Exome Sequencing Defines the Molecular Pathogenesis of Vein of Galen Malformation

Abstract INTRODUCTION Vein of Galen malformations (VOGMs) are morbid arteriovenous malformations, with poorly described genetis. 1 Despite improvement in endovascular treatment, VOGM mortality remains high. 2 VOGM has been reported as a rare finding in Capillary Malformation-Arteriovenous Malformation Syndrome (RASA1; OMIM #605384) and Hereditary Hemorrhagic Telangiectasia (ENG, ACVRL1; OMIM #187300, #600376). 3-4 Our previous work has identified EPH receptor tyrosine kinase as also playing a role in VOGM pathogenesis. 5 Here, we report a larger cohort of probands and identify a new gene in the same pathway as EPHB4 as playing a role in VOGM. METHODS Germline DNA was isolated from 84 unrelated probands harboring radiographically confirmed VOGMs Both parents were available for 69/84 probands. Exome capture and paired-end WES was performed on DNA samples from participating individuals (n = 237). Data was bioinformatically analyzed to identify rare de-novo and transmitted mutations. Binomial analysis tested for exome-wide significance of mutational burden. RESULTS Only 3/75 patients harbored mutations in previously reported VOGM-associated genes (2.3%; RASA1 n = 2, 1.1%; ACVRL1 n = 1). Significant enrichment of rare damaging mutations was found for a member of the EPH receptor tyrosine kinase family (EPHB4, n = 5; 6.0%; P = 3.31 × 10–7, 36.62-fold enrichment). Entirely novel mutations in the integrin family were also identified (n = 2; 1.1%, P = 6.03 × 10–5, 179.6-fold enrichment). Both of these mutations are located in the c-terminal domain and disrupt a binding motif. Furthermore, this integrin protein mutation is involved in the same pathway as EPHB4, although whether or not they directly interact remains unknown. CONCLUSION This work represents an expansion upon the largest phenotyped, exome-sequenced VOGM cohort in the world. Having discovered a new gene in the same pathway as EPHB4 strongly implicates said pathway in VOMG development. We are currently pioneering tissue sampling from endovascular instruments used during treatment to explore potential somatic mutations. Our findings continue to uncover genetic determinants of VOGM pathogenesis, providing novel insight into vascular developmental biology.

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A39 NOVEL GAIN OF FUNCTION NON-RECEPTOR TYROSINE KINASE MUTATIONS ARE LINKED TO THE PATHOGENESIS OF VEOIBD

Journal of the Canadian Association of Gastroenterology ◽

10.1093/jcag/gwz047.038 ◽

2020 ◽

Vol 3 (Supplement_1) ◽

pp. 46-48

Author(s):

M Mehta ◽

L Wang ◽

C Guo ◽

N Warner ◽

Q Li ◽

...

Keyword(s):

Tyrosine Kinase ◽

Western Blot ◽

Receptor Tyrosine Kinase ◽

Blot Analysis ◽

Western Blot Analysis ◽

Intestinal Inflammation ◽

De Novo ◽

Single Gene ◽

Gain Of Function

Abstract Background Very early-onset inflammatory bowel disease (VEOIBD) is an emerging global disease, that results in inflammation of the digestive tract. Severe forms of VEOIBD can be caused by mutations in a single gene (monogenic variants) and, can result in death. A candidate gene which codes for a non-receptor tyrosine kinase (nRTK) has recently been implicated as a monogenic cause of IBD (unpublished). Whole exome sequencing was performed in two unrelated children who presented with symptoms of IBD identifying two distinct de novo gain of function mutations (S550Y and P342T). Both mutations are located in the highly conserved region of the nRTK, and were predicted to have similar downstream effects. Furthermore, four other patients with a variety of adult-onset immune disorders have recently been identified with rare variants in the same gene (M450I, R42P, A353T, V433M, S550F) but, their potential gain of function status remains to be determined. Studies show that this nRTK is an essential mediator in inflammation. It is expressed in both intestinal epithelial and immune cells however, its role in infantile IBD is unclear. This protein is first activated by phosphorylation and is linked to activating downstream transcription factors such as ERK and JNK. All these target proteins play a meaningful role in intestinal inflammation in patients with IBD. Aims Since we identified P342T and S550Y to be gain of function, we wanted to determine if the new variants exhibit a similar downstream impact on target protein expression levels when compared with S550Y and P342T. We also wanted to identify if all variants can be rescued with a known nRTK inhibitor. It is hypothesized that the new variants are gain of function and that all variants can be rescued with the inhibitor. Methods Using western blot analysis, the activation of ERK, JNK and nRTK was compared between wildtype (WT) and mutants. This in vitro method helped identify the degree of activation. For the second part of the study, HEK293T cells were treated with inhibitor to test for a rescue of phenotypes via western blot analysis. Results Results show an increased activation of nRTK, ERK and JNK in all variants with S550Y and S550F having the highest activation. Furthermore, pharmacological inhibition using small molecular kinase inhibitors resulted in decreased activation of nRTK, ERK and JNK suggesting a rescue of phenotypes. Conclusions Characterizing the downstream functional impact of these nRTK variants is an important first step to determine if gain of function nRTK mutations drive IBD. With a rising prevalence of IBD worldwide, these findings may lead to the development of pharmacological nRTK inhibitors as a novel personalized therapeutic approach for these patients and possibly for the broader IBD population. Funding Agencies CIHR

