Role of Chd7 in zebrafish: a model for CHARGE syndrome

CHARGE syndrome is caused by mutations in the CHD7 gene. Several organ systems including the retina, cranial nerves, inner ear and heart are affected in CHARGE syndrome. However, the mechanistic link between mutations in CHD7 and many of the organ systems dysfunction remains elusive. Here, we show that Chd7 is required for the organization of the neural retina in zebrafish. We observe an abnormal expression or a complete absence of molecular markers for the retinal ganglion cells and photoreceptors, indicating that Chd7 regulates the differentiation of retinal cells and plays an essential role in retinal cell development. In addition, zebrafish with reduced Chd7 display an abnormal organization and clustering of cranial motor neurons. We also note a pronounced reduction in the facial branchiomotor neurons and the vagal motor neurons display aberrant positioning. Further, these fish exhibit a severe loss of the facial nerves. Knock-down of Chd7 results in a curvature of the long body axis and these fish develop irregular shaped vertebrae and have a reduction in bone mineralization. Chd7 knockdown also results in a loss of proper segment polarity illustrated by flawed efnb2a and ttna expression, which is associated with later vascular segmentation defects. These critical roles for Chd7 in retinal and vertebral development were previously unrecognized and our results provide new insights into the role of Chd7 during development and in CHARGE syndrome pathogenesis.     

CHD7 Deficiency in “Looper”, a New Mouse Model of CHARGE Syndrome,Results in Ossicle Malformation,Otosclerosis and Hearing Impairment

CHARGE syndrome is a rare human disorder caused by mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). Characteristics of CHARGE are varied and include developmental ear and hearing anomalies. Here we report a novel mouse model of CHD7 dysfunction, termed Looper. The Looper strain harbours a nonsense mutation (c.5690C>A, p.S1897X) within the Chd7 gene. Looper mice exhibit many of the clinical features of the human syndrome, consistent with previously reported CHARGE models, including growth retardation, facial asymmetry, vestibular defects, eye anomalies, hyperactivity, ossicle malformation, hearing loss and vestibular dysfunction. Looper mice display an otosclerosis-like fusion of the stapes footplate to the cochlear oval window and blepharoconjunctivitis but not coloboma. Looper mice are hyperactive and have vestibular dysfunction but do not display motor impairment.     

Morphological and sensorimotor phenotypes in a zebrafish CHARGE syndrome model are domain-dependent

CHARGE syndrome is a heterogeneous disorder characterized by a spectrum of defects affecting multiple tissues and behavioral difficulties such as autism, attention-deficit/hyperactivity disorder, obsessive–compulsive disorder, anxiety, and sensory deficits. Most CHARGE cases arise from de novo, loss-of-function mutations in chromodomain-helicase-DNA-binding-protein-7 (CHD7). CHD7 is required for processes such as neuronal differentiation and neural crest cell migration, but how CHD7 affects neural circuit function to regulate behavior is unclear. To investigate the pathophysiology of behavioral symptoms in CHARGE, we established a mutant chd7 zebrafish line that recapitulates multiple CHARGE phenotypes including ear, cardiac, and craniofacial defects. Using a panel of behavioral assays, we found that chd7 mutants have specific auditory and visual behavior deficits that are independent of defects in sensory structures. Mauthner cell-dependent short-latency acoustic startle responses are normal in chd7 mutants, while Mauthner-independent long-latency responses are reduced. Responses to sudden decreases in light are also reduced in mutants, while responses to sudden increases in light are normal, suggesting that the retinal OFF pathway may be affected. Furthermore, by analyzing multiple chd7 alleles we observed that the penetrance of morphological and behavioral phenotypes is influenced by genetic background but that it also depends on the mutation location, with a chromodomain mutation causing the highest penetrance. This pattern is consistent with analysis of a CHARGE patient dataset in which symptom penetrance was highest in subjects with mutations in the CHD7 chromodomains. These results provide new insight into the heterogeneity of CHARGE and will inform future work to define CHD7-dependent neurobehavioral mechanisms.     

