RESUMEN
The hair bundle of cochlear hair cells comprises specialized microvilli, the stereocilia, which fulfil the role of mechanotransduction. Genetic defects and environmental noise challenge the maintenance of hair bundle structure, critically contributing to age-related hearing loss. Stereocilia fusion is a major component of the hair bundle pathology in mature hair cells, but its role in hearing loss and its molecular basis are poorly understood. Here, we utilized super-resolution expansion microscopy to examine the molecular anatomy of outer hair cell stereocilia fusion in mouse models of age-related hearing loss, heightened endoplasmic reticulum stress and prolonged noise exposure. Prominent stereocilia fusion in our model of heightened endoplasmic reticulum stress, Manf (Mesencephalic astrocyte-derived neurotrophic factor)-inactivated mice in a background with Cadherin 23 missense mutation, impaired mechanotransduction and calcium balance in stereocilia. This was indicated by reduced FM1-43 dye uptake through the mechanotransduction channels, reduced neuroplastin/PMCA2 expression and increased expression of the calcium buffer oncomodulin inside stereocilia. Sparse BAIAP2L2 and myosin 7a expression was retained in the fused stereocilia but mislocalized away from their functional sites at the tips. These hair bundle abnormalities preceded cell soma degeneration, suggesting a sequela from stereociliary molecular perturbations to cell death signalling. In the age-related hearing loss and noise-exposure models, stereocilia fusion was more restricted within the bundles, yet both models exhibited oncomodulin upregulation at the fusion sites, implying perturbed calcium homeostasis. We conclude that stereocilia fusion is linked with the failure to maintain cellular proteostasis and with disturbances in stereociliary calcium balance. KEY POINTS: Stereocilia fusion is a hair cell pathology causing hearing loss. Inactivation of Manf, a component of the endoplasmic reticulum proteostasis machinery, has a cell-intrinsic mode of action in triggering outer hair cell stereocilia fusion and the death of these cells. The genetic background with Cadherin 23 missense mutation contributes to the high susceptibility of outer hair cells to stereocilia fusion, evidenced in Manf-inactivated mice and in the mouse models of early-onset hearing loss and noise exposure. Endoplasmic reticulum stress feeds to outer hair cell stereocilia bundle pathology and impairs the molecular anatomy of calcium regulation. The maintenance of the outer hair cell stereocilia bundle cohesion is challenged by intrinsic and extrinsic stressors, and understanding the underlying mechanisms will probably benefit the development of interventions to promote hearing health.
Asunto(s)
Cadherinas , Células Ciliadas Auditivas Externas , Mecanotransducción Celular , Estereocilios , Animales , Estereocilios/metabolismo , Estereocilios/patología , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/patología , Ratones , Cadherinas/metabolismo , Cadherinas/genética , Estrés del Retículo Endoplásmico , Ratones Endogámicos C57BL , Masculino , Calcio/metabolismo , Miosina VIIa/metabolismo , Femenino , Pérdida Auditiva/patología , Pérdida Auditiva/genética , Pérdida Auditiva/metabolismo , Mutación Missense , Proteínas de Unión al CalcioRESUMEN
Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause type 1 Usher syndrome, the most common and severe form of deafblindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily because of the difficulty of purifying stable intact protein. Here, we recombinantly produce the complete human myosin-7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog that primarily associates with calmodulin (CaM), we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, CaM, and CaM-like protein 4. Our results further reveal that CaM-like protein 4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arm's structural-functional integrity. Using our recombinant protein system, we purified two myosin-7a splicing isoforms that have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechanoenzymatic properties despite differing by only 11 amino acids at their N termini. Using single-molecule in vitro motility assays, we demonstrate that human myosin-7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins. These results suggest that myosin-7a can serve multiple roles in sensory systems such as acting as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin-7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions.
