CHD7 Mutations Research Articles

Mature middle and inner ears express Chd7 and exhibit distinctive pathologies in a mouse model of CHARGE syndrome

Heterozygous mutations in the gene encoding chromodomain-DNA-binding-protein 7 (CHD7) cause CHARGE syndrome, a multiple anomaly condition which includes vestibular dysfunction and hearing loss. Mice with heterozygous Chd7 mutations exhibit semicircular canal dysgenesis and abnormal inner ear neurogenesis, and are an excellent model of CHARGE syndrome. Here we characterized Chd7 expression in mature middle and inner ears, analyzed morphological features of mutant ears and tested whether Chd7 mutant mice have altered responses to noise exposure and correlated those responses to inner and middle ear structure. We found that Chd7 is highly expressed in mature inner and outer hair cells, spiral ganglion neurons, vestibular sensory epithelia and middle ear ossicles. There were no obvious defects in individual hair cell morphology by Prestin immunostaining or scanning electron microscopy, and cochlear innervation appeared normal in Chd7 Gt /+ mice. Hearing thresholds by auditory brainstem response (ABR) testing were elevated at 4 and 16 kHz in Chd7 Gt /+ mice, and there were reduced distortion product otoacoustic emissions (DPOAE). Exposure of Chd7 Gt /+ mice to broadband noise resulted in variable degrees of hair cell loss which inversely correlated with severity of stapedial defects. The degrees of hair cell loss and threshold shifts after noise exposure were more severe in wild type mice than in mutants. Together, these data indicate that Chd7 Gt /+ mice have combined conductive and sensorineural hearing loss, correlating with changes in both middle and inner ears.

Incidence, Phenotypic Features and Molecular Genetics of Kallmann Syndrome in Finland

BackgroundKallmann syndrome (KS), comprised of congenital hypogonadotropic hypogonadism (HH) and anosmia, is a clinically and genetically heterogeneous disorder. Its exact incidence is currently unknown, and a mutation in one of the identified KS genes has only been found in ~30% of the patients.MethodsHerein, we investigated epidemiological, clinical, and genetic features of KS in Finland.ResultsThe minimal incidence estimate of KS in Finland was 1:48 000, with clear difference between males (1:30 000) and females (1:125 000) (p = 0.02). The reproductive phenotype of 30 probands (25 men; 5 women) ranged from severe HH to partial puberty. Comprehensive mutation analysis of all 7 known KS genes (KAL1, FGFR1, FGF8, PROK2, PROKR2, CHD7, and WDR11) in these 30 well-phenotyped probands revealed mutations in KAL1 (3 men) and FGFR1 (all 5 women vs. 4/25 men), but not in other genes.ConclusionsOur results suggest that Finnish KS men harbor mutations in gene(s) yet-to-be discovered with sex-dependent penetrance of the disease phenotype. In addition, some KS patients without CHD7 mutations display CHARGE-syndrome associated phenotypic features (e.g. ear or eye anomalies), possibly implying that, in addition to CHD7, there may be other genes associated with phenotypes ranging from KS to CHARGE.

CHD7 mutations causing CHARGE syndrome are predominantly of paternal origin

CHARGE (coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, ear anomalies and deafness) syndrome is a congenital malformation syndrome caused by mutations in the CHD7 gene in approximately 2/3 of cases. In the vast majority of cases, CHARGE syndrome is sporadic. There are only a few reports of parent-to-child transmission and somatic or gonadal mosaicism. To determine the parental origin of CHD7 mutations in sporadic CHARGE syndrome, we screened 30 families for informative exonic or intronic polymorphisms located near the detected CHD7 mutation. An informative polymorphism could be identified in 13 out of 30 families. Linkage analysis was performed between the CHD7 mutation and the polymorphism in the child. In 12 out of 13 families, the mutation affected the paternal allele (92.3%). In our cohort, the mean paternal age at birth was 32.92 years. Comparing the age of fathers of an affected CHARGE patient with the paternal age of the German population in general, we could not observe any paternal age effect. Taken together, we show in this study that de novo CHD7 mutations occur predominantly in the male germ line.

