A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia

Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder characterized by pleiotropic developmental anomalies of the limbs, teeth, face and eyes that was shown recently to be caused by mutations in the gap junction protein alpha 1 gene (GJA1), encoding connexin 43 (Cx43). In the course of performing an N-ethyl-N-nitrosourea mutagenesis screen, we identified a dominant mouse mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1 leading to the substitution of a highly conserved amino acid (G60S) in Cx43. In vivo and in vitro studies revealed that the mutant Cx43 protein acts in a dominant-negative fashion to disrupt gap junction assembly and function. In addition to the classic features of ODDD, these mutant mice also showed decreased bone mass and mechanical strength, as well as altered hematopoietic stem cell and progenitor populations. Thus, these mice represent an experimental model with which to explore the clinical manifestations of ODDD and to evaluate potential intervention strategies.     

A novelGJA1 missense mutation in a Polish child with oculodentodigital dysplasia

Oculodentodigital dysplasia (ODDD) (OMIM #164200) is a rare congenital, autosomal dominant disorder comprising craniofacial, ocular, dental, and digital anomalies. The syndrome is caused byGJA1 mutations. The clinical phenotype of ODDD involves a characteristic dysmorphic facies, ocular findings (microphthalmia, microcornea, glaucoma), syndactyly type III of the hands, phalangeal abnormalities, diffuse skeletal dysplasia, enamel dysplasia, and hypotrichosis. In a Polish child with the clinical symptoms typical of ODDD, we demonstrated a novel missense mutation c.C31T resulting in p.L11F substitution. Our report provides evidence on the importance of this highly conserved amino acid residue for the proper functioning of GJA1 protein.     

Gap junctions in inherited human disorders of the central nervous system

CNS glia and neurons express connexins, the proteins that form gap junctions in vertebrates. We review the connexins expressed by oligodendrocytes and astrocytes, and discuss their proposed physiologic roles. Of the 21 members of the human connexin family, mutations in three are associated with significant central nervous system manifestations. For each, we review the phenotype and discuss possible mechanisms of disease. Mutations in GJB1, the gene for connexin 32 (Cx32) cause the second most common form of Charcot-Marie-Tooth disease (CMT1X). Though the only consistent phenotype in CMT1X patients is a peripheral demyelinating neuropathy, CNS signs and symptoms have been found in some patients. Recessive mutations in GJC2, the gene for Cx47, are one cause of Pelizaeus-Merzbacher-like disease (PMLD), which is characterized by nystagmus within the first 6months of life, cerebellar ataxia by 4years, and spasticity by 6years of age. MRI imaging shows abnormal myelination. A different recessive GJC2 mutation causes a form of hereditary spastic paraparesis, which is a milder phenotype than PMLD. Dominant mutations in GJA1, the gene for Cx43, cause oculodentodigital dysplasia (ODDD), a pleitropic disorder characterized by oculo-facial abnormalities including micropthalmia, microcornia and hypoplastic nares, syndactyly of the fourth to fifth fingers and dental abnormalities. Neurologic manifestations, including spasticity and gait difficulties, are often but not universally seen. Recessive GJA1 mutations cause Hallermann-Streiff syndrome, a disorder showing substantial overlap with ODDD. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and functions.     

Differential potency of dominant negative connexin43 mutants in oculodentodigital dysplasia

Oculodentodigital dysplasia (ODDD) is a congenital autosomal dominant disorder with phenotypic variability, which has been associated with mutations in the GJA1 gene encoding connexin43 (Cx43). Given that Cx43 mutants are thought to be equally co-expressed with wild-type Cx43 in ODDD patients, it is imperative to examine the consequence of these mutants in model systems that reflect this molar ratio. To that end, we used differential fluorescent protein tagging of mutant and wild-type Cx43 to quantitatively monitor the ratio of mutant/wild-type within the same putative gap junction plaques and co-immunoprecipitation to determine if the mutants interact with wild-type Cx43. Together the fluorescence-based assay was combined with patch clamp analysis to assess the dominant negative potency of Cx43 mutants. Our results revealed that the ODDD-linked Cx43 mutants, G21R and G138R, as well as amino terminus green fluorescent protein-tagged Cx43, were able to co-localize with wild-type Cx43 at the gap junction plaque-like structures and to co-immunoprecipitate with wild-type Cx43. All Cx43 mutants demonstrated dominant negative action on gap junctional conductance of wild-type Cx43 but not that of Cx32. More interestingly, these Cx43 mutants demonstrated different potencies in inhibiting the function of wild-type Cx43 with the G21R mutant being two times more potent than the G138R mutant. The potency difference in the dominant negative properties of ODDD-linked Cx43 mutants may have clinical implications for the various symptoms and disease severity observed in ODDD patients.     

