Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly

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, 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.

Multidisciplinary guidelines for initial evaluation of complicated lymphatic anomalies—expert opinion consensus

“…Though basic science has made significant progress in identifying molecular pathways and genes associated with CLAs, at the present time there are no specific gene defects that are diagnostic. However, the conference participants emphasized the need for continuing these studies and suggested the possibility of collecting tissue left over after clinical diagnoses for use in lymphatic research.…”

Generalized congenital epithelioid blue nevi (pigmented epithelioid melanocytomas) in an infant: Report of case and review of the literature

“…We performed whole exome sequencing on the patient together with his parents using samples of whole blood following protocols similar to those previously published . Variants were filtered against public variant databases (dbSNP, 1000 Genomes Project, NHLBI ESP6500SI, gnomAD, and Kaviar) and >5000 in‐house exomes.…”

“…We performed whole exome sequencing on the patient together with his parents using samples of whole blood following protocols similar to those previously published. 9,10 Variants were filtered against public variant databases (dbSNP, 1000 Genomes Project, NHLBI ESP6500SI, gnomAD, and Kaviar) and >5000 in-house exomes. Subsequent gene prioritization was on basis of deleterious predication and biology relevance by referring to Online Mendelian Inheritance in Man, which did not reveal any pathogenic variants in other candidate genes, including genes residing in chromosome 2p16, which has been linked to the autosomal dominant form of Carney complex.…”

Novel mutation in CCBE 1 as a cause of recurrent hydrops fetalis from Hennekam lymphangiectasia‐lymphedema syndrome‐1

“…However, CCBE1 variants are only found in 25% of suspected cases . Isolated or syndromic primary lymphedema has been associated with variants in 27 additional genes, including EPHB4, FLT4, FOXC2, GATA2, GJA1, GJC2, HGF, HRAS, IKBKG, ITGA9, KIF11, KRAS, PTPN11, PTPN14, RAF1, RASA1, SOS1, SOX18, VEGFC, CELSR1, EPHB4, FAT4, PIEZO1, RELN, RIT1, TSC1, TSC2 , and ADAMTS3 …”

“…4 Isolated or syndromic primary lymphedema has been associated with variants in 27 additional genes, including EPHB4, FLT4, FOXC2, GATA2, GJA1, GJC2, HGF, HRAS, IKBKG, ITGA9, KIF11, KRAS, PTPN11, PTPN14, RAF1, RASA1, SOS1, SOX18, VEGFC, CELSR1, EPHB4, FAT4, PIEZO1, RELN, RIT1, TSC1, TSC2, and ADAMTS3. [5][6][7] Hydrops fetalis is defined as excessive fluid collections within fetal cavities and extravascular compartments: skin edema, ascites, pleural or pericardial effusions, thickened placenta, and polyhydramnios. It represents the end stage of a variety of disorders.…”