Reproductive dysfunction and decreased GnRH neurogenesis in a mouse model of CHARGE syndrome

CHARGE is a multiple congenital anomaly disorder and a common cause of pubertal defects, olfactory dysfunction, growth delays, deaf-blindness, balance disorders and congenital heart malformations. Mutations in CHD7, the gene encoding chromodomain helicase DNA binding protein 7, are present in 60-80% of individuals with the CHARGE syndrome. Mutations in CHD7 have also been reported in the Kallmann syndrome (olfactory dysfunction, delayed puberty and hypogonadotropic hypogonadism). CHD7 is a positive regulator of neural stem cell proliferation and olfactory sensory neuron formation in the olfactory epithelium, suggesting that the loss of CHD7 might also disrupt development of other neural populations. Here we report that female Chd7(Gt/+) mice have delays in vaginal opening and estrus onset, and erratic estrus cycles. Chd7(Gt/+) mice also have decreased circulating levels of luteinizing hormone and follicle-stimulating hormone but apparently normal responsiveness to gonadotropin-releasing hormone (GnRH) agonist and antagonist treatment. GnRH neurons in the adult Chd7(Gt/+) hypothalamus and embryonic nasal region are diminished, and there is decreased cellular proliferation in the embryonic olfactory placode. Expression levels of GnRH1 and Otx2 in the hypothalamus and GnRHR in the pituitary are significantly reduced in adult Chd7(Gt/+) mice. Additionally, Chd7 mutant embryos have CHD7 dosage-dependent reductions in expression levels of Fgfr1, Bmp4 and Otx2 in the olfactory placode. Together, these data suggest that CHD7 has critical roles in the development and maintenance of GnRH neurons for regulating puberty and reproduction.

Genetic and expressional alterations of CHD genes in gastric and colorectal cancers

Chromodomain helicase DNA-binding protein (CHD) is a regulator of the chromatin remodelling process. The aim was to determine the CHD1, CHD2, CHD3, CHD4, CHD7, CHD8 and CHD9 mutational status of mononucleotide repeats in gastric and colorectal cancers with microsatellite instability (MSI). The repeats were determined in 28 gastric cancers (GCs) with high MSI (MSI-H), 45 GCs with low MSI (MSI-L)/stable MSI (MSS), 35 colorectal cancers (CRCs) with MSI-H and 45 CRCs with MSI-L/MSS by single-strand conformation polymorphism analysis. CHD4 and CHD8 expression was also examined in GCs and CRCs by immunohistochemistry. CHD1, CHD2, CHD3, CHD4, CHD7, CHD8 and CHD9 mutations were found in five, 19, three, five, seven, 10 and seven cancers, respectively. They were detected in MSI-H cancers, but not in MSI-L/MSS cancers. Loss of CHD4 expression was observed in 56.4% of the GCs and 55.7% of the CRCs, and loss of CHD8 was observed in 35.7% of the GCs and 28.6% of the CRCs. The cancers with CHD4 and CHD8 mutations showed loss of CHD4 and CHD8 expression, respectively. Frameshift mutation and loss of expression of CHD genes are common in GCs and CRCs with MSI-H.These alterations might contribute to cancer pathogenesis by deregulating CHD-mediated chromatin remodelling.

Role of rare cases in deciphering the mechanisms of congenital anomalies: CHARGE syndrome research

In this review, our work on CHARGE syndrome will be used to exemplify the role of rare cases in birth defects research. The analysis of 29 cases with mutations of CHD7, the causative gene for CHARGE syndrome, clarified the relative importance of the cardinal features, including facial nerve palsy and facial asymmetry. Concurrently, in situ hybridization using chick embryos studies were performed to delineate the expression pattern of Chd7. The Chd7-positive regions in the chick embryos and the anatomical defects commonly seen in patients with CHARGE syndrome were well correlated: expression in the optic placode corresponded with defects such as coloboma, neural tube with mental retardation, and otic placode with ear abnormalities. The correlation between expression in the branchial arches and nasal placode with the clinical symptoms of CHARGE syndrome, however, became apparent when we encountered two unique CHARGE syndrome patients: one with a DiGeorge syndrome phenotype and the other with a Kallman syndrome phenotype. A unifying hypothesis that could explain both the DiGeorge syndrome phenotype and the Kallman syndrome phenotype in patients with CHARGE syndrome may be that the mutation in CHD7 is likely to exert its effect in the common branch of the two pathways of neural crest cells. As exemplified in CHARGE syndrome research, rare cases play a critical role in deciphering the mechanisms of human development. Close collaboration among animal researchers, epidemiologists and clinicians hopefully will enhance and maximize the scientific value of rare cases.