Genetic analysis of the CHD7 gene in Korean patients with CHARGE syndrome

CHARGE syndrome is an autosomal dominant congenital disorder known to be caused by the haploinsufficiency of the CHD7 gene. Heterozygous mutations in the CHD7 gene have been identified in approximately 60–70% of patients clinically diagnosed with CHARGE syndrome. Although there have been many reports on the mutational spectrum of the CHD7 gene in patients with CHARGE syndrome worldwide, little is known about this syndrome in the Korean population. In this study, three Korean patients with CHARGE syndrome including one patient with Patau syndrome were evaluated for genetic analysis of the CHD7 gene using direct sequencing of all 38 exons and the flanking intronic regions. One nonsense and two novel missense mutations were identified in the CHD7 gene. Clinical symptoms caused by the missense mutations were much milder compared to the nonsense mutation, confirming the previously determined genotype–phenotype correlation in CHARGE syndrome. Our study demonstrates the importance of mutational screening of CHD7 in patients who have been diagnosed with other syndromes but display clinical features of CHARGE syndrome.     

Identification and Characterization of FAM124B as a Novel Component of a CHD7 and CHD8 Containing Complex

Background

Mutations in the chromodomain helicase DNA binding protein 7 gene (CHD7) lead to CHARGE syndrome, an autosomal dominant multiple malformation disorder. Proteins involved in chromatin remodeling typically act in multiprotein complexes. We previously demonstrated that a part of human CHD7 interacts with a part of human CHD8, another chromodomain helicase DNA binding protein presumably being involved in the pathogenesis of neurodevelopmental (NDD) and autism spectrum disorders (ASD). Because identification of novel CHD7 and CHD8 interacting partners will provide further insights into the pathogenesis of CHARGE syndrome and ASD/NDD, we searched for additional associated polypeptides using the method of stable isotope labeling by amino acids in cell culture (SILAC) in combination with mass spectrometry.

Principle findings

The hitherto uncharacterized FAM124B (Family with sequence similarity 124B) was identified as a potential interaction partner of both CHD7 and CHD8. We confirmed the result by co-immunoprecipitation studies and showed a direct binding to the CHD8 part by direct yeast two hybrid experiments. Furthermore, we characterized FAM124B as a mainly nuclear localized protein with a widespread expression in embryonic and adult mouse tissues.

Conclusion

Our results demonstrate that FAM124B is a potential interacting partner of a CHD7 and CHD8 containing complex. From the overlapping expression pattern between Chd7 and Fam124B at murine embryonic day E12.5 and the high expression of Fam124B in the developing mouse brain, we conclude that Fam124B is a novel protein possibly involved in the pathogenesis of CHARGE syndrome and neurodevelopmental disorders.     

Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.     

Deletion of CHD8 in cerebellar granule neuron progenitors leads to severe cerebellar hypoplasia,ataxia, and psychiatric behavior in mice

CHD8 is a candidate gene for autism spectrum disorders and neurological development delay. It has been reported to be essential for neurogenesis in the cerebral cortex, but the function of CHD8 in cerebellum has not been comprehensively investigated. The potential relationship of cerebellum dysplasia with psychiatric disorders in patients with CHD8 mutations is still not clear. In this study, we establish different conditional knockout mouse models to investigate the roles of CHD8 in cerebellar development. Mice with neural stem cell-specific Chd8 deletion exhibit significant reduction of cerebellum volume and no layering structure is detected. Genetic deletion of Chd8 in cerebellar granule neuron progenitors (GNPs) leads to cerebellar hypoplasia, absent of proliferation layer and ectopic of Purkinje neuron. However, no substantial cerebellar dysplasia is detected in mice with Purkinje neuron- or oligodendrocyte-specific Chd8 ablation. Single-cell RNA sequencing indicates that ribosome-related genes and pathways are most significantly disrupted in GNPs, indicating the potential mechanism. Importantly, in addition to the ataxia phenotype, mice with GNP-specific Chd8 ablation present a neuropsychiatric phenotype in three-chamber and light/dark tests. Taken together, our results provide insights not only into the function of CHD8 in cerebellar development, but also the pathogenesis of neuropsychiatric disorders in patients with CHD8 mutations.     

