GJA1 Research Articles

Identification of GJC2 gene mutations in Chinese patients with Pelizaeus-Merzbacher-like disease.

Clinical and genetic features were analyzed in five pedigrees with Pelizaeus-Merzbacher-like disease (PMLD) to provide bases for genetic counseling and prenatal diagnosis. Six patients from five pedigrees were diagnosed with PMLD based on their clinical data. Six GJC2 novel mutations were found in this study, expanding the spectrum of GJC2 mutations. This is the second group of GJC2 mutations reported from six Chinese patients with PMLD. Clinical data including medical history, physical signs, and auxiliary examinations were collected from six patients and their family numbers in five pedigrees with PMLD. Polymerase chain reaction and sequence analysis were used to amplify GJC2 and PLP1 alterations, while multiplex ligation-dependent probe amplification (MLPA) was performed to detect PLP1 dosage changes. The gene mutations were diagnosed for further analysis of the genetic features. A total of seven GJC2 mutations were identified in these patients, including two novel missense mutations (c.217C>T, p.Pro73Ser; c.1199C>A, p.Ala400Glu), one nonsense mutation (c.735C>A, p.Cys245X), three novel frameshift mutations (c.579delC, p.Gly193fsX17 and c.1296_1297insG, p.Gly433fsX59; c.689delG, p.Gly230AlafsX241), and one known missense mutation (c.814T>G, p.Tyr272Asp). Compound heterozygotes were found for P1-3, while homozygotes were found for P4-6 that were inherited from their parents with normal phenotypes except for P5 and P6, respectively. The c.814T>G (p.Tyr272Asp) mutation in P5 was de novo. A c.1199C>A (p.Ala400Glu) homozygous mutation in GJC2 was identified in P6. A heterozygous variation was found in his father and the wild type was seen in his mother.

Increased Hemichannel Activity Displayed by a Connexin43 Mutation Causing a Familial Connexinopathy Exhibiting Hypotrichosis with Follicular Keratosis and Hyperostosis.

Mutations in the GJA1 gene that encodes connexin43 (Cx43) cause several rare genetic disorders, including diseases affecting the epidermis. Here, we examined the in vitro functional consequences of a Cx43 mutation, Cx43-G38E, linked to a novel human phenotype of hypotrichosis, follicular keratosis and hyperostosis. We found that Cx43-G38E was efficiently translated in Xenopus oocytes and localized to gap junction plaques in transfected HeLa cells. Cx43-G38E formed functional gap junction channels with the same efficiency as wild-type Cx43 in Xenopus oocytes, although voltage gating of the gap junction channels was altered. Notably, Cx43-G38E significantly increased membrane current flow through the formation of active hemichannels when compared to wild-type Cx43. These data demonstrate the association of increased hemichannel activity to a connexin mutation linked to a skeletal-cutaneous phenotype, suggesting that augmented hemichannel activity could play a role in skin and skeletal disorders caused by human Cx43 mutations.

Evaluating gap junction variants for a role in pediatric cataract: an overview of the genetic landscape and clinical classification of variants in the GJA3 and GJA8 genes

ABSTRACT Introduction Variants in the two lens-expressed gap junction genes GJA3 and GJA8 are among the most common causes of inherited pediatric cataract. These two genes alone account for up to 18% of the cases, second only to the crystallin gene family. Areas covered All published cataract-associated variants in the GJA3 and GJA8 genes were reviewed for a role in pediatric cataract. Autosomal dominant inheritance was most frequently reported, alongside instances of reduced penetrance and autosomal recessive disease. Variant curation using the ACMG-AMP guidelines identified that many variants do not meet the modern standards for clinical interpretation of pathogenicity. There is broad phenotypic heterogeneity of cataract associated with gap junction gene variants. Pathogenic variants are located throughout both proteins with an enrichment in the N-terminal, first two transmembrane domains, and two extracellular loops. Expert opinion Nearly half the gap junction gene variants observed in cataract patients lack sufficient evidence of pathogenicity to form a useful clinical opinion. For many variants, this may be rectified over time as the variant is observed in additional patients but would be vastly accelerated by the generation of well-characterized and standardized functional data evaluating the specific effect of each variant on protein function.

Identification of a Missense Mutation in GJA8 Gene in an Iranian Family with Autosomal Dominant Congenital Cataract.

To identify the causative mutations of autosomal dominant (AD) congenital cataracts in a large Iranian family. The complete and accurate family history and clinical information of participants were collected. A total of 51 family members, including 22 affected and 29 unaffected individuals, were recruited in this study. We performed whole exome sequencing to reveal pathogenic mutation. We used amplification refractory mutation system polymerase chain reaction and Sanger sequencing techniques to confirm segregation in patients and also to rule it out in the healthy participants. A known missense mutation, c.827C>T (S276F), in GJA8 was identified. This mutation was confirmed in all patients. Neither all healthy family members nor 100 healthy individuals who served as controls from general population had this mutation. The missense mutation c. 827C>T in the GJA8 gene is associated with AD congenital lamellar cataract with complete penetrance in a six-generation Iranian family.

Pelizaeus-Merzbacher disease-like disorder in an Indian girl with a missense variant in GJC2 gene

Pelizaeus-Merzbacher-like disease 1 (PMLD1) is a slowly progressive leukodystrophy that typically presents during the neonatal or early infantile period with nystagmus, commonly associated with hypotonia, delayed acquisition of motor milestones, speech delay and dysarthria. We present a 7-year-old female born to a nonconsanguineous marriage with developmental delay. On examination, she had 22 teeth, and nystagmus with pseudophakia. Neurological examination showed spasticity with increased deep tendon reflexes. On investigation, MRI of the brain done at 3 years showed hypomyelination. Targeted exome sequencing revealed a homozygous non-synonymous variation c.138C>G in exon 2 of the GJC2 gene. Sanger sequencing was done which showed the presence of a variant in the heterozygous state in both parents. PMLD1 should be suspected in any child presenting with diffuse hypomyelination with abnormal eye movements, especially in a girl child with PelizaeusMerzbacher disease phenotype with hypomyelination in the pons.

Internal translation of Gja1 (Connexin43) to produce GJA1-20k: Implications for arrhythmia and ischemic-preconditioning.

Internal translation is a form of post-translation modification as it produces different proteins from one mRNA molecule by beginning translation at a methionine coding triplet downstream of the first methionine. Internal translation can eliminate domains of proteins that otherwise restrict movement or activity, thereby creating profound functional diversity. Connexin43 (Cx43), encoded by the gene Gja1, is the main gap junction protein necessary for propagating action potentials between adjacent cardiomyocytes. Gja1 can be internally translated to produce a peptide 20kD in length named GJA1-20k. This review focuses on the role of GJA1-20k in maintaining cardiac electrical rhythm as well as in ischemic preconditioning (IPC). Connexin43 is the only ion channel we are aware that has been reported to be subject to internal translation. We expect many other ion channels also undergo internal translation. The exploration of post-translational modification of ion channels, and in particular of internal translation, has the potential to greatly increase our understanding of both canonical and non-canonical ion channel biology.

Новая мутация p.Glu48Gly в гене GJA8 в компаунд-гетерозиготном состоянии у пациента со спорадической врожденной зонулярной катарактой

Значительная клиническая и генетическая гетерогенность наследственных катаракт существенно затрудняет их раннюю ДНК-диагностику и определяет актуальность изучения эпидемиологии заболевания, популяционных особенностей спектра и частот мутаций в ответственных генах, клинико-генетических корреляций. Мутации в гене коннексина 50 (GJA8) относятся к частым причинам наследственных, преимущественно аутосомно-доминантных, врожденных катаракт. У пациента с врожденной двусторонней зонулярной катарактой в гене GJA8 идентифицированы две нуклеотидные замены в компаунд-гетерозиготном состоянии - ранее не описанный миссенс-вариант с.143A>G (p.Glu48Gly) и известный нуклеотидный вариант с.741Т>G (p.Ile24Met). На основании клинико-генеалогического и молекулярно-генетического анализа установлен спорадический характер заболевания в семье. Впервые выявленный вариант p.Glu48Gly, предположительно, является патогенной мутацией, проявляющей рецессивный характер. Significant clinical and genetic heterogeneity of hereditary cataracts significantly complicates their early DNA diagnosis and determines the relevance of studying the epidemiology of the disease, population features of the spectrum and frequencies of mutations in responsible genes, clinical and genetic correlations. Mutations in the connexin 50 (GJA8) gene are common causes of hereditary, mainly autosomal dominant, congenital cataracts. In a patient with congenital bilateral zonular cataract in the GJA8 gene two nucleotide substitutions were identified in the compound heterozygous state - the previously undescribed missense variant C.143A>G (p.Glu48Gly) and the known nucleotide variant C.741T>G (p.Ile24Met). Based on clinical genealogical and molecular genetic analysis, the sporadic nature of the disease in the family was established. The newly identified variant of p.Glu48Gly is presumably a pathogenic mutation showing a recessive character.

GJA1/CX43 High Expression Levels in the Cervical Spinal Cord of ALS Patients Correlate to Microglia-Mediated Neuroinflammatory Profile.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motoneurons (MNs) with a fatal outcome. The typical degeneration of cortico-spinal, spinal, and bulbar MNs, observed in post-mortem biopsies, is associated with the activation of neuroimmune cells. GJA1, a member of the connexins (Cxs) gene family, encodes for connexin 43 (Cx43), a core gap junctions (GJs)- and hemichannels (HCs)-forming protein, involved in cell death, proliferation, and differentiation. Recently, Cx43 expression was found to play a role in ALS pathogenesis. Here, we used microarray and RNA-seq datasets from the NCBI of the spinal cord of control (NDC) and ALS patients, which were stratified according to the GJA1 gene expression. Genes that positively or negatively correlated to GJA1 expression were used to perform a genomic deconvolution analysis (GDA) using neuroimmune signatures. Expression analysis revealed a significantly higher GJA1 expression in the MNs of ALS patients as compared to NDC. Gene deconvolution analysis revealed that positively correlated genes were associated with microglia activation, whereas negatively correlated genes were associated with neuronal activation profiles. Moreover, gene ontology analysis, performed on genes characterizing either microglia or neuronal signature, indicated immune activation or neurogenesis as main biological processes. Finally, using a synthetic analysis of drugs able to revert the GJA1 transcriptomic signatures, we found a specific drug profile for ALS patients with high GJA1 expression levels, composed of amlodipine, sertraline, and prednisolone. In conclusion, our exploratory study suggests GJA1 as a new neuro-immunological gene correlated to microglial cellular profile in the spinal cord of ALS patients. Further studies are warranted to confirm these results and to evaluate the therapeutic potential of drugs able to revert typical GJA1/CX43 signature in ALS patients

GJA1 gene polymorphism is a genetic predictor of recurrence after pulmonary vein isolation in patients with paroxysmal atrial fibrillation

Atrial fibrillation (AF) and recurrence of AF after pulmonary vein isolation (PVI) have been linked to sinus node dysfunction. The purpose of this study was to investigate the association between the heart rate-associated single nucleotide polymorphisms (SNPs) identified in genome-wide association studies and recurrence of AF after PVI. In this study, patients with paroxysmal AF who underwent initial PVI, including 522 patients for screening and 172 patients for replication, were recruited and 21 heart rate-associated SNPs identified in genome-wide association studies were genotyped. The association between these SNPs and the recurrence of AF was investigated. Throughout the follow-up period of 21 ± 12 months, 119 patients with paroxysmal AF (22.8%) exhibited AF recurrences in the screening set. The rate of AF recurrence was significantly associated with the minor allele C of the gap junction alpha-1 protein (GJA1) rs1015451 (additive model: odds ratio 2.07; P = 9.32 × 10-7), but not with other SNPs. This association was confirmed in the replication set (allelic model: odds ratio 1.81; P = 2.70 × 10-2). Multivariate analysis revealed that the recurrence of AF after AF ablation was independently related to the GJA1 SNP rs1015451 additive model, duration of AF >1 year, AF from non-pulmonary vein foci, and thicker interventricular septum. The GJA1 SNP rs1015451, coding for a gap junction protein (connexin-43), may be considered a novel genetic marker for AF recurrence after PVI.

