结论

在所分析的大多数大脑动静脉畸形的组织样品中，我们检测到了KRAS激活突变，提示这些畸形发展是KRAS诱导的脑内皮细胞中MAPK-ERK信号传导通路激活的结果。

Background

Sporadic arteriovenous malformations of the brain, which are morphologically abnormal connections between arteries and veins in the brain vasculature, are a leading cause of hemorrhagic stroke in young adults and children. The genetic cause of this rare focal disorder is unknown.

Methods

We analyzed tissue and blood samples from patients with arteriovenous malformations of the brain to detect somatic mutations. We performed exome DNA sequencing of tissue samples of arteriovenous malformations of the brain from 26 patients in the main study group and of paired blood samples from 17 of those patients. To confirm our findings, we performed droplet digital polymerase-chain-reaction (PCR) analysis of tissue samples from 39 patients in the main study group (21 with matching blood samples) and from 33 patients in an independent validation group. We interrogated the downstream signaling pathways, changes in gene expression, and cellular phenotype that were induced by activating KRAS mutations, which we had discovered in tissue samples.

Results

We detected somatic activating KRAS mutations in tissue samples from 45 of the 72 patients and in none of the 21 paired blood samples. In endothelial cell–enriched cultures derived from arteriovenous malformations of the brain, we detected KRAS mutations and observed that expression of mutant KRAS (KRAS^G12V) in endothelial cells in vitro induced increased ERK (extracellular signal-regulated kinase) activity, increased expression of genes related to angiogenesis and Notch signaling, and enhanced migratory behavior. These processes were reversed by inhibition of MAPK (mitogen-activated protein kinase)–ERK signaling.

Conclusions

We identified activating KRAS mutations in the majority of tissue samples of arteriovenous malformations of the brain that we analyzed. We propose that these malformations develop as a result of KRAS-induced activation of the MAPK–ERK signaling pathway in brain endothelial cells. (Funded by the Swiss Cancer League and others.)

Supported by research grants to Dr. Nikolaev from the Swiss Cancer League (LSCC 2939-02-2012 and KSF-3985-08-2016), Dinu Lipatti (2014), and Novartis (14B065). Ms. Khyzha was supported by a Canada Graduate Scholarship from the Natural Sciences and Research Council of Canada. Dr. DiStefano was supported by a Postdoctoral Fellowship from the Toronto General Hospital Research Institute. Dr. Suutarinen was supported by a research grant from the Petri Honkanen Foundation. Dr. Herman was supported by a grant from the National Institutes of Health (2T32HL007676). Dr. Wythe was supported by an American Heart Association Grant-in-Aid (16GRNT31330023). Dr. Antonarakis was supported by a grant from the European Research Council. Dr. Frösen was supported by research grants from the Finnish Medical Foundation and Kuopio University Hospital. Dr. Fish was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) (MOP-119506) and a Team Project Award from the University of Toronto Medicine by Design initiative, which receives funding from the Canada First Research Excellence Fund and a Canada Foundation for Innovation equipment grant; he also received an Early Researcher Award from the Ontario Ministry of Research and Innovation and funding from the Canada Research Chair Program from the CIHR. Dr. Radovanovic was supported by the Timothy P. Susco Chair of Research Award from the Brain Aneurysm Foundation, the Toronto General and Western Hospital Foundation, and received seed support from the Department of Surgery and Division of Neurosurgery at the University Health Network.

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

Drs. Nikolaev, Vetiska, Frösen, Fish, and Radovanovic contributed equally to this article.

This article was published on January 3, 2018, at NEJM.org.

We thank the staff of the Princess Margaret Genomics Centre and Bioinformatics Services (C. Virtanen and Z. Lu) for generating the RNA and DNA sequencing data used in this study; the staff of the Centre for Applied Genomics at the Toronto Hospital for Sick Children (T. Paton) for performing droplet digital polymerase-chain-reaction analyses; Zhiqi Chen from University Health Network for help with cell biology assays; and Melanie Peralta from the Pathology Research Program, University Health Network, Toronto, for technical help with immunohistochemical analyses.

