Investigating the effects of compound paralogous EPHB receptor mutations on mouse facial development.

BACKGROUND: Variation in facial shape may arise from the combinatorial or overlapping actions of paralogous genes. Given its many members, and overlapping expression and functions, the EPH receptor family is a compelling candidate source of craniofacial morphological variation. We performed a detailed morphometric analysis of an allelic series of E14.5 Ephb1-3 receptor mutants to determine the effect of each paralogous receptor gene on craniofacial morphology. RESULTS: We found that Ephb1, Ephb2, and Ephb3 genotypes significantly influenced facial shape, but Ephb1 effects were weaker than Ephb2 and Ephb3 effects. Ephb2-/- and Ephb3-/- mutations affected similar aspects of facial morphology, but Ephb3-/- mutants had additional facial shape effects. Craniofacial differences across the allelic series were largely consistent with predicted additive genetic effects. However, we identified a potentially important nonadditive effect where Ephb1 mutants displayed different morphologies depending on the combination of other Ephb paralogs present, where Ephb1+/- , Ephb1-/- , and Ephb1-/- ; Ephb3-/- mutants exhibited a consistent deviation from their predicted facial shapes. CONCLUSIONS: This study provides a detailed assessment of the effects of Ephb receptor gene paralogs on E14.5 mouse facial morphology and demonstrates how the loss of specific receptors contributes to facial dysmorphology.

PDGF-A suppresses contact inhibition during directional collective cell migration.

The leading-edge mesendoderm (LEM) of the Xenopus gastrula moves as an aggregate by collective migration. However, LEM cells on fibronectin in vitro show contact inhibition of locomotion by quickly retracting lamellipodia upon mutual contact. We found that a fibronectin-integrin-syndecan module acts between p21-activated kinase 1 upstream and ephrin B1 downstream to promote the contact-induced collapse of lamellipodia. To function in this module, fibronectin has to be present as puncta on the surface of LEM cells. To overcome contact inhibition in LEM cell aggregates, PDGF-A deposited in the endogenous substratum of LEM migration blocks the fibronectin-integrin-syndecan module at the integrin level. This stabilizes lamellipodia preferentially in the direction of normal LEM movement and supports cell orientation and the directional migration of the coherent LEM cell mass.

Functional Consequences of Synapse Remodeling Following Astrocyte-Specific Regulation of Ephrin-B1 in the Adult Hippocampus.

Astrocyte-derived factors can control synapse formation and functions, making astrocytes an attractive target for regulating neuronal circuits and associated behaviors. Abnormal astrocyte-neuronal interactions are also implicated in neurodevelopmental disorders and neurodegenerative diseases associated with impaired learning and memory. However, little is known about astrocyte-mediated mechanisms that regulate learning and memory. Here, we propose astrocytic ephrin-B1 as a regulator of synaptogenesis in adult hippocampus and mouse learning behaviors. We found that astrocyte-specific ablation of ephrin-B1 in male mice triggers an increase in the density of immature dendritic spines and excitatory synaptic sites in the adult CA1 hippocampus. However, the prevalence of immature dendritic spines is associated with decreased evoked postsynaptic firing responses in CA1 pyramidal neurons, suggesting impaired maturation of these newly formed and potentially silent synapses or increased excitatory drive on the inhibitory neurons resulting in the overall decreased postsynaptic firing. Nevertheless, astrocyte-specific ephrin-B1 knock-out male mice exhibit normal acquisition of fear memory but enhanced contextual fear memory recall. In contrast, overexpression of astrocytic ephrin-B1 in the adult CA1 hippocampus leads to the loss of dendritic spines, reduced excitatory input, and impaired contextual memory retention. Our results suggest that astrocytic ephrin-B1 may compete with neuronal ephrin-B1 and mediate excitatory synapse elimination through its interactions with neuronal EphB receptors. Indeed, a deletion of neuronal EphB receptors impairs the ability of astrocytes expressing functional ephrin-B1 to engulf synaptosomes in vitro Our findings demonstrate that astrocytic ephrin-B1 regulates long-term contextual memory by restricting new synapse formation in the adult hippocampus.SIGNIFICANCE STATEMENT These studies address a gap in our knowledge of astrocyte-mediated regulation of learning and memory by unveiling a new role for ephrin-B1 in astrocytes and elucidating new mechanisms by which astrocytes regulate learning. Our studies explore the mechanisms underlying astrocyte regulation of hippocampal circuit remodeling during learning using new genetic tools that target ephrin-B signaling in astrocytes in vivo On a subcellular level, astrocytic ephrin-B1 may compete with neuronal ephrin-B1 and trigger astrocyte-mediated elimination of EphB receptor-containing synapses. Given the role EphB receptors play in neurodevelopmental disorders and neurodegenerative diseases, these findings establish a foundation for future studies of astrocyte-mediated synaptogenesis in clinically relevant conditions that can help to guide the development of clinical applications for a variety of neurological disorders.