8q12.1q12.3 de novo microdeletion involving the CHD7 gene in a patient without the major features of CHARGE syndrome: Case report and critical review of the literature

CHARGE syndrome is an autosomal dominant inherited disorder characterized by a specific and recognizable pattern of anomalies. De novo mutations or deletions of the gene encoding chromodomain helicase DNA binding protein 7 (CHD7) are the major cause of CHARGE syndrome. In this report, we describe a patient with a typical phenotype characterized by psychomotor retardation, hypertrichosis, facial asymmetry, synophria, failure to thrive, developmental delay and gastro-esophageal reflux, carrying a de novo 6.04 Mb interstitial deletion in 8q12.1q12.3 detected by single nucleotide polymorphism (SNP) array analysis. Despite the deletion includes CHD7 and although the patient shares some of the clinical features of the CHARGE syndrome, she does not fulfill the clinical criteria for this syndrome. To the best of our knowledge, this is the second case with an entire deletion of the CHD7 gene not leading to CHARGE syndrome and, for this reason, useful to expand and further delineate the clinical features associated with the 8q12.1q12.3 deletion. Furthermore, the literature review revealed that the phenotype secondary to duplications of the same region partially overlaps with the phenotype reported in this study. Selected genes that are present in the hemizygous state and which might be important for the phenotype of this patient, are discussed in context of the clinical features.     

CHD7 mutational analysis and clinical considerations for auditory rehabilitation in deaf patients with CHARGE syndrome

Background

Otologic manifestations are one of the most consistent findings of CHARGE syndrome found in more than 90%. Since genetic analysis of the CHD7 gene has rarely been performed in previous reports dealing with ear abnormalities, the genotypic spectrum of CHD7 mutations was analyzed in deaf patients with CHARGE syndrome, and the clinical considerations concerning auditory rehabilitation were investigated.

Methods

Nine Korean patients with CHARGE syndrome showing profound hearing loss and semicircular canal aplasia were included. All 38 exons of CHD7 were analyzed by direct sequencing. For splice site variations, in silico and exon-trapping analyses were performed to verify the pathogenicity of nucleotide variations. Clinical features and the outcome of auditory rehabilitation were also analyzed.

Results

Eight of 9 patients revealed alterations of the CHD7 gene including 3 frameshift, 2 nonsense, 2 splice site, and 1 missense mutations. Five of 9 patients were clinically diagnosed as atypical CHARGE syndrome but demonstrated various mutations of the CHD7 gene. One familial case showed intra-familial variability. Radiologic findings suggesting cochleovestibular nerve deficiency were identified in most of the patients. Of the 8 patients who underwent cochlear implantation, 5 patients demonstrated favorable outcome. Larger diameter of the cochleovestibular nerve on imaging and absence of severe mental retardation were factors related to better outcome after cochlear implantation rather than the type of CHD7 mutations. Auditory brainstem implantation was performed in two patients who did not benefit from cochlear implantation.

Conclusions

Genetic analysis of the CHD7 gene should be performed in cases with semicircular canal aplasia even when other typical features of CHARGE syndrome are absent. For auditory rehabilitation in CHARGE syndrome, cochlear implantation should be strongly recommended in selected cases with favorable prognostic factors. Auditory brainstem implantation may be a viable option in patients with CHARGE syndrome who have failed to benefit from cochlear implantation.     

Spectrum of CHD7 mutations in 110 individuals with CHARGE syndrome and genotype-phenotype correlation

CHARGE syndrome is a well-established multiple-malformation syndrome with distinctive consensus diagnostic criteria. Characteristic associated anomalies include ocular coloboma, choanal atresia, cranial nerve defects, distinctive external and inner ear abnormalities, hearing loss, cardiovascular malformations, urogenital anomalies, and growth retardation. Recently, mutations of the chromodomain helicase DNA-binding protein gene CHD7 were reported to be a major cause of CHARGE syndrome. We sequenced the CHD7 gene in 110 individuals who had received the clinical diagnosis of CHARGE syndrome, and we detected mutations in 64 (58%). Mutations were distributed throughout the coding exons and conserved splice sites of CHD7. Of the 64 mutations, 47 (73%) predicted premature truncation of the protein. These included nonsense and frameshift mutations, which most likely lead to haploinsufficiency. Phenotypically, the mutation-positive group was more likely to exhibit cardiovascular malformations (54 of 59 in the mutation-positive group vs. 30 of 42 in the mutation-negative group; P=.014), coloboma of the eye (55 of 62 in the mutation-positive group vs. 30 of 43 in the mutation-negative group; P=.022), and facial asymmetry, often caused by seventh cranial nerve abnormalities (36 of 56 in the mutation-positive group vs. 13 of 39 in the mutation-negative group; P=.004). Mouse embryo whole-mount and section in situ hybridization showed the expression of Chd7 in the outflow tract of the heart, optic vesicle, facio-acoustic preganglion complex, brain, olfactory pit, and mandibular component of the first branchial arch. Microarray gene-expression analysis showed a signature pattern of gene-expression differences that distinguished the individuals with CHARGE syndrome with CHD7 mutation from the controls. We conclude that cardiovascular malformations, coloboma, and facial asymmetry are common findings in CHARGE syndrome caused by CHD7 mutation.     

