RESUMO
Hearing depends on intricate morphologies and mechanical properties of diverse inner ear cell types. The individual contributions of various inner ear cell types into mechanical properties of the organ of Corti and the mechanisms of their integration are yet largely unknown. Using sub-100-nm spatial resolution atomic force microscopy (AFM), we mapped the Young's modulus (stiffness) of the apical surface of the different cells of the freshly dissected P5-P6 cochlear epithelium from wild-type and mice lacking either Trio and F-actin binding protein (TRIOBP) isoforms 4 and 5 or isoform 5 only. Variants of TRIOBP are associated with deafness in human and in Triobp mutant mouse models. Remarkably, nanoscale AFM mapping revealed unrecognized bidirectional radial stiffness gradients of different magnitudes and opposite orientations between rows of wild-type supporting cells and sensory hair cells. Moreover, the observed bidirectional radial stiffness gradients are unbalanced, with sensory cells being stiffer overall compared to neighboring supporting cells. Deafness-associated TRIOBP deficiencies significantly disrupted the magnitude and orientation of these bidirectional radial stiffness gradients. In addition, serial sectioning with focused ion beam and backscatter scanning electron microscopy shows that a TRIOBP deficiency results in ultrastructural changes of supporting cell apical phalangeal microfilaments and bundled cortical F-actin of hair cell cuticular plates, correlating with messenger RNA and protein expression levels and AFM stiffness measurements that exposed a softening of the apical surface of the sensory epithelium in mutant mice. Altogether, this additional complexity in the mechanical properties of the sensory epithelium is hypothesized to be an essential contributor to frequency selectivity and sensitivity of mammalian hearing.
Assuntos
Citoesqueleto de Actina , Surdez , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Cóclea/metabolismo , Surdez/metabolismo , Células Ciliadas Auditivas/metabolismo , Mamíferos/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Órgão Espiral , Isoformas de Proteínas/metabolismoRESUMO
PURPOSE: Genome sequencing (GS) is one of the most comprehensive assays that interrogate single-nucleotide variants, copy number variants, mitochondrial variants, repeat expansions, and structural variants in a single assay. Despite the clear technical superiority, the full clinical utility of GS has yet to be determined. METHODS: We systematically evaluated 2100 clinical GS index cases performed in our laboratory to explore the diagnostic yield of GS as first-tier and as follow-up testing. RESULTS: The overall diagnostic yield was 28% (585/2100). The diagnostic yield for GS as the first-tier test was 26% (294/1146). Among cases with prior non-diagnostic genetic tests, GS provided a diagnosis for 27% (247/910) of cases, including 56 cases with prior exome sequencing (ES). Although re-analysis of previous ES might have resolved the diagnosis in 29 cases, diagnoses for 27 cases would have been missed because of the technical inferiority of ES. Moreover, GS further disclosed additional genetic etiology in 3 out of 44 cases with existing partial diagnosis. CONCLUSION: We present the largest-to-date GS data set of a clinically heterogeneous cohort from a single clinical laboratory. Our data demonstrate that GS should be considered as the first-tier genetic test that has the potential to shorten the diagnostic odyssey.
Assuntos
Exoma , Testes Genéticos , Humanos , Exoma/genética , Sequência de Bases , Mapeamento Cromossômico , Sequenciamento do ExomaRESUMO
Hereditary deafness and retinal dystrophy are each genetically heterogenous and clinically variable. Three small unrelated families segregating the combination of deafness and retinal dystrophy were studied by exome sequencing (ES). The proband of Family 1 was found to be compound heterozygous for NM_004525.3: LRP2: c.5005A > G, p.(Asn1669Asp) and c.149C > G, p.(Thr50Ser). In Family 2, two sisters were found to be compound heterozygous for LRP2 variants, p.(Tyr3933Cys) and an experimentally confirmed c.7715 + 3A > T consensus splice-altering variant. In Family 3, the proband is compound heterozygous for a consensus donor splice site variant LRP2: c.8452_8452 + 1del and p.(Cys3150Tyr). In mouse cochlea, Lrp2 is expressed abundantly in the stria vascularis marginal cells demonstrated by smFISH, single-cell and single-nucleus RNAseq, suggesting that a deficiency of LRP2 may compromise the endocochlear potential, which is required for hearing. LRP2 variants have been associated with Donnai-Barrow syndrome and other multisystem pleiotropic phenotypes different from the phenotypes of the four cases reported herein. Our data expand the phenotypic spectrum associated with pathogenic variants in LRP2 warranting their consideration in individuals with a combination of hereditary hearing loss and retinal dystrophy.
