Alternative titles; symbols
HGNC Approved Gene Symbol: EPHB4
Cytogenetic location: 7q22.1 Genomic coordinates (GRCh38): 7:100,802,565-100,827,523 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 7q22.1 | Capillary malformation-arteriovenous malformation 2 | 618196 | Autosomal dominant | 3 |
| Lymphatic malformation 7 | 617300 | Autosomal dominant | 3 |
In CD34+ human bone marrow cells and a human hepatocellular carcinoma cell line, Bennett et al. (1994) identified a novel transmembrane tyrosine kinase, which they called hepatoma transmembrane kinase, or HTK. They reported that the predicted 987-amino acid sequence of HTK includes a transmembrane region and signal sequence. The predicted extracellular domain contains a cysteine-rich region and tandem fibronectin type III repeats, while the intracellular domain contains the catalytic domain. Northern blot analysis demonstrated a single HTK transcript abundantly expressed in placenta and in a range of primary tissues and malignant cell lines. It is expressed in fetal, but not adult, brain, and in primitive and myeloid, but not lymphoid, hematopoietic cells. The protein shared amino acid similarity with the Eph subfamily of tyrosine kinases.
Martin-Almedina et al. (2016) found expression of Ephb4 in embryonic venous and lymphatic endothelial cells in the skin and mesenteries of mice.
See 179610 for information on the Eph receptor gene family.
Using 2 independent sets of primers specific for human HTK to amplify DNA from a panel of human-hamster hybrid cell lines, Bennett et al. (1994) demonstrated that the human EPHB4 gene is located on chromosome 7.
By sequencing genomic clones and database analysis, Wilson et al. (2001) mapped the EPHB4 gene to chromosome 7q22. They mapped the mouse Ephb4 gene to a region of conserved synteny on mouse chromosome 5.
Hartz (2017) mapped the EPHB4 gene to chromosome 7q22.1 based on an alignment of the EPHB4 sequence (GenBank AY056048) with the genomic sequence (GRCh38).
Berclaz et al. (1996) examined the expression of HTK in normal and malignant breast tissue. They found that in normal breast, expression is confined to secretory luminal epithelial cells. They found elevated expression of HTK in several human breast carcinoma cell lines as well as in primary ductal carcinomas of the breast. The authors suggested that HTK may have a role in the differentiation or maintenance of secretory epithelia.
Using mouse and human erythroleukemia cell lines, Sakamoto et al. (2004) showed that interaction of EPHB4 with ephrin B2 (EFNB2; 600527) did not require EPHB4 kinase activity. Activation of EPHB4 via ligation to ephrin B2 resulted in formation of filopodia, phosphorylation of the signal transduction protein CBL (165360), and recruitment of CRKL (602007) to CBL.
EPHB4 and ephrin B2 show bidirectional signaling, whereby activation of EPHB4 results in forward signaling in the receptor-expressing cell, and activation of ephrin B2 results in reverse signaling in the ligand-expressing cell. Erber et al. (2006) found that overexpression of wildtype or kinase-dead EPHB4 in human SF126 glioblastoma cells resulted in phosphorylation of endogenous ephrin B2 and reverse signaling. SF126 xenograft tumors expressed ectopic EPHB4 and endogenous ephrin B2 in the same blood vessels, and reverse signaling resulted in abnormally large and dense vessels with reduced permeability. The apparent switch from sprouting angiogenesis to circumferential vessel growth and tightening of the permeability barrier was accompanied by elevated expression of Ang1 (ANGPT1; 601667) relative to Ang2 (ANGPT2; 601922) and elevated activation of the Ang1 receptor Tie2 (TEK; 600221). A similar phenomenon was detected in retinas of newborn mice overexpressing Ephb4. Erber et al. (2006) concluded that activation of ephrin B2 by reverse signaling in turn activates the ANG1/TIE2 axis between endothelial cells and perivascular mural cells and pericytes.
Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from preexisting blood vessels (angiogenesis). Using high-resolution imaging of zebrafish vascular development, Herbert et al. (2009) uncovered a third mode of blood vessel formation whereby the first embryonic artery and vein, 2 unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. Herbert et al. (2009) found that these processes were regulated by the ligand ephrin B2 and its receptor Ephb4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, Herbert et al. (2009) concluded that directional control of progenitor migration drives arterial-venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.