Asunto(s)
Actinas , Trastornos Sordoceguera , Miosina VIIa , Humanos , Actinas/metabolismo , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Miosina VIIa/genética , Miosina VIIa/metabolismo , Calmodulina/metabolismo , Proteínas de Unión al Calcio/metabolismoRESUMEN
BACKGROUND: Usher syndrome 1 (USH1) is the most severe subtype of Usher syndrome characterized by severe sensorineural hearing impairment, retinitis pigmentosa, and vestibular areflexia. USH1 is usually induced by variants in MYO7A, a gene that encodes the myosin-VIIa protein. Myosin-VIIA is effectively involved in intracellular molecular traffic essential for the proper function of the cochlea, the retinal photoreceptors, and the retinal pigmented epithelial cells. METHODS AND RESULTS: In this study, we report a new homozygous missense variant (NM_000260.4: c.1657 C > T p.(His553Tyr)) in MYO7A of a 28-year-old female with symptoms consistent with USH1. This variant, c.1657 C > T p.(His553Tyr) is positioned in the highly conserved myosin-VIIA motor domain. Previous studies showed that variants in this domain might disrupt the ability of the protein to bind to actin and thus cause the disorder. CONCLUSIONS: Our findings contribute to our understanding of the phenotypic and mutational spectrum of USH1 associated with autosomal recessive MYO7A variants and emphasize the important role of molecular testing in accurately diagnosing this syndrome. More advanced research is required to understand the functional effect of the identified variant and the genotype-phonotype correlations of MYO7A-related Usher syndrome 1.
Asunto(s)
Homocigoto , Mutación Missense , Miosina VIIa , Síndromes de Usher , Síndromes de Usher/genética , Miosina VIIa/metabolismo , Miosina VIIa/genética , Humanos , Femenino , Mutación Missense/genética , Adulto , Miosinas/genética , LinajeRESUMEN
Usher syndrome 1B (USH1B) is a devastating genetic disorder with congenital deafness, loss of balance, and blindness caused by mutations in the myosin-VIIa (MYO7A) gene, for which there is currently no cure. We developed a gene therapy approach addressing the vestibulo-cochlear deficits of USH1B using a third-generation, high-capacity lentiviral vector system capable of delivering the large 6,645-bp MYO7A cDNA. Lentivirally delivered MYO7A and co-encoded dTomato were successfully expressed in the cochlear cell line HEI-OC1. In normal-hearing mice, both cochlea and the vestibular organ were efficiently transduced, and ectopic MYO7A overexpression did not show any adverse effects. In Shaker-1 mice, an USH1B disease model based on Myo7a mutation, cochlear and vestibular hair cells, the main inner ear cell types affected in USH1B, were successfully transduced. In homozygous mutant mice, delivery of MYO7A at postnatal day 16 resulted in a trend for partial recovery of auditory function and in strongly reduced balance deficits. Heterozygous mutant mice were found to develop severe hearing loss at 6 months of age without balance deficits, and lentiviral MYO7A gene therapy completely rescued hearing to wild-type hearing thresholds. In summary, this study demonstrates improved hearing and balance function through lentiviral gene therapy in the inner ear.
Asunto(s)
Miosinas , Síndromes de Usher , Ratones , Animales , Miosinas/genética , Miosinas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Miosina VIIa/genética , Síndromes de Usher/genética , Síndromes de Usher/terapia , Modelos Animales de Enfermedad , Mutación , Terapia GenéticaRESUMEN
Myosin VIIA (MYO7A)-associated Usher syndrome type 1B (USH1B) is a severe disorder that impacts the auditory, vestibular, and visual systems of affected patients. Due to the large size (~7.5 kb) of the MYO7A coding sequence, we have designed a dual adeno-associated virus (AAV) vector-based approach for the treatment of USH1B-related vision loss. Due to the added complexity of dual-AAV gene therapy, careful attention must be paid to the protein products expressed following vector recombination. In order to improve the sensitivity and quantifiability of our immunoassays, we adapted our traditional western blot protocol for use with the Jess™ Simple Western System. Following several rounds of testing, we optimized our protocol for the detection of MYO7A in two of our most frequently used sample types, mouse eyes, and infected HEK293 cell lysates.
