Connexin Genes Research Articles

Expression of connexin-43 in the cardiac muscle of children diagnosed with hypoplastic left heart syndrome: a Western blot and confocal laser scanning microscopy study.

Hypoplastic left heart syndrome consists of several structural abnormalities in the left side of the heart and may be associated with a hereditary genetic cause, possibly related to the connexin gene GJA1; however, only a few studies have investigated it. The present study aimed to analyse the expression of connexin-43 in the cardiac muscle of hypoplastic left heart syndrome children by Western blot method and confocal laser scanning microscopy. For that, tissue samples were taken during corrective surgery to treat heart defects. Patients of control group (8) presented any type of heart defect not related to hypoplastic left heart syndrome, connexin-43, or its gene and those of hypoplastic left heart syndrome group (9) presented this disease singly, without any other associated congenital diseases. By means of confocal laser scanning microscopy, it was noticed no connexin-43 qualitative differences in positioning and location pattern between both groups. From Western blot analysis, the connexin-43 expression did not show a statistically significant difference (p = 0.0571) as well. Within the limits of this study, it is suggested that cardiomyocytes of hypoplastic left heart syndrome children are similar in connexin-43 location, distribution, and structural and conformational patterns to those of children with heart defects not related to this protein and its genes.

Connexin 43 Mutations Lead to Increased Hemichannel Functionality in Skin Disease.

Gap junctional channels are specialized components of the cellular membrane that allow the intercellular passage of small metabolites, ions, and second messengers to maintain homeostasis. They are comprised of members of the connexin gene family that encode a wide array of proteins that are expressed in nearly every tissue type. Cx43 is perceived to be the most broadly expressed connexin in humans, with several genetic skin diseases being linked to Cx43 mutations specifically. These mutations, in large, produce a gain of functional hemichannels that contribute to the phenotypes of Erythrokeratoderma Variabilis et Progressiva (EKVP), Palmoplantar Keratodemra Congenital Alopecia-1 (PPKCA1), and others that produce large conductance and increased permselectivity in otherwise quiescent structures. Gaining functional hemichannels can have adverse effects in the skin, inducing apoptosis via Ca2+ overload or increased ATP permeability. Here, we review the link between Cx43 and skin disease. We aim to provide insight into the mechanisms regulating the normal and pathophysiological gating of these essential proteins, as well as address current therapeutic strategies. We also demonstrate that transient transfection of neuro-2a (N2a) cells with mutant Cx43 cDNA resulted in increased hemichannel activity compared to wild-type Cx43 and untransfected cells, which is consistent with other studies in the current literature.

Comparative genomic analysis of eutherian connexin genes

The eutherian connexins were characterized as protein constituents of gap junctions implicated in cell-cell communications between adjoining cells in multiple cell types, regulation of major physiological processes and disease pathogeneses. However, conventional connexin gene and protein classifications could be regarded as unsuitable in descriptions of comprehensive eutherian connexin gene data sets, due to ambiguities and inconsistencies in connexin gene and protein nomenclatures. Using eutherian comparative genomic analysis protocol and 35 public eutherian reference genomic sequence data sets, the present analysis attempted to update and revise comprehensive eutherian connexin gene data sets, and address and resolve major discrepancies in their descriptions. Among 631 potential coding sequences, the tests of reliability of eutherian public genomic sequences annotated, in aggregate, 349 connexin complete coding sequences. The most comprehensive curated eutherian connexin gene data set described 21 major gene clusters, 4 of which included evidence of differential gene expansions. For example, the present gene annotations initially described human CXNK1 gene and annotated 22 human connexin genes. Phylogenetic tree calculations and calculations of pairwise nucleotide sequence identity patterns proposed revised and updated phylogenetic classification of eutherian connexin genes. Therefore, the present study integrating gene annotations, phylogenetic analysis and protein molecular evolution analysis proposed new nomenclature of eutherian connexin genes and proteins.