Assuntos
Surdez , Perda Auditiva Neurossensorial , Perda Auditiva , Miopia , Distrofias Retinianas , Animais , Camundongos , Humanos , Perda Auditiva Neurossensorial/genética , Surdez/genética , Miopia/genética , Mutação , Linhagem , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genéticaRESUMO
Hereditary deafness is clinically and genetically heterogeneous. We investigated deafness segregating as a recessive trait in two families. Audiological examinations revealed an asymmetric mild to profound hearing loss with childhood or adolescent onset. Exome sequencing of probands identified a homozygous c.475G>A;p.(Glu159Lys) variant of CLDN9 (NM_020982.4) in one family and a homozygous c.370_372dupATC;p.(Ile124dup) CLDN9 variant in an affected individual of a second family. Claudin 9 (CLDN9) is an integral membrane protein and constituent of epithelial bicellular tight junctions (TJs) that form semipermeable, paracellular barriers between inner ear perilymphatic and endolymphatic compartments. Computational structural modeling predicts that substitution of a lysine for glutamic acid p.(Glu159Lys) alters one of two cis-interactions between CLDN9 protomers. The p.(Ile124dup) variant is predicted to locally misfold CLDN9 and mCherry tagged p.(Ile124dup) CLDN9 is not targeted to the HeLa cell membrane. In situ hybridization shows that mouse Cldn9 expression increases from embryonic to postnatal development and persists in adult inner ears coinciding with prominent CLDN9 immunoreactivity in TJs of epithelia outlining the scala media. Together with the Cldn9 deaf mouse and a homozygous frameshift of CLDN9 previously associated with deafness, the two bi-allelic variants of CLDN9 described here point to CLDN9 as a bona fide human deafness gene.
Assuntos
Claudinas , Surdez , Adolescente , Animais , Criança , Claudinas/genética , Surdez/genética , Células HeLa , Homozigoto , Humanos , Camundongos , Mutação , LinhagemRESUMO
Usher syndrome has been historically categorized into one of three classical types based on the patient phenotype. However, the vestibular phenotype does not infallibly predict which Usher genes are mutated. Conversely, the Usher syndrome genotype is not sufficient to reliably predict vestibular function. Here we present a characterization of the vestibular phenotype of 90 patients with clinical presentation of Usher syndrome (59 females), aged 10.9 to 75.5 years, with genetic variants in eight Usher syndromic genes and expand the description of atypical Usher syndrome. We identified unexpected horizontal semicircular canal reactivity in response to caloric and rotational stimuli in 12.5% (3 of 24) and 41.7% (10 of 24), respectively, of our USH1 cohort. These findings are not consistent with the classical phenotypic definition of vestibular areflexia in USH1. Similarly, 17% (6 of 35) of our cohort with USH2A mutations had saccular dysfunction as evidenced by absent cervical vestibular evoked myogenic potentials in contradiction to the classical assumption of normal vestibular function. The surprising lack of consistent genotypic to vestibular phenotypic findings as well as no clear vestibular phenotypic patterns among atypical USH cases, indicate that even rigorous vestibular phenotyping data will not reliably differentiate the three USH types.