Asunto(s)
Miosinas , Síndromes de Usher , Ratones , Animales , Humanos , Miosinas/genética , Miosinas/metabolismo , Células HEK293 , Síndromes de Usher/genética , Síndromes de Usher/terapia , Miosina VIIa/genética , MutaciónRESUMEN
Actin filament crosslinking, bundling and molecular motor proteins are necessary for the assembly of epithelial projections such as microvilli, stereocilia, hairs, and bristles. Mutations in such proteins cause defects in the shape, structure, and function of these actin - based protrusions. One protein necessary for stereocilia formation, Myosin VIIA, is an actin - based motor protein conserved throughout phylogeny. In Drosophila melanogaster, severe mutations in the MyoVIIA homolog crinkled (ck) are "semi - lethal" with only a very small percentage of flies surviving to adulthood. Such survivors show morphological defects related to actin bundling in hairs and bristles. To better understand ck/MyoVIIA's function in bundled - actin structures, we used dominant female sterile approaches to analyze the loss of maternal and zygotic (M/Z) ck/MyoVIIA in the morphogenesis of denticles, small actin - based projections on the ventral epidermis of Drosophila embryos. M/Z ck mutants displayed severe defects in denticle morphology - actin filaments initiated in the correct location, but failed to elongate and bundle to form normal projections. Using deletion mutant constructs, we demonstrated that both of the C - terminal MyTH4 and FERM domains are necessary for proper denticle formation. Furthermore, we show that ck/MyoVIIA interacts genetically with dusky - like (dyl), a member of the ZPD family of proteins that links the extracellular matrix to the plasma membrane, and when mutated also disrupts normal denticle formation. Loss of either protein alone does not alter the localization of the other; however, loss of the two proteins together dramatically enhances the defects in denticle shape observed when either protein alone was absent. Our data indicate that ck/MyoVIIA plays a key role in the formation and/or organization of actin filament bundles, which drive proper shape of cellular projections.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Extensiones de la Superficie Celular/ultraestructura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Miosina VIIa/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Epidermis/embriología , Femenino , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Morfogénesis , Proteínas Mutantes/metabolismo , Mutación , Miosina VIIa/genéticaRESUMEN
BACKGROUND: Variants in the MYO7A gene are increasingly identified among patients suffering from Usher syndrome type 1B (USH1B). However, such mutations are less commonly detected among patients suffering from nonsyndromic hearing loss (NSHL), including autosomal recessive deafness (DFNB2) and autosomal dominant deafness (DFNA11). This research attempts to clarify the genetic base of DFNB2 in a Chinese family and determine the pathogenicity of the identified mutations. METHOD: Targeted next-generation sequencing (TGS) of 127 known deafness genes was performed for the 14-year-old proband. Then, Sanger sequencing was performed on the available family members. A minigene splicing assay was performed to verify the impact of the novel MYO7A synonymous variant. After performing targeted next-generation sequencing (TGS) of 127 existing hearing loss-related genes in a 14-year-old proband, Sanger sequencing was carried out on the available family members. Then, to confirm the influence of the novel MYO7A synonymous variants, a minigene splicing assay was performed. RESULTS: Two heteroallelic mutants of MYO7A (NM_000260.3) were identified: a maternally inherited synonymous variant c.2904G > A (p.Glu968=) in exon 23 and a paternally inherited missense variant c.5994G > T (p.Trp1998Cys) in exon 44. The in vitro minigene expression indicated that c.2904G > A may result in skipping of exon 23 resulting in a truncated protein. CONCLUSIONS: We reported a novel missense (c.5994G > T) and identified, for the first time, a novel pathogenic synonymous (c.2904G > A) variant within MYO7A in a patient with DFNB2. These findings enrich our understanding of the MYO7A variant spectrum of DFNB2 and can contribute to accurate genetic counseling and diagnosis of NSHL patients.
Asunto(s)
Miosinas , Síndromes de Usher , Humanos , Adolescente , Miosina VIIa , Linaje , Miosinas/genética , Síndromes de Usher/genética , Secuenciación de Nucleótidos de Alto Rendimiento , ChinaRESUMEN
Recent research has revealed that an adhesion complex based on cadherins and the motor protein myosin-7b (MYO7B) links the tips of intestinal microvilli. Choi et al. now report that a largely uncharacterized protein known as calmodulin-like protein 4 (CALML4) is a component of this adhesion complex and functions as a light chain for myosin-7b. Because the intermicrovillar adhesion complex is homologous to the myosin-7a (MYO7A)-based Usher syndrome complex and Choi et al. also report that CALML4 can bind to myosin-7a, this work also has important implications for research on myosin-7a and hereditary deaf-blindness.
