Genesis and Progression of Cerebral Cavernous Malformations

描述（由申请人提供）：超过100万美国人患有脑海绵状畸形（CCMs），由增生扩张的出血性毛细血管簇组成，使患者易患中风、癫痫发作和其他神经系统后遗症。CCMs可能是偶发的，但发生CCMs的风险也可能通过最近发现的三种基因中的一种突变而遗传。散发性病例中存在孤立病变，常染色体显性病例中存在多个随机分散病变，这使我们假设CCM病变发生遵循Knudson双靶机制，需要体细胞丢失野生型等位基因，导致内皮细胞增殖和CCM生长。我们可以通过分析人类CCM病变组织中各自CCM基因的双等位基因突变来验证我们的假设。不幸的是，在人类病变发展的早期阶段，对体细胞突变和增殖活动的评估受到组织可用性的强烈选择偏差的阻碍。较小的ccm，特别是在病变发生的早期阶段，很少被手术切除。因此，我们无法确定在人类病变中发现的双等位基因突变是病变生长的原因还是仅仅是病变生长的结果。为了验证病变发生的双重打击假说，我们创建了具有Ccm1或Ccm2靶向突变的小鼠模型，其中杂合子动物仅在肿瘤抑制基因Trp53 （p53）基因的遗传背景中发生CCM病变。我们同时正在开发第二代模型，其中Ccm基因类似地交叉到错配修复缺陷的遗传背景中，以便将体细胞突变偏向于易于通过DNA测序识别的简单点突变。我们同时开发了高分辨率磁共振（MR）成像和立体定向定位技术，可以检测和表征小鼠病变，并在病变的早期阶段进行细胞解剖。利用这一新颖的平台和一组储存的人类切除病变，我们提出了三个具体的目标，研究以下假设：(1)适当Ccm基因的野生型拷贝的体细胞突变发生在病变发生的早期（扩张的毛细血管）阶段，并在靶向突变的小鼠晚期（多海绵状）Ccm病变的海绵状血管中克隆扩增；(2)病变随着时间的推移而发展和生长，早期和晚期小鼠CCM病变中的内皮细胞均表现出增殖活性和血脑屏障的改变；(3)切除的人类CCM病变的体细胞突变验证了人类病变发生的双重打击机制。这项工作将阐明CCM发生的分子机制，并有助于我们寻找预防和治疗CCM的新方法。公共卫生相关性：本研究与公共卫生的相关性在于，在本研究中，我们将调查CCM病变随时间发展和生长的假设，以及它们与典型的双打击肿瘤抑制机制相关，导致血管组织增生，血脑屏障有缺陷，导致随后的出血和相关的神经系统后遗症。这些问题的答案将允许对这些临床重要病变进行适当的分类，并导致未来的研究沿着适当的道路发展以生物为基础的治疗。

DESCRIPTION (provided by applicant): More than one million Americans harbor cerebral cavernous malformations (CCMs), consisting of clusters of proliferating dilated hemorrhagic capillaries, predisposing patients to a lifetime risk of stroke, seizures, and other neurologic sequelae. CCMs can develop sporadically, but the risk to develop CCMs can also be inherited by mutation in one of three recently identified genes. The presence of solitary lesions in sporadic cases and multiple, randomly-scattered lesions in autosomal dominant cases have led us to hypothesize that CCM lesion genesis follows a Knudson two-hit mechanism requiring somatic loss of the wild-type allele, leading to endothelial cell proliferation and CCM growth. We can test our hypothesis by analyzing human CCM lesion tissue for the presence of bi-allelic mutations in the respective CCM gene. Unfortunately, the assessment of somatic mutations and proliferative activity at earliest stages of lesion development in humans is hindered by a strong selection bias in tissue availability. Smaller CCMs, especially at earliest phases of lesion genesis, are rarely if ever surgically resected. Thus, we cannot determine whether bi-allelic mutations identified in the human lesions are the cause or merely an effect of lesion growth. In order to test our two-hit hypothesis for lesion genesis, we have created mouse models with targeted mutations of either Ccm1 or Ccm2, where heterozygote animals develop CCM lesions only in a genetic background null for the tumor suppressor Trp53 (p53) gene. We are concurrently developing second generation models, where the Ccm genes are similarly crossed into a genetic background deficient in mismatch repair, in order to bias somatic mutations towards simple point mutations easily identified by DNA sequencing. We have in parallel developed high resolution magnetic resonance (MR) imaging and stereotactic localization techniques which enable the detection and characterization of murine lesions, and cellular dissection from lesions at earliest stages of development. Using this novel platform and a collection of banked excised human lesions, we propose three specific aims investigating the following hypotheses: (1) that somatic mutation in the wild type copy of the appropriate Ccm gene occurs at early (dilated capillary) stage of lesion genesis, and is expanded clonally in cavernous vessels of later stage (multi-cavernous) CCM lesions in mice with targeted mutagenesis; (2) that lesions develop and grow over time, and endothelial cells in both early and late stage murine CCM lesions exhibit proliferative activity and alterations of the blood-brain-barrier; and (3) that somatic mutations in resected human CCM lesions validate the two-hit mechanisms of lesion genesis in man. This work will shed light on the molecular mechanism of CCM genesis, and aid in our long term goal of finding new approaches to their prevention and treatment. PUBLIC HEALTH RELEVANCE: The relevance of this research to public health is that in this study we will investigate hypotheses that CCM lesions develop and grow over time, and that they are associated with a classic two-hit tumor suppressor mechanism leading to proliferation of vascular tissue with a defective blood brain barrier, leading to subsequent hemorrhage and associated neurological sequelae. Answers to these questions will allow the proper classification of these clinically important lesions and lead to future investigation down the appropriate path for development of biologically-based therapy.