Chronic hypoxia-induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin-B1.

Advanced solid tumors are exposed to hypoxic conditions over longer periods of time as they grow. Tumor hypoxia is a major factor that induces malignant progression, but most previous studies on tumor hypoxia were performed under short-term hypoxia for up to 72 hours and few studies have focused on tumor response to chronic hypoxic conditions. Here we show a molecular mechanism by which chronic hypoxia promotes invasive behavior in prostate cancer cells. We found that an epithelial-mesenchymal transition (EMT)-driving transcription factor, slug, is specifically upregulated under chronic hypoxia and promotes tumor cell migration and invasion. Unexpectedly, processes associated with EMT, such as loss of E-cadherin, are not observed under chronic hypoxia. Instead, expression of ephrin-B1, a ligand of Eph-related receptor tyrosine kinases, is markedly induced by slug through E-box motifs and promotes cell migration and invasion. Furthermore, slug and ephrin-B1 are highly coexpressed in chronic hypoxic cells of human prostate adenocarcinoma tissues after androgen deprivation, which is known to cause tumor hypoxia. Taken together, these results indicate that chronic hypoxia-induced slug promotes invasive behavior of prostate cancer cells by activating the expression of ephrin-B1. In addition, ephrin-B1 may be a novel therapeutic target in combination with androgen deprivation therapy for aggressive prostate cancer.

Craniofrontonasal syndrome in a male due to chromosomal mosaicism involving EFNB1: further insights into a genetic paradox.

Craniofrontonasal syndrome (CFNS) is an X-linked disorder caused by inactivating mutations in the gene for ephrin-B1 (EFNB1). Paradoxically it shows a more severe phenotype in females than in males. As a result of X inactivation cell populations with and without EFNB1 expression are found in EFNB1+/- females. This is thought to initiate a process termed cellular interference which may be responsible for the phenotype in females. We present a boy with severe clinical features of CFNS. In ∼42% of his blood cells we found a supernumerary ring X chromosome containing EFNB1 but lacking XIST. Mosaicism for cell populations with different levels of EFNB1 expression can explain the severe phenotype of this patient. In vitro experiments in Xenopus tissue showed that cells overexpress ephrinB1 cluster and sort out from wild-type cells. Our report provides further evidence that cellular interference contributes to the paradoxical inheritance pattern of CFNS.

Ephrin B1 Regulates Inflammatory Pathways in Retinal Müller Cells.