Assuntos
Síndromes de Usher/genética , Síndromes de Usher/fisiopatologia , Vestíbulo do Labirinto/fisiopatologia , Adolescente , Adulto , Idoso , Criança , Estudos de Coortes , Ingestão de Energia , Potenciais Evocados Auditivos , Feminino , Estudos de Associação Genética , Humanos , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound sensorineural hearing loss locus, DFNB100 on chromosome 5q13.2-q23.2. Exome enrichment followed by massive parallel sequencing revealed a c.2510G>A transition variant in PPIP5K2 that segregated with DFNB100-associated hearing loss in two large apparently unrelated Pakistani families. PPIP5Ks enzymes interconvert 5-IP7 and IP8, two key members of the inositol pyrophosphate (PP-IP) cell-signaling family. Their actions at the interface of cell signaling and bioenergetic homeostasis can impact many biological processes. The c.2510G>A transition variant is predicted to substitute a highly invariant arginine residue with histidine (p.Arg837His) in the phosphatase domain of PPIP5K2. Biochemical studies revealed that the p.Arg837His variant reduces the phosphatase activity of PPIP5K2 and elevates its kinase activity. We found that in mouse inner ear, PPIP5K2 is expressed in the cochlear and vestibular sensory hair cells, supporting cells and spiral ganglion neurons. Mice homozygous for a targeted deletion of the Ppip5k2 phosphatase domain exhibit degeneration of cochlear outer hair cells and elevated hearing thresholds. Our demonstration that PPIP5K2 has a role in hearing in humans indicates that PP-IP signaling is important to hair cell maintenance and function within inner ear.
Assuntos
Perda Auditiva Neurossensorial/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Animais , Cromossomos Humanos Par 5 , Orelha Interna/fisiopatologia , Exoma , Feminino , Genes Recessivos , Ligação Genética , Células Ciliadas Auditivas Internas , Homeostase , Humanos , Masculino , Camundongos , Linhagem , Mutação PuntualRESUMO
The DFNB74 locus for autosomal-recessive, nonsyndromic deafness segregating in three families was previously mapped to a 5.36 Mb interval on chromosome 12q14.2-q15. Subsequently, we ascertained five additional consanguineous families in which deafness segregated with markers at this locus and refined the critical interval to 2.31 Mb. We then sequenced the protein-coding exons of 18 genes in this interval. The affected individuals of six apparently unrelated families were homozygous for the same transversion (c.265T>G) in MSRB3, which encodes a zinc-containing methionine sulfoxide reductase B3. c.265T>G results in a substitution of glycine for cysteine (p.Cys89Gly), and this substitution cosegregates with deafness in the six DFNB74 families. This cysteine residue of MSRB3 is conserved in orthologs from yeast to humans and is involved in binding structural zinc. In vitro, p.Cys89Gly abolished zinc binding and MSRB3 enzymatic activity, indicating that p.Cys89Gly is a loss-of-function allele. The affected individuals in two other families were homozygous for a transition mutation (c.55T>C), which results in a nonsense mutation (p.Arg19X) in alternatively spliced exon 3, encoding a mitochondrial localization signal. This finding suggests that DFNB74 deafness is due to a mitochondrial dysfunction. In a cohort of 1,040 individuals (aged 53-67 years) of European ancestry, we found no association between 17 tagSNPs for MSRB3 and age-related hearing loss. Mouse Msrb3 is expressed widely. In the inner ear, it is found in the sensory epithelium of the organ of Corti and vestibular end organs as well as in cells of the spiral ganglion. Taken together, MSRB3-catalyzed reduction of methionine sulfoxides to methionine is essential for hearing.