The identification and characterization of the p.G91 deletion in CRYBA1 in a Chinese family with congenital cataracts

BackgroundMutations in more than 52 genes have been identified in isolated congenital cataracts, the majority of which are located in crystalline and connexin (gap junction) genes. An in-frame one amino acid deletion in the beta-crystalline gene CRYBA1 has been reported in several different Chinese, Caucasian and Iranian families of congenital cataracts. Further functional studies are needed to confirm the variant pathogenicity.MethodsThe purpose of this study is to identify the genetic causes that contribute to congenital cataracts with esotropia and nystagmus in a Chinese family. Whole-exome sequencing was performed on samples from all five family members. The two brothers of the father and their daughters were then enrolled in the study, and 40 suspected variants were sequenced among the 9 subjects using Sanger sequencing. The mRNA and protein levels of CRYBA1 in the lens epithelium from cataract patients and normal controls were compared using quantitative polymerase chain reaction (qPCR) and Western blot analyses. The wild-type and mutated forms (p.G91del) of CRYBA1 cDNA were transfected into two types of cell lines, and the expression level of exogenous CRYBA1 was measured by Western blot analysis. The exogenous CRYBA1 proteins were visualized by immunofluorescence staining.ResultsIn this two-generation family, all three descendants inherited congenital cataracts with esotropia and nystagmus from the father, while the mother’s lens was normal. After two rounds of sequencing, CRYBA1 (c. 269–271 del, p.G91del) was identified as the mutation responsible for the autosomal dominant congenital cataract in the Chinese family. CRYBA1 showed lower expression in cataract lenses than in control lenses. The deleted form (p.G91del) of CRYBA1 showed lower expression and was more aggregate to the cell membrane than the wild-type CRYBA1.ConclusionsWe performed molecular experiments to confirm that the p.G91del mutation in CRYBA1 results in abnormal expression and distribution of CRYBA1 protein, and this study could serve as an example of the pathogenicity of an in-frame small deletion in an inherited eye disorder.

The Role of Desmoglein 1 in Gap Junction Turnover Revealed through the Study of SAM Syndrome

An effective epidermal barrier requires structural and functional integration of adherens junctions, tight junctions, gap junctions (GJ), and desmosomes. Desmosomes govern epidermal integrity while GJs facilitate small molecule transfer across cell membranes. Some patients with severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome, caused by biallelic desmoglein 1 (DSG1) mutations, exhibit skin lesions reminiscent of erythrokeratodermia variabilis, caused by mutations in connexin (Cx) genes. We, therefore, examined whether SAM syndrome-causing DSG1 mutations interfere with Cx expression and GJ function. Lesional skin biopsies from SAM syndrome patients (n= 7) revealed decreased Dsg1 and Cx43 plasma membrane localization compared with control and nonlesional skin. Cultured keratinocytes and organotypic skin equivalents depleted of Dsg1 exhibited reduced Cx43 expression, rescued upon re-introduction of wild-type Dsg1, but not Dsg1 constructs modeling SAM syndrome-causing mutations. Ectopic Dsg1 expression increased cell-cell dye transfer, which Cx43 silencing inhibited, suggesting that Dsg1 promotes GJ function through Cx43. As GJA1 gene expression was not decreased upon Dsg1 loss, we hypothesized that Cx43 reduction was due to enhanced protein degradation. Supporting this, PKC-dependent Cx43 S368 phosphorylation, which signals Cx43 turnover, increased after Dsg1 depletion, while lysosomal inhibition restored Cx43 levels. These data reveal a role for Dsg1 in regulating epidermal Cx43 turnover.

Intermittent restraint stress induces circadian misalignment in the mouse bladder, leading to nocturia

