Mice harbouring an oculodentodigital dysplasia-linked Cx43 G60S mutation have severe hearing loss.

Given the importance of connexin43 (Cx43, encoded by GJA1) function in the central nervous system and sensory organ processing, we proposed that it would also be crucial in auditory function. To that end, hearing was examined in two mouse models of oculodentodigital dysplasia that globally express GJA1 mutations resulting in mild or severe loss of Cx43 function. Although Cx43I130T/+ mutant mice, with ∼50% Cx43 channel function, did not have any hearing loss, Cx43G60S/+ mutant mice, with ∼20% Cx43 channel function, had severe hearing loss. There was no evidence of inner ear sensory hair cell loss, suggesting that the mechanism for Cx43-linked hearing loss lies downstream in the auditory pathway. Since evidence suggests that Cx26 function is essential for hearing and may be protective against noise-induced hearing loss, we challenged Cx43I130T/+ mice with a loud noise and found that they had a similar susceptibility to noise-induced hearing loss to that found in controls, suggesting that decreased Cx43 function does not sensitize the mice for environmentally induced hearing loss. Taken together, this study suggests that Cx43 plays an important role in baseline hearing and is essential for auditory processing.This article has an associated First Person interview with the first author of the paper.

Effects of Reduced Connexin43 Function on Mandibular Morphology and Osteogenesis in Mutant Mouse Models of Oculodentodigital Dysplasia.

Mutations in the gene encoding the gap-junctional protein connexin43 (Cx43) are the cause of the human disease oculodentodigital dysplasia (ODDD). The mandible is often affected in this disease, with clinical reports describing both mandibular overgrowth and conversely, retrognathia. These seemingly opposing observations underscore our relative lack of understanding of how ODDD affects mandibular morphology. Using two mutant mouse models that mimic the ODDD phenotype (I130T/+ and G60S/+), we sought to uncover how altered Cx43 function may affect mandibular development. Specifically, mandibles of newborn mice were imaged using micro-CT, to enable statistical comparisons of shape. Tissue-level comparisons of key regions of the mandible were conducted using histomorphology, and we quantified the mRNA expression of several cartilage and bone cell differentiation markers. Both G60S/+ and I130T/+ mutant mice had altered mandibular morphology compared to their wildtype counterparts, and the morphological effects were similarly localized for both mutants. Specifically, the biggest phenotypic differences in mutant mice were focused in regions exposed to mechanical forces, such as alveolar bone, muscular attachment sites, and articular surfaces. Histological analyses revealed differences in ossification of the intramembranous bone of the mandibles of both mutant mice compared to their wildtype littermates. However, chondrocyte organization within the secondary cartilages of the mandible was unaffected in the mutant mice. Overall, our results suggest that the morphological differences seen in G60S/+ and I130T/+ mouse mandibles are due to delayed ossification and suggest that mechanical forces may exacerbate the effects of ODDD on the skeleton.

Manipulating Cx43 expression triggers gene reprogramming events in dermal fibroblasts from oculodentodigital dysplasia patients.

Oculodentodigital dysplasia (ODDD) is primarily an autosomal dominant disorder linked to over 70 GJA1 gene [connexin43 (Cx43)] mutations. For nearly a decade, our laboratory has been investigating the relationship between Cx43 and ODDD by expressing disease-linked mutants in reference cells, tissue-relevant cell lines, 3D organ cultures and by using genetically modified mouse models of human disease. Although salient features of Cx43 mutants have been revealed, these models do not necessarily reflect the complexity of the human context. To further overcome these limitations, we have acquired dermal fibroblasts from two ODDD-affected individuals harbouring D3N and V216L mutations in Cx43, along with familial controls. Using these ODDD patient dermal fibroblasts, which naturally produce less GJA1 gene product, along with RNAi and RNA activation (RNAa) approaches, we show that manipulating Cx43 expression triggers cellular gene reprogramming. Quantitative RT-PCR, Western blot and immunofluorescent analysis of ODDD patient fibroblasts show unusually high levels of extracellular matrix (ECM)-interacting proteins, including integrin α5ß1, matrix metalloproteinases as well as secreted ECM proteins collagen-I and laminin. Cx43 knockdown in familial control cells produces similar effects on ECM expression, whereas Cx43 transcriptional up-regulation using RNAa decreases production of collagen-I. Interestingly, the enhanced levels of ECM-associated proteins in ODDD V216L fibroblasts is not only a consequence of increased ECM gene expression, but also due to an apparent deficit in collagen-I secretion which may further contribute to impaired collagen gel contraction in ODDD fibroblasts. These findings further illuminate the altered function of Cx43 in ODDD-affected individuals and highlight the impact of manipulating Cx43 expression in human cells.