Delayed Puberty Due to a Novel Mutation in CHD7 Causing CHARGE Syndrome

We report the case of a 15-year-old girl who presented to a pediatric endocrinology clinic for delayed puberty with no signs of secondary sexual development. Her past medical history was significant for bilateral colobomas, inner-ear anomalies, hearing loss, and anosmia. Genetic testing revealed a novel de novo mutation in the CHD7 gene, one of the causative genes in CHARGE syndrome (coloboma, heart disease, choanal atresia, retarded growth and development and/or central nervous system anomalies, genital anomalies and/or hypogonadism, and ear anomalies and/or deafness). We review the distinction between hypogonadotrophic hypogonadism and hypergonadotrophic hypogonadism and discuss the availability of molecular genetic testing for idiopathic hypogonadotrophic hypogonadism. CHD7 mutations have also been found in some patients with Kallmann syndrome, hypogonadotrophic hypogonadism, and anosmia, and we discuss the overlap between this syndrome and CHARGE syndrome. With the increased availability of genetic testing for a variety of disorders, it is important for pediatricians to become familiar with interpreting genetic test results. Finally, we illustrate that Bayes' theorem is a useful statistical tool for interpreting novel missense mutations of unknown significance.

Mutations in the CHD7 Gene: The Experience of a Commercial Laboratory

CHARGE syndrome is an autosomal dominant multisystem disorder caused by mutation in the CHD7 gene, encoding chromodomain helicase DNA-binding protein 7. Molecular diagnostic testing for CHD7 mutation has been available in a clinical setting since 2005. We report here the results from the first 642 unrelated proband samples submitted for testing. Thirty-two percent (n = 203) of patient samples had a heterozygous pathogenic variant identified. The lower mutation rate than that published for well-characterized clinical samples is likely due to referral bias, as samples submitted for clinical testing may be for "rule-out" diagnoses, rather than solely to confirm clinical suspicion. We identified 159 unique pathogenic mutations, and of these, 134 mutations were each seen in a single individual and 25 mutations were found in two to five individuals (n =69). Of the 203 mutations, only 9 were missense, with 107 nonsense, 69 frameshift, and 15 splice-site mutations likely leading to haploinsufficiency at the cellular level. An additional 72 variations identified in the 642 tested samples (11%) were considered to have unknown clinical significance. Copy number changes (deletion/duplication of the entire gene or one/several exons) were found to account for a very small number of cases (n = 3). This cohort represents the largest CHARGE syndrome sample size to date and is intended to serve as a resource for clinicians, genetic counselors, researchers, and other diagnostic laboratories.

Characterization of a de novo balanced t(4;20)(q33;q12) translocation in a patient with mental retardation

CHD6 is an ATP-dependent chromatin-remodeling enzyme, which has been implicated as a crucial component for maintaining and regulating chromatin structure. CHD6 belongs to the largest subfamily, subfamily III (CHD6-9), of the chromodomain helicase DNA (CHD-binding protein) family of enzymes (CHD1-9). Here we report on a female patient with a balanced translocation t(4;20)(q33;q12) presenting with severe mental retardation and brachydactyly of the toes. We identified the translocation breakpoint in intron 27 of CHD6 at 20q12, while the 4q33 breakpoint was intergenic. Northern blot analysis demonstrated the CHD6 mRNA in the patient's lymphoblastoid cells was decreased to ∼50% of the control cells. To investigate the cellular mechanism of diseases resulting from decreased CHD subfamily III proteins, we knocked down CHD6 or CHD7 by RNA interference in HeLa cells and analyzed chromosome alignment. The both CHD6- and CHD7-knockdown cells showed increased frequency of misaligned chromosomes on metaphase plates. Moreover, an elevated frequency of aneuploidy, the major cause of miscarriages and mental retardation, was observed in patients with CHD6 and CHD7 haploinsufficiency. These results suggest that CHD6 and CHD7 play important roles in chromatin assembly during mitosis and that mitotic delay and/or impaired cell proliferation may be associated with pathogenesis of the diseases caused by CHD6 or CHD7 mutations.