Lymphatic Malformation 7
In 11 members of 2 unrelated families with nonimmune hydrops fetalis and postnatal lymphatic dysfunction (LMPHM7; 617300), Martin-Almedina et al. (2016) identified different heterozygous missense mutations in the EPHB4 gene (600011.0001 and 600011.0002). The mutations, which were found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the families, although there was variable expressivity of the phenotype. Both mutations occurred at highly conserved residues in the tyrosine kinase domain, and in vitro functional expression studies in HEK293 cells and lymphatic endothelial cells showed that the mutant proteins were devoid of tyrosine kinase activity, suggesting altered EPHB4 signaling. The findings indicated that the mutations had a negative effect on receptor activity after ligand stimulation. There was no evidence of a dominant-negative effect.
In a large 4-generation family with hydrops fetalis, lymphedema, and central conduction lymphatic anomalies, Li et al. (2018) performed whole-exome sequencing and identified heterozygosity for a splice site mutation in the EPHB4 gene (600011.0013) that segregated fully with disease in the family and was not found in in-house control samples or in public variant databases.
Capillary Malformation-Arteriovenous Malformation 2
In an 8-year-old white boy with multiple capillary malformations (CMAVM2; 618196), Yu et al. (2017) identified a de novo heterozygous missense mutation in the EPHB4 gene (D802G; 600011.0003).
From a cohort of 365 index patients with CMAVM, Amyere et al. (2017) identified 47 distinct heterozygous mutations in the EPHB4 gene in 54 probands (see, e.g., 600011.0004-600011.0008). The authors noted that the mutations were located throughout the gene and they found no genotype/phenotype correlations.
In a cohort of 51 patients with vein of Galen aneurysmal malformation, Vivanti et al. (2018) identified 5 with heterozygous mutations in the EPHB4 gene (see, e.g., 600011.0009-600011.0012). Two of the patients also exhibited capillary malformations. Reduced penetrance was observed, with unaffected carrier parents in 3 of the families, and the authors suggested that other genes play a role in the clinical expression of the disease phenotype.
Gerety et al. (1999) generated mice with a targeted disruption of Ephb4 by introducing a tau-lacZ marker into the gene. Unlike the broadly expressed ephrin B2 gene (EFNB2; 600527), Ephb4 is uniquely expressed in vascular endothelial and endocardial cells. The authors' analysis also confirmed that Ephb4 is preferentially expressed on veins. Remarkably, the phenotype of homozygous Ephb4 mutants was virtually symmetric to that of Efnb2 mutants. These data identified EPHB4 as the major essential interaction partner of EFNB2 in angiogenesis and further indicated that the requisite function of this receptor is intrinsic to the circulatory system. In addition, these data indicated that EFNB2 and EPHB4 mediate reciprocal interactions between arteries and veins that are essential for proper angiogenic remodeling of the capillary beds.
Wang et al. (2015) developed a line of mice with targeted deletion of Ephb4 in smooth muscle cells (SMCs). Mutant mice showed sex differences in the vascular effects of SMC-specific Ephb4 deletion. Mutant males, but not females, showed significantly reduced systolic blood pressure and mean arterial pressure compared with wildtype, whereas mutant females, but not males, showed significantly reduced heart rate compared with wildtype. Male Ephb4 -/- mesenteric arteries showed abnormally low contractility in response to phenylephrin when grown on substrate containing Ephb4 or ephrin B2, suggesting that both forward and reverse signaling between Ephb4 and ephrin B2 are involved in blood pressure regulation. Male Ephb4 -/- vascular SMCs showed reduced Mlc (see 609931) phosphorylation and elevated Mlck (MYLK; 600922) and CAMKII (see 114078) phosphorylation. Wang et al. (2015) concluded that EPHB4 is involved in the CAMKII/MLCK/MLC signaling cascade in vascular SMCs.
Martin-Almedina et al. (2016) found that deletion of the Ephb4 gene specifically in the lymphatic vasculature of embryonic mice between days E10 to E12 resulted in subcutaneous edema and abnormal lymphatic development. Tissue from mutant mice showed tortuous and dilated dermal lymphatic vessels associated with defective formation of lymphovenous valves.
Vivanti et al. (2018) examined different brain layers of transgenic zebrafish embryos with knockdown of ephb4a, the EPHB4 ortholog, and observed marked vascular anomalies of the dorsal cranial vessels including both dorsal longitudinal and mesencephalic veins by 3 days postfertilization. Abnormal number of dorsal longitudinal vein (duplicated or triplicated) was observed in 82% of the mutant embryos, and abnormal morphology of the mesencephalic vein (absent or interrupted, unilaterally or bilaterally) was observed in 76%. Coinjection of wildtype EPHB4 showed significant rescue of both vascular phenotypes.