The Pax3 and Pax7 paralogs cooperate in neural and neural crest patterning using distinct molecular mechanisms, in Xenopus laevis embryos

Pax3 and Pax7 paralogous genes have functionally diverged in vertebrate evolution, creating opportunity for a new distribution of roles between the two genes and the evolution of novel functions. Here we focus on the regulation and function of Pax7 in the brain and neural crest of amphibian embryos, which display a different pax7 expression pattern, compared to the other vertebrates already described. Pax7 expression is restricted to the midbrain, hindbrain and anterior spinal cord, and Pax7 activity is important for maintaining the fates of these regions, by restricting otx2 expression anteriorly. In contrast, pax3 displays broader expression along the entire neuraxis and Pax3 function is important for posterior brain patterning without acting on otx2 expression. Moreover, while both genes are essential for neural crest patterning, we show that they do so using two distinct mechanisms: Pax3 acts within the ectoderm which will be induced into neural crest, while Pax7 is essential for the inducing activity of the paraxial mesoderm towards the prospective neural crest.     

Sema6B,Sema6C,and Sema6D Expression and Function during Mammalian Retinal Development

In the vertebrate retina, the formation of neural circuits within discrete laminae is critical for the establishment of retinal visual function. Precise formation of retinal circuits requires the coordinated actions of adhesive and repulsive molecules, including repulsive transmembrane semaphorins (Sema6A, Sema5A, and Sema5B). These semaphorins signal through different Plexin A (PlexA) receptors, thereby regulating distinct aspects of retinal circuit assembly. Here, we investigate the physiological roles of three Class 6 transmembrane semaphorins (Sema6B, Sema6C, and Sema6D), previously identified as PlexA receptor ligands in non-retinal tissues, in mammalian retinal development. We performed expression analysis and also phenotypic analyses of mice that carry null mutations in each of genes encoding these proteins using a broad range of inner and outer retinal markers. We find that these Class 6 semaphorins are uniquely expressed throughout postnatal retinal development in specific domains and cell types of the developing retina. However, we do not observe defects in stereotypical lamina-specific neurite stratification of retinal neuron subtypes in Sema6B^−/− or Sema6C^−/−; Sema6D^−/− retinas. These findings indicate these Class 6 transmembrane semaphorins are unlikely to serve as major PlexA receptor ligands for the assembly of murine retinal circuit laminar organization.     

Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development

Cranial neural crest cells migrate into the periocular region and later contribute to various ocular tissues including the cornea, ciliary body and iris. After reaching the eye, they initially pause before migrating over the lens to form the cornea. Interestingly, removal of the lens leads to premature invasion and abnormal differentiation of the cornea. In exploring the molecular mechanisms underlying this effect, we find that semaphorin3A (Sema3A) is expressed in the lens placode and epithelium continuously throughout eye development. Interestingly, neuropilin-1 (Npn-1) is expressed by periocular neural crest but down-regulated, in a manner independent of the lens, by the subpopulation that migrates into the eye and gives rise to the cornea endothelium and stroma. In contrast, Npn-1 expressing neural crest cells remain in the periocular region and contribute to the anterior uvea and ocular blood vessels. Introduction of a peptide that inhibits Sema3A/Npn-1 signaling results in premature entry of neural crest cells over the lens that phenocopies lens ablation. Furthermore, Sema3A inhibits periocular neural crest migration in vitro. Taken together, our data reveal a novel and essential role of Sema3A/Npn-1 signaling in coordinating periocular neural crest migration that is vital for proper ocular development.