Assuntos
Surdez/enzimologia , Surdez/genética , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Idoso , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Transporte/genética , Estudos de Coortes , Orelha Interna/enzimologia , Éxons/genética , Feminino , Genes Recessivos , Ligação Genética , Loci Gênicos , Perda Auditiva/genética , Homozigoto , Humanos , Masculino , Metionina Sulfóxido Redutases , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Polimorfismo de Nucleotídeo Único , População Branca/genéticaRESUMO
Deafness in vertebrates is associated with variants of hundreds of genes. Yet, many mutant genes causing rare forms of deafness remain to be discovered. A consanguineous Pakistani family segregating nonsyndromic deafness in two sibships were studied using microarrays and exome sequencing. A 1.2 Mb locus (DFNB128) on chromosome 5q11.2 encompassing six genes was identified. In one of the two sibships of this family, a novel homozygous recessive variant NM_005921.2:c.4460G>A p.(Arg1487His) in the kinase domain of MAP3K1 co-segregated with nonsyndromic deafness. There are two previously reported Map3k1-kinase-deficient mouse models that are associated with recessively inherited syndromic deafness. MAP3K1 phosphorylates serine and threonine and functions in a signaling pathway where pathogenic variants of HGF, MET, and GAB1 were previously reported to be associated with human deafness DFNB39, DFNB97, and DFNB26, respectively. Our single-cell transcriptome data of mouse cochlea mRNA show expression of Map3k1 and its signaling partners in several inner ear cell types suggesting a requirement of wild-type MAP3K1 for normal hearing. In contrast to dominant variants of MAP3K1 associated with Disorders of Sex Development 46,XY sex-reversal, our computational modeling of the recessive substitution p.(Arg1487His) predicts a subtle structural alteration in MAP3K1, consistent with the limited phenotype of nonsyndromic deafness.
Assuntos
Surdez , Genes Recessivos , MAP Quinase Quinase Quinase 1 , Linhagem , Animais , Camundongos , Humanos , Feminino , Masculino , Surdez/genética , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 1/metabolismo , Modelos Animais de Doenças , Perda Auditiva/genética , Sequenciamento do Exoma , ConsanguinidadeRESUMO
The mammalian inner ear contains six sensory patches that allow detection of auditory stimuli as well as movement and balance. Much research has focused on the organ of Corti, the sensory organ of the cochlea that detects sound. Unfortunately, these cells are difficult to access in vivo, especially in the mature animal, but the development of genetically modified mouse models, including Cre/Lox mice, has improved the ability to label, purify or manipulate these cells. Here, we describe a new tamoxifen-inducible CreER mouse line, the Fbxo2CreERT2 mouse, that can be used to specifically manipulate cells throughout the cochlear duct of the neonatal and mature cochlear epithelium. In vestibular sensory epithelia, Fbxo2CreERT2-mediated recombination occurs in many hair cells and more rarely in supporting cells of neonatal and adult mice, with a higher rate of Fbxo2CreERT2 induction in type 1 versus type 2 hair cells in adults. Fbxo2CreERT2 mice, therefore, are a new tool for the specific manipulation of epithelial cells of the inner ear and targeted manipulation of vestibular type 1 hair cells.
Assuntos
Cóclea , Células Ciliadas Vestibulares , Camundongos , Animais , Células Ciliadas Auditivas , Epitélio , Células Ciliadas Auditivas Internas , MamíferosRESUMO
Importance: Although the clinical utility of genome sequencing for critically ill children is well recognized, its utility for proactive pediatric screening is not well explored. Objective: To evaluate molecular findings from screening ostensibly healthy children with genome sequencing compared with a gene panel for medically actionable pediatric conditions. Design, Setting, and Participants: This case series study was conducted among consecutive, apparently healthy children undergoing proactive genetic screening for pediatric disorders by genome sequencing (n = 562) or an exome-based panel of 268 genes (n = 606) from March 1, 2018, through July 31, 2022. Exposures: Genetic screening for pediatric-onset disorders using genome sequencing or an exome-based panel of 268 genes. Main Outcomes and Measures: Molecular findings indicative of genetic disease risk. Results: Of 562 apparently healthy children (286 girls [50.9%]; median age, 29 days [IQR, 9-117 days]) undergoing screening by genome sequencing, 46 (8.2%; 95% CI, 5.9%-10.5%) were found to be at risk for pediatric-onset disease, including 22 children (3.9%) at risk for high-penetrance disorders. Sequence analysis uncovered molecular diagnoses among 32 individuals (5.7%), while copy number variant analysis uncovered molecular diagnoses among 14 individuals (2.5%), including 4 individuals (0.7%) with chromosome scale abnormalities. Overall, there were 47 molecular diagnoses, with 1 individual receiving 2 diagnoses; of the 47 potential diagnoses, 22 (46.8%) were associated with high-penetrance conditions. Pathogenic variants in medically actionable pediatric genes were found in 6 individuals (1.1%), constituting 12.8% (6 of 47) of all diagnoses. At least 1 pharmacogenomic variant was reported for 89.0% (500 of 562) of the cohort. In contrast, of 606 children (293 girls [48.3%]; median age, 26 days [IQR, 10-67 days]) undergoing gene panel screening, only 13 (2.1%; 95% CI, 1.0%-3.3%) resulted in potential childhood-onset diagnoses, a significantly lower rate than those screened by genome sequencing (P < .001). Conclusions and Relevance: In this case series study, genome sequencing as a proactive screening approach for children, due to its unrestrictive gene content and technical advantages in comparison with an exome-based gene panel for medically actionable childhood conditions, uncovered a wide range of heterogeneous high-penetrance pediatric conditions that could guide early interventions and medical management.