In 7 affected members of a 3-generation family from the United Kingdom with nonimmune hydrops fetalis and postnatal lymphatic dysfunction (LMPHM7; 617300), Martin-Almedina et al. (2016) identified a heterozygous c.2216G-A transition (c.2216G-A, NM_004444.4) in exon 13 of the EPHB4 gene, resulting in an arg739-to-glu (R739E) substitution at a highly conserved residue in the tyrosine kinase domain. The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family, although there was variable expressivity of the phenotype. The variant was filtered against the dbSNP (build 135) and 1000 Genomes Project databases and was not found in 900 in-house control exomes. In vitro functional expression studies in HEK293 cells and lymphatic endothelial cells showed that the mutant protein was devoid of tyrosine kinase activity, suggesting altered EPHB4 signaling. The findings indicated that the mutation has a negative effect on receptor activity after ligand stimulation. There was no evidence of a dominant-negative effect.
In 4 affected members of a 2-generation Norwegian family with nonimmune hydrops fetalis and postnatal lymphatic dysfunction (LMPHM7; 617300), Martin-Almedina et al. (2016) identified a heterozygous c.2345T-G transversion (c.2345T-G, NM_004444.4) in exon 14 of the EPHB4 gene, resulting in an ile782-to-ser (I782S) substitution at a highly conserved residue in the tyrosine kinase domain. The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family, although there was variable expressivity of the phenotype. The variant was filtered against the dbSNP (build 135) and 1000 Genomes Project databases and was not found in 900 in-house control exomes. There were 2 affected mothers, who were monozygotic twins, and each had an affected son. The mothers, whose phenotype was milder than that of the sons, were found to be mosaic for the I782S mutation. In vitro functional expression studies in HEK293 cells and lymphatic endothelial cells showed that the mutant protein was devoid of tyrosine kinase activity, suggesting altered EPHB4 signaling. The findings indicated that the mutation has a negative effect on receptor activity after ligand stimulation. There was no evidence of a dominant-negative effect.
In an 8-year-old white boy with multiple capillary malformations (CMAVM2; 618196), Yu et al. (2017) identified heterozygosity for a de novo c.2405A-G transition in the EPHB4 gene, resulting in an asp802-to-gly (D802G) substitution. His unaffected parents did not carry the mutation. The mutation was identified by whole-exome sequencing.
In 7 affected members of a 5-generation family (CM-13-HO) with multiple capillary malformations (CMAVM2; 618196), Amyere et al. (2017) identified heterozygosity for a 1-bp deletion (c.33delG) in exon 1 of the EPHB4 gene, causing a frameshift predicted to result in a premature termination codon (Leu12TrpfsTer10). The mutation segregated fully with disease in the family and was not found in the ExAC database.
In 2 brothers and their affected father and paternal grandmother (family CM-90) with multiple capillary malformations (CMAVM2; 618196), Amyere et al. (2017) identified heterozygosity for a c.802T-C transition in exon 4 of the EPHB4 gene, resulting in a cys268-to-arg (C268R) substitution. The mutation segregated fully with disease in the family and was not found in the ExAC database. The 4 mutation carriers all exhibited capillary malformations, and 1 also showed a Parkes Weber lesion on the right leg.
In 2 related patients (family CM-291) with capillary malformations (CMAVM2; 618196), Amyere et al. (2017) identified heterozygosity for a 2-bp deletion (c.632_633delTG) in exon 4 of the EPHB4 gene, causing a frameshift predicted to result in a premature termination codon (Val211AlafsTer11). The mutation was not found in the ExAC database.
In 4 members of a family (CM-397) with capillary malformations and vein of Galen aneurysmal malformations (CMAVM2; 618196), Amyere et al. (2017) identified heterozygosity for a c.1990G-A transition in exon 12 of the EPHB4 gene, resulting in a glu664-to-lys (E664K) substitution. Three members of the family exhibited cutaneous capillary malformations, and 2 had vein of Galen aneurysmal malformations. The mutation was not found in the ExAC database. Analysis of transfected COS-7 cells showed a low level of mutant protein compared to wildtype EPHB4, and the typical membranous staining was not seen with the mutant on immunofluorescence, indicating that E664K represents a loss-of-function mutation.
In a patient (family CM-490) with capillary malformations and a perimedullary arteriovenous malformation of the spine at D8-L1 (CMAVM2; 618196), Amyere et al. (2017) identified heterozygosity for a splice site mutation (c.2484+1G-A) in intron 14 of the EPHB4 gene. The mutation was not found in the ExAC database.