Asunto(s)
Miosina VIIa , Síndromes de Usher , Cadherinas/metabolismo , Dineínas , Humanos , Cadenas Ligeras de MiosinaRESUMEN
The pig breeding system provides a unique framework to study recessive defects and the consequence on the phenotype. We examined a commercial synthetic Duroc population for recessive defects and identified a haplotype on chromosome 9 significantly affecting pre-weaning mortality. To identify the causal variant underlying the mortality, we examined sequence data of four carrier animals and 21 non-carrier animals from the same population. The results yield a strong candidate causal stop-gained variant (NM_001099928.1:c.541C>T) affecting the MYO7A gene in complete linkage disequilibrium with the lethal haplotype. The variant leads to an impaired (p.Gln181*) MYO7A protein that truncates 2032 amino acids from the protein. We examined a litter from a carrier sow inseminated by a carrier boar. From the resulting piglets, two confirmed homozygous piglets suffered from severe balance difficulties and the inability to walk properly. The variant segregates at a carrier frequency of 8.2% in the evaluated population and will be gradually purged from the population, improving animal welfare. Finally, this 'natural knockout' will increase our understanding of the functioning of the MYO7A gene and provides a potential model for Usher syndrome in humans.
Asunto(s)
Longevidad/genética , Miosina VIIa/deficiencia , Sus scrofa/fisiología , Animales , Sus scrofa/genética , DesteteRESUMEN
Background: Approximately 70% of congenital deafness is attributable to genetic causes. Incidence of congenital deafness is known to be higher in families with consanguineous marriage. In this study, we investigated the genetic causes in three consanguineous Pakistani families segregating with prelingual, severe-to-profound deafness. Results: Through targeted next-generation sequencing of 414 genes known to be associated with deafness, homozygous variants c.536del (p. Leu180Serfs∗20) in TECTA, c.3719 G>A (p. Arg1240Gln) in MYO7A, and c.482+1986_1988del in HGF were identified as the pathogenic causes of enrolled families. Interestingly, in one large consanguineous family, an additional c.706G>A (p. Glu236Lys) variant in the X-linked POU3F4 gene was also identified in multiple affected family members causing deafness. Genotype-phenotype cosegregation was confirmed in all participating family members by Sanger sequencing. Conclusions: Our results showed that the genetic causes of deafness are highly heterogeneous. Even within a single family, the affected members with apparently indistinguishable clinical phenotypes may have different pathogenic variants.
Asunto(s)
Sordera/genética , Proteínas de la Matriz Extracelular/genética , Factor de Crecimiento de Hepatocito/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Miosina VIIa/genética , Factores del Dominio POU/genética , Adulto , Anciano , Consanguinidad , Femenino , Proteínas Ligadas a GPI/genética , Genes Ligados a X/genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Pakistán , Linaje , FenotipoRESUMEN
Usher syndrome is an autosomal recessive disorder characterized by congenital hearing loss combined with retinitis pigmentosa, and in some cases, vestibular areflexia. Three clinical subtypes are distinguished, and MYO7A and USH2A represent the two major causal genes involved in Usher type I, the most severe form, and type II, the most frequent form, respectively. Massively parallel sequencing was performed on a cohort of patients in the context of a molecular diagnosis to confirm clinical suspicion of Usher syndrome. We report here 231 pathogenic MYO7A and USH2A genotypes identified in 73 Usher type I and 158 Usher type II patients. Furthermore, we present the ACMG classification of the variants, which comprise all types. Among them, 68 have not been previously reported in the literature, including 12 missense and 16 splice variants. We also report a new deep intronic variant in USH2A. Despite the important number of molecular studies published on these two genes, we show that during the course of routine genetic diagnosis, undescribed variants continue to be identified at a high rate. This is particularly pertinent in the current era, where therapeutic strategies based on DNA or RNA technologies are being developed.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Genotipo , Mutación Missense , Miosina VIIa/genética , Sitios de Empalme de ARN , Síndromes de Usher , Adulto , Femenino , Francia , Humanos , Masculino , Síndromes de Usher/clasificación , Síndromes de Usher/genéticaRESUMEN
Usher syndrome (USH) is an autosomal recessive syndromic ciliopathy characterized by sensorineural hearing loss, retinitis pigmentosa and, sometimes, vestibular dysfunction. There are three clinical types depending on the severity and age of onset of the symptoms; in addition, ten genes are reported to be causative of USH, and six more related to the disease. These genes encode proteins of a diverse nature, which interact and form a dynamic protein network called the "Usher interactome". In the organ of Corti, the USH proteins are essential for the correct development and maintenance of the structure and cohesion of the stereocilia. In the retina, the USH protein network is principally located in the periciliary region of the photoreceptors, and plays an important role in the maintenance of the periciliary structure and the trafficking of molecules between the inner and the outer segments of photoreceptors. Even though some genes are clearly involved in the syndrome, others are controversial. Moreover, expression of some USH genes has been detected in other tissues, which could explain their involvement in additional mild comorbidities. In this paper, we review the genetics of Usher syndrome and the spectrum of mutations in USH genes. The aim is to identify possible mutation associations with the disease and provide an updated genotype-phenotype correlation.