Some Oculodentodigital Dysplasia-Associated Cx43 Mutations Cause Increased Hemichannel Activity in Addition to Deficient Gap Junction Channels

Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in ODDD patients who are deficient in Cx43 gap junction channels. Radoslaw Dobrowolski and Annette Sommershof contributed equally to this work.     

Closing the gap on autosomal dominant connexin-26 and connexin-43 mutants linked to human disease

Cells within the vast majority of human tissues communicate directly through clustered arrays of intercellular channels called gap junctions. Gene ablation studies in mouse models have revealed that these intercellular channels are necessary for a variety of organ functions and that some of these genes are essential for survival. Molecular genetics has uncovered that germ line mutations in nearly half of the genes that encode the 21-member connexin family of gap junction proteins are linked to one or more human diseases. Frequently, these mutations are autosomal recessive, whereas in other cases, autosomal dominant mutations manifest as disease. Given the broad and overlapping distribution of connexins in a wide arrangement of tissues, it is hard to predict where connexin-linked diseases will clinically manifest. For instance, the most prevalent connexin in the human body is connexin-43 (Cx43), yet autosomal dominant mutations in the GJA1 gene, which encodes Cx43, exhibit modest developmental disorders resulting in a disease termed oculodentodigital dysplasia. Autosomal recessive mutations in the gene encoding Cx26 result in moderate to severe sensorineural hearing loss, whereas autosomal dominant mutations produce hearing loss and a wide range of skin diseases, including palmoplantar keratoderma. Here, we will focus on autosomal dominant mutations of the genes encoding Cx26 and Cx43 in relation to models that link genotypes to phenotypic outcomes with particular reference to how these approaches provide insight into human disease.     

Decreased levels of Cx43 gap junctions result in ameloblast dysregulation and enamel hypoplasia in Gja1Jrt/+ mice

Coordinated differentiation of the ameloblast cell layer is essential to enamel matrix protein deposition and subsequent mineralization. It has been hypothesized that this process is governed by Cx43‐based gap junctional intercellular communication as oculodentodigital dysplasia (ODDD) patients harboring autosomal‐dominant mutations in Cx43 exhibit enamel defects typically resulting in early adulthood tooth loss. To assess the role of Cx43 in tooth development we employ a mouse model of ODDD that harbors a G60S Cx43 mutant, Gja1^Jrt/+, and appears to exhibit tooth abnormalities that mimic the human disease. We found that total Cx43 plaques at all stages of ameloblast differentiation, as well as within the supporting cell layers, were greatly reduced in Gja1^Jrt/+ incisors compared to wild‐type littermate controls. To characterize the Gja1^Jrt/+ mouse tooth phenotype, mice were sacrificed prior to tooth eruption (postnatal day 7), weaning (postnatal day 21), and adulthood (2 months postnatal). A severely disorganized Gja1^Jrt/+ mouse ameloblast layer and abnormal accumulation of amelogenin were observed at stages when the cells were active in secretion and mineralization. Differences in enamel thickness became more apparent after tooth eruption and incisor exposure to the oral cavity suggesting that enamel integrity is compromised, leading to rapid erosion. Additional analysis of incisors from mutant mice revealed that they were longer with a thicker dentin layer than their wild‐type littermates, which may reflect a mechanical stress response to the depleted enamel layer. Together, these data show that reduced levels of Cx43 gap junctions result in ameloblast dysregulation, enamel hypoplasia, and secondary tissue responses. J. Cell. Physiol. 223:601–609, 2010. © 2010 Wiley‐Liss, Inc.     

