Loss-of-Function Mutations in

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Loss-of-Function Mutations in ELMO2 Cause Intraosseous Vascular Malformation by Impeding RAC1 Signaling

Arda Cetinkaya,1,15 Jingwei Rachel Xiong,2,15 İbrahim Vargel,3 Kemal Kösemehmetoğlu,4 Halil İbrahim Canter,5 Ömer Faruk Gerdan,6 Nicola Longo,7 Ahmad Alzahrani,8 Mireia Perez Camps,2 Ekim Zihni Taşkıran,1 Simone Laupheimer,2 Lorenzo D Botto,7 Eeswari Paramalingam,2 Zeliha Gormez,6 Elif Uz,1,9 Bayram Yuksel,10 Şevket Ruacan,11 Mahmut Şamil Sağıroğlu,6 Tokiharu Takahashi,12 Bruno Reversade,2,13* Nurten Ayse Akarsu1,14**

1Department of Medical Genetics, Gene Mapping Laboratory, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey

2Institute of Medical Biology, Human Genetics and Embryology Laboratory, A*STAR, Singapore 138648, Singapore

3Department of Plastic and Reconstructive Surgery, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey

4Department of Pathology, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey

5Department of Plastic and Reconstructive Surgery, Faculty of Medicine, Acıbadem University, Istanbul 34457, Turkey

6Advanced Genomics and Bioinformatics Research Center (IGBAM), BILGEM, TUBITAK, Kocaeli 41400, Turkey

7Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, UT 84108, USA

8Maternity Children Hospital, Makkah, 24246, The Kingdom of Saudi Arabia

9Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Uludag University, Bursa 16059, Turkey

10Genetic Engineering and Biotechnology Institute, TUBITAK Marmara Research Center, Kocaeli 41400, Turkey

11Department of Pathology, Faculty of Medicine, Koç University, Istanbul34450, Turkey

12Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester M13 9PT, UK

13Institute of Molecular and Cell Biology, ASTAR, Singapore 138673, Singapore

14Center for Biobanking and Genomics, Hacettepe University, Ankara 06100, Turkey

15Equally contributed
** Nurten A. Akarsu, MD, PhD

Professor of Medical Genetic

Hacettepe University Medical Faculty

Department of Medical Genetics

Sihhiye, 06100

Ankara, Turkey

Phone: +90-312-305 2559

Fax: +90-312-426 8592

E-mail: nakarsu@hacettepe.edu.tr;


* Bruno Reversade, PhD

Institute of Medical Biology, A *STAR

Laboratory of Human Embryology and Genetics

8A Biomedical Grove, Singapore 138648

E-mail: bruno@reversade.com


Vascular malformations are non-neoplastic expansions of blood vessels that arise due to errors during angiogenesis. They are a heterogeneous group of sporadic or inherited vascular disorders characterized by localized lesions of arteriovenous, capillary, or lymphatic origin. Vascular malformations that occur inside bone tissue are rare. Herein, we report loss-of-function mutations in ELMO2, which translates extracellular signals into cellular movements, that are causative for autosomal recessive intraosseous vascular malformation (VMOS) in 5 different families. Individuals with VMOS suffer from life-threatening progressive expansion of the jaw, craniofacial and other intramembranous bones caused by malformed blood vessels that lack a mature vascular smooth muscle layer. Analysis of primary fibroblasts from an affected individual showed that absence of ELMO2 correlated with a significant down regulation of binding partner DOCK1, resulting in deficient RAC1-dependent cell migration. Unexpectedly, elmo2-knockout zebrafish appeared phenotypically norm al, suggesting there might be human-specific ELMO2 requirements in bone vasculature homeostasis or genetic compensation by related genes. Comparative phylogenetic analysis indicated that elmo2
originated upon the appearance of intramembranous bones and the jaw in ancestral vertebrates, implying that elmo2 might have been involved in the evolution of these novel traits. The present findings highlight the necessity of ELMO2 for maintaining vascular integrity, specifically in intramembranous bones.