Source Information

From the Department of Genetic Medicine and Development, University of Geneva Medical School (S.I.N., X.B., S.E.A.), Service of Genetic Medicine, University Hospitals of Geneva (S.I.N., S.E.A.), and iGE3, Institute of Genetics and Genomics of Geneva (S.E.A.) — all in Geneva; the Department of Fundamental Neurobiology, Krembil Research Institute (S.V., M.T., I.R.), Toronto General Hospital Research Institute (E.B., N.K., P.V.D., J.E.F.), the Department of Pathology (T.-R.K.), the Division of Neurosurgery, Department of Surgery (V.M.P., T.K., H.A.-B., T.T., T.V., G.Z., M.T., I.R.), and the Joint Division of Medical Imaging, Department of Medical Imaging (V.M.P., T.K.), Toronto Western Hospital, University Health Network, the Department of Laboratory Medicine and Pathobiology, University of Toronto (E.B., N.K., P.V.D., T.-R.K., J.E.F.), and the Heart and Stroke Richard Lewar Centre of Excellence in Cardiovascular Research (E.B., N.K., P.V.D., J.E.F.) — all in Toronto; the Department of Molecular Medicine, AIV Institute, University of Eastern Finland (S.J., B.R.J., S.S., S.Y.-H., J.F.), and the Hemorrhagic Brain Pathology Research Group, Department of Neurosurgery and NeuroCenter (S.J., B.R.J., S.S., T.R., J.F.), and the Department of Pathology (T.R.), Kuopio University Hospital — all in Kuopio, Finland; and the Cardiovascular Research Institute and the Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston (A.M.H., J.D.W.).

Address reprint requests to Dr. Nikolaev at sergey.nikolaev@unige.ch, to Dr. Frösen at juhana.frosen@kuh.fi, to Dr. Fish at jason.fish@utoronto.ca, or to Dr. Radovanovic at ivan.radovanovic@uhn.ca.

中文翻译：

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大脑动静脉畸形的体细胞激活KRAS突变 脑动静脉畸形中的体细胞KRAS激活突变

摘要

在所分析的大多数大脑动静脉畸形的组织样品中，我们检测到了KRAS激活突变，提示这些畸形发展是KRAS诱导的脑脊髓细胞中MAPK-ERK信号传递传递激活的结果。

背景

大脑偶发性动静脉畸形是大脑血管系统中的动脉与静脉之间的形态异常连接，是年轻人和儿童出血性中风的主要原因。这种罕见的局灶性疾病的遗传原因尚不清楚。

方法

我们分析了来自脑动静脉畸形患者的组织和血液样本，以检测体细胞突变。我们对主要研究组中26位患者的脑动静脉畸形组织样本和其中17位患者的配对血液样本进行了外显子组DNA测序。为了证实我们的发现，我们对主要研究组的39名患者（21名血液样本相匹配）和独立验证组的33名患者的组织样品进行了液滴数字聚合酶链反应（PCR）分析。我们询问了激活KRAS突变诱导的下游信号传导途径，基因表达的变化和细胞表型，我们已经在组织样本中发现了这种突变。

结果

我们在72位患者中的45位患者的组织样品中检测到了体细胞激活性KRAS突变，而在21对配对的血液样品中均未检测到。在源自脑动静脉畸形的富含内皮细胞的培养物中，我们检测到KRAS突变，并观察到体外内皮细胞中突变KRAS（KRAS ^G12V）的表达可诱导ERK（细胞外信号调节激酶）活性增加，基因表达增加与血管生成和Notch信号传导有关，并增强了迁徙行为。通过抑制MAPK（促分裂原激活的蛋白激酶）-ERK信号可以逆转这些过程。

结论

我们在我们分析过的大脑动静脉畸形的大多数组织样本中确定了激活的KRAS突变。我们认为这些畸形是由于KRAS诱导的脑内皮细胞MAPK-ERK信号通路活化而发展的。（由瑞士癌症联盟和其他组织资助。）