Intermittent stress disrupts the circadian rhythm in clock genes such as Per2 only in peripheral organs without any effect on the central circadian clock in the suprachiasmatic nucleus. Here, the effect of restraint stress (RS) on circadian bladder function was investigated based on urination behavior and gene expression rhythms. Furthermore, PF670462 (PF), a Per2 phosphorylation enzyme inhibitor, was administered to investigate the effects on circadian bladder re-alignment after RS. Two-hour RS during the light (sleep) phase was applied to mice (RS mice) for 5 days. The following parameters were then examined: urination behaviors; clock gene expression rhythms and urinary sensory-related molecules such as piezo type mechanosensitive ion channel component 1 (Piezo1), transient receptor potential cation channel subfamily V member 4 (TRPV4), and Connexin26 (Cx26) in the bladder mucosa; Per2 expression in the excised bladder of Per2luciferase knock-in mice (Per2::luc); in vivo Per2 expression rhythms in the bladder of Per2::luc mice. Control mice did not show altered urination behavior in the light phase, whereas RS mice exhibited a higher voiding frequency and lower bladder capacity. In the bladder mucosa, RS mice also showed abrogated or misaligned Piezo1, TRPV4, Connexin26, and clock gene expression. The rhythmic expression of Per2 was also altered in RS mice both in excised- and in vivo bladder, compared with control mice. After PF administration, voiding frequency was reduced and bladder capacity was increased during the light phase in RS mice; the in vivo Per2 expression rhythm was also fully restored. Therefore, RS can alter circadian gene expression in the bladder during the light phase and might cause nocturia via changes in circadian bladder function due the dysregulation of clock genes. Amending the circadian rhythm therapeutically could be applied for nocturia.

Connexin Gap Junction Proteins in Development and Disease

Connexins form tightly‐regulated large‐pore channels that facilitate the passage of various ions, metabolites, and signaling molecules directly between cells, or in some cases and/or pathologies, to the extracellular milieu. The human connexin encoding gene family consists of 21 members and mutations in half of these genes leads to over two dozen diseases ranging in severity from manageable developmental abnormalities to life‐shortening organ failure. Developmentally, connexin expression and gap junctional intercellular communication begins at the 8 cell stage and persists throughout organogenesis resulting in most adult cells expressing two or more connexin subtypes. Connexin genes are exquisitely regulated during all developmental processes before birth, and in many tissues like the skin, after birth. Connexins regulate their function by channel gating processes and notably through their turnover owing to their short half‐life of only a few hours. Given their ubiquitous distribution it is not at all surprising that gene mutations lead to disease. In fact, it is probably more surprising that patients harbouring connexin gene mutations do not present with more clinical morbidities. Our laboratory has used tissue‐relevant cells, primary cells, organotypic cultures, mouse models of human diseases, and induced pluripotent stem cells from connexin‐linked disease patients to uncover ten distinct mechanisms by which connexin gene mutations cause disease. Collectively, connexin‐disease linked mutants have been classified into distinct gain‐ and loss‐of function molecular mechanisms of action. Our long‐term goals include developing strategies to compensate or overcome the cellular and tissue defects triggered by these mutants. This presentation will discuss how connexins have emerged as therapeutic targets in disease and in injury repair. We anticipate that once it is better understood how connexin gene mutations cause disease and abnormalities, which often present more acutely during aging, these findings could be translated into pre‐clinical studies leading to possible treatments for gap junction‐linked diseases.Support or Funding InformationSupported by the Canadian Institutes of Health Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparison of Predictive In Silico Tools on Missense Variants in GJB2, GJB6, and GJB3 Genes Associated with Autosomal Recessive Deafness 1A (DFNB1A).

In silico predictive software allows assessing the effect of amino acid substitutions on the structure or function of a protein without conducting functional studies. The accuracy of in silico pathogenicity prediction tools has not been previously assessed for variants associated with autosomal recessive deafness 1A (DFNB1A). Here, we identify in silico tools with the most accurate clinical significance predictions for missense variants of the GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) connexin genes associated with DFNB1A. To evaluate accuracy of selected in silico tools (SIFT, FATHMM, MutationAssessor, PolyPhen-2, CONDEL, MutationTaster, MutPred, Align GVGD, and PROVEAN), we tested nine missense variants with previously confirmed clinical significance in a large cohort of deaf patients and control groups from the Sakha Republic (Eastern Siberia, Russia): Сх26: p.Val27Ile, p.Met34Thr, p.Val37Ile, p.Leu90Pro, p.Glu114Gly, p.Thr123Asn, and p.Val153Ile; Cx30: p.Glu101Lys; Cx31: p.Ala194Thr. We compared the performance of the in silico tools (accuracy, sensitivity, and specificity) by using the missense variants in GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) genes associated with DFNB1A. The correlation coefficient (r) and coefficient of the area under the Receiver Operating Characteristic (ROC) curve as alternative quality indicators of the tested programs were used. The resulting ROC curves demonstrated that the largest coefficient of the area under the curve was provided by three programs: SIFT (AUC = 0.833, p = 0.046), PROVEAN (AUC = 0.833, p = 0.046), and MutationAssessor (AUC = 0.833, p = 0.002). The most accurate predictions were given by two tested programs: SIFT and PROVEAN (Ac = 89%, Se = 67%, Sp = 100%, r = 0.75, AUC = 0.833). The results of this study may be applicable for analysis of novel missense variants of the GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) connexin genes.