Embryonic development of blood vessels involves two sequential processes: vasculogenesis and angiogenesis. Vasculogenesis is the de novo vascular growth from mesoderm-derived angioblasts, and subsequent formation of the primary capillary plexus in vascular islets.1 Angiogenesis refers to the subsequent process whereby the primary capillary plexus undergoes remodeling and extension via endothelial cell (EC) proliferation and vascular smooth muscle cell (vSMC) recruitment to form mature blood vessels.2 Errors in molecular control of angiogenesis cause vascular anomalies—localized lesions of arteriovenous, capillary, or lymphatic origin. The International Society for the Study of Vascular Anomalies (ISSVA) classifies vascular anomalies into 2 major categories:—tumors and malformations, according to their clinical behavior and endothelial properties.3-5 Vascular tumors are characterized by actively proliferating neoplastic ECs, whereas vascular malformations are non-neoplastic abnormal expansions of vascular tissue, without prominent endothelial proliferation.3 The most common vascular tumor—infantile hemangioma—is a rapidly growing benign tumor that regresses during the first decade of life. In contrast, vascular malformations never regress and grow proportionally with age.

Both sporadic and inherited forms of vascular malformations have been described.5 Hereditary vascular malformations are caused by dominant mutations in several genes, with some indication of the contribution of a second-hit, whose protein products regulate EC-vSMC communication, as well as recruitment and migration of vSMCs to the vascular bed.6-8 Mutations in TEK [MIM: 600221] cause multiple cutaneous and mucosal venous malformations [MIM: 600195], mutations in GLML [MIM: 601749] cause glomuvenous malformation [MIM: 138000], mutations in RASA1 [MIM: 139150] cause capillary malformation-arteriovenous malformation [MIM: 608354] and Parkes-Weber Syndrome [MIM: 608355], mutations in ENG [MIM: 131195] and ACVRL1 [MIM: 601284] cause hereditary hemorrhagic telangiectasia type 1 [MIM: 187300], and type 2 [MIM: 600376], respectively, and mutations in KRIT1 [MIM: 604214], CCM2 [MIM: 607929], and PDCD10 [MIM: 609118] cause cerebral cavernous malformations type 1 [MIM: 116860], type 2 [MIM: 603284], and type 3 [MIM: 603285], respectively.

Intraosseous vascular malformations are rare abnormalities that account for <0.2% of all bony tumors.9 They are almost exclusively described in sporadic cases involving the skull and vertebral column; however, we identified the first 2 autosomal recessive families with primary intraosseous vascular malformation, VMOS [MIM: 606893], which severely affected cranial bones.10,11 The malformation is characterized by severe and progressive blood vessel expansion within the craniofacial bones, variably accompanied by midline abnormalities such as diastasis recti and supraumbilical raphe.10 Prior to the onset of puberty, the vascular malformation and bone enlargement is restricted to the mandibular and maxillary region; thereafter, rapid expansion occurs with extension to all cranial bones. Clinically, the gradual increase in intracranial pressure or massive bleeding—either spontaneous or induced by surgery—can be life-threatening. Treatment is extremely challenging, as embolization, sclerotherapy, or surgical manipulations are only effective for slowing disease progression. The facial appearance of individuals with VMOS and their CT images resemble cherubism, as mandibular bone is replaced by excessive amounts of fibrous tissue.10,12,13 Pathological findings are critical for differential diagnosis, because there are no pathognomonic radiographic findings for VMOSs.11,13,14

Various terms have been used to describe malformations similar to VMOSs, including intraosseous cavernous hemangioma, extraspinal osseous hemangioma, central hemangioma, cavernous angiomata of skull, and cystic angiomatosis.12,14-16 Recently, the nomenclature of vascular bone lesions was updated and earlier cases were re-classified according to the new ISSVA classification.4,5,17 With this reclassification the diverse and sometimes contradictory terminology used for intraosseous vascular lesions was largely standardized; however, as no mode of inheritance for the aforementioned malformations were addressed, it remains unclear if VMOS and similar conditions reported in the literature have a common genetic etiology. 18

The present study used homozygosity mapping and massively parallel sequencing in combination to identify 4 distinct germline mutations in ELMO2 [MIM: 606421], which encodes for Engulfment and cell motility protein 2 (ELMO2), in 5 consanguineous families affected by VMOS. Functional testing using affected cells showed that there was a significant reduction in ELMO2 transcript and loss of ELMO2 with concomitant downregulation of DOCK1. Overexpressed ELMO2 mutant proteins derived from an affected individual’s mutant transcripts were unable to stably recruit interacting protein DOCK1 or fully enhance downstream RAC1 activation. The fibroblasts from an affected individual showed a deficiency in cell migration that could be partially rescued by exogenous ELMO2. Attempts to model VMOS in zebrafish by generating CRISPR/Cas9-elmo2 null alleles showed that unlike in humans, elmo2 may not be essential in zebrafish for proper vasculature development and homeostasis.

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