Prevalence of Genetic Testing in CHARGE Syndrome

Parents of 145 individuals with a clinical diagnosis of CHARGE syndrome, ages 2 to 39 years, indicated in a survey whether their child had been tested for the CHD7 mutation, which is the only gene presently known to be associated with CHARGE. More than two thirds (68%) of the affected individuals had never been gene tested. Of the 46 who had been tested, 74% tested positive for the mutation. Half (50%) of those who underwent testing did so as a part of a conference blood draw by Baylor College of Medicine in 1999. Children who were tested were significantly younger than those who had not been tested. A second group of 43 parents were informally surveyed at a conference in 2009. More than half of their children had been tested, and nearly 70% were positive for the mutation. Reasons given by these parents for testing included confirming the diagnosis and assisting research. Reasons given for not testing included lack of opportunity, no known benefit, and lack of insurance coverage.

The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear

Inner ear neurogenesis is positively regulated by the pro-neural bHLH transcription factors Ngn1 and NeuroD, but the factors that act upstream of this regulation are not well understood. Recent evidence in mouse and Drosophila suggests that neural development depends on proper chromatin remodeling, both for maintenance of neural stem cells and for proper neuronal differentiation. Here, we show that CHD7, an ATP-dependent chromatin remodeling enzyme mutated in human CHARGE syndrome, is necessary for proliferation of inner ear neuroblasts and inner ear morphogenesis. Conditional deletion of Chd7 in the developing otocyst using Foxg1-Cre resulted in cochlear hypoplasia and complete absence of the semicircular canals and cristae. Conditional knockout and null otocysts also had reductions in vestibulo-cochlear ganglion size and neuron number in combination with reduced expression of Ngn1, Otx2 and Fgf10, concurrent with expansion of the neural fate suppressor Tbx1 and reduced cellular proliferation. Heterozygosity for Chd7 mutations had no major effects on expression of otic patterning genes or on cell survival, but resulted in decreased proliferation within the neurogenic domain. These data indicate that epigenetic regulation of gene expression by CHD7 must be tightly coordinated for proper development of inner ear neuroblasts.

Combined microdeletions and CHD7 mutation causing severe CHARGE/DiGeorge syndrome: clinical presentation and molecular investigation by array-CGH

Phenotypic variation in CHARGE syndrome remains unexplained. A subcategory of CHARGE patients show overlapping phenotypic characteristics with DiGeorge syndrome (thymic hypo/aplasia, hypocalcemia, T-cell immunodeficiency). Very few have been tested or reported to carry a mutation of the CHD7 (chromodomain helicase DNA-binding domain) gene detected in two-thirds of CHARGE patients. In an attempt to explore the genetic background of a severe CHARGE/DiGeorge phenotype, we performed comparative genomic array hybridization in an infant carrier of a CHD7 mutation. The high-resolution comparative genomic array hybridization revealed interesting findings, including a deletion distal to the DiGeorge region and disruptions in other chromosomal regions of genes implicated in immunological and other functions possibly contributing to the patient's severe phenotype and early death.