Milk-derived small extracellular vesicles: nanomaterials to promote bone formation

Small extracellular vesicles (sEVs) are an important component in the paracrine pathway. They can be used as a substitute for seed cells and have shown good application prospects in promoting bone regeneration. Cow’s milk could be used as a source of sEVs with good biocompatibility and cost-effectiveness, with easy availability, low cost and low toxicity. This study focused on the role and mechanism of small extracellular vesicles derived from milk in bone repair. In order to explore the mechanism via which Milk-sEVs promote bone repair, we screened the differential gene GJA1 in Milk-sEV-treated osteoblasts through transcriptome chips, and verified the transcript AP3B1 of GJA1 through chromatin immunoprecipitation (CHIP). We have proved by in vivo and in vitro experiments that milk-derived sEVs (Milk-sEVs) increase the repair ability of bone tissue, and promote expression of the osteogenic gene GJA1 through the transcript AP3B1.Graphical abstract

GJA1遺伝子多型マイナーアレルは心房細動中の高心拍数に関与している

GJA1遺伝子多型マイナーアレルは心房細動中の高心拍数に関与している

Ephaptic Coupling Is a Mechanism of Conduction Reserve During Reduced Gap Junction Coupling.

Many cardiac pathologies are associated with reduced gap junction (GJ) coupling, an important modulator of cardiac conduction velocity (CV). However, the relationship between phenotype and functional expression of the connexin GJ family of proteins is controversial. For example, a 50% reduction of GJ coupling has been shown to have little impact on myocardial CV due to a concept known as conduction reserve. This can be explained by the ephaptic coupling (EpC) theory whereby conduction is maintained by a combination of low GJ coupling and increased electrical fields generated in the sodium channel rich clefts between neighboring myocytes. At the same time, low GJ coupling may also increase intracellular charge accumulation within myocytes, resulting in a faster transmembrane potential rate of change during depolarization (dV/dt_max) that maintains macroscopic conduction. To provide insight into the prevalence of these two phenomena during pathological conditions, we investigated the relationship between EpC and charge accumulation within the setting of GJ remodeling using multicellular simulations and companion perfused mouse heart experiments. Conduction along a fiber of myocardial cells was simulated for a range of GJ conditions. The model incorporated intercellular variations, including GJ coupling conductance and distribution, cell-to-cell separation in the intercalated disc (perinexal width—WP), and variations in sodium channel distribution. Perfused heart studies having conditions analogous to those of the simulations were performed using wild type mice and mice heterozygous null for the connexin gene Gja1. With insight from simulations, the relative contributions of EpC and charge accumulation on action potential parameters and conduction velocities were analyzed. Both simulation and experimental results support a common conclusion that low GJ coupling decreases and narrowing WP increases the rate of the AP upstroke when sodium channels are densely expressed at the ends of myocytes, indicating that conduction reserve is more dependent on EpC than charge accumulation during GJ uncoupling.

Transcriptional profile of cumulus associated GJA1, PTX3, PRSS35, and SERPINE2 genes with oocytes and embryonic development in water buffalo.

In the present study, the potential of different groups of cumulus-oocyte complexes (COC's) for in vitro maturation (IVM) and embryonic development was assessed in two groups of COC's of water buffalo. Further, the expression pattern of cumulus-associatedGJA1,PTX3, PRSS35,andSERPINE2genes and their effects on embryonic development was analyzed. Slaughterhouse-derived buffalo COC's were graded into groups A and B. An equal number of 410 COC's were taken in both groups. IVM was carried out using Slaughterhouse-derived buffalo epididymis. A remarkable degree of cumulus expansion was noticed in group A (92.68%) as compared to group B (81.25%) oocytes. On in vitro fertilization (IVF) and embryo culture, group A produced a significantly higher rate of cleavage and blastocyst (92.682 ± 0.7179% and 42.682 ± 0.9683%) as compared to group B (85.365 ± 0.7608% and 31.707 ± 0.9688%). Also, the transcriptional analysis of cumulus-associated genes revealed significantly higher expression in group A as compared to group B. It was revealed that oocytes having good cumulus mass had a higher developmental potential. Based on differential gene expression of cumulus-associated genes, different quality of COC's, and the resultant embryos after IVF, it was concluded that these genes could be used as a marker for predicting the developmental competence of the oocytes. We concluded that morphologically good quality of COC's had a higher developmental competence, and also the differential expressions of cumulus-associated genes in cumulus cells and embryos. So, we can conclude that these genes could be used as marker genes for predicting the developmental competence of buffalo's oocytes.