Autosomal recessive GJA1 (Cx43) gene mutations cause oculodentodigital dysplasia by distinct mechanisms.

Oculodentodigital dysplasia (ODDD) is mainly an autosomal dominant human disease caused by mutations in the GJA1 gene, which encodes the gap junction protein connexin43 (Cx43). Surprisingly, there have been two autosomal recessive mutations reported that cause ODDD: a single amino acid substitution (R76H) and a premature truncation mutation (R33X). When expressed in either gap junctional intercellular communication (GJIC)-deficient HeLa cells or Cx43-expressing NRK cells, the R76H mutant trafficked to the plasma membrane to form gap junction-like plaques, whereas the R33X mutant remained diffusely localized throughout the cell, including the nucleus. As expected, the R33X mutant failed to form functional channels. In the case of the R76H mutant, dye transfer studies in HeLa cells and electrical conductance analysis in GJIC-deficient N2a cells revealed that this mutant could form functional gap junction channels, albeit with reduced macroscopic and single channel conductance. Alexa 350 dye transfer studies further revealed that the R76H mutant had no detectable negative effect on the function of co-expressed Cx26, Cx32, Cx37 or Cx40, whereas the R33X mutant exhibited significant dominant or trans-dominant effects on Cx43 and Cx40 as manifested by a reduction in wild-type connexin gap junction plaques. Taken together, our results suggest that the trans-dominant effect of R33X together with its complete inability to form a functional channel may explain why patients harboring this autosomal recessive R33X mutant exhibit greater disease burden than patients harboring the R76H mutant.

Myogenic bladder defects in mouse models of human oculodentodigital dysplasia.

To date, over 65 mutations in the gene encoding Cx43 (connexin43) have been linked to the autosomal-dominant disease ODDD (oculodentodigital dysplasia). A subset of these patients experience bladder incontinence which could be due to underlying neurogenic deterioration or aberrant myogenic regulation. BSMCs (bladder smooth muscle cells) from wild-type and two Cx43 mutant lines (Cx43(G60S) and Cx43(I130T)) that mimic ODDD exhibit a significant reduction in total Cx43. Dye transfer studies revealed that the G60S mutant was a potent dominant-negative inhibitor of co-expressed Cx43, a property not equally shared by the I130T mutant. BSMCs from both mutant mouse strains were defective in their ability to contract, which is indicative of phenotype changes due to harbouring the Cx43 mutants. Upon stretching, Cx43 levels were significantly elevated in controls and mutants containing BSMCs, but the non-muscle myosin heavy chain A levels were only reduced in cells from control mice. Although the Cx43(G60S) mutant mice showed no difference in voided urine volume or frequency, the Cx43(I130T) mice voided less frequently. Thus, similar to the diversity of morbidities seen in ODDD patients, genetically modified mice also display mutation-specific changes in bladder function. Furthermore, although mutant mice have compromised smooth muscle contraction and response to stretch, overriding bladder defects in Cx43(I130T) mice are likely to be complemented by neurogenic changes.

The severity of mammary gland developmental defects is linked to the overall functional status of Cx43 as revealed by genetically modified mice.

Genetically modified mice mimicking ODDD (oculodentodigital dysplasia), a disease characterized by reduced Cx43 (connexin 43)-mediated gap junctional intercellular communication, represent an in vivo model to assess the role of Cx43 in mammary gland development and function. We previously reported that severely compromised Cx43 function delayed mammary gland development and impaired milk ejection in mice that harboured a G60S Cx43 mutant, yet there are no reports of lactation defects in ODDD patients. To address this further, we obtained a second mouse model of ODDD expressing an I130T Cx43 mutant to assess whether a mutant with partial gap junction channel activity would be sufficient to retain mammary gland development and function. The results of the present study show that virgin Cx43I130T/+ mice exhibited a temporary delay in ductal elongation at 4 weeks. In addition, Cx43I130T/+ mice develop smaller mammary glands at parturition due to reduced cell proliferation despite similar overall gland architecture. Distinct from Cx43G60S/+ mice, Cx43I130T/+ mice adequately produce and deliver milk to pups, suggesting that milk ejection is unaffected. Thus the present study suggests that a loss-of-function mutant of Cx43 with partial gap junction channel coupling conductance results in a less severe mammary gland phenotype, which may partially explain the lack of reported lactation defects associated with ODDD patients.

Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects.

The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.

