Sergey I Nikolaev1, Sandra Vetiska1, Ximena Bonilla1, Emilie Boudreau1, Suvi Jauhiainen1, Behnam Rezai Jahromi1, Nadiya Khyzha1, Peter V DiStefano1, Santeri Suutarinen1, Tim-Rasmus Kiehl1, Vitor Mendes Pereira1, Alexander M Herman1, Timo Krings1, Hugo Andrade-Barazarte1, Takyee Tung1, Taufik Valiante1, Gelareh Zadeh1, Mike Tymianski1, Tuomas Rauramaa1, Seppo Ylä-Herttuala1, Joshua D Wythe1, Stylianos E Antonarakis1, Juhana Frösen1, Jason E Fish1, Ivan Radovanovic1. 1. 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.).
Abstract
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 (KRASG12V) 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.).
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 (KRASG12V) 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.).
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