Melatonin treatment improves ventricular conduction via upregulation of Nav1.5 channel proteins and sodium current in the normal rat heart

Melatonin treatment was reported to reduce the risk of cardiac arrhythmias, and crucial for this antiarrhythmic action was the effect of melatonin on activation spread. The aim of the present study was evaluation of the mechanisms of this activation enhancement.Experiments were performed in a total of 123 control and melatonin-treated (10 mg/kg, daily, for 7 days) male Wistar rats. In epicardial mapping studies (64 leads, interlead distance 0.5 mm) in the anesthetized animals, activation times (ATs) were determined in each lead as dV/dt minimum during QRS complex under sinus rhythm. Epicardial pacing was performed to measure conduction velocity (CV) across the mapped area. Average left ventricular ATs were shorter in the treated animals as compared to the controls, whereas the minimal epicardial ATs indicating the duration of activation propagation via the ventricular conduction system did not differ between the groups. CV was higher in the treated groups indicating that melatonin affected conduction via contractile myocardium The area of Cx43-derived fluorescence, as well as the expression of Cx43 protein, was similar in ventricles in the control and melatonin-treated groups. Expression of Gja1 gene transcripts encoding Cx43, was increased in the last group. An uncoupling agent octanol modified myocardial conduction properties (time of activation, action potential upstroke velocity, passive electrotonic phase duration) similarly in both groups. On the other hand, the expression of both Scn5a gene transcripts encoding Nav1.5 proteins, as well as peak density of transmembrane sodium current were increased in the ventricular myocytes from the melatonin-treated animals. Thus, a week-long melatonin treatment caused the increase of conduction velocity via enhancement of sodium channel proteins expression and increase of sodium current in the ventricular myocytes.

GJA1-20k and Mitochondrial Dynamics.

Connexin 43 (Cx43) is the primary gap junction protein of mammalian heart ventricles and is encoded by the gene Gja1 which has a single coding exon and therefore cannot be spliced. We previously identified that Gja1 mRNA undergoes endogenous internal translation initiated at one of several internal AUG (M) start codons, generating N-terminal truncated protein isoforms that retain the C-terminus distal to the start site. GJA1-20k, whose translation initiates at mRNA M213, is usually the most abundant isoform in cells and greatly increases after ischemic and metabolic stress. GJA1-20k consists of a small segment of the last transmembrane domain and the complete C-terminus tail of Cx43, with a total size of about 20 kDa. The original role identified for GJA1-20k is as an essential subunit that facilitates the trafficking of full-length Cx43 hexameric hemichannels to cell-cell contacts, generating traditional gap junctions between adjacent cells facilitating, in cardiac muscle, efficient spread of electrical excitation. GJA1-20k deficient mice (generated by a M213L substitution in Gja1) suffer poor electrical coupling between cardiomycytes and arrhythmogenic sudden death two to 4 weeks after their birth. We recently identified that exogenous GJA1-20k expression also mimics the effect of ischemic preconditioning in mouse heart. Furthermore, GJA1-20k localizes to the mitochondrial outer membrane and induces a protective and DRP1 independent form of mitochondrial fission, preserving ATP production and generating less reactive oxygen species (ROS) under metabolic stress, providing powerful protection of myocardium to ischemic insult. In this manuscript, we focus on the detailed roles of GJA1-20k in mitochondria, and its interaction with the actin cytoskeleton.