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A Semisynthetic Eph Receptor Tyrosine Kinase Provides Insight into Ligand- Induced Kinase Activation

Chemistry & Biology ◽

10.1016/j.chembiol.2011.01.011 ◽

2011 ◽

Vol 18 (3) ◽

pp. 361-371 ◽

Cited By ~ 19

Author(s):

Nikhil Singla ◽

Hediye Erdjument-Bromage ◽

Juha P. Himanen ◽

Tom W. Muir ◽

Dimitar B. Nikolov

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Eph Receptor ◽

Kinase Activation ◽

Insight Into

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Receptor tyrosine kinase signaling required for integrin alpha v beta 5-directed cell motility but not adhesion on vitronectin.

The Journal of Cell Biology ◽

10.1083/jcb.127.3.859 ◽

1994 ◽

Vol 127 (3) ◽

pp. 859-866 ◽

Cited By ~ 177

Author(s):

R L Klemke ◽

M Yebra ◽

E M Bayna ◽

D A Cheresh

Keyword(s):

Cell Migration ◽

Tyrosine Kinase ◽

Cell Motility ◽

Receptor Tyrosine Kinase ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

De Novo ◽

Growth Factor Receptor ◽

Dependent Manner ◽

Pkc Activation

FG human pancreatic carcinoma cells adhere to vitronectin using integrin alpha v beta 5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an alpha 2 beta 1-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo alpha v beta 5-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to alpha v beta 5-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGF. These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.

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Normal ontogenic observations on the expression of Eph receptor tyrosine kinase, Cek8, in chick embryos

Anatomy and Embryology ◽

10.1007/s004290050130 ◽

1998 ◽

Vol 197 (3) ◽

pp. 187-197 ◽

Cited By ~ 9

Author(s):

S. Hirano ◽

Hideaki Tanaka ◽

Kunimasa Ohta ◽

Masao Norita ◽

Kaeko Hoshino ◽

...

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Chick Embryos ◽

Eph Receptor

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EphB1 in Endothelial Cells Regulates Caveolae Formation and Endocytosis

10.1101/619510 ◽

2019 ◽

Author(s):

Chinnaswamy Tiruppathi ◽

Sushil C. Regmi ◽

Dong-Mei Wang ◽

Gary C.H. Mo ◽

Peter T. Toth ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Binding Motif ◽

Caveolin 1 ◽

Lung Vessels ◽

Native Ligand

AbstractCaveolae, the cave-like structures abundant in endothelial cells (ECs), are important in regulating key functions such as caveolae-mediated endocytosis and generation of nitric oxide. Here we show that deletion of the receptor tyrosine kinase EphB1 (EphB1−/−) in mice markedly reduced the caveolae number in ECs of heart and lung vessels and prevented caveolae-mediated endocytosis. EphB1 expressed in adult ECs was shown to bind the caveolin-1 (Cav-1) scaffold domain (CSD) via the CSD binding motif (CSDBM) on EphB1. We demonstrated that activation of EphB1 by the native ligand Ephrin B1 uncoupled EphB1 from Cav-1, and licensed Src-dependent Y-14 Cav-1 phosphorylation. Deletion of CSDBM on EphB1 prevented EphB1/Cav-1 interaction and the activation of Src and Src mediated Y-14 Cav-1 phosphorylation. These studies identify the central role of endothelium expressed EphB1 in regulating caveolae biogenesis and caveolae-mediated endocytosis.

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