Cardiac phenotype in ATP1A3-related syndromes: A multicenter cohort study.

OBJECTIVE: To define the risks and consequences of cardiac abnormalities in ATP1A3-related syndromes. METHODS: Patients meeting clinical diagnostic criteria for rapid-onset dystonia-parkinsonism (RDP), alternating hemiplegia of childhood (AHC), and cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) with ATP1A3 genetic analysis and at least 1 cardiac assessment were included. We evaluated the cardiac phenotype in an Atp1a3 knock-in mouse (Mashl+/-) to determine the sequence of events in seizure-related cardiac death. RESULTS: Ninety-eight patients with AHC, 9 with RDP, and 3 with CAPOS (63 female, mean age 17 years) were included. Resting ECG abnormalities were found in 52 of 87 (60%) with AHC, 2 of 3 (67%) with CAPOS, and 6 of 9 (67%) with RDP. Serial ECGs showed dynamic changes in 10 of 18 patients with AHC. The first Holter ECG was abnormal in 24 of 65 (37%) cases with AHC and RDP with either repolarization or conduction abnormalities. Echocardiography was normal. Cardiac intervention was required in 3 of 98 (≈3%) patients with AHC. In the mouse model, resting ECGs showed intracardiac conduction delay; during induced seizures, heart block or complete sinus arrest led to death. CONCLUSIONS: We found increased prevalence of ECG dynamic abnormalities in all ATP1A3-related syndromes, with a risk of life-threatening cardiac rhythm abnormalities equivalent to that in established cardiac channelopathies (≈3%). Sudden cardiac death due to conduction abnormality emerged as a seizure-related outcome in murine Atp1a3-related disease. ATP1A3-related syndromes are cardiac diseases and neurologic diseases. We provide guidance to identify patients potentially at higher risk of sudden cardiac death who may benefit from insertion of a pacemaker or implantable cardioverter-defibrillator.

Heterozygous Mutations in SMARCA2 Reprogram the Enhancer Landscape by Global Retargeting of SMARCA4.

Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase (either SMARCA4 or SMARCA2). Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that the AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate that SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.

Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase.

The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na+,K+-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na+ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ∼30-fold reduction of apparent Na+ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E1P and E2P. The apparent affinity for K+ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na+-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E1-E2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K+ than the WT enzyme, which together with the reduced Na+ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na+ interaction at site III.

Advances in computer-assisted syndrome recognition by the example of inborn errors of metabolism.

Significant improvements in automated image analysis have been achieved in recent years and tools are now increasingly being used in computer-assisted syndromology. However, the ability to recognize a syndromic facial gestalt might depend on the syndrome and may also be confounded by severity of phenotype, size of available training sets, ethnicity, age, and sex. Therefore, benchmarking and comparing the performance of deep-learned classification processes is inherently difficult. For a systematic analysis of these influencing factors we chose the lysosomal storage diseases mucolipidosis as well as mucopolysaccharidosis type I and II that are known for their wide and overlapping phenotypic spectra. For a dysmorphic comparison we used Smith-Lemli-Opitz syndrome as another inborn error of metabolism and Nicolaides-Baraitser syndrome as another disorder that is also characterized by coarse facies. A classifier that was trained on these five cohorts, comprising 289 patients in total, achieved a mean accuracy of 62%. We also developed a simulation framework to analyze the effect of potential confounders, such as cohort size, age, sex, or ethnic background on the distinguishability of phenotypes. We found that the true positive rate increases for all analyzed disorders for growing cohorts (n = [10...40]) while ethnicity and sex have no significant influence. The dynamics of the accuracies strongly suggest that the maximum distinguishability is a phenotype-specific value, which has not been reached yet for any of the studied disorders. This should also be a motivation to further intensify data sharing efforts, as computer-assisted syndrome classification can still be improved by enlarging the available training sets.

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.

Relative anterior microphthalmos in oculodentodigital dysplasia.

Here, we report a patient with oculodentodigital dysplasia (ODDD) caused by the c. 413G>A, p.Gly138Asp mutation in the gap junction protein alpha-1 gene. The patient suffered from characteristic dysmorphic features of ODDD. Ophthalmological investigation disclosed microcornea and a shallow anterior chamber, as expected. Surprisingly, the patient had a normal axial length and moderate myopia on both eyes. To the best of our knowledge, this is the first report on ODDD associated with relative anterior microphthalmos and myopia.

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.

A New Subtype of Multiple Synostoses Syndrome Is Caused by a Mutation in GDF6 That Decreases Its Sensitivity to Noggin and Enhances Its Potency as a BMP Signal.