Enhancement of connexin30.3 expression in mouse embryonic stem cell line EB3 in response to cell-cell contacts.

To clarify the potential role of gap junction in cell-cell contact response, the expression of connexin30.3 gene (Cx30.3), a specifically expressed isoform in undifferentiated state of mouse embryonic stem (ES) cell line EB3 was investigated under different cell-cell contact conditions. ES cells were cultured by hanging drop culture method to increase cell-cell contact frequency. As control, a single cell culture was conducted. After culture for 12h, the Cx30.3 expression level in hanging drop culture reached 1.73-fold that of the control (p < 0.001). By contrast, connexin43 gene (Cx43), a ubiquitously expressed gene, showed no difference between both cultures. The experiment of E-cadherin inhibition and β-catenin knockdown suggested the action of E-cadherin upstream of the Cx30.3 regulating pathway. The cell-cell contacts with different cell lines such as HeLa cells and B16/BL6 caused no effect on the Cx30.3 in ES cells. These suggest a potential role of Cx30.3 as a cell-cell contact signal mediator partially regulated by E-cadherin signaling.

Selective Cre-mediated gene deletion identifies connexin 43 as the main connexin channel supporting olfactory ensheathing cell networks.

Many functions of glial cells depend on the formation of selective glial networks mediated by gap junctions formed by members of the connexin family. Olfactory ensheathing cells (OECs) are specialized glia associated with olfactory sensory neuron axons. Like other glia, they form selective networks, however, the connexins that support OEC connectivity in vivo have not been identified. We used an in vivo mouse model to selectively delete candidate connexin genes with temporal control from OECs and address the physiological consequences. Using this model, we effectively abolished the expression of connexin 43 (Cx43) in OECs in both juvenile and adult mice. Cx43-deleted OECs exhibited features consistent with the loss of gap junctions including reduced membrane conductance, largely reduced sensitivity to the gap junction blocker meclofenamic acid and loss of dye coupling. This indicates that Cx43, a typically astrocytic connexin, is the main connexin forming functional channels in OECs. Despite these changes in functional properties, the deletion of Cx43 deletion did not alter the density of OECs. The strategy used here may prove useful to delete other candidate genes to better understand the functional roles of OECs in vivo.

Cancer Prevention and Therapy of Two Types of Gap Junctional Intercellular Communication⁻Deficient "Cancer Stem Cell".

Early observations showed a lack of growth control and terminal differentiation with a lack of gap junctional intercellular communication (GJIC). Subsequent observations showed that epigenetic tumor promoters and activated oncogenes, which block gap junction function, provide insights into the multi-stage, multi-mechanism carcinogenic process. With the isolation of embryonic induced pluri-potent stem cells and organ-specific adult stem cells, gap junctions were linked to early development. While tumors and tumor cell lines are a heterogeneous mixture of “cancer stem cells” and “cancer non-stem cells”, the cancer stem cells seem to be of two types, namely, they express (a) no connexin genes or (b) connexin genes, but do not have functional GJIC. These observations suggest that these “cancer stem cells” originate from normal adult stem cells or from the de-differentiation or re-programming of somatic differentiated cells. This “Concept Paper” provides a hypothesis that “cancer stem cells” either originate from (a) organ-specific adult stem cells before the expression of the connexin genes or (b) organ-specific adult stem cells that just express gap junction genes but that the connexin proteins are rendered dysfunctional by activated oncogenes. Therefore, cancer prevention and therapeutic strategies must account for these two different types of “cancer stem cell”.

