EPHB4 Mutation Suppresses PROX1 Expression and Disrupts Lymphatic Development in Neonatal Hydrops

See the Supplemental Materials for the methods and materials used in this study.

A 35-week gestational age male infant with a birth weight of 4.11 kg was prenatally diagnosed with nonimmune hydrops. The infant was born to a 33-year-old mother with a history of abortion at 11 weeks gestation with cystic hygroma. Mother and grandmother from maternal side had a history of early-onset varicose veins. Aside from varicose veins, mother had swelling of lower extremities only during pregnancy. There was no family history of echocardiography-diagnosed cardiac defects. The infant was hydropic at birth, requiring intubation, and developed recurrent, chylous pleural effusions bilaterally, requiring thoracentesis. The infant was transferred to the level 4 NICU at ∼2 months of age. He had significant subcutaneous edema, and MRI lymphangiogram revealed the absence of central lymphatic vessels and ectasia of subcutaneous lymphatic channels (Fig 1 A and B). A repeat MRI lymphangiogram 60 days later revealed worsening of lymphatic flow to the bowel wall and lumen (Fig 1C). On day of life 145, the infant developed sepsis and succumbed to septic shock, despite intensive cardiovascular and respiratory support.

Genetic testing initially started with a karyotype (46XY) and microarray that were noncontributory. WES revealed heterozygous variants in the EPHB4 (locus 7q22.1; GenBank under accession no. NM_004444), HLCS, PKLR, and SOS1 genes. The EPHB4 c.2098G>A (p.Ala700Thr) variant was a missense substitution, with the alanine amino acid being highly conserved in 12 species (up to zebrafish). This maternally inherited EPHB4 variant is predicted to be deleterious/probably damaging, affecting the serine-threonine/tyrosine-protein kinase catalytic domain. HLCS and SOS1 variants were predicted bioinformatically to be tolerant/benign. PKLR variant was intronic and not near a splice site.

Autopsy revealed anasarca, absent thoracic duct, diaphragm microcalcifications, depletion of lymphoid tissue of the reticuloendothelial system, acute global hypoxic-ischemic brain changes with acute meningitis, lungs with extensive bilateral pleural adhesions and pulmonary lymphangiectasia, and postmortem identification of methicillin-resistant Staphylococcus aureus in spleen and Cutibacterium acnes in lung. Immunohistochemistry (IHC) studies showed loss of PROX1 staining in the ECs of the large lymphatic channels in the lung and small intestinal villi, with preservation of podoplanin expression in the same vessels. In comparison, the two postmortem control cases revealed intact PROX1 and podoplanin staining of the lymphatic channels (Figs 2 and 3).

The EPHB4 p.Ala700Thr variant localizes to the kinase domain of EPHB4^7,9  (Fig 4A). We therefore examined EPHB4 phosphorylation in native conditions in HEK293 cells. Although wild-type EPHB4 was significantly phosphorylated (Tyr987) in HEK293 cells, there was an almost complete loss of phosphorylation of the EPHB4 p.Ala700Thr variant (Fig 4B). Similar studies pursued in immortalized human pulmonary microvascular ECs (HPMEC-Im)¹⁰  also showed significant attenuation of EPHB4 phosphorylation with the EPHB4 p.Ala700Thr variant (Fig 4 C and D).

EPHB4 phosphorylation can activate extracellular signal-related kinase (ERK), a mitogen-activated protein kinase important for lymphatic fate specification.⁷  In HPMEC-Im, although the wild-type allele induced ERK phosphorylation, the EPHB4 p.Ala700Thr variant was not able to further induce ERK phosphorylation (Fig 4 C and D). The transcription factor PROX1 is essentially for maintaining lymphatic EC specification as well as for thoracic and central lymphatic development from venous blood vessels.^8,11  We postulated that loss of EPHB4 and ERK phosphorylation would result in decreased PROX1 expression. Immunoblotting studies revealed low PROX1 expression in HPMEC-Im when transfected with empty plasmids (Fig 4 C and D), by strong induction of PROX1 by the EPHB4 wild-type allele, but not by the variant allele (Fig 4 C and D).

Nonimmune hydrops fetalis is frequently associated with major anomalies of the lymphatic system, but the underlying genetic etiology is often not investigated or identified. In this report, we describe a case of fatal nonimmune hydrops with striking radiologic and pathological findings of absence of the central lymphatic system with dermal ectasia of lymphatic vessels consistent with diagnosis of CCLA and associated with a functional mutation in the kinase domain of EPHB4 p.Ala700Thr variant that suppresses self-phosphorylation.¹²  The EPHB4 mutation is novel because it is not found in the Genome Aggregation Database (https://gnomad.broadinstitute.org). We demonstrate that the variant attenuates ERK phosphorylation and EPHB4-induced expression of PROX1, a transcription factor required for lymphatic EC specification and development of central lymphatic vessels.^7,9  Our study reveals a novel role for the EPHB4- PROX1 axis in lymphatic vasculature development that is disrupted by the EPHB4 p.Ala700Thr variant contributing to hydrops fetalis.