由瑞士癌症联盟（LSCC 2939-02-2012和KSF-3985-08-2016），Dinu Lipatti（2014）和诺华（14B065）的尼古拉耶夫博士提供研究资助。Khyzha女士得到了加拿大自然科学与研究委员会的加拿大研究生奖学金的支持。DiStefano博士得到了多伦多综合医院研究所的博士后奖学金的支持。Suutarinen博士得到了Petri Honkanen基金会的研究资助。Herman博士得到了美国国立卫生研究院（2T32HL007676）的资助。Wythe博士得到了美国心脏协会的资助（16GRNT31330023）的支持。Antonarakis博士得到了欧洲研究理事会的资助。Frösen博士得到了芬兰医学基金会和库奥皮奥大学医院的研究资助。博士 Fish得到了加拿大卫生研究院（CIHR）（MOP-119506）的运营赠款和多伦多大学设计医学计划的团队项目奖的支持，该计划获得了加拿大第一研究卓越基金和加拿大的资助创新设备赠款基金会；他还获得了安大略省研究与创新部的早期研究员奖，并获得了CIHR加拿大研究主席计划的资助。Radovanovic博士得到了脑动脉瘤基金会，多伦多总医院和西部医院基金会的蒂莫西·苏斯科研究奖主席的支持，并获得了大学卫生网络外科和神经外科分部的种子支持。

作者提供的披露表可在NEJM.org上获得本文的全文。

博士 尼古拉耶夫（Nikolaev），维蒂斯卡（Vetiska），弗洛森（Frösen），菲什（Fish）和拉多瓦诺维奇（Radovanovic）对本文的贡献均相同。

本文于2018年1月3日在NEJM.org上发布。

我们感谢玛格丽特公主基因组学中心和生物信息学服务中心（C. Virtanen和Z. Lu）的工作人员提供了本研究中使用的RNA和DNA测序数据。多伦多病童医院（T. Paton）应用基因组学中心的工作人员进行液滴数字聚合酶链反应分析；来自大学健康网的Chenzhiqi Chen帮助进行细胞生物学测定；多伦多大学健康网病理研究计划的Melanie Peralta提供了免疫组织化学分析的技术帮助。

源信息

来自日内瓦大学医学院（SIN，XB，SEA）遗传医学与发展系，日内瓦大学医院（SIN，SEA）的遗传医学服务，以及日内瓦遗传与基因组学研究所（SEA）的iGE3 -全部在日内瓦；克林比尔研究所（SV，MT，IR），多伦多综合医院研究所（EB，NK，PVD，JEF）基础神经生物学系，病理学系（T.-RK），神经外科多伦多西部医院医学影像学系（VMP，TK，HA-B。，TT，TV，GZ，MT，IR）和医学影像学联合科（VMP，TK），大学卫生网络，医学系多伦多大学检验医学与病理生物学系（EB，NK，PVD，T.-RK，JEF），以及心脏和中风理查德·勒瓦尔心血管研究卓越中心（EB，NK，PVD，JEF）–全部位于多伦多；东欧芬兰大学AIV研究所分子医学系（SJ，BRJ，SS，SY-H。，JF）和出血性脑病理研究组，神经外科和神经中心系（SJ，BRJ，SS，TR， JF）和Kuopio大学医院病理科（TR）-全部位于芬兰的Kuopio；休斯敦贝勒医学院的心血管研究所和分子生理学与生物物理系（AMH，JDW）。-H。，JF）和出血性脑病理学研究组，神经外科和NeuroCenter系（SJ，BRJ，SS，TR，JF）和Kuopio大学医院病理系（TR）—全部位于芬兰的Kuopio ; 休斯敦贝勒医学院的心血管研究所以及分子生理学和生物物理系（AMH，JDW）。-H。，JF）和出血性脑病理学研究组，神经外科和NeuroCenter系（SJ，BRJ，SS，TR，JF）和Kuopio大学医院病理系（TR）—全部位于芬兰的Kuopio ; 休斯敦贝勒医学院的心血管研究所以及分子生理学和生物物理系（AMH，JDW）。

将重印请求发送至sergey.nikolaev@unige.ch，发送至Nikolaev博士，发送至juhana.frosen@kuh.fi，发送至Frösen博士，发送至jason.fish@utoronto.ca的Fish博士，或发送至ivan的Radovanovic博士。 radovanovic@uhn.ca。