The role of TCPTP on leptin effects on astrocyte morphology

Leptin and LPS has been implicated in the development of hypothalamic astrogliosis in rodents. Astrocytes, which are interconnected by gap junction proteins, have emerged as important players in the control of energy homeostasis exerted by the hypothalamus. To investigate the hypothesis of action of T-cell protein tyrosine phosphatase (TCPTP) on the astrocyte morphology, astrocytes from the hypothalamus of one-day-old rats were stimulated with leptin and LPS (used as a positive control). Leptin and LPS induced a marked increase in astrocyte size, an increase in Ptpn2 (TCPTP gene) and gap junction alpha-1 protein, - Gja1 (connexin 43 - CX43 gene) mRNA expression and a decrease in gap junction protein, alpha 6 - Gja6 (CX30 gene) mRNA expression. Remarkably, these effects on astrocytes morphology and connexins were prevented by Ptpn2 siRNA. Astrocytes are known to produce cytokines; here we show that TCPTP acts as an important regulator of the cytokines and it possesses a reciprocal interplay with protein tyrosine phosphatase 1B (PTP1B). Our findings demonstrate that leptin and LPS alter astrocyte morphology by increasing TCPTP, which in turn modulates connexin 30 (CX30) and connexin 43 (CX43) expression. TCPTP and PTP1B seem to act in the regulation of cytokine production in astrocytes.

116 Differential expression of connexin 43 and 37 mRNA transcripts during the estrous cycle in canines

Gap junctions are intercellular channels that mediate cell-to-cell communication, allowing the passage of small signalling molecules. In the ovary, connexin 43 (Cx43) and connexin 37 (Cx37) are important gap junctional proteins expressed in the granulosa and cumulus cells or oocytes of several species. Gap junctions and connexins are required for the regulation of the oocytes meiotic resumption in preovulatory follicles after the surge of LH. However, unlike other species, canine oocytes do not resume meiosis before ovulation, which could be related to expression patterns of Cx43 and Cx37 during oocyte development and ovulation. Therefore, this study aimed to address the canine Cx37 and Cx43 gene expressions throughout the oestrous cycle, including the preovulatory period. The ovaries were obtained from bitches 1-6 years old (n=72) following ovariohysterectomy. The stage of the oestrous cycle was assessed according the ovarian structures and by measurements of serum progesterone (P4) levels obtained from blood samples on the day of surgery. Anestrus was &amp;lt;0.1 ng/mL P4 and absence of follicles or corpus luteum in the ovarian surface; proestrus was 0.2-2 ng/mL P4 and growing small to medium follicles on the surface of the ovaries; oestrus was 2-19 mg/mL P4 and large follicles on the surface of the ovaries; and diestrus was &amp;gt;20 ng/mL P4 and mainly predominant corpus luteum on the ovaries. For Cx43 analysis, follicular cells (granulosa and theca) were mechanically recovered from follicles (n=620) distributed into 4 types: prenatal (1 layer of granulosa cells up to the onset of antrum formation), small antral (~0.2-0.39mm), medium antral (~0.4-5.9mm), and large antral (~6-10mm). For Cx37 study, the cumulus-oocytes complexes (COC) from the same follicles were used. Total RNA extraction was performed, and the evaluation of gene expression levels was achieved by relative quantification quantitative PCR analysis in follicular cells and COC. The data from at least 3 independent experiments for each gene were evaluated by ANOVA. The gene expression of both Connexins were observed in all stages of follicular development; however, the mRNA levels varied over the oestrous cycle. Both Cx43 and Cx37 transcripts showed the highest (P&amp;lt;0.05) levels at anestrus when compared to other phases. The mRNA levels of both genes remained without changes in large follicles at oestrus stage, suggesting that, in contrast to other mammals where LH down-regulates connexins expression leading to the subsequent loss of intercellular coupling, the communication between the oocyte and follicular cells was maintained in canines. In conclusion, these 2 connexin genes were differentially expressed in canine follicular cells and COC during the follicular development. The maintenance of the gene expression of these connexins at the final follicular growth may be involved in the prolonged meiotic arrest in this species. Supported by Ga grant from FONDECYT (1171670).

