OTX2 mutations contribute to the otocephaly-dysgnathia complex.

BACKGROUND: Otocephaly or dysgnathia complex is characterised by mandibular hypoplasia/agenesis, ear anomalies, microstomia, and microglossia; the molecular basis of this developmental defect is largely unknown in humans. METHODS AND RESULTS: This study reports a large family in which two cousins with micro/anophthalmia each gave birth to at least one child with otocephaly, suggesting a genetic relationship between anophthalmia and otocephaly. OTX2, a known microphthalmia locus, was screened in this family and a frameshifting mutation was found. The study subsequently identified in one unrelated otocephalic patient a sporadic OTX2 mutation. Because OTX2 mutations may not be sufficient to cause otocephaly, the study assayed the potential of otx2 to modify craniofacial phenotypes in the context of known otocephaly gene suppression in vivo. It was found that otx2 can interact genetically with pgap1, prrx1, and msx1 to exacerbate mandibular and midline defects during zebrafish development. However, sequencing of these loci in the OTX2-positive families did not unearth likely pathogenic lesions, suggesting further genetic heterogeneity and complexity. CONCLUSION: Identification of OTX2 involvement in otocephaly/dysgnathia in humans, even if loss of function mutations at this locus does not sufficiently explain the complex anatomical defects of these patients, suggests the requirement for a second genetic hit. Consistent with this notion, trans suppression of otx2 and other developmentally related genes recapitulate aspects of the otocephaly phenotype in zebrafish. This study highlights the combined utility of genetics and functional approaches to dissect both the regulatory pathways that govern craniofacial development and the genetics of this disease group.

GJA1 mutations, variants, and connexin 43 dysfunction as it relates to the oculodentodigital dysplasia phenotype.

The predominantly autosomal dominant disorder, oculodentodigital dysplasia (ODDD) has high penetrance with intra- and interfamilial phenotypic variability. Abnormalities observed in ODDD affect the eye, dentition, and digits of the hands and feet. Patients present with a characteristic facial appearance, narrow nose, and hypoplastic alae nasi. Neurological problems, including dysarthria, neurogenic bladder disturbances, spastic paraparesis, ataxia, anterior tibial muscle weakness, and seizures, are known to occur as well as conductive hearing loss, cardiac defects, and anomalies of the skin, hair, and nails. In 2003, our analysis of 17 ODDD families revealed that each had a different mutation within the human gap junction alpha 1 (GJA1) gene which encodes the protein connexin 43 (Cx43). Since then at least 17 publications have identified an additional 26 GJA1 mutations and in this study, we present 28 new cases with 18 novel GJA1 mutations. We include tables summarizing the 62 known GJA1 nucleotide changes leading to Cx43 protein alterations and the phenotypic information available on 177 affected individuals from 54 genotyped families. Mutations resulting in ODDD occur in each of the nine domains of the Cx43 protein, and we review our functional experiments and those in the literature, examining the effects of 13 different Cx43 mutations upon gap junction activity.

Functional characterization of connexin43 mutations found in patients with oculodentodigital dysplasia.

Specific mutations in GJA1, the gene encoding the gap junction protein connexin43 (Cx43), cause an autosomal dominant disorder called oculodentodigital dysplasia (ODDD). Here, we characterize the effects of 8 of these mutations on Cx43 function. Immunochemical studies have shown that most of the mutant proteins formed gap junction plaques at the sites of cell-cell apposition. However, 2 of the mutations (a codon duplication in the first extracellular loop, F52dup, and a missense mutation in the second extracellular loop, R202H, produced full-length connexins that failed to properly form gap junction plaques. Cx43 proteins containing ODDD mutations found in the N-terminus (Y17S), first transmembrane domain (G21R, A40V), second transmembrane domain (L90V), and cytoplasmic loop (I130T, K134E) do form gap junction plaques but show compromised channel function. L90V, I130T, and K134E demonstrated a significant decrease in junctional conductance relative to Cx43WT. Mutations Y17S, G21R, and A40V demonstrated a complete lack of functional electrical coupling even in the presence of significant plaque formation between paired cells. Heterologous channels formed by coexpression of Cx43WT and mutation R202H resulted in electrically functional gap junctions that were not permeable to Lucifer yellow. Therefore, the mutations found in ODDD not only cause phenotypic variability, but also result in various functional consequences. Overall, our data show an extensive range of molecular phenotypes, consistent with the pleiotropic nature of the clinical syndrome as a whole.

Receptor tyrosine kinases activate canonical WNT/ß-catenin signaling via MAP kinase/LRP6 pathway and direct ß-catenin phosphorylation.

Receptor tyrosine kinase signaling cooperates with WNT/ß-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/ß-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate ß-catenin at Tyr142, which is known to increase cytoplasmic ß-catenin concentration via release of ß-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct ß-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling.

Tyrosine-dependent basolateral targeting of human connexin43-eYFP in Madin-Darby canine kidney cells can be disrupted by the oculodentodigital dysplasia mutation L90V.

