PUBLICATION

Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly

Authors
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., Chiavacci, R., Hou, C., Wang, T., Bakay, M., Pellegrino da Silva, R., Perkins, J.A., Sleiman, P., Levine, M.A., Hicks, P.J., Itkin, M., Dori, Y., Hakonarson, H.
ID
ZDB-PUB-180616-10
Date
2018
Source
Human molecular genetics   27(18): 3233-3245 (Journal)
Registered Authors
Seiler, Christoph
Keywords
none
MeSH Terms
  • Animals
  • Disease Models, Animal
  • Exome Sequencing*
  • HEK293 Cells
  • Heterozygote
  • Humans
  • Lymphatic Abnormalities/genetics*
  • Lymphatic Abnormalities/metabolism
  • Lymphatic Abnormalities/pathology
  • Lymphatic Vessels/metabolism*
  • Lymphatic Vessels/pathology
  • Mechanistic Target of Rapamycin Complex 1/genetics
  • Pedigree
  • Phosphorylation
  • Receptor Protein-Tyrosine Kinases/genetics
  • Receptor, EphB4/genetics*
  • Signal Transduction
  • Zebrafish/genetics
PubMed
29905864 Full text @ Hum. Mol. Genet.
Abstract
Central conducting lymphatic anomaly (CCLA) is one of the complex lymphatic anomalies characterized by dilated lymphatic channels, lymphatic channel dysmotility, and distal obstruction affecting lymphatic drainage. We performed whole exome sequencing (WES) of DNA from a four-generation pedigree and examined the consequences of the variant by transfection of mammalian cells and morpholino and rescue studies in zebrafish. WES revealed a heterozygous mutation in EPHB4 (RefSeq NM_004444.4; c.2334 + 1G>C) and RNA-Seq demonstrated that the EPHB4 mutation destroys the normal donor site, which leads to the use of a cryptic splice donor that results in retention of the intervening 12-bp intron sequence. Transient co-expression of the wild type and mutant EPHB4 proteins showed reduced phosphorylation of tyrosine, consistent with a loss-of-function effect. Zebrafish ephb4a morpholino resulted in vessel misbranching and deformities in the lymphatic vessel development, indicative of possible differentiation defects in lymphatic vessels, mimicking the lymphatic presentations of the patients. Immunoblot analysis using zebrafish lysates demonstrated over-activation of mTORC1 as a consequence of reduced EPHB4 signaling. Strikingly, drugs that inhibit mTOR signaling or RAS-MAPK signaling effectively rescued the misbranching phenotype in a comparable manner. Moreover, knockin of EPHB4 mutation in HEK293T cells also induced mTORC1 activity. Our data demonstrate the pathogenicity of the identified EPHB4 mutation as a novel cause of CCLA and suggesting that ERK inhibitors may have therapeutic benefits in such patients with complex lymphatic anomalies.
Genes / Markers
Figures
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Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping