Male patients affected by mosaic PCDH19 mutations: five new cases.

Pathogenic variants in the PCDH19 gene are associated with epilepsy, intellectual disability (ID) and behavioural disturbances. Only heterozygous females and mosaic males are affected, likely due to a disease mechanism named cellular interference. Until now, only four affected mosaic male patients have been described in literature. Here, we report five additional male patients, of which four are older than the oldest patient reported so far. All reported patients were selected for genetic testing because of developmental delay and/or epilepsy. Custom-targeted next generation sequencing gene panels for epilepsy genes were used. Clinical data were collected from medical records. All patients were mosaic in blood for likely pathogenic variants in the PCDH19 gene. In most, clinical features were very similar to the female phenotype, with normal development before seizure onset, which occurred between 5 and 10 months of age, clustering of seizures and sensitivity to fever. Four out of five patients had mild to severe ID and behavioural problems. We reaffirm the similarity between male and female PCDH19-related phenotypes, now also in a later phase of the disorder (ages 10-14 years).

Clinical and genetic aspects of PCDH19-related epilepsy syndromes and the possible role of PCDH19 mutations in males with autism spectrum disorders.

Epilepsy and mental retardation limited to females (EFMR), caused by PCDH19 mutations, has a variable clinical expression that needs further exploration. Onset of epilepsy may be provoked by fever and can resemble Dravet syndrome. Furthermore, transmitting males have no seizures, but are reported to have rigid personalities suggesting possible autism spectrum disorders (ASD). Therefore, this study aimed to determine the phenotypic spectrum associated with PCDH19 mutations in Dravet-like and EFMR female patients and in males with ASD. We screened 120 females suffering from Dravet-like epilepsy, 136 females with EFMR features and 20 males with ASD. Phenotypes and genotypes of the PCDH19 mutation carriers were compared with those of 125 females with EFMR reported in the literature. We report 15 additional patients with a PCDH19 mutation. Review of clinical data of all reported patients showed that the clinical picture of EFMR is heterogeneous, but epilepsy onset in infancy, fever sensitivity and occurrence of seizures in clusters are key features. Seizures remit in the majority of patients during teenage years. Intellectual disability and behavioural disturbances are common. Fifty percent of all mutations are missense mutations, located in the extracellular domains only. Truncating mutations have been identified in all protein domains. One ASD proband carried one missense mutation predicted to have a deleterious effect, suggesting that ASD in males can be associated with PCDH19 mutations.

Endothelial-specific deletion of connexin40 promotes atherosclerosis by increasing CD73-dependent leukocyte adhesion.

BACKGROUND: Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. In the present report, we sought to determine whether Cx40 contributes to the development of the disease. METHODS AND RESULTS: Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Cx40del mice). Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in Cx40del mice after 5 and 10 weeks of a high-cholesterol diet, and spontaneous lesions were observed in the aortic sinuses of young mice without such a diet. These lesions showed monocyte infiltration into the intima, increased expression of vascular cell adhesion molecule-1, and decreased expression of the ecto-enzyme CD73 in the endothelium. The proinflammatory phenotype of Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. Endothelial CD73 is known to induce antiadhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with small interfering RNA or antisense decreased CD73 expression and activity and increased leukocyte adhesion to mouse endothelial cells. These effects were reversed by an adenosine receptor agonist. CONCLUSIONS: Cx40-mediated gap junctional communication contributes to a quiescent nonactivated endothelium by propagating adenosine-evoked antiinflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.

Impaired conduction in the bundle branches of mouse hearts lacking the gap junction protein connexin40.

BACKGROUND: Connexin (Cx)40 and Cx45 are the major protein subunits of gap junction channels in the conduction system of mammals. To determine the role of Cx40, we correlated cardiac activation with Connexin distribution in normal and Cx40-deficient mice hearts. METHODS AND RESULTS: Epicardial and septal activation was recorded in Langendorff-perfused adult mice hearts with a 247-point compound electrode (interelectrode distance, 0.3 mm). After electrophysiological measurements, hearts were prepared for immunohistochemistry and histology to determine Connexin distribution and fibrosis. In both wild-type and Cx40-deficient animals, epicardial activation patterns were similar. The right and left ventricular septum was invariably activated from base to apex. Histology revealed a continuity of myocytes from the common bundle to the septal myocardium. Within this continuity, colocalization was found of Cx43 and Cx45 but not of Cx40 and Cx43. Both animals showed similar His-bundle activation. In Cx40-deficient mice, the proximal bundle branches expressed Cx45 only. The absence of Cx40 in the proximal bundles correlated with right bundle-branch block. Conduction in the left bundle branch was impaired as compared with wild-type animals. CONCLUSIONS: Our data show that (1) in mice, a continuity exists between the common bundle and the septum, and (2) Cx40 deficiency results in right bundle-branch block and impaired left bundle-branch conduction.

Connexin expression in cultured neonatal rat myocytes reflects the pattern of the intact ventricle.

OBJECTIVE: Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts. METHODS: Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain > 75, approximately 50%, and < 25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 were detected by immunofluorescence and immunoblotting. RESULTS: Immunohistochemical stainings confirmed that gap junctions in the atrium and His-Purkinje system were composed of at least Cx43 and Cx40. From the subendocardium towards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wall. In ventricular myocytes cultured at high density (> 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (< 25% confluency) the relative number of cell-cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities. CONCLUSIONS: These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and function.

Developmental changes of connexin40 and connexin43 mRNA distribution patterns in the rat heart.

OBJECTIVES: Gap junctions have been demonstrated ultrastructurally in cardiac regions where connexin40 (Cx40) and connexin43 (Cx43) protein could not be detected immunohistochemically. We investigated therefore the distribution of their mRNAs with more sensitive techniques. METHODS: In situ hybridizations with Cx40 and Cx43 cRNA probes were performed on sections of rat hearts from 9 embryonic days (ED 9) to adults. RESULTS: From ED 13, Cx40 and Cx43 mRNA are detectable in atria and ventricles, but not in their flanking myocardium (inflow tract, atrioventricular canal and outflow tract). Even though Cx40 and Cx43 mRNA eventually become expressed in the inflow tract, they remain undetectable in the sinoatrial node, the atrioventricular canal (including atrioventricular node) and outflow tract. Expression of Cx40 is maximal in the fetal period and declines towards birth. Cx40 expression in the left and right ventricles evolves independently, its mRNA disappearing 4 days earlier from the right than from the left ventricle, and earlier from the free wall than from the trabeculations. Expression of Cx43 mRNA increases during development and changes postnatally from uniform to punctate. Prenatally, Cx43 mRNA was strongest in the subepicardial layer of the ventricular free wall. Nevertheless, we did not detect protein in this layer. CONCLUSIONS: Cardiac regions without detectable Cx40 or Cx43 mRNA either have extremely low levels of expression or express a different connexin. The temporally separate disappearance of Cx40 mRNA from the fetal ventricles implies that left and right ventricles mature independently with respect to gap-junctional communication. The division of the developing heart in compartments where Cx40 and Cx43 mRNA can and cannot be detected, implies pretranslationally regulated gene expression. The postnatally observed subcellular redistribution of Cx43 mRNA coincides with a reported increase in protein expression.

Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution.

OBJECTIVE: The sinoatrial (SA) node consists of a relatively small number of poorly coupled cells. It is not well understood how these pacemaker cells drive the surrounding atrium and at the same time are protected from its hyperpolarizing influence. To explore this issue on a small tissue scale we studied the activation pattern of the mouse SA node region and correlated this pattern with the distribution of different gap junction proteins, connexin (Cx)37, Cx40, Cx43 and Cx45. METHODS AND RESULTS: The mouse SA node was electrophysiologically mapped using a conventional microelectrode technique. The primary pacemaker area was located in the corner between the lateral and medial limb of the crista terminalis. Unifocal pacemaking occurred in a group of pacemaking fibers consisting of 450 cells. In the nodal area transitions of nodal and atrial waveform were observed over small distances ( approximately 100 microm). Correlation between the activation pattern and connexin distribution revealed extensive labeling by anti-Cx45 in the primary and secondary pacemaker area. Within these nodal areas no gradient in Cx45 labeling was found. A sharp transition was found between Cx40- and Cx43-expressing myocytes of the crista terminalis and the Cx45-expressing myocytes of the node. In addition, strands of myocytes labeled for Cx43 and Cx40 protrude into the nodal area. Cx37 labeling was only present between endothelial cells. Furthermore, a band of connective tissue largely separates the nodal from the atrial tissue. CONCLUSIONS: Our results demonstrate strands of Cx43 and Cx40 positive atrial cells protruding into the Cx45 positive nodal area and a band of connective tissue largely separating the nodal and atrial tissue. This organization of the mouse SA node provides a structural substrate that both shields the nodal area from the hyperpolarizing influence of the atrium and allows fast action potential conduction from the nodal area into the surrounding atrium.

Viral rhinitis and asthma.

Upper respiratory tract infections are among the most common infectious diseases. Approximately 80% of the common colds are caused by rhinoviruses. Recently, rhinovirus colds have been linked with lower airway illnesses such as asthma exacerbations resulting in a considerable interest in the pathogenesis of lower respiratory tract pathology. The important role that allergic airway disease plays in virally induced changes in airway function has been experimentally shown in several studies. Unfortunately, the precise mechanisms by which viruses could induce lower airway symptoms have not yet been determined.

Differential connexin distribution accommodates cardiac function in different species.

Using immunohistochemical staining, the distribution of connexin40 (Cx40) and connexin43 (Cx43) was studied in rat, guinea pig, porcine, bovine and human hearts. These species display differences in the degree of morphological differentiation of the conduction system. This study was performed in the anticipation that comparison of the distributions of Cx40 and Cx43 in young and adult specimens may provide clues as to the physiological role of connexins in the heart. To a large extent, the distribution patterns of Cx40 and Cx43 are comparable between species. In neonates and adults, Cx43 was immunolocalized throughout the working myocardium, but in the conduction system Cx43 was detected only after birth. Cx40 was found to appear slightly earlier in development than Cx43 and to disappear when levels of Cx43 became more abundant. This time course was seen in working myocardium and in the ventricular conduction system. Together these data suggest that expression of Cx40 induces or facilitates expression of Cx43, while abundant expression of Cx43 in turn leads to suppression of Cx40 expression. The exceptions to this may represent blocks in this potential regulatory sequence. A second conclusion is that Cx40 and Cx43 containing gap junctions appear in the ventricular conduction system from distal to proximal and only after birth. This indicates that terminal differentiation of the conduction system occurs unexpectedly late in development.

External rhinoplasty approach for septal perforation.

The external rhinoplasty approach was used in nine patients for closure of septal perforations varying from 8 to 30 mm in diameter. Nasal obstruction was the most prominent symptom. An autologous graft was always inserted between the mucoperichondrial flaps. Seven perforations were closed successfully. Symptomatic improvement was achieved in all patients. The closure of nasal septal perforations via external rhinoplasty approach has a high percentage of success due to excellent exposure of the septum.

Distribution of connexin37, connexin40 and connexin43 in the aorta and coronary artery of several mammals.

Intercellular communication between cells of the vessel wall is established by a combination of diffusion and convection of humoral and endothelial factors in the extracellular fluid or by direct intercellular contacts present in the form of gap junctions composed of proteins called connexins. At least connexin (Cx)37, Cx40 and Cx43 are expressed in the vessel wall, but disparate findings with regard to the cell specific localisation of connexins in the vasculature indicate that the distribution of connexins may be species and vessel specific. Moreover, differences in expression exist between cells in culture and tissue sections. We performed an inventory immunohistochemical study on the localisation of Cx37, Cx40 and Cx43 on tissue sections of the bovine, micropig and rat aorta and coronary system, which represent morphologically and functionally different types of vessels in the arterial system. We could observe Cx40 labelling most commonly, although with various intensities, between endothelial and smooth muscle cells of the species studied, with the exception of rat aortic smooth muscle cells. The distribution of Cx43 is more differentiated and mostly confined to smooth muscle cells, although it can be detected scarcely between endothelial cells. Cx37, when detectable, is predominantly expressed between endothelial cells in a heterogeneous pattern. We conclude that Cx40 is the constitutive vascular gap junction protein in situ and guarantees cell coupling between cells in the vessel wall. The differentiated distribution of both Cx37 and Cx43 suggests they are involved in more dynamic processes.

The common cold at the turn of the millennium.

Upper respiratory tract infections are one of the most common infectious diseases in man and are characterized by transient, relatively mild symptoms. Human rhinoviruses are known to be the major causative agent in adult common colds and their relative importance has further increased with the use of the sensitive RT-PCR technique. Characteristic for a common cold is the selective neutrophil recruitment and time-limited increase in mediator, cytokine, and chemokine concentrations that orchestrate chemotaxis, transmigration, and activation of inflammatory and immunocompetent cells. Common cold symptoms are found to correlate to rhinovirus-induced IL-8 elaboration and neutrophil activation. Treatment of rhinoviral upper respiratory tract infections consists of an inhibition of viral infection by antiviral agents and/or a reduction of symptoms by damping the host inflammatory response.

The immune response in adenoids and tonsils.