In a male patient (family AA5615) with capillary malformations and vein of Galen aneurysmal malformation (CMAVM2; 618196), Vivanti et al. (2018) identified heterozygosity for a splice site mutation (c.2484+1G-T, NM_004444.4) in intron 14 of the EPHB4 gene, predicted to result in a met814_val829 deletion within the catalytic domain of protein tyrosine kinases. The mutation was inherited from his mother, who exhibited only capillary malformations. The variant was not found in the 1000 Genomes Project or ExAC databases.
In a male patient (family AA5616) with vein of Galen aneurysmal malformation (CMAVM2; 618196), Vivanti et al. (2018) identified heterozygosity for a splice site insertion (c.2484+2insT, NM_004444.4) in intron 14 of the EPHB4 gene. The mutation, which was inherited from his unaffected father, was not found in the 1000 Genomes Project or ExAC databases. The patient's venous anomaly was discovered at 4 days of life upon evaluation for cardiac failure.
In a female patient (family AA5614) with capillary malformation and vein of Galen aneurysmal malformation (CMAVM2; 618196), Vivanti et al. (2018) identified heterozygosity for a de novo 1-bp duplication (c.570dupG, NM_004444.4) in the EPHB4 gene, causing a frameshift predicted to result in a premature termination codon (His191AlafsTer32). The mutation, which was not present in her unaffected parents, was not found in the 1000 Genomes Project or ExAC databases. In a zebrafish knockdown model (see ANIMAL MODEL), injection with mut-mRNA mimicking the c.570dupG mutation did not rescue the cranial venous malformation phenotype, whereas there was statistically significant rescue with wildtype EPHB4.
In a female patient (family AA5718) with vein of Galen aneurysmal malformation (CMAVM2; 618196), Vivanti et al. (2018) identified heterozygosity for a c.319T-C transition (c.319T-C, NM_004444.4) in the EPHB4 gene, resulting in a cys107-to-arg (C107R) substitution within the ligand-binding domain. The mutation, which was inherited from her unaffected mother, was not found in the 1000 Genomes Project or ExAC databases.
In 6 affected members of a large 4-generation family with hydrops fetalis, lymphedema, and central conduction lymphatic anomalies (LMPHM7; 617300), Li et al. (2018) identified heterozygosity for a splice site mutation (c.2334+1G-C, NM_004444.4) in intron 13 of the EPHB4 gene. The mutation was not found in 9 unaffected family members, in more than 4,000 in-house control samples, or in the 1000 Genomes Project, COSMIC v80, ESP6500SI, or gnomAD databases. RNAseq with skin biopsies from the proband demonstrated use of a cryptic splice donor site, causing retention of the intervening 12 bp of the intron, resulting in an in-frame insertion of 4 amino acids (Leu778_Gly779insLeuMetLeuGly) within the highly conserved catalytic loop of the protein kinase domain. Transfection of wildtype EPHB4 into HEK293T cells resulted in constitutive tyrosine phosphorylation of the protein, whereas markedly reduced phosphorylation occurred with the mutant EPHB4. In transfected A375 cells, stimulation by the EPHB4 ligand ephrin-B2 induced phosphorylation of wildtype but not mutant EPHB4, consistent with a loss-of-function effect. Injection into zebrafish of an ephb4a morpholino inhibiting the same splice junction of exon 13 resulted in vessel misbranching and deformities in lymphatic vessel development, indicating possible differentiation defects in lymphatic vessels and mimicking the lymphatic presentations of the patients.
Amyere, M., Revencu, N., Helaers, R., Pairet, E., Baselga, E., Cordisco, M., Chung, W., Dubois, J., Lacour, J.-P., Martorell, L., Mazereeuw-Hautier, J., Pyeritz, R. E., and 33 others. Germline loss-of-function mutations in EPHB4 cause a second form of capillary malformation-arteriovenous malformation (CM-AVM2) deregulating RAS-MAPK signaling. Circulation 136: 1037-1048, 2017. [PubMed: 28687708] [Full Text: https://doi.org/10.1161/CIRCULATIONAHA.116.026886]
Bennett, B. D., Wang, Z., Kuang, W.-J., Wang, A., Groopman, J. E., Goeddel, D. V., Scadden, D. T. Cloning and characterization of HTK, a novel transmembrane tyrosine kinase of the EPH subfamily. J. Biol. Chem. 269: 14211-14218, 1994. [PubMed: 8188704]
Berclaz, G., Andres, A.-C., Albrecht, D., Dreher, E., Ziemiecki, A., Gusterson, B. A., Crompton, M. R. Expression of the receptor protein tyrosine kinase myk-1/htk in normal and malignant mammary epithelium. Biochem. Biophys. Res. Commun. 226: 869-875, 1996. [PubMed: 8831703] [Full Text: https://doi.org/10.1006/bbrc.1996.1442]
Erber, R., Eichelsbacher, U., Powajbo, V., Korn, T., Djonov, V., Lin, J., Hammes, H.-P., Grobholz, R., Ullrich, A., Vajkoczy, P. EphB4 controls blood vascular morphogenesis during postnatal angiogenesis. EMBO J. 25: 628-641, 2006. [PubMed: 16424904] [Full Text: https://doi.org/10.1038/sj.emboj.7600949]
Gerety, S. S., Wang, H. U., Chen, Z.-F., Anderson, D. J. Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Molec. Cell 4: 403-414, 1999. [PubMed: 10518221] [Full Text: https://doi.org/10.1016/s1097-2765(00)80342-1]
Hartz, P. A. Personal Communication. Baltimore, Md. 8/31/2017.
