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Background: Non-immune hydrops fetalis is often a fatal condition and in 20% of cases related to lymphatic anomalies. Maldevelopment of lymphatic valves, atretic or absence of central lymphatic vessels has been described in hydrops fetalis, but the genetic basis of these anomalies remains undefined. Recent reports suggest that Ephrin type B receptor 4 (EPHB4) gene has a significant role in embryonic development of lymphatic system. To test the hypothesis that EPHB4 mutations can cause hydrops fetalis, we combined whole exome sequencing (WES) with functional and pathological analysis of an infant who succumbed to neonatal hydrops.

Objective: To discover the possible mechanism of non-immune hydrops in EPHB4 variant by WES, histopathologic analysis and in vitro functional analysis of genetic mutation in human lung endothelial cells (HLEC).

Design/Methods: 35-week, 4.11kg male infant developed hydrops prenatally requiring multiple thoracentesis postnatally due to bilateral chylous pleural effusions and ascites, and eventually succumbed to infection and immunodeficiency from T-lymphocyte depletion. WES was performed to identify genetic causes of non-immune hydrops. Immunohistochemistry (IHC) was performed on autopsy specimens of lung and intestinal tissue. In vitro functional analysis was done using HLEC, and immunoblotting used of investigating signaling events.

Results: Magnetic resonance imaging (MRI) lymphangiogram showed complete absence of central lymphatic ducts (Figure 1). WES showed a rare heterozygous missense mutation in EPHB4 (p.Ala700Thr) that was maternally inherited. Autopsy revealed pulmonary lymphangiectasia, depletion of lymphoid tissue, and staphylococcus aureus in spleen. IHC showed loss of PROX1 expression (prospero homeobox protein 1) in large lymphatic channels in lung and small intestinal villi (Figure 2). In vitrofunctional studies showed the EPHB4 mutation resulted in loss of phosphorylation, decreased ERK phosphorylation and suppressed PROX1 expression, necessary for lymphatic valve development (Figure 3).

Conclusion(s): We report a novel case of fatal non-immune hydrops with lymphatic anomalies associated with an EPHB4functional mutation that suppresses its phosphorylation and PROX1 expression. This report highlights the importance of screening for EPHB4 variant in infants with the diagnosis of non-immune hydrops fetalis and lymphatic anomalies. This discovery will trigger further studies to find genetic basis of non-immune hydrops and novel therapies for this fatal disease.

Presented at the 2021 PAS Virtual Conference


Pathology | Pediatrics

A Deleterious EPHB4 Mutation Suppresses PROX1 Expression and Disrupts Lymphatic Development in Neonatal Non-immune Hydrops