The role of inflammation has been accepted as a factor in the complications of diabetic retinopathy. Discovery of the upstream regulation of these inflammatory factors has remained a challenge. In this study, we explored the actions of ephrin B1 in retinal Müller cells and their actions on inflammatory proteins. We used diabetic human and mouse samples, as well as Müller cells in culture to measure ephrin B1 in Müller cells. We then generated Müller cell specific ephrin B1 knockout mice. We measure levels of key inflammatory proteins, including high mobility group box 1 (HMGB1) and NOD-like receptor protein 3 (NLRP3) pathway proteins in retinal lysates from the ephrin B1 floxed and ephrin B1 Müller cell specific knockout mice. Data show that ephrin B1 is significantly increased in the retina of diabetic humans and mice, as well as in Müller cells grown in high glucose. Elimination of ephrin B1 in mouse Müller cells led to a significant decline in all inflammatory proteins studied. In conclusion, a reduction in ephrin B1 in the diabetic retina may offer a new therapeutic modality for diabetic retinopathy.

Ephrin-B1 regulates the adult diastolic function through a late postnatal maturation of cardiomyocyte surface crests.

The rod-shaped adult cardiomyocyte (CM) harbors a unique architecture of its lateral surface with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) with unknown role. Here, we investigated the development and functional role of CM crests during the postnatal period. We found in rodents that CM crest maturation occurs late between postnatal day 20 (P20) and P60 through both SSM biogenesis, swelling and crest-crest lateral interactions between adjacent CM, promoting tissue compaction. At the functional level, we showed that the P20-P60 period is dedicated to the improvement of relaxation. Interestingly, crest maturation specifically contributes to an atypical CM hypertrophy of its short axis, without myofibril addition, but relying on CM lateral stretching. Mechanistically, using constitutive and conditional CM-specific knock-out mice, we identified ephrin-B1, a lateral membrane stabilizer, as a molecular determinant of P20-P60 crest maturation, governing both the CM lateral stretch and the diastolic function, thus highly suggesting a link between crest maturity and diastole. Remarkably, while young adult CM-specific Efnb1 KO mice essentially exhibit an impairment of the ventricular diastole with preserved ejection fraction and exercise intolerance, they progressively switch toward systolic heart failure with 100% KO mice dying after 13 months, indicative of a critical role of CM-ephrin-B1 in the adult heart function. This study highlights the molecular determinants and the biological implication of a new late P20-P60 postnatal developmental stage of the heart in rodents during which, in part, ephrin-B1 specifically regulates the maturation of the CM surface crests and of the diastolic function.

Cooccurring Type 1 Diabetes Mellitus and Autoimmune Thyroiditis in a Girl with Craniofrontonasal Syndrome: Are EFNB1 Variants Associated with Autoimmunity?

Craniofrontonasal syndrome (CFNS), also known as craniofrontonasal dysplasia, is an X-linked inherited developmental malformation caused by mutations in the ephrin B1 (EFNB1) gene. The main phenotypic features of the syndrome are coronal synostosis, hypertelorism, bifid nasal tip, dry and curly hair, and longitudinal splitting of nails. A 9-year-and-11-month-old girl with CFNS was admitted due to polyuria, polydipsia, fatigue, and abdominal pain. On physical examination, she had the classical phenotypical features of CFNS. Genetic tests revealed a c.429_430insT (p.Gly144TrpfsTer31) heterozygote variant in the EFNB1 coding region. The patient was diagnosed with type 1 diabetes mellitus (T1DM) and autoimmune thyroiditis based on laboratory findings and symptoms. The mother of the patient, who had the same CFNS phenotype and EFNB1 variant, was screened for autoimmune diseases and was also with autoimmune thyroiditis. This is the first report describing the association of CFNS with T1DM and autoimmune thyroiditis in patients with EFNB1 mutation.

Expression and localisation of ephrin-B1 and EphB4 in steroidogenic cells in the naturally cycling mouse ovary.

Ephrin receptors and ligands are membrane-bound molecules that modulate diverse cellular functions such as cell adhesion, epithelial-mesenchymal transition, motility, differentiation and proliferation. We recently reported the co-expression of ephrin-B1 and EphB4 in adult and foetal Leydig cells of the mouse testis, and thus speculated that their co-expression is a common property in gonadal steroidogenic cells. Therefore, in this study we examined the expression and localisation of ephrin-B1 and EphB4 in the naturally cycling mouse ovary, as their expression patterns in the ovary are virtually unknown. We found that ephrin-B1 and EphB4 were co-expressed in steroidogenic cells of all kinds, i.e. granulosa cells and CYP17A1-positive steroidogenic theca cells as well as in 3ß-HSD-positive luteal cells and the interstitial glands; their co-expression potentially serves as a good marker to identify sex steroid-producing cells even in extra-gonadal organs/tissues. We also found that ephrin-B1 and EphB4 expression in granulosa cells was faint and strong, respectively; ephrin-B1 expression in luteal cells was weak in developing and temporally mature corpora lutea (those of the current cycle) and likely strong in regressing corpora lutea (those of the previous cycle) and EphB4 expression in luteal cells was weak in corpora lutea of the current cycle and likely faint/negative in the corpora lutea of the previous cycle. These findings suggest that a luteinising hormone surge triggers the upregulation of ephrin-B1 and downregulation of EphB4, as this expression fluctuation occurs after the surge. Overall, ephrin-B1 and EphB4 expression patterns may represent benchmarks for steroidogenic cells in the ovary.

ß-Tubulin Isotype, TUBB4B, Regulates The Maintenance of Cancer Stem Cells.

Recent advancements in cancer research have shown that cancer stem cell (CSC) niche is a crucial factor modulating tumor progression and treatment outcomes. It sustains CSCs by orchestrated regulation of several cytokines, growth factors, and signaling pathways. Although the features defining adult stem cell niches are well-explored, the CSC niche is poorly characterized. Since membrane trafficking proteins have been shown to be essential for the localization of critical proteins supporting CSCs, we investigated the role of TUBB4B, a probable membrane trafficking protein that was found to be overexpressed in the membranes of stem cell enriched cultures, in sustaining CSCs in oral cancer. Here, we show that the knockdown of TUBB4B downregulates the expression of pluripotency markers, depletes ALDH1A1+ population, decreases in vitro sphere formation, and diminishes the tumor initiation potential in vivo. As TUBB4B is not known to have any role in transcriptional regulation nor cell signaling, we suspected that its membrane trafficking function plays a role in constituting a CSC niche. The pattern of its expression in tissue sections, forming a gradient in and around the CSCs, reinforced the notion. Later, we explored its possible cooperation with a signaling protein, Ephrin-B1, the abrogation of which reduces the self-renewal of oral cancer stem cells. Expression and survival analyses based on the TCGA dataset of head and neck squamous cell carcinoma (HNSCC) samples indicated that the functional cooperation of TUBB4 and EFNB1 results in a poor prognosis. We also show that TUBB4B and Ephrin-B1 cohabit in the CSC niche. Moreover, depletion of TUBB4B downregulates the membrane expression of Ephrin-B1 and reduces the CSC population. Our results imply that the dynamics of TUBB4B is decisive for the surface localization of proteins, like Ephrin-B1, that sustain CSCs by their concerted signaling.

RNF186 regulates EFNB1 (ephrin B1)-EPHB2-induced autophagy in the colonic epithelial cells for the maintenance of intestinal homeostasis.

Although genome-wide association studies have identified the gene RNF186 encoding an E3 ubiquitin-protein ligase as conferring susceptibility to ulcerative colitis, the exact function of this protein remains unclear. In the present study, we demonstrate an important role for RNF186 in macroautophagy/autophagy activation in colonic epithelial cells and intestinal homeostasis. Mechanistically, RNF186 acts as an E3 ubiquitin-protein ligase for EPHB2 and regulates the ubiquitination of EPHB2. Upon stimulation by ligand EFNB1 (ephrin B1), EPHB2 is ubiquitinated by RNF186 at Lys892, and further recruits MAP1LC3B for autophagy. Compared to control mice, rnf186-/- and ephb2-/- mice have a more severe phenotype in the DSS-induced colitis model, which is due to a defect in autophagy in colon epithelial cells. More importantly, treatment with ephrin-B1-Fc recombinant protein effectively relieves DSS-induced mouse colitis, which suggests that ephrin-B1-Fc may be a potential therapy for human inflammatory bowel diseases.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG16L1: autophagy related 16 like 1; ATP: adenosine triphosphate; Cas9: CRISPR associated protein 9; CD: Crohn disease; CQ: chloroquine; Csf2: colony stimulating factor 2; Cxcl1: c-x-c motif chemokine ligand 1; DMSO: dimethyl sulfoxide; DSS: dextran sodium sulfate; EFNB1: ephrin B1; EPHB2: EPH receptor B2; EPHB3: EPH receptor B3; EPHB2K788R: lysine 788 mutated to arginine in EPHB2; EPHB2K892R: lysine 892 mutated to arginine in EPHB2; ER: endoplasmic reticulum; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; GWAS: genome-wide association studies; HRP: horseradish peroxidase; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IBD: inflammatory bowel diseases; Il1b: interleukin 1 beta; Il6: interleukin 6; IRGM:immunity related GTPase M; i.p.: intraperitoneally; IPP: inorganic pyrophosphatase; KD: knockdown; KO: knockout; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; NOD2: nucleotide binding oligomerization domain containing 2; PI3K: phosphoinositide 3-kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; RNF186: ring finger protein 186; RNF186A64T: alanine 64 mutated to threonine in RNF186; RNF186R179X: arginine 179 mutated to X in RNF186; RPS6: ribosomal protein S6; Tnf: tumor necrosis factor; SQSTM1: sequestosome 1; Ub: ubiquitin; UBE2D2: ubiquitin conjugating enzyme E2 D2; UBE2H: ubiquitin conjugating enzyme E2 H; UBE2K: ubiquitin conjugating enzyme E2 K; UBE2N: ubiquitin conjugating enzyme E2 N; UC: ulcerative colitis; ULK1:unc-51 like autophagy activating kinase 1; WT: wild type.

Clinical and molecular characterization of craniofrontonasal syndrome: new symptoms and novel pathogenic variants in the EFNB1 gene.

BACKGROUND: Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder that results from pathogenic variants in the EFNB1 gene. The syndrome paradoxically presents with greater severity of the symptoms in heterozygous females than hemizygous males. RESULTS: We have recruited and screened a female cohort affected with CFNS. Our primary finding was the description of monozygotic twins, i.e., patients 5 and 6, discordant for the CFNS phenotype. Intriguingly, patient 5 presented classical CFNS gestalt, whereas patient 6 manifested only very subtle craniofacial features, not resembling CFNS. Besides, we have expanded the mutational spectrum of the EFNB1 gene through reporting four novel pathogenic variants-p.(Trp12*), p.(Cys64Phe), p.(Tyr73Metfs*86), p.(Glu210*). All those alterations were found applying either targeted NGS of a custom gene panel or PCR followed by Sanger sequencing and evaluated using in silico predictors. Lastly, we have also expanded the CFNS phenotypic spectrum by describing in patient 3 several novel features of the syndrome, such as bifid hallux, bicornuate uterus, and abnormal right ovary segmented into six parts. CONCLUSIONS: We have described the unreported so far differences of the clinical phenotype in the monozygotic twin patients 5 and 6 harboring an identical p.(Glu210*) variant located in the EFNB1 gene. With our finding, we have pointed to an unusual phenomenon of mildly affected females with CFNS, who may not manifest features suggestive of the syndrome. Consequently, this study may be valuable for geneticists consulting patients with craniofacial disorders.

Ephrin-B1 Is a Novel Biomarker of Bladder Cancer Aggressiveness. Studies in Murine Models and in Human Samples.