Chromodomain proteins in development: lessons from CHARGE syndrome

In humans, heterozygous mutations in the adenosine triphosphate-dependent chromatin remodeling gene CHD7 cause CHARGE syndrome, a common cause of deaf-blindness, balance disorders, congenital heart malformations, and olfactory dysfunction with an estimated incidence of approximately 1 in 10,000 newborns. The clinical features of CHARGE in humans and mice are highly variable and incompletely penetrant, and most mutations appear to result in haploinsufficiency of functional CHD7 protein. Mice with heterozygous loss of function mutations in Chd7 are a good model for CHARGE syndrome, and analyses of mouse mutant phenotypes have begun to clarify a role for CHD7 during development and into adulthood. Chd7 heterozygous mutant mice have postnatal delayed growth, inner ear malformations, anosmia/hyposmia, and craniofacial defects, and Chd7 homozygous mutants are embryonic lethal. A central question in developmental biology is how chromodomain proteins like CHD7 regulate important developmental processes, and whether they directly activate or repress downstream gene transcription or act more globally to alter chromatin structure and/or function. CHD7 is expressed in a wide variety of tissues during development, suggesting that it has tissue-specific and developmental stage-specific roles. Here, we review recent and ongoing analyses of CHD7 function in mouse models and cell-based systems. These studies explore tissue-specific effects of CHD7 deficiency, known CHD7 interacting proteins, and downstream target sites for CHD7 binding. CHD7 is emerging as a critical regulator of important developmental processes in organs affected by human CHARGE syndrome.

The inner ear phenotype of Volchok (Vlk): An ENU-induced mouse model for CHARGE syndrome

Objective: The Volchok (Vlk) mouse originated as a part of a large-scale ENU program on a C3HeB/FeJ background. The mice present a dominant pattern of inheritance refl ected mainly by circling activity, which led us to search for the causative mutation underlying the vestibular phenotype. Study design: Linkage analysis and subsequent sequencing were used to narrow down the genomic region and uncover the mutation, respectively. Behavioral analysis and examination of the inner ear phenotype was performed using the modifi ed SHIRPA protocol, auditory brainstem response (ABR), immunofl uorescence, light microscopy, scanning electron microscopy (SEM), paint-fi ll and neurofi lament staining techniques. Results: A nonsense mutation was discovered in the Chd7 gene responsible for CHARGE syndrome in humans, which leads to a decrease in Chd7 expression in the nuclei of the vestibular and cochlear cells and structural abnormalities in the inner ear. A mild hearing loss was found in the mutant mice. The malformations detected include a truncated lateral semicircular canal and smaller lateral crista, a decrease in size or absence of the round window in the cochlea and lack of innervations to the posterior crista. Conclusion: Vlk joins a series of previously characterized mice bearing a Chd7 mutation, linking abnormalities found both in ENU-induced mutants and a knock-out mouse model. Combining the information from multiple Chd7 alleles provides a more comprehensive understanding of the mechanisms underlying this human disease.

A candidate gene for choanal atresia in alpaca

Choanal atresia (CA) is a common nasal craniofacial malformation in New World domestic camelids (alpaca and llama). CA results from abnormal development of the nasal passages and is especially debilitating to newborn crias. CA in camelids shares many of the clinical manifestations of a similar condition in humans (CHARGE syndrome). Herein we report on the regulatory gene CHD7 of alpaca, whose homologue in humans is most frequently associated with CHARGE. Sequence of the CHD7 coding region was obtained from a non-affected cria. The complete coding region was 9003 bp, corresponding to a translated amino acid sequence of 3000 aa. Additional genomic sequences corresponding to a significant portion of the CHD7 gene were identified and assembled from the 2x alpaca whole genome sequence, providing confirmatory sequence for much of the CHD7 coding region. The alpaca CHD7 mRNA sequence was 97.9% similar to the human sequence, with the greatest sequence difference being an insertion in exon 38 that results in a polyalanine repeat (A12). Polymorphism in this repeat was tested for association with CA in alpaca by cloning and sequencing the repeat from both affected and non-affected individuals. Variation in length of the poly-A repeat was not associated with CA. Complete sequencing of the CHD7 gene will be necessary to determine whether other mutations in CHD7 are the cause of CA in camelids.

Molecular and phenotypic aspects of CHD7 mutation in CHARGE syndrome

CHARGE syndrome [coloboma of the eye, heart defects, atresia of the choanae, retardation of growth and/or development, genital and/or urinary abnormalities, and ear abnormalities (including deafness)] is a genetic disorder characterized by a specific and a recognizable pattern of anomalies. De novo mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7) are the major cause of CHARGE syndrome. Here, we review the clinical features of 379 CHARGE patients who tested positive or negative for mutations in CHD7. We found that CHARGE individuals with CHD7 mutations more commonly have ocular colobomas, temporal bone anomalies (semicircular canal hypoplasia/dysplasia), and facial nerve paralysis compared with mutation negative individuals. We also highlight recent genetic and genomic studies that have provided functional insights into CHD7 and the pathogenesis of CHARGE syndrome.