Growth and differentiation factors (GDFs) are secreted signaling molecules within the BMP family that have critical roles in joint morphogenesis during skeletal development in mice and humans. Using genetic data obtained from a six-generation Chinese family, we identified a missense variant in GDF6 (NP_001001557.1; p.Y444N) that fully segregates with a novel autosomal dominant synostoses (SYNS) phenotype, which we designate as SYNS4. Affected individuals display bilateral wrist and ankle deformities at birth and progressive conductive deafness after age 40 years. We find that the Y444N variant affects a highly conserved residue of GDF6 in a region critical for binding of GDF6 to its receptor(s) and to the BMP antagonist NOG, and show that this mutant GDF6 is a more potent stimulator of the canonical BMP signaling pathway compared with wild-type GDF6. Further, we determine that the enhanced BMP activity exhibited by mutant GDF6 is attributable to resistance to NOG-mediated antagonism. Collectively, our findings indicate that increased BMP signaling owing to a GDF6 gain-of-function mutation is responsible for loss of joint formation and profound functional impairment in patients with SYNS4. More broadly, our study highlights the delicate balance of BMP signaling required for proper joint morphogenesis and reinforces the critical role of BMP signaling in skeletal development.

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.

Intrafamilial variability of ZRS-associated syndrome: characterization of a mosaic ZRS mutation by pyrosequencing.

During limb development, the spatio-temporal expression of sonic hedgehog (SHH) is driven by the Zone of polarizing activity Regulatory Sequence (ZRS), located 1 megabase upstream from SHH. Gain-of-function mutations of this enhancer, which cause ectopic expression of SHH, are known to be responsible for congenital limb malformations with variable expressivity, ranging from preaxial polydactyly or triphalangeal thumbs to polysyndactyly, which may also be associated with mesomelic deficiency. In this report, we describe a patient affected with mirror-image polydactyly of the four extremities and bilateral tibial deficiency. The proband's father had isolated preaxial polydactyly type II (PPD2). Using Sanger sequencing, a ZRS point mutation (NC_000007.14, g.156584153A>G, UCSC, Build hg.19) was only identified in the patient. However, pyrosequencing analysis enabled the detection of a 10% somatic mosaic in the blood and saliva from the father. To our knowledge, this is the first description of a ZRS mosaic mutation. This report highlights the complexity of genotype-phenotype correlation in ZRS-associated syndromes and the importance of detecting somatic mosaicism for accurate genetic counselling.

The genetics of cognitive epigenetics.

Cognitive disorders (CDs) are a heterogeneous group of disorders for which the genetic foundations are rapidly being uncovered. The large number of CD-associated gene mutations presents an opportunity to identify common mechanisms of disease as well as molecular processes that are of key importance to human cognition. Given the disproportionately high number of epigenetic genes associated with CD, epigenetic regulation of gene transcription is emerging as a process of major importance in cognition. The cognate protein products of these genes often co-operate in shared protein complexes or pathways, which is reflected in similarities between the neurodevelopmental phenotypes corresponding to these mutant genes. Here we provide an overview of the genes associated with CDs, and highlight some of the epigenetic regulatory complexes involving multiple CD genes. Such common gene networks may provide a handle for designing therapeutic interventions applicable to a number of cognitive disorders with variable genetic etiology.

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.

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.

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.

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.

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.

Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development.

Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.

Gap junctions and hemichannels in signal transmission, function and development of bone.

Gap junctional intercellular communication (GJIC) mediated by connexins, in particular connexin 43 (Cx43), plays important roles in regulating signal transmission among different bone cells and thereby regulates development, differentiation, modeling and remodeling of the bone. GJIC regulates osteoblast formation, differentiation, survival and apoptosis. Osteoclast formation and resorptive ability are also reported to be modulated by GJIC. Furthermore, osteocytes utilize GJIC to coordinate bone remodeling in response to anabolic factors and mechanical loading. Apart from gap junctions, connexins also form hemichannels, which are localized on the cell surface and function independently of the gap junction channels. Both these channels mediate the transfer of molecules smaller than 1.2kDa including small ions, metabolites, ATP, prostaglandin and IP(3). The biological importance of the communication mediated by connexin-forming channels in bone development is revealed by the low bone mass and osteoblast dysfunction in the Cx43-null mice and the skeletal malformations observed in occulodentodigital dysplasia (ODDD) caused by mutations in the Cx43 gene. The current review summarizes the role of gap junctions and hemichannels in regulating signaling, function and development of bone cells. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.