified de novo and inherited gene mutations that account for ~30% of sporadic VOGM cases (Duran et al., Neuron, 2019). These included a genome-wide significant burden of rare, damaging mutations in EPHB4 (EphB4), a critical regulator of arterio-venous specification also mutated in the familial AVM syndrome, capillary malformation (CM)-AVM type II (CM-AVM2). We also discovered new mutations in other genes that function in the same Ephrin signaling interactome, including RASA1 (also mutated in CM-AVM1). We further demonstrated that EphB4 exists in a physical complex with RASA1, and have now solved the first multi-domain crystal structure of RASA1. Nonetheless, most VOGM cases remain genetically unsolved, and the molecular mechanisms of VOGM-associated mutations are poorly understood. To address these knowledge gaps, we propose a functional genomics approach to discover and mechanistically elucidate VOGM-associated mutations with atomic-level resolution. We hypothesize WES will identify novel VOGM genes and mutations, including mosaic and somatic “second-hit” mutations, which disrupt the regulated activity of an EphB4-RASA1 signaling complex essential for arterio-venous development. Based on our successful experience in identifying structural brain disorder genes over the past several years, Aim 1 will ascertain additional VOGM case-parent trios and perform WES on our growing cohort (already the largest in the world) to discover novel de novo and transmitted germline VOGM gene mutations, mosaic variants, and somatic mutations in lesional tissue. In Aim 2, we will determine the structural and functional impact of VOGM mutations using biochemical, biophysical, structural biology and cell biology techniques, with validation experiments in autopsied VOGM tissue, and in skin biopsies of VOGM patients with associated cutaneous vascular malformations. Successful completion of these Aims will increase our understanding of human cerebrovascular development and VOGM pathophysiology. These advances will improve disease management and genetic counseling, and will stimulate development of targeted therapeutics for VOGMs that may be broadly relevant for other vascular lesions, including AVMs and intracranial aneurysms.