Successful cord blood transplantation for a CHARGE syndrome with CHD7 mutation showing DiGeorge sequence including hypoparathyroidism

It is rare that coloboma, heart anomalies, choanal atresia, retarded growth and development, and genital and ear anomalies (CHARGE) syndrome patients have DiGeorge sequence showing severe immunodeficiency due to the defect of the thymus. Although the only treatment to achieve immunological recovery for these patients in countries where thymic transplantation is not ethically approved would be hematopoietic cell transplantation, long-term survival has not been obtained in most patients. On the other hand, it is still not clarified whether hypoparathyroidism is one of the manifestations of CHARGE syndrome. We observed a CHARGE syndrome patient with chromodomain helicase DNA-binding protein 7 mutation showing DiGeorge sequence including the defect of T cells accompanied with the aplasia of the thymus, severe hypoparathyroidism, and conotruncal cardiac anomaly. He received unrelated cord blood transplantation without conditioning at 4 months of age. Recovery of T cell number and of proliferative response against mitogens was achieved by peripheral expansion of mature T cells in cord blood without thymic output. Although he is still suffering from severe hypoparathyroidism, he is alive without serious infections for 10 months.

321 Congenital heart defects in CHARGE syndrome patients with CHD7 mutations

CHARGE syndrome (MIM 214800) consists of a combination of congenital malformations including Coloboma, Heart defects, Atresia of choanae, Retardation of growth and developmental delay, Genital anomalies and Ear anomalies. Diagnosis criteria have been recently refined and several other features have been described such as semi-circular canals and cranial nerve anomalies. A CHD7 gene mutation is found in 60% of patients with a clinical diagnosis of CHARGE syndrome. Although frequently found, cardiovascular malformations (CVM) are not specific.

Novel CHD7 and FBN1 mutations in an infant with multiple congenital anamolies

The first case of an infant with a dual genetic diagnosis of CHARGE and Marfan syndrome is reported here. The patient had multiple congenital anamolies, many of them consistent with CHARGE syndrome and genetic testing identified a heterozygous mutation c.3806_11del6insA in the CHD7 gene. In addition, his father had physical features consistent with Marfan syndrome. Fibrillin-1 (FBN1) mutation screening identified a heterozygous c.3990insC mutation in both father and the patient.

Study of smell and reproductive organs in a mouse model for CHARGE syndrome

CHARGE syndrome is a multiple congenital anomaly syndrome characterised by Coloboma, Heart defects, Atresia of choanae, Retardation of growth and/or development, Genital hypoplasia, and Ear anomalies often associated with deafness. It is caused by heterozygous mutations in the CHD7 gene and shows a highly variable phenotype. Anosmia and hypogonadotropic hypogonadism occur in the majority of the CHARGE patients, but the underlying pathogenesis is unknown. Therefore, we studied the ability to smell and aspects of the reproductive system (reproductive performance, gonadotropin-releasing hormone (GnRH) neurons and anatomy of testes and uteri) in a mouse model for CHARGE syndrome, the whirligig mouse (Chd7Whi/+). We showed that Chromodomain Helicase DNA-binding protein 7 (Chd7) is expressed in brain areas involved in olfaction and reproduction during embryonic development. We observed poorer performance in the smell test in adult Chd7Whi/+ mice, secondary either to olfactory dysfunction or to balance disturbances. Olfactory bulb and reproductive organ abnormalities were observed in a proportion of Chd7Whi/+ mice. Hypothalamic GnRH neurons were slightly reduced in Chd7Whi/+ females and reproductive performance was slightly less in Chd7Whi/+ mice. This study shows that the penetrance of anosmia and hypogonadotropic hypogonadism is lower in Chd7Whi/+ mice than in CHARGE patients. Interestingly, many phenotypic features of the Chd7 mutation showed incomplete penetrance in our model mice, despite the use of inbred, genetically identical mice. This supports the theory that the extreme variability of the CHARGE phenotype in both humans and mice might be attributed to variations in the fetal microenvironment or to purely stochastic events.