Bladder cancer (BC) is the ninth most common cancer worldwide, but molecular changes are still under study. During tumor progression, Epithelial cadherin (E-cadherin) expression is altered and ß-catenin may be translocated to the nucleus, where it acts as co-transcription factor of tumor invasion associated genes. This investigation further characterizes E-cadherin and ß-catenin associated changes in BC, by combining bioinformatics, an experimental murine cell model (MB49/MB49-I) and human BC samples. In in silico studies, a DisGeNET (gene-disease associations database) analysis identified CDH1 (E-cadherin gene) as one with highest score among 130 BC related-genes. COSMIC mutation analysis revealed CDH1 low mutations rates. Compared to MB49 control BC cells, MB49-I invasive cells showed decreased E-cadherin expression, E- to P-cadherin switch, higher ß-catenin nuclear signal and lower cytoplasmic p-Ser33-ß-catenin signal, higher Ephrin-B1 ligand and EphB2 receptor expression, higher Phospho-Stat3 and Urokinase-type Plasminogen Activator (UPA), and UPA receptor expression. MB49-I cells transfected with Ephrin-B1 siRNA showed lower migratory and invasive capacity than control cells (scramble siRNA). By immunohistochemistry, orthotopic MB49-I tumors had lower E-cadherin, increased nuclear ß-catenin, lower pSer33-ß-catenin cytoplasmic signal, and higher Ephrin-B1 expression than MB49 tumors. Similar changes were found in human BC tumors, and 83% of infiltrating tumors depicted a high Ephrin-B1 stain. An association between higher Ephrin-B1 expression and higher stage and tumor grade was found. No association was found between abnormal E-cadherin signal, Ephrin-B1 expression or clinical-pathological parameter. This study thoroughly analyzed E-cadherin and associated changes in BC, and reports Ephrin-B1 as a new marker of tumor aggressiveness.

Astrocytic Ephrin-B1 Controls Synapse Formation in the Hippocampus During Learning and Memory.

Astrocytes play a fundamental role in synapse formation, pruning, and plasticity, which are associated with learning and memory. However, the role of astrocytes in learning and memory is still largely unknown. Our previous study showed that astrocyte-specific ephrin-B1 knock-out (KO) enhanced but ephrin-B1 overexpression (OE) in hippocampal astrocytes impaired contextual memory recall following fear conditioning. The goal of this study was to understand the mechanism by which astrocytic ephrin-B1 influences learning; specifically, learning-induced remodeling of synapses and dendritic spines in CA1 hippocampus using fear-conditioning paradigm. While we found a higher dendritic spine density and clustering on c-Fos-positive (+) neurons activated during contextual memory recall in both wild-type (WT) and KO mice, overall spine density and mEPSC amplitude were increased in CA1 neurons of KO compared to WT. In contrast, ephrin-B1 OE in hippocampal astrocytes impaired dendritic spine formation and clustering, specifically on c-Fos(+) neurons, coinciding with an overall decrease in vGlut1/PSD95 co-localization. Although astrocytic ephrin-B1 influenced learning-induced spine formation, the changes in astrocytic ephrin-B1 levels did not affect spine enlargement as no genotype differences in spine volume were observed between trained WT, KO, and OE groups. Our results suggest that a reduced formation of new spines rather than spine maturation in activated CA1 hippocampal neurons is most likely responsible for impaired contextual learning in OE mice due to abundantly high ephrin-B1 levels in astrocytes. The ability of astrocytic ephrin-B1 to negatively influence new spine formation during learning can potentially regulate new synapse formation at specific dendritic domains and underlie memory encoding.

Compartments with predominant ephrin-B1 and EphB2/B4 expression are present alternately along the excurrent duct system in the adult mouse testis and epididymis.

