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（CCM），由增殖扩张的出血性毛细血管簇组成，使患者终生面临中风、癫痫发作和其他神经系统后遗症的风险。CCM可以零星发生，但发生CCM的风险也可以通过最近发现的三种基因之一的突变遗传。在散发病例中存在孤立性病变，在常染色体显性遗传病例中存在多个随机分散的病变，这使我们假设CCM病变发生遵循Knudson两次打击机制，需要野生型等位基因的体细胞丢失，导致内皮细胞增殖和CCM生长。我们可以通过分析人CCM病变组织中相应CCM基因中双等位基因突变的存在来测试我们的假设。不幸的是，在人类病变发展的最早阶段评估体细胞突变和增殖活性受到组织可用性的强烈选择偏倚的阻碍。较小的CCM，特别是在病变发生的早期阶段，很少手术切除。因此，我们不能确定在人类病变中鉴定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影响。为了测试我们的两次打击假说的病变发生，我们已经创建了小鼠模型与靶向突变的Ccm 1或Ccm 2，杂合子动物发展CCM病变只有在一个遗传背景空的肿瘤抑制基因Trp 53（p53）基因。我们正在同时开发第二代模型，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）成像和立体定位技术，使检测和表征小鼠病变，并从病变的细胞解剖在发展的最早阶段。使用这个新的平台和一组库存的切除的人类病变，我们提出了三个具体的目标，调查以下假设：（1）适当的Ccm基因的野生型拷贝中的体细胞突变发生在早期，（扩张的毛细血管）阶段，并在后期的海绵状血管中克隆性扩张（2）病变随时间发展和生长，早期和晚期小鼠CCM病变中的内皮细胞表现出增殖活性和血脑屏障的改变;（3）人CCM切除病灶中的体细胞突变证实了CCM发生的两次打击机制，这将有助于阐明CCM发生的分子机制，并有助于我们寻找防治CCM的新方法。公共卫生相关性：这项研究与公共卫生的相关性在于，在这项研究中，我们将调查CCM病变随时间发展和生长的假设，并且它们与经典的两次打击肿瘤抑制机制相关，导致血管组织增殖，血脑屏障缺陷，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这些临床上重要的病变的正确分类，并导致未来的调查，为发展生物为基础的治疗的适当路径。

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.