PURPOSE:Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformationswith arteriovenous malformations 2 (CM-AVM2) have been associated withEPHB4(Ephrin type B receptor 4) variants, demandingnew approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified inEPHB4, and for theidentification 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, whosedistinct clinical phenotypes are described in detail in this study, presented with a variant inEPHB4. In vitro functional studies wereperformed to confirm pathogenicity.RESULTS:Pathogenicity was demonstrated for six of the seven novelEPHB4VUS investigated. A heterogeneity of molecular diseasemechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of thephosphorylation capability of the receptor. There was some phenotype–genotype correlation; however, previously unreportedintrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family wereobserved.CONCLUSION:This study highlights the usefulness of protein expression and subcellular localization studies to predictEPHB4variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum ofEPHB4-relateddisorders, introducing the discovery of cases with overlapping phenotypes.