BACKGROUND: Ephrin receptors (Eph) and ligands are membrane-bound cell-cell communication molecules that regulate the spatial organization of various tissues and organs by repulsive or adhesive signals arising from contact between Eph- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the testis and epididymis are virtually unknown. OBJECTIVES: We aimed to investigate the expression of several EphB receptors and ephrin-B ligands in the testis and epididymis of adult mice. MATERIALS AND METHODS: mRNA and protein expression was detected via reverse transcription-polymerase chain reaction amplification and immunostaining, respectively. RESULTS: Complementary expression patterns were observed in the epithelia along the excurrent duct system in the testis and epididymis; ephrin-B1 was strongly expressed in the epithelia of the rete testis and segment I in the ductus epididymis, whereas EphB2 and/or EphB4 were strongly expressed in the epithelia of the straight tubules and efferent ductules. Moreover, ephrin-B1 was expressed in the spermatogonia, Leydig cells, and peritubular myoid cells in the testis, whereas EphB2 was expressed in elongated spermatids and EphB4 was expressed in the spermatogonia and Leydig cells. Furthermore, these receptors were found to be tyrosine-phosphorylated in the testis and/or epididymis. DISCUSSION: Receptor localization and phosphorylation patterns suggested that EphB/ephrin-B signaling might occur in the seminiferous tubules and epithelial junctions among the straight tubules, rete testis, efferent ductules, and ductus epididymis. Therefore, we propose that EphB/ephrin-B signaling may regulate epithelial boundary formation in the excurrent tubule/ductule/duct system as well as modulate spermatogenesis and spermiation. CONCLUSION: Overall, this study represents the first analysis of EphB receptor and ephrin-B ligand expression in the normal adult testis and epididymis.

A Family with Craniofrontonasal Syndrome and a Mutation (p.G151S) in the EFNB1 Gene: Expanding the Phenotype.

Craniofrontonasal syndrome (CFNS) is a rare genetic entity with X-linked dominant inheritance. CFNS is due to mutations in the Ephrin-B1 (EFNB1) gene. It is characterized by brachycephaly, frontonasal dysplasia, palate/lip defects, dental malocclusion, short neck, split nails, syndactyly, toe and finger defects, and minor skeletal defects. Intelligence is usually unaffected. CFNS exhibits unexpected manifestations between males and females as the latter are more affected. Cellular or metabolic interference due to X inactivation explains the more severe phenotype in heterozygous females. One family with several members affected with CFNS and 100 healthy controls were examined. DNA from leukocytes was isolated to analyze the EFNB1 gene. We did molecular modeling to assess the impact of the mutation on the EFNB1-encoded protein. DNA sequencing analysis of the EFNB1 gene of the affected members showed the heterozygous missense mutation c.451G>A in the EFNB1 gene (GRcH38, chrX: 68,839,708; GERP score in hg38 of 9.961). This transition mutation resulted in the substitution of Gly at position 151 by Ser. Analysis of the healthy members of the family and 100 unrelated controls showed a normal sequence of the EFNB1 gene. Phenotypes of the patients in this family differ from the classical CFNS due to the decreased size of sulci and fissures, subarachnoid space and ventricles, and the absence of a cleft lip/palate.

Use of targeted next-generation sequencing for molecular diagnosis of craniosynostosis: Identification of a novel de novo mutation of EFNB1.

Craniofrontonasal syndrome (CFNS; MIM#304110) is characterized by asymmetric facial features with hypertelorism and a broad bifid nose due to synostosis of the coronal suture. CFNS shows a unique X-linked inheritance pattern (most affected patients are female and obligate male carriers exhibit a mild manifestation or no typical features at all) associated with the ephrin-B1 gene (EFNB1) located in the Xq13.1 region. In this study, we performed targeted, massively parallel sequencing using a next-generation sequencer, and identified a novel EFNB1 mutation, c.270_271delCA, in a Japanese female patient with craniosynostosis. Because subsequent Sanger sequencing identified no mutation in either parent, this mutation was determined to be de novo in origin. After obtaining molecular diagnosis, a retrospective clinical evaluation confirmed the clinical diagnosis of CFNS in this patient. Comprehensive molecular diagnosis using a next-generation sequencer would be beneficial for early diagnosis of the patients with undiagnosed craniosynostosis.

Claudin-5 levels are reduced from multiple cell types in human failing hearts and are associated with mislocalization of ephrin-B1.

