Somatic mutation detection in brain AVM by massively high-throughput sequencing

描述(申请人提供)：脑血管畸形影响300多万美国人，并在相当大比例的受影响个人中导致出血性中风和严重的神经残疾或死亡。脑动静脉畸形(AVM)是两种最常见的血管畸形之一，是一种复杂的血管病变，由错综复杂的血管组成，这些血管将血液从动脉分流到静脉循环，而没有真正的毛细血管床。脑动静脉畸形的人群流行率为每10万人中有10到18人，是年轻人出血性中风的重要原因。病变的局部性和常常是零星的和孤立的性质表明，有害的体细胞突变是脑AVM病因学的基础。对于发生在家族性综合征HHT的动静脉畸形，已经提出了两次打击假说，即在同一基因中发生杂合突变的第二次体细胞突变或“击中”，才能形成动静脉动静脉畸形病变。在散发性脑动静脉畸形中，第一次命中可能是新生的，第二次可能是躯体命中，或者可能有两次躯体命中。AVM病变是复杂和异质性的；体细胞突变可能只存在于一小部分病变细胞中。因此，直到最近，检测动静脉畸形的体细胞突变一直是困难的。最近大规模并行的下一代测序技术的出现为脑动静脉畸形和其他复杂病变的体细胞突变检测提供了一种新的、高灵敏度和高通量的方法。为了验证体细胞突变是脑AVM病因学的基础的假设，无论是遗传性的(HHT)还是散发性的AVM疾病，我们将实施大规模并行的下一代测序技术来检测从AVM病变组织中提取的DNA的体细胞突变。我们将利用这项技术对100例散发性脑AVM和6例HHT患者的AVM病变组织中至少40个候选基因的250 kb DNA进行重测序，其中包括3个在HHT中发生杂合性突变的基因。大量高通量测序用于血管病变的体细胞突变检测将是一个重要的进展，适用于各种血管和肿瘤病变。体细胞突变假说的证明将是理解脑AVM病因学机制的一个重要里程碑。从长远来看，我们将研究已发现的体细胞突变与疾病临床病程之间的关系，长期目标是弥合AVM临床管理中理解基本生物学机制和风险分层之间的分歧。 公共卫生相关性：脑动静脉畸形(AVM)是年轻人出血性中风的重要原因，导致相当大的公共卫生负担，包括高昂的医疗成本和生产力损失。脑动静脉畸形的病因目前知之甚少。我们将调查脑动静脉畸形，无论是散发性的还是发生在遗传性出血性毛细血管扩张症中的脑动静脉畸形，是否是由动静脉畸形病变细胞的体细胞突变引起的。这项工作将加强对动静脉动静脉畸形形成和出血的分子机制的了解，长期目标是改善临床管理和开发新的治疗方法，以减轻动静脉动静脉畸形对健康的负面负担。

DESCRIPTION (provided by applicant): Vascular malformations of the brain affect over 3 million Americans, and cause hemorrhagic stroke and serious neurological disability or death in a significant proportion of affected individuals. Brain arteriovenous malformations (AVMs), one of the two most common types of vascular malformation, are complex vascular lesions, comprised of a tangle of blood vessels that shunts blood from the arterial to the venous circulation without an intervening true capillary bed. Brain AVMs have a population prevalence of 10 to 18 per 100,000 adults, and are an important cause of hemorrhagic stroke in young adults. The localized and often sporadic and solitary nature of the lesion suggests the hypothesis that deleterious somatic mutations underlie brain AVM etiology. For AVMs occurring in the familial syndrome HHT, the two- hit hypothesis has been proposed, where a second, somatic mutation or "hit" in the same gene that is heterozygously mutated in the germline is required for the AVM lesion to form. In sporadic brain AVMs, the first, germline, hit may be de novo; followed by a second, somatic hit, or there may be two somatic hits. AVM lesions are complex and heterogeneous; somatic mutations may only be present in a fraction of lesion cells. Therefore, until recently, detection of somatic mutations in AVMs has been difficult. The recent advent of massively-parallel next generation sequencing technology now suggests a novel, highly sensitive and high- throughput approach to somatic mutation detection in brain AVMs and other complex lesions. To test the hypothesis that somatic mutations underlie brain AVM etiology, both in the inherited (HHT) and the sporadic form of the disease, we will implement massively-parallel next generation sequencing technology to detect somatic mutations in DNA extracted from AVM lesion tissue. We will use this technology to resequence 250 kilobases of DNA from at least 40 candidate genes, including the 3 genes heterozygously mutated in HHT, in AVM lesion tissue from 100 sporadic brain AVM patients and 6 HHT patients. Validation of massively high throughput sequencing for somatic mutation detection in vascular lesions will be an important advance applicable to a variety of vascular and neoplastic lesions. Demonstration of the somatic mutation hypothesis will represent a significant milestone in understanding the mechanisms of brain AVM etiology. In the long term, we will study the relationship between somatic mutations identified and the clinical course of the disease, with a long-term goal of bridging the divide between understanding basic biological mechanisms and risk stratification for AVM clinical management. PUBLIC HEALTH RELEVANCE: Brain arteriovenous malformations (AVMs) are an important cause of hemorrhagic stroke in young adults, resulting in a considerable public health burden encompassing high healthcare costs and lost productivity. The causes of brain AVMs are poorly understood. We will investigate whether brain AVMs, both sporadic and those occurring in the familial disease Hereditary Hemorrhagic Telangiectasia, are caused by somatic mutations in the cells of the AVM lesion. This work will enhance understanding of the molecular mechanisms of AVM formation and hemorrhage, with the long-term goal of improving clinical management and developing new treatment approaches to lessen the negative burden AVMs on health.