Compound mutations of PEO1 and TYMP in a progressive external ophthalmoplegia patient with incomplete mitochondrial neurogastrointestinal encephalomyopathy phenotype

The Mendelian inherited progressive external ophthalmoplegia (PEO) and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) are genetically heterogeneous mitochondrial diseases caused by nuclear-mitochondrial intergenomic defects. The PEO1 and TYMP nuclear genes are closely related in the machinery of the mitochondrial DNA (mtDNA) replication. Mutations in PEO1 and TYMP genes usually cause autosomal dominant PEO, and autosomal recessive MNGIE. We identified a PEO family of Korean origin with additional phenotype of incomplete MNGIE symptom (Family ID: MT16). The entire mitochondrial genome and all coding exons of PEO1, TYMP, ANT1, POLG1, POLG2, DGUOK, and TK2 nuclear genes were sequenced. Clinical information was obtained through history taking, physical examinations, clinical observations, and electrophysiological investigations. Muscle biopsy of left biceps brachii and shoulder magnetic resonance imaging (MRI) were undertaken. We found two heterozygous mutations, Arg374Gln in PEO1 and Glu106Gln in TYMP from the proband who showed complex phenotypes of a typical PEO and late-onset incomplete MNGIE. The PEO1 Arg374Gln has been reported in several PEO patients, but TYMP Glu106Gln has not been reported. Neither large deletion nor causative point mutations were observed in the mtDNA. We suggest that the heterozygous TYMP mutation might affect complex phenotypes as a secondary genetic cause in the co-presence of PEO1 mutation.     

Structure and functional studies of N-terminal Cx43 mutants linked to oculodentodigital dysplasia

Mutations in the gene encoding connexin-43 (Cx43) cause the human development disorder known as oculodentodigital dysplasia (ODDD). In this study, ODDD-linked Cx43 N-terminal mutants formed nonfunctional gap junction-like plaques and exhibited dominant-negative effects on the coupling conductance of coexpressed endogenous Cx43 in reference cell models. Nuclear magnetic resonance (NMR) protein structure determination of an N-terminal 23-amino acid polypeptide of wild-type Cx43 revealed that it folded in to a kinked α-helical structure. This finding predicted that W4 might be critically important in intramolecular and intermolecular interactions. Thus we engineered and characterized a W4A mutant and found that this mutant formed a regular, nonkinked α-helix but did not form functional gap junctions. Furthermore, a G2V variant peptide of Cx43 showed a kinked helix that now included V2 interactions with W4, resulting in the G2V mutant forming nonfunctional gap junctions. Also predicted from the NMR structures, a G2S mutant was found to relieve these interactions and allowed the protein to form functional gap junctions. Collectively, these studies suggest that the nature of the mutation conveys loss of Cx43 function by distinctly different mechanisms that are rooted in the structure of the N-terminal region.     

Functional characterization of a GJA1 frameshift mutation causing oculodentodigital dysplasia and palmoplantar keratoderma

A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms.     

Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling

Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.     

Effect of LEPR Gln223Arg polymorphism on breast cancer risk in different ethnic populations: a meta-analysis

Leptin and leptin receptor have been implicated in processes leading to breast cancer initiation and progression. An A to G transition mutation in codon 223, in exon 6 of the leptin receptor gene (LEPR) can result in glutamine to arginine substitution (Gln223Arg). A variety of case–control studies have been published evaluating the association between LEPR Gln223Arg polymorphism and breast cancer. However, published studies have yielded contradictory conclusions. This meta-analysis enrolled eight studies to estimate the overall risk of LEPR Gln223Arg polymorphism associated with breast cancer. The pooled ORs were performed for codominant model (Arg/Arg versus Gln/Gln; Arg/Gln versus Gln/Gln), dominant model (Arg/Arg + Arg/Gln versus Gln/Gln), recessive model (Arg/Arg versus Arg/Gln + Gln/Gln). Overall significantly elevated breast cancer risk was found for recessive model (OR 1.32, 95% CI 1.03–1.69) and for genotype Arg/Gln versus Gln/Gln (OR 1.16, 95% CI 1.01–1.34). In the stratified analysis by ethnicity, significantly increased risks were also found among Africans for genotype Arg/Arg versus Gln/Gln: OR 1.86, 95% CI 1.28–2.71, Arg/Gln versus Gln/Gln: OR 1.48, 95% CI 1.10–1.99, dominant model: OR 1.60, 95% CI 1.21–2.11 and recessive model: OR 1.48, 95% CI 1.07–2.05; for Asians, Arg/Arg versus Gln/Gln: OR 6.79, 95% CI 3.42–13.47 and dominant model: OR 2.03, 95% CI 1.42–2.90. However, no significantly increased risk was found among Europeans for all genetic models. In conclusion, the LEPR 223Arg is a low-penetrant risk for developing breast cancer, especially for black African women.     