Identification of Potential Driver Genes and Pathways Based on Transcriptomics Data in Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. To identify AD-related genes from transcriptomics and help to develop new drugs to treat AD. In this study, firstly, we obtained differentially expressed genes (DEG)-enriched coexpression networks between AD and normal samples in multiple transcriptomics datasets by weighted gene co-expression network analysis (WGCNA). Then, a convergent genomic approach (CFG) integrating multiple AD-related evidence was used to prioritize potential genes from DEG-enriched modules. Subsequently, we identified candidate genes in the potential genes list. Lastly, we combined deepDTnet and SAveRUNNER to predict interaction among candidate genes, drug and AD. Experiments on five datasets show that the CFG score of GJA1 is the highest among all potential driver genes of AD. Moreover, we found GJA1 interacts with AD from target-drugs-diseases network prediction. Therefore, candidate gene GJA1 is the most likely to be target of AD. In summary, identification of AD-related genes contributes to the understanding of AD pathophysiology and the development of new drugs.

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein.

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.

Absence of Connexin 43 Results in Smaller Retinas and Arrested, Depolarized Retinal Progenitor Cells in Human Retinal Organoids.

The development of the vertebrate retina relies on complex regulatory mechanisms to achieve its characteristic layered morphology containing multiple neuronal cell types. While connexin 43 (CX43) is not expressed by mature retinal neurons, mutations in its gene GJA1 are associated with microphthalmia and low vision in patients. To delineate how lack of CX43 affects retinal development, GJA1 was disrupted in human induced pluripotent stem cells (hiPSCs) (GJA1-/-) using CRISPR/Cas9 editing, and these were subsequently differentiated into retinal organoids. GJA1-/- hiPSCs do not display defects in self-renewal and pluripotency, but the resulting organoids are smaller with a thinner neural retina and decreased abundance of many retinal cell types. CX43-deficient organoids express lower levels of the neural marker PAX6 and the retinal progenitor cell (RPC) markers PAX6, SIX3, and SIX6. Conversely, expression of the early neuroectoderm markers SOX1 and SOX2 remains high in GJA1-/- organoids throughout their development. The lack of CX43 results in an increased population of CHX10-positive RPCs that are smaller, disorganized, do not become polarized, and possess a limited ability to commit to retinal fate specification. Our data indicate that lack of CX43 causes a developmental arrest in RPCs that subsequently leads to pan-retinal defects and stunted ocular growth.

Interrogation of Carboxy-Terminus Localized GJA1 Variants Associated with Erythrokeratodermia Variabilis et Progressiva.

Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell–cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.

Broadening the Mutation Spectrum in GJA8 and CHMP4B: Novel Missense Variants and the Associated Phenotypes in Six Chinese Han Congenital Cataracts Families.

Purpose: To broaden the mutation and phenotype spectrum of the GJA8 and CHMP4B genes and to reveal genotype-phenotype correlations in a cohort of Chinese patients with congenital cataracts (CCs).Methods: Six Chinese Han families with CCs inherited in an autosomal dominant (AD) pattern were recruited for this study. All patients underwent full ocular examinations. Genomic DNA was extracted from the leukocytes of peripheral blood collected from all available patients and their unaffected family members. Whole-exome sequencing (WES) was performed on all probands and at least one of their parents. Candidate variants were further confirmed by Sanger sequencing. Bioinformatic analysis with several computational predictive programs was performed to assess the impacts of the candidate variants on the structure and function of the proteins.Results: Four heterozygous candidate variants in three different genes (CRYBB2, GJA8, and CHMP4B) were identified in affected individuals from the six families, including two novel missense variants (GJA8: c.64G > C/p. G22R, and CHMP4B: c.587C > G/p. S196C), one missense mutation (CRYBB2: c.562C > T/p. R188C), and one small deletion (GJA8: c.426_440delGCTGGAGGGGACCCT/p.143_147delLEGTL). The three missense mutations were predicted as deleterious in all four computational prediction programs. In the homologous model, the GJA8: p.143_147delLEGTL mutation showed a sequence deletion of five amino acids at the cytoplasmic loop of the Cx50 protein, close to the third transmembrane domain. Patients carrying mutations in the same gene showed similar cataract phenotypes at a young age, including total cataracts, Y-sutural with fetal nuclear cataracts, and subcapsular cataracts.Conclusion: This study further expands the mutation spectrum and genotype-phenotype correlation of CRYBB2, GJA8, and CHMP4B underlying CCs. This study sheds light on the importance of comparing congenital cataract phenotypes in patients at the same age stage. It offers clues for the pathogenesis of CCs and allows for an early prenatal diagnosis for families carrying these genetic variants.