Asunto(s)
Mutación , Síndromes de Usher/genética , Animales , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Proteínas de Ciclo Celular/genética , Ciliopatías/etiología , Ciliopatías/patología , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Humanos , Proteínas de la Membrana/genética , Miosina VIIa/genética , Mapas de Interacción de Proteínas/genética , Síndromes de Usher/patologíaRESUMEN
BACKGROUND: Hearing loss (HL) is the most common sensory disorder in humans, which affects individuals in both inherited and acquired forms. MYO15A and MYO7A gene mutations have a significant role in the development of deafness. In this study, we assessed the prevalence of MYO15A and MYO7A mutations in one hundred non-relative deaf Iranians. Materials and methods: The existence of MYO15A and MYO7A mutations were assessed using the tetra-primer ARMS-PCR method, High Resolution Melting (HRM) and sequencing method. Results: A heterozygote missense mutation, p.V2135L (c.6403G > T) in the MYO15A gene, was found in a patient using the sequencing method. Conclusion: These results explain the negligible prevalence of selected mutations among Iranian patients. Identifying common mutations in patients of an ethnic group can reduce the financial costs and time needed for identifying the causes of deafness.
Asunto(s)
Sordera , Miosina VIIa/genética , Miosinas , Sordera/genética , Humanos , Irán , Mutación , Miosinas/genética , LinajeRESUMEN
Objective: To analyze the clinical characteristics and identify the causative gene of a case with congenital deafness. Methods: Detailed medical history and clinical examination of a 4-year-old male child with congenital deafness were conducted in the First Affiliated Hospital of Army Military Medical University in June 2016. He was diagnosed with sensorineural deafness. The venous blood of the child and his parents was drawn, and genomic DNA was extracted. Proband's DNA was performed with targeted capture of high-throughput sequencing, then Sanger sequencing was used to verify the suspected mutation and segregation in this pedigree. According to the genetic diagnosis of the proband's deafness, ophthalmic examinations were performed. Genetic prenatal diagnosis was performed when the proband's mother was pregnant again. Results: The patient was detected with p.Trp1466Ter/p.Tyr2042Ter compound heterozygous mutations of MYO7A gene with targeted high-throughput sequencing. The mutation of p.Trp1466Ter was a reported mutation, while p.Tyr2042Ter has not been reported. In addition to congenital deafness, retinitis pigmentosa was also found by ophthalmologic examination, and the patient was clinically diagnosed with Usher syndrome type 1. Amniocentesis and fetal DNA sequencing were performed on the repregnancy fetus of this family at 18 weeks of gestation. The heterozygous mutation of MYO7A gene p.Tyr2042Ter was found, and the other allele was the wild type, indicating that the child will not exhibit clinical manifestations of Usher syndrome type 1. Indeed, the second child passed neonatal hearing screening. Conclusions: The clinical features and genetic variants were delineated in this family with Usher syndrome type 1. The results of the current study have enriched the phenotype and genotype data of the disease and provided a basis for genetic counseling.
Asunto(s)
Síndromes de Usher , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Mutación , Miosina VIIa , Miosinas/genética , Linaje , Embarazo , Diagnóstico Prenatal , Síndromes de Usher/genéticaRESUMEN
Ion pairs are key stabilizing interactions between oppositely charged amino acid side chains in proteins. They are often depicted as single conformer salt bridges (hydrogen-bonded ion pairs) in crystal structures, but it is unclear how dynamic they are in solution. Ion pairs are thought to be particularly important in stabilizing single α-helix (SAH) domains in solution. These highly stable domains are rich in charged residues (such as Arg, Lys, and Glu) with potential ion pairs across adjacent turns of the helix. They provide a good model system to investigate how ion pairs can contribute to protein stability. Using NMR spectroscopy, small-angle X-ray light scattering (SAXS), and molecular dynamics simulations, we provide here experimental evidence that ion pairs exist in a SAH in murine myosin 7a (residues 858-935), but that they are not fixed or long lasting. In silico modeling revealed that the ion pairs within this α-helix exhibit dynamic behavior, rapidly forming and breaking and alternating between different partner residues. The low-energy helical state was compatible with a great variety of ion pair combinations. Flexible ion pair formation utilizing a subset of those available at any one time avoided the entropic penalty of fixing side chain conformations, which likely contributed to helix stability overall. These results indicate the dynamic nature of ion pairs in SAHs. More broadly, thermodynamic stability in other proteins is likely to benefit from the dynamic behavior of multi-option solvent-exposed ion pairs.