Polarized membrane sorting of connexin 43 (Cx43) has not been well-characterized. Based on the presence of a putative sorting signal, YKLV(286-289), within its C-terminal cytoplasmic domain, we hypothesized that Cx43 is selectively expressed on the basolateral surface of Madin-Darby canine kidney (MDCK) cells in a tyrosine-dependent manner. We generated stable MDCK cell lines expressing human wild-type and mutant Cx43-eYFP, and analyzed the membrane localization of Cx43-eYFP within polarized monolayers using confocal microscopy and selective surface biotinylation. We found that wild-type Cx43-eYFP was selectively targeted to the basolateral membrane domain of MDCK cells. Substitution of alanine for Y286 disrupted basolateral targeting of Cx43-eYFP. Additionally, substitution of a sequence containing the transferrin receptor internalization signal, LSYTRF, for PGYKLV(284-289) also disrupted basolateral targeting. Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43-eYFP to the basolateral membrane domain of MDCK cells. To determine whether the F52dup or L90V oculodentodigital dysplasia-associated mutations could affect polarized sorting of Cx43-eYFP, we analyzed the expression of these Cx43-eYFP mutant constructs and found that the L90V mutation disrupted basolateral expression. These findings raise the possibility that some oculodentodigitial dysplasia-associated mutations contribute to disease by altering polarized targeting of Cx43.

Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells.

Oculodentodigital dysplasia (ODDD) is a rare developmental disorder characterized by craniofacial and limb abnormalities. Over 35 separate mutations in human connexin43 (Cx43) causing ODDD have been identified. Several mutations are also associated with central nervous system involvement, including white-matter changes detected by magnetic resonance imaging. As Cx43 is abundantly expressed in astrocytes, we hypothesized that the mutant Cx43 proteins that produce neurological dysfunction have abnormal functional characteristics in astrocytes. To understand how ODDD-associated mutations affect Cx43 signaling in cells of glial origin, we conducted studies in rat C6 glioma cells, a communication-deficient glial cell line that expresses low levels of Cx43. We generated stable cell lines expressing enhanced yellow fluorescent protein (eYFP)-tagged human Cx43 constructs encoding wild-type and six eYFP-tagged mutant Cx43 mutants: Y17S, G21R, A40V, F52dup, L90V and I130T. Of these, Y17S, L90V and I130T are associated with neurological abnormalities. We found that all mutants could be detected on the cell surface. Y17S, G21R, A40V, L90V and I130T formed triton-resistant plaques representing gap junctions, although the relative ability to form plaques was decreased in these mutants compared with the wild type. F52dup formed dramatically reduced numbers of plaques. Propidium iodide uptake experiments demonstrated that all mutants were associated with reduced connexin hemichannel function compared with wild type. Scrape-loading experiments performed on the same stable cell lines showed reduced gap junctional dye transfer in all mutants compared with the wild type. These studies demonstrated that ODDD-associated Cx43 mutations result in non-functional connexin hemichannels and gap junction functions in a glial cell line regardless of whether the particular mutant is associated with neurological dysfunction.

Increased risk for developmental delay in Saethre-Chotzen syndrome is associated with TWIST deletions: an improved strategy for TWIST mutation screening.

The majority of patients with Saethre-Chotzen syndrome have mutations in the TWIST gene, which codes for a basic helix-loop-helix transcription factor. Of the genetic alterations identified in TWIST, nonsense mutations, frameshifts secondary to small deletions or insertions, and large deletions implicate haploinsufficiency as the pathogenic mechanism. We identified three novel intragenic mutations and six deletions in our patients by using a new strategy to screen for TWIST mutations. We used polymerase chain reaction (PCR) amplification with subsequent sequencing to identify point mutations and small insertions or deletions in the coding region, and real-time PCR-based gene dosage analysis to identify large deletions encompassing the gene, with confirmation by microsatellite and fluorescence in situ hybridization (FISH) analyses. The size of the deletions can also be analyzed by using the gene dosage assay with "PCR walking" across the critical region. In 55 patients with features of Saethre-Chotzen syndrome, 11% were detected to have deletions by real-time gene dosage analysis. Two patients had a translocation or inversion at least 260 kb 3' of the gene, suggesting they had position-effect mutations. Of the 37 patients with classic features of Saethre-Chotzen syndrome, the overall detection rate for TWIST mutations was 68%. The risk for developmental delay in patients with deletions involving the TWIST gene is approximately 90% or eight times more common than in patients with intragenic mutations.

Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia.

Gap junctions are assemblies of intercellular channels that regulate a variety of physiologic and developmental processes through the exchange of small ions and signaling molecules. These channels consist of connexin family proteins that allow for diversity of channel composition and conductance properties. The human connexin 43 gene, or GJA1, is located at human chromosome 6q22-q23 within the candidate region for the oculodentodigital dysplasia locus. This autosomal dominant syndrome presents with craniofacial (ocular, nasal, and dental) and limb dysmorphisms, spastic paraplegia, and neurodegeneration. Syndactyly type III and conductive deafness can occur in some cases, and cardiac abnormalities are observed in rare instances. We found mutations in the GJA1 gene in all 17 families with oculodentodigital dysplasia that we screened. Sixteen different missense mutations and one codon duplication were detected. These mutations may cause misassembly of channels or alter channel conduction properties. Expression patterns and phenotypic features of gja1 animal mutants, reported elsewhere, are compatible with the pleiotropic clinical presentation of oculodentodigital dysplasia.