Herbert, S. P., Huisken, J., Kim, T. N., Feldman, M. E., Houseman, B. T., Wang, R. A., Shokat, K. M., Stainier, D. Y. R. Arterial-venous segregation by selective cell sprouting: an alternative mode of blood vessel formation. Science 326: 294-298, 2009. [PubMed: 19815777] [Full Text: https://doi.org/10.1126/science.1178577]
Li, D., Wenger, T. L., Seiler, C., March, M. E., Gutierrez-Uzquiza, A., Kao, C., Bhoj, E., Tian, L., Rosenbach, M., Liu, Y., Robinson, N., Behr, M., and 12 others. Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly. Hum. Molec. Genet. 27: 3233-3245, 2018. [PubMed: 29905864] [Full Text: https://doi.org/10.1093/hmg/ddy218]
Martin-Almedina, S., Martinez-Corral, I., Holdhus, R., Vicente, A., Fotiou, E., Lin, S., Petersen, K., Simpson, M. A., Hoischen, A., Gilissen, C., Jeffery, H., Atton, G., and 13 others. EPHB4 kinase-inactivating mutations cause autosomal dominant lymphatic-related hydrops fetalis. J. Clin. Invest. 126: 3080-3088, 2016. [PubMed: 27400125] [Full Text: https://doi.org/10.1172/JCI85794]
Sakamoto, H., Zhang, X.-Q., Suenobu, S., Ohbo, K., Ogawa, M., Suda, T. Cell adhesion to ephrinb2 is induced by EphB4 independently of its kinase activity. Biochem. Biophys. Res. Commun. 321: 681-687, 2004. [PubMed: 15358160] [Full Text: https://doi.org/10.1016/j.bbrc.2004.07.016]
Vivanti, A., Ozanne, A., Grondin, C., Saliou, G., Quevarec, L., Maurey, H., Aubourg, P., Benachi, A., Gut, M., Gut, I., Martinovic, J., Senat, M. C., Tawk, M., Melki, J. Loss of function mutations in EPHB4 are responsible for vein of Galen aneurysmal malformation. Brain 141: 979-988, 2018. [PubMed: 29444212] [Full Text: https://doi.org/10.1093/brain/awy020]
Wang, Y., Thorin, E., Luo, H., Tremblay, J., Lavoie, J. L., Wu, Z., Peng, J., Qi, S., Wu, J. EPHB4 protein expression in vascular smooth muscle cells regulates their contractility, and EPHB4 deletion leads to hypotension in mice. J. Biol. Chem. 290: 14235-14244, 2015. [PubMed: 25903126] [Full Text: https://doi.org/10.1074/jbc.M114.621615]
Wilson, M. D., Riemer, C., Martindale, D. W., Schnupf, P., Boright, A. P., Cheung, T. L., Hardy, D. M., Schwartz, S., Scherer, S. W., Tsui, L.-C., Miller, W., Koop, B. F. Comparative analysis of the gene-dense ACHE/TFR2 region on human chromosome 7q22 with the orthologous region on mouse chromosome 5. Nucleic Acids Res. 29: 1352-1365, 2001. [PubMed: 11239002] [Full Text: https://doi.org/10.1093/nar/29.6.1352]
Yu, J.,Streicher, J. L., Medne, L., Krantz, I. D., Yan, A. C. EPHB4 mutation implicated in capillary malformation-arteriovenous malformation syndrome: a case report. Pediat. Derm. 34: e227-e230, 2017. Note: Electronic Article. [PubMed: 28730721] [Full Text: https://doi.org/10.1111/pde.13208]