Assuntos
Testes Genéticos , Genômica , Feminino , Criança , Humanos , Recém-Nascido , Penetrância , ExomaRESUMO
Although variant alleles of hundreds of genes are associated with sensorineural deafness in children, the genes and alleles involved remain largely unknown in the Sub-Saharan regions of Africa. We ascertained 56 small families mainly of Yoruba ethno-lingual ancestry in or near Ibadan, Nigeria, that had at least one individual with nonsyndromic, severe-to-profound, prelingual-onset, bilateral hearing loss not attributed to nongenetic factors. We performed a combination of exome and Sanger sequencing analyses to evaluate both nuclear and mitochondrial genomes. No biallelic pathogenic variants were identified in GJB2, a common cause of deafness in many populations. Potential causative variants were identified in genes associated with nonsyndromic hearing loss (CIB2, COL11A1, ILDR1, MYO15A, TMPRSS3, and WFS1), nonsyndromic hearing loss or Usher syndrome (CDH23, MYO7A, PCDH15, and USH2A), and other syndromic forms of hearing loss (CHD7, OPA1, and SPTLC1). Several rare mitochondrial variants, including m.1555A>G, were detected in the gene MT-RNR1 but not in control Yoruba samples. Overall, 20 (33%) of 60 independent cases of hearing loss in this cohort of families were associated with likely causal variants in genes reported to underlie deafness in other populations. None of these likely causal variants were present in more than one family, most were detected as compound heterozygotes, and 77% had not been previously associated with hearing loss. These results indicate an unusually high level of genetic heterogeneity of hearing loss in Ibadan, Nigeria and point to challenges for molecular genetic screening, counseling, and early intervention in this population.
Assuntos
Heterogeneidade Genética , Perda Auditiva Neurossensorial/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Frequência do Gene , Loci Gênicos , Heterozigoto , Humanos , Povos Indígenas/genética , Masculino , NigériaRESUMO
Reprogramming of the cochlea with hair-cell-specific transcription factors such as ATOH1 has been proposed as a potential therapeutic strategy for hearing loss. ATOH1 expression in the developing cochlea can efficiently induce hair cell regeneration but the efficiency of hair cell reprogramming declines rapidly as the cochlea matures. We developed Cre-inducible mice to compare hair cell reprogramming with ATOH1 alone or in combination with two other hair cell transcription factors, GFI1 and POU4F3. In newborn mice, all transcription factor combinations tested produced large numbers of cells with the morphology of hair cells and rudimentary mechanotransduction properties. However, 1 week later, only a combination of ATOH1, GFI1 and POU4F3 could reprogram non-sensory cells of the cochlea to a hair cell fate, and these new cells were less mature than cells generated by reprogramming 1 week earlier. We used scRNA-seq and combined scRNA-seq and ATAC-seq to suggest at least two impediments to hair cell reprogramming in older animals. First, hair cell gene loci become less epigenetically accessible in non-sensory cells of the cochlea with increasing age. Second, signaling from hair cells to supporting cells, including Notch signaling, can prevent reprogramming of many supporting cells to hair cells, even with three hair cell transcription factors. Our results shed light on the molecular barriers that must be overcome to promote hair cell regeneration in the adult cochlea.