Asunto(s)
Miosinas/química , Miosinas/metabolismo , Animales , Cristalografía por Rayos X , Ratones , Simulación de Dinámica Molecular , Miosina VIIa , Conformación Proteica en Hélice alfa , Estabilidad ProteicaRESUMEN
Unconventional myosin VIIA (Myo7a) is an actin-based motor molecule that normally functions in the cochlear hair cells of the inner ear. Mutations of MYO7A/Myo7a have been implicated in inherited deafness in both humans and mice. However, there is limited information about the functions of Myo7a outside of the specialized cells of the ears. Herein, we report a previously unidentified function of Myo7a by demonstrating that it plays an important role in melanoma progression. We found that silencing Myo7a by means of RNAi inhibited melanoma cell growth through upregulation of cell cycle regulator p21 (also known as CDKN1A) and suppressed melanoma cell migration and invasion through downregulation of RhoGDI2 (also known as ARHGDIB) and MMP9. Furthermore, Myo7a depletion suppressed melanoma cell metastases to the lung, kidney and bone in mice. In contrast, overexpression of Myo7a promoted melanoma xenograft growth and lung metastasis. Importantly, Myo7a levels are remarkably elevated in human melanoma patients. Collectively, we demonstrated for the first time that Myo7a is able to function in non-specialized cells, a finding that reveals the complicated disease-related roles of Myo7a, especially in melanomas.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Melanoma/genética , Miosinas/genética , Inhibidor beta de Disociación del Nucleótido Guanina rho/genética , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Oído Interno/metabolismo , Oído Interno/patología , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patología , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Neoplasias Renales/secundario , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Metaloproteinasa 9 de la Matriz/genética , Melanoma/patología , Ratones , Mutación , Miosina VIIa , Miosinas/antagonistas & inhibidores , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To document the rod-cone dystrophy phenotype of patients with Usher syndrome type 1 (USH1) harboring MYO7A mutations. METHODS: Retrospective cohort study of 53 patients (42 families) with biallelic MYO7A mutations who underwent comprehensive examination, including functional visual tests and multimodal retinal imaging. Genetic analysis was performed either using a multiplex amplicon panel or through direct sequencing. Data were analyzed with IBM SPSS Statistics software v. 21.0. RESULTS: Fifty different genetic variations including 4 novel were identified. Most patients showed a typical rod-cone dystrophy phenotype, with best-corrected visual acuity and central visual field deteriorating linearly with age. At age 29, binocular visual field demonstrated an average preservation of 50 central degrees, constricting by 50% within 5 years. Structural changes based on spectral domain optical coherence tomography, short wavelength autofluorescence, and near-infrared autofluorescence measurements did not however correlate with age. Our study revealed a higher percentage of epiretinal membranes and cystoid macular edema in patients with MYO7A mutations compared with rod-cone dystrophy patients with other mutations. Subgroup analyses did not reveal substantial genotype-phenotype correlations. CONCLUSION: To the best of our knowledge, this is the largest French cohort of patients with MYO7A mutations reported to date. Functional visual characteristics of this subset of patients followed a linear decline as in other typical rod-cone dystrophy, but structural changes were variable indicating the need for a case-by-case evaluation for prognostic prediction and choice of potential therapies.