Novel ocular findings in oculodentodigital dysplasia (ODDD): a case report and literature review.

BACKGROUND: Oculodentodigitaldysplasia (ODDD; MIM no. 164200) is a rare hereditary disorder caused by mutations in the gene GJA1.Ocular disorders included microcornea, cornea opacity and glaucoma. However, few studies described fundus findings. MATERIALS AND METHODS: Ophthalmic examination included visual acuity measurement, intraocular pressure (IOP) measurements, slit-lamp biomicroscopy, B-scan ultrasonography, Ultrasound biomicroscopy (UBM), spectral-domain optical coherence tomography (SD-OCT), ERG and retcam fluorescein angiogram. In addition, blood samples were taken from this patient for mutation analyze of GJA1. RESULT: The ophthalmic features of this patient were microcornea, cornea opacity, glaucoma as expected. Interestingly, the patient had a normal axial length with refractive status of emmetropia, but extremely retinal dysplasia and severe choroid thinning was noted. Flash electroretinogram (ERG) was extinguished in both eyes. This study identified a novel mutation c.91A>T in the GJA1 gene associated with fundus abnormalities. Bioinformatics and structural modeling suggested the mutation to be pathogenic. CONCLUSION: Our research expanded not only the mutation spectrum, but also the clinical characteristics of ODDD. To the best of our knowledge, this is the first report on anatomical and functional chorioretinal changes in ODDD patients. These novel ocular features highlight the importance of fundus morphological and functional evaluation in ODDD. ABBREVIATIONS: ODDD: oculodentodigital dysplasia; OCT: optical coherence tomography; ERG: electroretinogram; TACT: teller acuity card test; UBM: ultrasound biomicroscopy; MW: molecular weights; AL: axial length; Cx43: connexin 43; RPE: retinal pigment epithelium; RGCs: retinal ganglion cells; FEVR: familial exudative vitreoretinopathy; ROP: retinopathy of prematurity.

GJA1 Variants Cause Spastic Paraplegia Associated with Cerebral Hypomyelination.

Oculodentodigital dysplasia is an autosomal dominant disorder due to GJA1 variants characterized by dysmorphic features. Neurologic symptoms have been described in some patients but without a clear neuroimaging pattern. To understand the pathophysiology underlying neurologic deficits in oculodentodigital dysplasia, we studied 8 consecutive patients presenting with hereditary spastic paraplegia due to GJA1 variants. Clinical disease severity was highly variable. Cerebral MR imaging revealed variable white matter abnormalities, consistent with a hypomyelination pattern, and bilateral hypointense signal of the basal ganglia on T2-weighted images and/or magnetic susceptibility sequences, as seen in neurodegeneration with brain iron accumulation diseases. Patients with the more prominent basal ganglia abnormalities were the most disabled ones. This study suggests that GJA1-related hereditary spastic paraplegia is a complex neurodegenerative disease affecting both the myelin and the basal ganglia. GJA1 variants should be considered in patients with hereditary spastic paraplegia presenting with brain hypomyelination, especially if associated with neurodegeneration and a brain iron accumulation pattern.

Oculodentodigital Dysplasia: A Hypomyelinating Leukodystrophy with a Characteristic MRI Pattern of Brain Stem Involvement.

Oculodentodigital dysplasia, a rare genetic disorder caused by mutations in the gene encoding gap junction protein 1, classically presents with typical facial features, dental and ocular anomalies, and syndactyly. Oligosymptomatic patients are common and difficult to recognize, in particular if syndactyly is absent. Neurologic manifestation occurs in approximately 30% of patients, and leukodystrophy or T2 hypointensity of gray matter structures or both have been noted in individual patients. To investigate MR imaging changes in oculodentodigital dysplasia, we retrospectively and systematically reviewed 12 MRIs from 6 genetically confirmed patients. Diffuse supratentorial hypomyelination, T2-hypointense Rolandic and primary visual cortex, and symmetric involvement of middle cerebellar peduncle, pyramidal tract, and medial lemniscus was present in all, T2-hypointense pallidum and dentate nucleus in 2 patients each. This consistent, characteristic pattern of diffuse supratentorial hypomyelination and brain stem involvement differs from other hypomyelinating and nonhypomyelinating leukodystrophies with brain stem involvement, and its recognition should trigger genetic testing for oculodentodigital dysplasia.

Two novel GJA1 variants in oculodentodigital dysplasia.