Functional defects of Cx26 resulting from a heterozygous missense mutation in a family with dominant deaf-mutism and palmoplantar keratoderma

Mutations in GJB2 encoding the gap junction protein connexin-26 (Cx26) have been established as the basis of autosomal recessive non-syndromic hearing loss. The involvement of GJB2 in autosomal dominant deafness has also been proposed, although the putative mutation identified in one family with both deafness and palmoplantar keratoderma has recently been suggested to be merely a non-disease associated polymorphism. We have observed a similar phenotype in an Egyptian family that segregated with a heterozygous missense mutation of GJB2, leading to a non-conservative amino acid substitution (R75W). The deleterious dominant-negative effect of R75W on gap channel function was subsequently demonstrated in the paired oocyte expression system. Not only was R75W alone incapable of inducing electrical conductance between adjacent cells, but it almost completely suppressed the activity of co-expressed wildtype protein. The Cx26 mutant W77R, which has been implicated in autosomal recessive deafness, also failed to form functional gap channels by itself but did not significantly interfere with the function of wildtype Cx26. These data provide compelling evidence for the serious functional consequences of Cx26 mutations in dominant and recessive deafness. Received: 22 June 1998 / Accepted: 15 July 1998     

Functional Characterization of Oculodentodigital Dysplasia-Associated Cx43 Mutants

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.     

Syndromic and non-syndromic disease-linked Cx43 mutations

There are now at least 14 distinct diseases linked to germ line mutations in the 21 genes that encode the connexin (Cx) family of gap junction proteins. This review focuses on the links between germ-line mutations in the gene encoding Cx43 (GJA1) and the human disease termed oculodentodigital dysplasia (ODDD). This disease is clinically characterized by soft tissue fusion of the digits, abnormal craniofacial bone development, small eyes and loss of tooth enamel. However, the disease is considerably more complex and somewhat degenerative as patients often suffer from other syndromic effects that include incontinence, glaucoma, skin diseases and neuropathies that become more pronounced during aging. The challenge continues to be understanding how distinct Cx43 gene mutations cause such a diverse range of tissue phenotypes and pathophysiological changes while other Cx43-rich organs are relatively unaffected. This review will provide an overview of many of these studies and distill some themes and outstanding questions that need to be addressed in the coming years.     

Functional characterization of oculodentodigital dysplasia-associated Cx43 mutants

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.     

Characterization of Gap Junction Proteins in the Bladder of Cx43 Mutant Mouse Models of Oculodentodigital Dysplasia

Oculodentodigital dysplasia (ODDD) is a rare developmental disease resulting from germline mutations in the GJA1 gene that encodes the gap junction protein connexin43 (Cx43). In addition to the classical ODDD symptoms that affect the eyes, teeth, bone and digits, in some cases ODDD patients have reported bladder impairments. Thus, we chose to characterize the bladder in mutant mouse models of ODDD that harbor two distinct Cx43 mutations, G60S and I130T. Histological assessment revealed no difference in bladder detrusor wall thickness in mutant compared to littermate control mice. The overall localization of Cx43 in the lamina propria and detrusor also appeared to be similar in the bladders of mutant mice with the exception that the G60S mice had more instances of intracellular Cx43. However, both mutant mouse lines exhibited a significant reduction in the phosphorylated P1 and P2 isoforms of Cx43, while only the I130T mice exhibited a reduction in total Cx43 levels. Interestingly, Cx26 levels and distribution were not altered in mutant mice as it was localized to intracellular compartments and restricted to the basal cell layers of the urothelium. Our studies suggest that these two distinct genetically modified mouse models of ODDD probably mimic patients who lack bladder defects or other factors, such as aging or co-morbidities, are necessary to reveal a bladder phenotype.