Claudin-5 is transcriptionally downregulated resulting in dramatically reduced protein levels in human heart failure. Studies in mice have demonstrated that reduced claudin-5 levels occur prior to cardiac damage and far before reduced whole heart function. Therefore, claudin-5 may be a useful early therapeutic target for human heart failure. However, the cell types in which claudin-5 is localized in human heart and from which claudin-5 is reduced in heart failure is not known. The recent identification of claudin-5's interaction with ephrin-B1 in mouse hearts has also not been investigated in non-failing or failing human hearts. In this study we collected human left ventricular mid-myocardium histological samples from 7 non-failing hearts and 16 end-stage failing hearts. Immunoblots demonstrate severe reductions of claudin-5 protein in 14 of 16 failing hearts compared to non-failing controls. Claudin-5 was observed to localize to cardiomyocytes, endothelial cells, and a subset of fibroblasts in non-failing human heart sections. In isolated cardiomyocytes, the transmembrane claudin-5 protein localized in longitudinal striations in lateral membranes. In failing heart, both cardiomyocyte and endothelial claudin-5 localization was severely reduced, but claudin-5 remained in fibroblasts. Absence of claudin-5 staining also correlated with the reduction of the endothelial cell marker CD31. Ephrin-B1 localization, but not protein levels, was altered in failing hearts supporting that claudin-5 is required for ephrin-B1 localization. These data support that loss of claudin-5 in cardiomyocytes and endothelial cells is prevalent in human heart failure. Investigating claudin-5/ephrin-B1 protein complexes and gene regulation may lead to novel therapies.

Therapeutic target for nephrotic syndrome: Identification of novel slit diaphragm associated molecules.

The slit diaphragm bridging the neighboring foot processes functions as a final barrier of glomerular capillary wall for preventing the leak of plasma proteins into primary urine. It is now accepted that the dysfunction of the sit diaphragm contributes to the development of proteinuria in several glomerular diseases. Nephrin, a gene product of NPHS1, a gene for a congenital nephrotic syndrome of Finnish type, constitutes an extracellular domain of the slit diaphragm. Podocin was identified as a gene product of NPHS2, a gene for a familial steroid-resistant nephrotic syndrome of French. Podocin binds the cytoplasmic domain of nephrin. After then, CD2 associated protein, NEPH1 and transient receptor potential-6 were also found as crucial molecules of the slit diaphragm. In order to explore other novel molecules contributing to the development of proteinuria, we performed a subtraction hybridization assay with a normal rat glomerular RNA and a glomerular RNA of rats with a puromycin aminonucleoside nephropathy, a mimic of a human minimal change type nephrotic syndrome. Then we have found that synaptic vesicle protein 2B, ephrin-B1 and neurexin were already downregulated at the early stage of puromycin aminonucleoside nephropathy, and that these molecules were localized close to nephrin. It is conceivable that these molecules are the slit diaphragm associated molecules, which participate in the regulation of the barrier function. These molecules could be targets to establish a novel therapy for nephrotic syndrome.

Ephrin B1 and RYK expression in DRG of rats after peripheral nerve injury / 基础医学与临床

Objective To study changes of expression in Ephrin and its receptors in DRG neurons after peripheral nerve injury. Methods A rat model of unilateral crushed sciatic nerve was prepared, and changes in the expression pattern of Ephrin B1, Eph B1、Eph B2、Eph B3、EphA4 and RYK in DRG neurons in the rats crushed unilateral sciatic nerve were estimated by the number of positive staining cells in DRG, and the percentage of positive cells as well as the cell size.Results After peripheral nerve injury, the expression of Ephrin B1 significantly decreased, but there was no change in expression of Eph B1、Eph B2、Eph B3 or Eph A4, RYK expression was intensely induced in DRG neurons. Conclusion Ephrin B1 and RYK showed very significant expression changes in ERG neurons after peripheral nerve injury, and may be a component factor process of peripheral never regeneration.