The role of gene GJB2 and connexin 26 in hearing impairment

The role of gene GJB2 and connexin 26 in hearing impairment

Site specific hypermethylation of CpGs in Connexin genes 30, 26 and 43 in different grades of glioma and attenuated levels of their mRNAs

Aim: Gliomas, the intracranial tumours are considered the deadliest malignancies. The gap junctional Connexins (Cxs) that maintain cellular homeostasis perform a unique function in glial tumour suppression. However, the differential methylation patterns of Cxs were not revealed in glioma so far. The current study attempts to categorise promoter methylation of Cx30 and Cx26 and intron methylation of Cx43 in different grades of human glioma.Materials and methods: About 85 glioma patients with pathologically confirmed grades and 15 control brain tissues were recruited in the study. Bisulphite-PCR-Single Stranded Conformation analysis(SSCA), Bisulphite sequencing and MeDIP-qPCR were carried out to assess methylation status and Cx mRNA levels were also analysed to evaluate the effect of methylation.Results: We found that promoter CpG islands(CpGs) reside in Sp1 and Ap2 sites of Cx30 and 26 were hypermethylated in high grades (HG) of glioma rather than low grades. The input % of both was significantly increased (p < 0.03) in progressive grades. Interestingly, Cx43 could exhibit a significant increase (p < 0.05) in input % only in grade IV. While, Cx30 and 26 mRNAs were downregulated according to their methylation status in progressive fashion with grades, Cx43 was downregulated irrespective of intron methylation.Conclusion: Thus, we suggest that the sites and extent of methylation of Cxs (30 and 26 but not in 43) are found to be altered. In different grades of glioma can provide better appreciation of the grade of the patient and might help in strategies based on epigenetic approaches.

3D Carbon-Nanotube-Based Composites for Cardiac Tissue Engineering.

Heart failure is a disease of epidemic proportion and a leading cause of mortality in the world. Because cardiac myocytes are terminally differentiated cells with minimal intrinsic ability to self-regenerate, cardiac tissue engineering has emerged as one of the most realistic therapeutic strategies for cardiac repair. We have previously proven the ability of carbon nanotube scaffolds to promote cardiomyocyte proliferation, maturation, and long-term survival. Here, we tested if three-dimensional scaffolds of carbon nanotube-based composites can also promote cardiomyocyte growth, electrophysiological maturation, and formation of functional syncytia. To this purpose, we developed an elastomeric scaffold that consists of a microporous and self-standing material made of polydimethylsiloxane (PDMS) containing micrometric cavities, and integrated multiwall carbon nanotubes (MWCNTs) into the scaffold. We combined microscopy, cell biology, and calcium imaging to investigate whether neonatal rat ventricular myocytes (NRVMs) cultured on the 3D-PDMS+MWCNT acquire a more viable and mature phenotype compared to control. We found that when cultured in the 3D-PDMS+MWCNTs, NRVMs showed improved viability (p < 0.005 at day 3) and more defined and mature sarcomeric phenotype compared to 3D-PDMS control. These modifications were associated with an increase of connexin-43 gene expression, gap junction areas (p < 0.005 at day 3), and a more mature electrophysiological phenotype of syncytia and calcium transients. Finally, 3D-PDMS+MWCNT boosted NRVMs proliferation (p < 0.005 at day 3) while hindering cardiac fibroblasts proliferation compared to control PDMS. Thus, 3D-PDMS+MWCNT has the ability to promote viability, proliferation and functional maturation of cardiac myocytes. These properties are essential in cardiac tissue engineering and offer novel perspectives in the development of innovative therapies for cardiac repair.

Gap junction β‑2 expression is negatively associated with the estrogen receptor status in breast cancer tissues and is a regulator of breast tumorigenesis.