After the differentiation of arterial and venous vessel identity, a subset of venous EC acquires the fate of lymphatic EC under the control of transcription factors such as PROX1.^11–13  Ephrin B2 expressed on arterial ECs works in tandem with its receptor EPHB4 expressed on venous ECs to establish arterial versus venous EC fate.¹⁴  EPHB4 plays an important role in maturation of the lymphatic network and lymphatic valve formation in mouse embryos.⁶  Previously, two EPHB4 mutations, p.Arg739Glu and p.I1e782Ser, were reported to occur in the tyrosine kinase domain of EPHB4 protein, leading to tyrosine kinase inactivity that is required for lymphatic valve development.²  Phenotypes arising from EPHB4 mutations have been reported to be frequently associated with venous malformation, including varicose veins associated with venous stasis and swelling of legs.⁶  Interestingly, the mother of our proband is a carrier for the EPHB4 mutation with a history of early-onset varicose veins.

In our proband, we demonstrate that the maternally inherited EPHB4 p.Ala700Thr variant was associated with hydrops fetalis. Previously, Li et al⁵  reported on an infant who developed nonimmune hydrops and was critical in the neonatal period but survived until his mid-20s with a significant history of effusions, ascites, and leg swelling. They performed WES on the proband and a 4-generation pedigree and found an EPHB4 mutation that disrupts a normal splice donor site, resulting in a cryptic splice site that incorporates an additional 4–amino acid sequence into the protein, resulting in loss of EPHB4 phosphorylation as in our proband. It is unclear why the mother did not have symptoms of hydrops in the neonatal period but the proband and the earlier sibling who died in utero had hydrops, as both carried the heterozygous EPHB4 mutation. That EPHB4 mutations can function in an autosomal dominant manner can be an explanation for the phenotype in the proband but not necessarily the milder phenotype in the mother. We speculate that the potential presence of other modifier gene defects or sex-determined effects may have played a role. Martin-Almedina et al²  performed WES in 2 families with a history of in utero neonatal deaths related to nonimmune hydrops and found 2 heterozygous missense variants in EPHB4, localizing to the tyrosine kinase domain, leading to tyrosine kinase inactivity.

Transcriptional programs in lymphatic development are regulated by endothelial ERK signaling.⁷  The differentiation of venous ECs into lymphatic ECs is regulated by ERK and the transcription factors SOX18, Coup-TFII, and PROX1.⁷  Our data showed that the EPHB4 variant does not induce further ERK phosphorylation, consistent with repression of ERK signaling. PROX1 is a transcriptional factor required for lymphatic fate specification, and its deletion in mice^7,15  prevents ECs from becoming lymphatic ECs, disrupting lymphatic vessel development.^7,8,16  Strikingly, EPHB4, but not the protein encoded by the EPHB4 p.Ala700Thr variant, was able to further induce PROX1 expression. To confirm in vivo relevance, IHC studies in our proband and postmortem control cases showed severely reduced PROX1 with EPHB4 mutation.

We describe a novel mutation in EPHB4 that disrupts the gene’s phosphorylation, resulting in decreased PROX1 expression, in a neonate with fatal CCLA. To our knowledge, this study is the first demonstrating a link between EPHB4 mutation and PROX1 expression and CCLA-related hydrops in neonates. Although we report novel data, the mechanisms by which EPHB4 expression regulates PROX1 expression and regulation of lymphatic development by the EPHB4/ERK/PROX1 axis remain to be elucidated. Geneticist referral is warranted because genetic testing algorithms for nonimmune hydrops indicate the performance of next-generation sequencing (whole-genome sequencing or WES) to look for specific genetic mutations in PIEZO1, CCBE1, EPHB4, FOXC2, VEGFR3, SOX18, and ITGA9 genes.^17,18  In addition to these genes, WES will inform the discovery of new susceptibility loci for nonimmune hydrops, aiding perinatal management and counseling.

CCLA

central conducting lymphatic anomaly

EC

endothelial cell

EPHB4

ephrin type B receptor 4

ERK

extracellular signal-related kinase

HEK

human embryonic kidney cell

HPMEC-Im

immortalized human pulmonary microvascular endothelial cells

IHC

immunohistochemistry

PROX1

prospero homeobox 1

WES

whole-exome sequencing