Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly

Dong Li, Tara L. Wenger, Christoph Seiler, Michael E. March, Alvaro Gutierrez-Uzquiza, Charlly Kao, Elizabeth Bhoj, Lifeng Tian, Misha Rosenbach, Yichuan Liu, Nora Robinson, Mechenzie Behr, Rosetta Chiavacci, Cuiping Hou, Tiancheng Wang, Marina Bakay, Renata Pellegrino Da Silva, Jonathan A. Perkins, Patrick Sleiman, Michael A. LevinePatricia J. Hicks, Maxim Itki, Yoav Dori, Hakon Hakonarson

Research output: Contribution to journalArticlepeer-review

54 Scopus citations


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 coexpression of the wild-type and mutant EPHB4 proteins showed reduced phosphorylation of tyrosine, consistent with a lossof- 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, knock-in 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.

Original languageEnglish (US)
Pages (from-to)3233-3245
Number of pages13
JournalHuman molecular genetics
Issue number18
StatePublished - Sep 15 2018
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Genetics(clinical)


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