Gap junction β‑2 gene (GJB2, also known as connexin26) is a member of the connexin family which forms gap junction channels. Many connexin genes have been considered to be tumor suppressor genes. However, the overexpression of GJB2 has been found to be associated with a poor prognosis in several human cancers. In our previous microarray study, we revealed the overexpression of GJB2 in breast cancer tissues. Hence, in this study, we investigated the expression of GJB2 in human breast cancer and its role in breast cancer cell proliferation and migration. The RT‑qPCR results revealed the upregulation of the GJB2 gene in invasive ductal carcinoma (P<0.001) of the breast. Immunohistochemical analysis revealed an intense cytoplasmic and membrane staining. We observed that the staining for GJB2 was more intense in the majority of the estrogen receptor (ER)‑negative breast cancer tissues compared to the normal breast tissues (P<0.0001). By contrast, the majority of the ER‑positive breast cancer samples exhibited weak to moderate staining; however, this difference was not statistically significant compared to the normal tisues. The knockdown of GJB2 in human breast cancer cell lines using shRNA led to a significant decrease in the proliferative ability and an increase in the migratory ability of breast cancer cells. In addition, the knockdown of GJB‑2 led to a significant reduction in tumor volume and proliferation (as demonstrated by MIB‑1 staining) in orthotopic xenografts in immunocompromised mice. On the whole, the findings of this study indicate that GJB2 may be an important regulator of breast tumorigenesis.

Novel ELOVL4 mutation associated with erythrokeratodermia and spinocerebellar ataxia (SCA 34).

Erythrokeratodermia (EK) is a rare skin disorder, likely genetic and usually present from infancy.1 There is patchy symmetrical involvement over the body surface, manifested in progressive figurate plaques of hyperkeratosis and more transient areas of erythema. There is significant overlap in the clinical and genetic features of the “variabilis” and “progressiva” forms of EK. Restricted cutaneous syndromes of EK have been described associated with mutations in the connexin ( GJB3, GJB4 , and GJA1 ) and loricrin ( LOR ) genes. The majority of patients with EK, however, have no pathogenic mutations in the GJB genes or LOR .

The role of connexins during early embryonic development: pluripotent stem cells, gene editing, and artificial embryonic tissues as tools to close the knowledge gap.

Since almost 4 decades, connexins have been discussed as important regulators of embryogenesis. Several different members of the gene family can be detected in the preimplantation embryo and during gastrulation. However, genetically engineered mice deficient for every connexin expressed during early development are available and even double-deficient mice were generated. Interestingly, all of these mice complete gastrulation without any abnormalities. This raises the question if the role of connexins has been overrated or if other gene family members compensate and mask their importance. To answer this question, embryos completely devoid of any gap junctional communication need to be investigated. This is challenging because a variety of connexin genes are co-expressed and some null mutations lead to a lethal phenotype. In addition, maternal connexin transcripts were described to persist until the blastocyst stage. In this review, we summarize the current knowledge about the role of connexins during preimplantation development and in embryonic stem cells. We propose that the use of pluripotent stem cells, trophoblast stem cells, as well as artificial embryo-like structures and organoid cultures in combination with multiplex CRISPR/Cas9-based genome editing provides a powerful platform to comprehensively readdress this issue and decipher the role of connexins during lineage decision, differentiation, and morphogenesis in a cell culture model for mouse and human development.

The first successful application of preimplantation genetic diagnosis for hearing loss in Iran

Hearing impairment (HI) caused by mutations in the connexin-26 gene (GJB2) accounts for the majority of cases with inherited, nonsyndromic sensorineural hearing loss. Due to the illegality of the abortion of deaf fetuses in Islamic countries, preimplantation genetic diagnosis (PGD) is a possible solution for afflicted families to have a healthy offspring. This study describes the first use of PGD for GJB2 associated non-syndromic deafness in Iran. GJB2 donor splicing site IVS1+1G&gt;A mutation analysis was performed using Sanger sequencing for a total of 71 Iranian families with at least 1 deaf child diagnosed with non-syndromic deafness. In Vitro Fertilization (IVF) was performed, followed by PGD for a cousin couple with a 50% chance of having an affected child. Bi-allelic pathogenic mutations were found in a total of 12 families (~17 %); of which a couple was a PGD volunteer. The deaf woman in this family was homozygous and her husband was a carrier of the IVS1+1G&gt;A gene mutation. Among 8 biopsied embryos, two healthy embryos were implanted which resulted in a single pregnancy and subsequent birth of a healthy baby boy. This is the first report of a successful application of PGD for hearing loss in Iran. Having a baby with a severe hearing impairment often imposes families with long-term disease burden and heavy therapy costs. Today PGD has provided an opportunity for high-risk individuals to avoid the birth of a deaf child.