The adenoid and tonsils are lymphoid tissues located in the pharynx that play an important role in host defense against invading antigens of the upper respiratory tract. Histologically, these structures consist of four well-defined microcompartments which all participate in the immune response: the cryptepithelium, the follicular germinal center with the mantle zone and interfollicular area. With the uptake of antigen by M-cells present in the cryptepithelium a process is initiated which ultimately results in the generation and dissemination of antigen-specific memory and mainly dimeric IgA-producing effector B-lymphocytes. This process requires successful cognate interactions between antigen-presenting cells and lymphocytes and mutually between lymphocytes, which depend not only on antigen-specific signals but also on the expression of various complementary adhesion and costimulatory molecules.

Tumour necrosis factor alpha alters the expression of connexin43, connexin40, and connexin37 in human umbilical vein endothelial cells.

Tumour necrosis factor alpha (TNF-alpha) plays an important role in orchestrating inflammatory responses with the vascular endothelium as main target cell type, and was found to promote migration of endothelial cells, as occurs in wound healing processes. Substantial evidence exists that endothelial cell migration in wound healing is related to changes in cell coupling by means of gap junctions. Gap junctions are agglomerates of cell-to-cell channels that allow direct electrical and metabolic communication between cells. The authors have investigated whether TNF-alpha alters the expression of gap junction proteins (connexins, Cx) between human umbilical vein endothelial cells (HUVEC), thereby changing the extent of intercellular communication, as measured by dye coupling. Under control conditions, Cx43, Cx40, and Cx37 protein and mRNA were present in HUVEC. After exposure to 0.5 nM TNF-alpha for 48 h, however, the authors were no longer able to detect Cx37 and Cx40 protein, whereas Cx43 levels seemed unaltered but showed more perinuclear staining. After 24 and 48 h exposure to TNF-alpha, levels of Cx37 and Cx40 mRNA, were reduced, while the level of Cx43 mRNA remained unaltered, suggesting transcriptional regulation. If TNF-alpha was removed from the medium, Cx37 and Cx40 expression was restored within 24 h. The modulation of connexin expression by TNF-alpha resulted in a decrease in dye coupling of 40%.

Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart.

The developmental appearance and spatial distribution pattern of gap junctions were studied in prenatal and adult rat hearts. Gap junctions were visualized immunohistochemically with an antibody raised against a unique cytoplasmic epitope of connexin43, and the spatial distribution pattern was determined by three-dimensional reconstruction. The results demonstrate that from embryonic day 13 onward, connexin43 becomes detectable immunohistochemically in the myocardium of atria and ventricles. No expression is initially detectable in the myocardium of the sinus venosus, the sinoatrial node, the posterior wall of the atrium and pulmonary veins, the interatrial septum, the atrioventricular canal, including atrioventricular node and bundle, the interventricular septum, and the outflow tract. The developmental increase in the density of gap junctions in atria and ventricles of prenatal hearts correlates well with the reported developmental increase in conduction velocity. Whereas connexin43 becomes expressed in the derivatives of the sinus venosus (except for the sinoatrial node) and in the subepicardial layer of the ventricular free wall shortly before birth, it remains undetectable in the atrioventricular node and bundle and the proximal part of the ventricular conduction tissue, even in the adult heart. The apparent absence of an abundant expression of connexin43 at a location with a supposedly high conduction velocity (i.e., the atrioventricular bundle and bundle branches) is unexpected. These observations were confirmed in studies of the adult mouse heart, which showed, in addition, that connexin32 is not expressed in any part of the heart.

The common cold.

Upper respiratory tract infections are one of the most common infectious diseases in man and are mainly caused by rhinoviruses. A rhinoviral cold is characterized by a neutrophilic inflammatory reaction with relatively mild symptoms that rather result from the host inflammatory response to the virus than from a direct viral cytotoxic effect. As regulators of chemotaxis, transmigration and activators of inflammatory and immunocompetent cells, cytokines and mediators were shown to play a crucial role in the pathogenesis of a rhinovirus infection.

Gap junctions in the rabbit sinoatrial node.

In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchronization and conduction in the sinoatrial node are poorly understood. Therefore, we have taken a combined immunohistochemical and electrophysiological approach to characterize gap junctions in the nodal area. We report that the pacemaker myocytes in the center of the rabbit sinoatrial node express the gap junction proteins connexin (Cx)40 and Cx46. In the periphery of the node, strands of pacemaker myocytes expressing Cx43 intermingle with strands expressing Cx40 and Cx46. Biophysical properties of gap junctions in isolated pairs of pacemaker myocytes were recorded under dual voltage clamp with the use of the perforated-patch method. Macroscopic junctional conductance ranged between 0.6 and 25 nS with a mean value of 7.5 nS. The junctional conductance did not show a pronounced sensitivity to the transjunctional potential difference. Single-channel recordings from pairs of pacemaker myocytes revealed populations of single-channel conductances at 133, 202, and 241 pS. With these single-channel conductances, the observed average macroscopic junctional conductance, 7.5 nS, would require only 30-60 open gap junction channels.

