Janus-faced EPHB4-associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes.

PURPOSE: Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformations with arteriovenous malformations 2 (CM-AVM2) have been associated with EPHB4 (Ephrin type B receptor 4) variants, demanding new approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified in EPHB4, and for the identification of differentiated disease mechanisms at the molecular level. METHODS: Ten index cases with various phenotypes, either fetal hydrops, CM-AVM2, or peripheral lower limb lymphedema, whose distinct clinical phenotypes are described in detail in this study, presented with a variant in EPHB4. In vitro functional studies were performed to confirm pathogenicity. RESULTS: Pathogenicity was demonstrated for six of the seven novel EPHB4 VUS investigated. A heterogeneity of molecular disease mechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of the phosphorylation capability of the receptor. There was some phenotype-genotype correlation; however, previously unreported intrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family were observed. CONCLUSION: This study highlights the usefulness of protein expression and subcellular localization studies to predict EPHB4 variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum of EPHB4-related disorders, introducing the discovery of cases with overlapping phenotypes.

A Novel Splice-Site Mutation in VEGFC Is Associated with Congenital Primary Lymphoedema of Gordon.

Lymphedema is characterized by chronic swelling of any body part caused by malfunctioning or obstruction in the lymphatic system. Primary lymphedema is often considered genetic in origin. VEGFC, which is a gene encoding the ligand for the vascular endothelial growth factor receptor 3 (VEGFR3/FLT4) and important for lymph vessel development during lymphangiogenesis, has been associated with a specific subtype of primary lymphedema. Through Sanger sequencing of a proband with bilateral congenital pedal edema resembling Milroy disease, we identified a novel mutation (NM_005429.2; c.361+5G>A) in VEGFC. The mutation induced skipping of exon 2 of VEGFC resulting in a frameshift and the introduction of a premature stop codon (p.Ala50ValfsTer18). The mutation leads to a loss of the entire VEGF-homology domain and the C-terminus. Expression of this Vegfc variant in the zebrafish floorplate showed that the splice-site variant significantly reduces the biological activity of the protein. Our findings confirm that the splice-site variant, c.361+5G>A, causes the primary lymphedema phenotype in the proband. We examine the mutations and clinical phenotypes of the previously reported cases to review the current knowledge in this area.

EPHB4 kinase-inactivating mutations cause autosomal dominant lymphatic-related hydrops fetalis.

Hydrops fetalis describes fluid accumulation in at least 2 fetal compartments, including abdominal cavities, pleura, and pericardium, or in body tissue. The majority of hydrops fetalis cases are nonimmune conditions that present with generalized edema of the fetus, and approximately 15% of these nonimmune cases result from a lymphatic abnormality. Here, we have identified an autosomal dominant, inherited form of lymphatic-related (nonimmune) hydrops fetalis (LRHF). Independent exome sequencing projects on 2 families with a history of in utero and neonatal deaths associated with nonimmune hydrops fetalis uncovered 2 heterozygous missense variants in the gene encoding Eph receptor B4 (EPHB4). Biochemical analysis determined that the mutant EPHB4 proteins are devoid of tyrosine kinase activity, indicating that loss of EPHB4 signaling contributes to LRHF pathogenesis. Further, inactivation of Ephb4 in lymphatic endothelial cells of developing mouse embryos led to defective lymphovenous valve formation and consequent subcutaneous edema. Together, these findings identify EPHB4 as a critical regulator of early lymphatic vascular development and demonstrate that mutations in the gene can cause an autosomal dominant form of LRHF that is associated with a high mortality rate.

Grape pomace extract exerts antioxidant effects through an increase in GCS levels and GST activity in muscle and endothelial cells.

In a previous study, we demonstrated that a grape pomace extract (GPE) exerted antioxidant activity in endothelial (EA.hy926) and muscle (C2C12) cells through an increase in glutathione (GSH) levels. In the present study, in order to elucidate the mechanisms responsible for the antioxidant activity of GPE, its effects on the expression of critical antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD)1, heme oxygenase 1 (HO-1) and gamma-glutamylcysteine synthetase (GCS) were assessed in EA.hy926 and C2C12 cells. Moreover, the effects of GPE on CAT, SOD and glutathione S-transferase (GST) enzymatic activity were evaluated. For this purpose, the C2C12 and EA.hy926 cells were treated with GPE at low and non-cytotoxic concentrations (2.5 and 10 µg/ml for the C2C12 cells; 0.068 and 0.250 µg/ml for the EA.hy926 cells) for 3, 6, 12, 18 and 24 h. Following incubation, enzymatic expression and activity were assessed. The results revealed that treatment with GPE significantly increased GCS levels and GST activity in both the C2C12 and EA.hy926 cells. However, GPE significantly decreased CAT levels and activity, but only in the muscle cells, while it had no effect on CAT levels and activity in the endothelial cells. Moreover, treatment with GPE had no effect on HO-1 and SOD expression and activity in both cell lines. Therefore, the present results provide further evidence of the crucial role of GSH systems in the antioxidant effects exerted by GPE. Thus, GPE may prove to be effective for use as a food supplement for the treatment of oxidative stress-induced pathological conditions of the cardiovascular and skeletal muscle systems, particularly those associated with low GSH levels.