BACKGROUND: Oculodentodigital dysplasia (ODDD) is a rare disorder with pleiotropic effects involving multiple body systems, caused by mutations in the gap junction protein alpha 1 (GJA1) gene. GJA1 gene encodes a polytopic connexin membrane protein, Cx43, that is a component of connexon membrane channels. METHODS: We describe two unrelated female probands referred for a genetic review in view of a dysmorphic clinical phenotype. RESULTS: Two novel missense mutations in GJA1 that substitute conserved amino acids in the first and second transmembrane domains (NM_000165.5: c.77T>C p.Leu26Pro and NM_000165.5:c.287T>G p.Val96Gly) were detected through targeted sequencing of GJA1. These variants were detected in the heterozygous state in the two Maltese probands and segregated with the disease phenotype. CONCLUSION: This report further expands the mutational spectrum of ODDD.

Laurin-Sandrow syndrome: review and redefinition.

We report on a newborn infant with characteristics of Laurin-Sandrow syndrome (LSS). She had hypertelorism, flat nose with grooved collumella, "V" shaped mouth with thin lips, 7 well-recognized and fused digits and 1 additional postaxial bilateral appendix on each hand. The right and left feet had 12 and 11 toes, respectively, the 4 external ones were recognizable, and the rest were fused in a uniform mass but with independent nails. There was also a 2.3 cm-long digitiform appendix in the internal part of both feet. Radiographs showed seven metacarpals and seven metatarsals with similar morphology; both hands lacking thumbs. The four lateral-most toes had regular shaped phalanges and the rest were irregular. The left digitiform appendix had three bones and the right only two. Tibiae were shorter than fibulae. Central Nervous System examination showed an abnormally shaped olivary nucleus, cerebellar cortical heterotopias, gray matter ectopias in both spinal cord and hemispheric white matter, marked ventricular dilatation, and moderate diffuse white matter gliosis. Karyotype was 46XX. A complete necropsy study is presented and all reported cases are reviewed focusing on their phenotypic differences and their nosologic classification. We propose the entity LSS only in cases with symmetric tetrameric polysyndactyly, especially cup-shaped hands and mirror feet, in association with nasal anomalies.

Connexin43 Mutant Patient-Derived Induced Pluripotent Stem Cells Exhibit Altered Differentiation Potential.

We present for the first time the generation of induced pluripotent stem cells (iPSCs) from a patient with a connexin-linked disease. The importance of gap junctional intercellular communication in bone homeostasis is exemplified by the autosomal dominant developmental disorder oculodentodigital dysplasia (ODDD), which is linked to mutations in the GJA1 (Cx43) gene. ODDD is characterized by craniofacial malformations, ophthalmic deficits, enamel hypoplasia, and syndactyly. In addition to harboring a Cx43 p.V216L mutation, ODDD iPSCs exhibit reduced Cx43 mRNA and protein abundance when compared to control iPSCs and display impaired channel function. Osteogenic differentiation involved an early, and dramatic downregulation of Cx43 followed by a slight upregulation during the final stages of differentiation. Interestingly, osteoblast differentiation was delayed in ODDD iPSCs. Moreover, Cx43 subcellular localization was altered during chondrogenic differentiation of ODDD iPSCs compared to controls and this may have contributed to the more compact cartilage pellet morphology found in differentiated ODDD iPSCs. These studies highlight the importance of Cx43 expression and function during osteoblast and chondrocyte differentiation, and establish a potential mechanism for how ODDD-associated Cx43 mutations may have altered cell lineages involved in bone and cartilage development. © 2017 American Society for Bone and Mineral Research.

Specific functional pathologies of Cx43 mutations associated with oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a rare genetic disease that affects the development of multiple organs in the human body. More than 70 mutations in the gap junction connexin43 (Cx43) gene, GJA1, are associated with ODDD, most of which are inherited in an autosomal dominant manner. Many patients exhibit similar clinical presentations. However, there is high intrafamilial and interfamilial phenotypic variability. To better understand this variability, we established primary human dermal fibroblast cultures from several ODDD patients and unaffected controls. In the present study, we characterized three fibroblast lines expressing heterozygous p.L7V, p.G138R, and p.G143S Cx43 variants. All ODDD fibroblasts exhibited slower growth, reduced migration, and defective cell polarization, traits common to all ODDD fibroblasts studied so far. However, we found striking differences in overall expression levels, with p.L7V down-regulated at the mRNA and protein level. Although all of the Cx43 variants could traffic to the cell surface, there were stark differences in gap junction plaque formation, gap junctional intercellular communication, Cx43 phosphorylation, and hemichannel activity among Cx43 variants, as well as subtle differences in myofibroblast differentiation. Together these findings enabled us to discover mutation-specific pathologies that may help to predict future clinical outcomes.