Elevated levels of myeloperoxidase, pro-inflammatory cytokines and chemokines in naturally acquired upper respiratory tract infections.

Upper respiratory tract infections (URTIs) are characterised by a neutrophilic mucosal infiltration. The purpose of this study was to investigate the time course of release of the cytokines/chemokines interleukins (IL) IL-1beta, IL-1ra, tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-8, interferon-gamma (IFN-gamma) and monocyte chemotactic protein (MCP-1), soluble intercellular adhesion molecule-1 (sICAM-1), myeloperoxidase (MPO) and bradykinin in nasal secretions of patients with a naturally acquired URTI. A total of 117 healthy adult volunteers were recruited for baseline nasal lavages, 39 of whom developed URTI symptoms within 6 months and returned to our centre within 48 h. Lavages were performed daily during the symptomatic period and 3 weeks thereafter, with symptoms no longer present. Compared to baseline, significantly elevated concentrations of total protein, bradykinin, IL-1beta, TNF-alpha, IL-6, IL-8, MCP-1, IFN-gamma, MPO and sICAM-1 were detected in nasal lavage fluids of symptomatic patients, whereas IL-1ra remained unaltered. All studied variables reached baseline 3 weeks after the URTI. Naturally acquired URTI represent a limited, neutrophilic inflammatory reaction, orchestrated by the release of pro-inflammatory cytokines and chemokines.

Immunohistochemical delineation of the conduction system. I: The sinoatrial node.

We have raised a mouse monoclonal antibody that reacts specifically with the myocytes of the sinoatrial node of the bovine heart. By use of this antibody (445-6E10) and antibodies against the gap junction protein connexin43, the periphery of the sinoatrial node and the distribution of gap junctions in the nodal region were studied. The reaction patterns of 445-6E10 and anti-connexin43 are exactly complementary; ie, connexin43 was not detected in the nodal myocytes but was clearly present in the atrial myocytes. Both reaction patterns demonstrate that nodal myocytes and atrial myocytes can unambiguously be distinguished by their characteristic molecular phenotype. The transitional nodal myocytes at the periphery of the node that have intermediate morphological and electrophysiological characteristics could now clearly be defined as nodal by our immunohistochemical criteria. The center of the node is surrounded by a region of interdigitating nodal and atrial bundles. Nodal bundles, coming from the center of the node, penetrate the atrial myocardium aligned at atrial bundles, forming histological connections between nodal and atrial myocytes at regular distances. This interdigitating arrangement of bundles of connexin43-negative nodal and connexin43-positive atrial myocytes is also found in the human and rat heart. We hypothesize that the architecture of the periphery of the node is important to prevent silencing of the pacemaking nodal myocytes by the atrium while ensuring a sufficient source loading of the nodal myocytes.

Characterization of gap junction channels in adult rabbit atrial and ventricular myocardium.

For effective cardiac output, it is essential that electrical excitation spread rapidly throughout the atria and ventricles. This is effected by electrical coupling through gap junction channels at contact sites between myocytes. These channels form a low-resistance pathway between adjacent myocytes and consist of connexin proteins. The connexin family is a large multigene family, and the channels formed by different members of this family have distinct electrical and regulatory properties. We have studied gap junction channels between adult rabbit atrial and ventricular myocytes using immunocytochemical and electrophysiological methods. Gap junctions of ventricular myocytes were immunoreactive to antibodies directed against connexin43 (Cx43) and Cx45, but not to antibodies against Cx37 or Cx40. Gap junctions between atrial myocytes showed immunostaining with anti-Cx40, -Cx43, and -Cx45 antibodies, but not with anti-Cx37 antibody. Endocardial and endothelial tissue were labeled with both Cx37 and Cx40 antibodies. The conductance of rabbit myocardial gap junctions was measured using the double whole-cell voltage-clamp method. The average macroscopic junctional conductance, corrected for series resistance, of atrial and ventricular cell pairs did not differ significantly (169+/-146 and 175+/-147 nS, respectively), and both were at most only slightly sensitive to the applied transjunctional potential difference. The difference in connexin expression between atrial and ventricular myocytes was reflected in the distribution of single gap junction channel conductances. A single population of unitary channel conductances with an average of 100 pS was observed between ventricular myocyte pairs. In addition to this population, a population with an average conductance of 185 pS was present between atrial myocyte pairs. The observed difference in connexin expression between atrial and ventricular myocardium may enable differential regulation of conduction in these tissues.