Asunto(s)
Distrofias de Conos y Bastones/genética , Mutación , Miosina VIIa/genética , Síndromes de Usher/genética , Adolescente , Adulto , Niño , Preescolar , Distrofias de Conos y Bastones/diagnóstico , Distrofias de Conos y Bastones/fisiopatología , Análisis Mutacional de ADN , Electrorretinografía , Femenino , Francia , Estudios de Asociación Genética , Humanos , Lactante , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , Tomografía de Coherencia Óptica , Síndromes de Usher/diagnóstico , Síndromes de Usher/fisiopatología , Agudeza Visual/fisiología , Pruebas del Campo Visual , Campos Visuales/fisiología , Adulto JovenRESUMEN
Unconventional myosin 7a (Myo7a), myosin 7b (Myo7b), and myosin 15a (Myo15a) all contain MyTH4-FERM domains (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in their cargo binding tails and are essential for the growth and function of microvilli and stereocilia. Numerous mutations have been identified in the MyTH4-FERM tandems of these myosins in patients suffering visual and hearing impairment. Although a number of MF domain binding partners have been identified, the molecular basis of interactions with the C-terminal MF domain (CMF) of these myosins remains poorly understood. Here we report the high-resolution crystal structure of Myo7b CMF in complex with the extended PDZ3 domain of USH1C (a.k.a., Harmonin), revealing a previously uncharacterized interaction mode both for MyTH4-FERM tandems and for PDZ domains. We predicted, based on the structure of the Myo7b CMF/USH1C PDZ3 complex, and verified that Myo7a CMF also binds to USH1C PDZ3 using a similar mode. The structure of the Myo7b CMF/USH1C PDZ complex provides mechanistic explanations for >20 deafness-causing mutations in Myo7a CMF. Taken together, these findings suggest that binding to PDZ domains, such as those from USH1C, PDZD7, and Whirlin, is a common property of CMFs of Myo7a, Myo7b, and Myo15a.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Complejos Multiproteicos/química , Miosinas/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células CACO-2 , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Dominios PDZ , Estructura Cuaternaria de ProteínaRESUMEN
OBJECTIVE: To detect potential variants in a family affected with Usher syndrome type I, and analyze its genotype-phenotype correlation. METHODS: Clinical data of the family was collected. Potential variants in the proband were detected by high-throughput sequencing. Suspected variants were verified by Sanger sequencing. RESULTS: The proband developed night blindness at 10 year old, in addition with bilateral cataract and retinal degeneration. Hearing loss occurred along with increase of age. High-throughput sequencing and Sanger sequencing revealed that she has carried compound heterozygous variants of the MYO7A gene, namely c.2694+2T>G and c.6028G>A. Her sister carried the same variants with similar clinical phenotypes. Her daughter was heterozygous for the c.6028G>A variant but was phenotypically normal. CONCLUSION: The clinical features and genetic variants were delineated in this family with Usher syndrome type I. The results have enriched the phenotype and genotype data of the disease and provided a basis for genetic counseling.
Asunto(s)
Genotipo , Fenotipo , Síndromes de Usher , Niño , Femenino , Variación Genética , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Miosina VIIa/genética , Ceguera Nocturna/etiología , Linaje , Síndromes de Usher/genética , Síndromes de Usher/patologíaRESUMEN
Mutations in the MYO7A gene, encoding the motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome in humans, and the shaker-1 phenotype, characterized by deafness, head tossing, and circling behavior, in mice. Myosin VIIa is responsible for tension bearing and the transduction mechanism in the stereocilia and for melanosome transport in the retina, in line with the phenotypic outcomes observed in mice. However, the effect of the shaker-1 mutation, a R502P amino acid substitution, on the motor function is unclear. To explore this question, we determined the kinetic properties and the effect on the filopodial tip localization of the recombinant mouse myosin VIIa-5IQ-SAH R502P (myoVIIa-sh1) construct. Interestingly, although residue 502 is localized to a region thought to be involved in interacting with actin, the kinetic parameters for actin binding changed only slightly for the mutant construct. However, the rate constant for ATP hydrolysis (k+H + k-H) was reduced by â¼200-fold from 12 s-1 to 0.05 s-1, making the hydrolysis step the rate-limiting step of the ATPase cycle in the presence and absence of actin. Given that wild-type mouse myosin VIIa is a slow, high-duty ratio, monomeric motor, this altered hydrolysis rate would reduce activity to extremely low levels. Indeed, the translocation to the filopodial tips was hampered by the diminished motor function of a dimeric construct of the shaker-1 mutant. We conclude that the diminished motor activity of this mutant is most likely responsible for impaired hearing in the shaker-1 mice.