Lobster-claw syndrome.

Lobster-Claw syndrome is a rare autosomal dominant, hand-foot malformation with Oro-dental features. This is a rare condition and from the available reports so far, cases being reported are less than 1%. Most reports have focused on the hand-foot deformity of this syndrome. This paper highlights the typical Oro-dental features associated with this syndrome such as retained deciduous teeth, hypodontia and variation in crown size, arch length and arch width.

Laurin-Sandrow syndrome with additional associated manifestations.

A Thai man with Laurin-Sandrow syndrome (LSS, MIM 135750), the ninth reported case, is described. He had an underdeveloped nasal bone, scar-like seams under the nose, large heads of mandibular condyles, and brachymesophalangy of toes as newly observed findings of the syndrome. He also had mental retardation. The patient had duplication of ulna, with triphalangeal thumbs, and polydactyly of one finger. The triphalangeal thumbs were non-opposable. Carpal bones were malformed. Mirror image polydactyly of the toes was present. There were nine toes on the right and eight on the left. Joint abnormalities were observed at his elbows, wrists, knees, ankles, fingers, and toes. Synostosis of severely malformed tarsal bones was noted. This appears to be the first case of LSS with anomalies not limited to the nose and limbs. The relationship between LSS, tibial hemimelia-polysyndactyly-triphalangeal thumbs syndrome, triphalangeal thumb-polysyndactyly syndrome, preaxial polydactyly types 2 and 3, and Haas-type syndactyly is discussed.

Digitotalar dysmorphism with craniofacial and other new associated abnormalities.

We report digitotalar dysmorphism in a grandfather, father, and a daughter. All the affected members had clasped thumbs. The father had a short stature, large zygomatic arch and a flat mandibular condyle. The newly recognized findings found in the affected girl were large maxillary deciduous central incisors, a short proximal phalanx of the second finger, and a large subcutaneous hemangioma of the back. Her paternal grandfather had only congenital clasped thumbs. Congenital clasped thumb is a very heterogeneous anomaly and related to many syndromes. The findings in the reported family which are consistent with digitotalar dysmorphism, include congenital clasped thumbs, ulnar deviation of fingers, and a congenital vertical tali.

Mild facial dysmorphism and quasidominant inheritance in Cenani-Lenz syndrome.

Cenani-Lenz syndrome (CLS; MIM 212780) is a rare autosomal recessive syndactyly/synostosis syndrome. No facial dysmorphism was previously noted. We studied two families; in the first an affected female had a previously affected brother and her father was said to have been similarly affected. Extensive inbreeding in this family suggests quasidominant inheritance. In the second family there was a history of a similarly affected sib who, in addition, had genital anomalies and cleft palate. The parents were first cousins. Both probands had similar mild facial dysmorphism; a high broad, prominent forehead, hypertelorism, a depressed nasal bridge, downslanting palpebral fissures, a short nose, a short prominent philtrum and malar hypoplasia. The present report suggests mild facial dysmorphism and quasidominant inheritance in one family with Cenani-Lenz syndrome.

Tibial aplasia, lower extremity mirror image polydactyly, brachyphalangy, craniofacial dysmorphism and genital hypoplasia: further delineation and mutational analysis.

Congenital anomalies involving tibial aplasia are rare. Recently, four children with an unusual combination of limb anomalies, facial dysmorphism and genital hypoplasia have been reported. All affected children reported were male. One case noted father to son transmission, implying autosomal dominant inheritance. We report the first female patient with this syndrome. The patient had tibial aplasia, mirror image preaxial polydactyly involving her feet, brachyphalangy, genital hypoplasia as well as facial dysmorphism including telecanthus, blepharophimosis, a flat nasal bridge with a small nose and a small mouth. Consistent with reports in males of a micropenis and hypoplastic scrotum, our patient had absent labia minora and a very small clitoris. Her father had very minor anomalies suggestive of somatic mosaicism or marked variability. Mouse models affecting limb development are powerful tools in the study of human syndromes. The clinical phenotype of patients with this syndrome is reminiscent of some luxoid mouse mutants suggesting Alx4 and related members of the paired homeodomain class as candidate genes. ALX4 haploinsufficiency in humans causes parietal foramina, which one patient with this syndrome was reported to have. Sequencing of coding exons of ALX4 and its related homologue, ALX3, in the proband failed to reveal coding sequence alterations. Our father/daughter pair is the second family reported, supporting a dominant mode of inheritance. Moreover, the very mild phenotype in the father suggests the need for very careful attention to parental examination in such cases.