Assuntos
Reprogramação Celular , Células Ciliadas Auditivas Internas , Mecanotransdução Celular , Animais , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Proteínas de Homeodomínio , Transdução de Sinais , Fator de Transcrição Brn-3C/genética , Fatores de Transcrição/genética , Células Ciliadas Auditivas Internas/citologiaRESUMO
The plant genome has conserved small non-coding microRNAs (miRNAs) genes about 20-24 nucleotides long. They play a vital role in the gene regulation at various stages of plant life. Their conserved nature among the various organisms not only suggests their early evolution in eukaryotes but also makes them a good source of new miRNA discovery by homology search using bioinformatics tools. A systematic search approach was used for interspecies orthologues of miRNA precursors, from known sequences of Gossypium in GenBank. The study resulted in 22 miRNAs belonging to 13 families. We found 7 miRNA families (miR160, 164, 827, 829, 836, 845 and 865) for the first time in cotton. All 22 miRNA precursors form stable minimum free energy (mfe) stem loop structure as their orthologues form in Arabidopsis and the mature miRNAs reside in the stem portion of the stem loop structure. Fifteen miRNAs belong to the world's most commercial fiber producing upland cotton (Gossypium hirsutum), five are from Gossypium raimondii and one each is from Gossypium herbaceum and Gossypium arboreum. Their targets consist of transcription factors, cell division regulating proteins and virus response gene. The discovery of 22 miRNAs will be helpful in future for detection of precise function of each miRNA at a particular stage in life cycle of cotton.
Assuntos
Genoma de Planta , Gossypium/genética , MicroRNAs/genética , RNA de Plantas/genética , Arabidopsis/genética , Sequência de Bases , Biologia Computacional , Sequência Conservada , Bases de Dados de Ácidos Nucleicos , Etiquetas de Sequências Expressas , Genes de Plantas , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Alinhamento de Sequência , Análise de Sequência de RNARESUMO
A modifier variant can abrogate the risk of a monogenic disorder. DFNM1 is a locus on chromosome 1 encoding a dominant suppressor of human DFNB26 recessive, profound deafness. Here, we report that DFNB26 is associated with a substitution (p.Gly116Glu) in the pleckstrin homology domain of GRB2-associated binding protein 1 (GAB1), an essential scaffold in the MET proto-oncogene, receptor tyrosine kinase/HGF (MET/HGF) pathway. A dominant substitution (p.Arg544Gln) of METTL13, encoding a predicted methyltransferase, is the DFNM1 suppressor of GAB1-associated deafness. In zebrafish, human METTL13 mRNA harboring the modifier allele rescued the GAB1-associated morphant phenotype. In mice, GAB1 and METTL13 colocalized in auditory sensory neurons, and METTL13 coimmunoprecipitated with GAB1 and SPRY2, indicating at least a tripartite complex. Expression of MET-signaling genes in human lymphoblastoid cells of individuals homozygous for p.Gly116Glu GAB1 revealed dysregulation of HGF, MET, SHP2, and SPRY2, all of which have reported variants associated with deafness. However, SPRY2 was not dysregulated in normal-hearing humans homozygous for both the GAB1 DFNB26 deafness variant and the dominant METTL13 deafness suppressor, indicating a plausible mechanism of suppression. Identification of METTL13-based modification of MET signaling offers a potential therapeutic strategy for a wide range of associated hearing disorders. Furthermore, MET signaling is essential for diverse functions in many tissues including the inner ear. Therefore, identification of the modifier of MET signaling is likely to have broad clinical implications.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Perda Auditiva Neurossensorial/metabolismo , Metiltransferases/metabolismo , Mutação de Sentido Incorreto , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Animais , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Humanos , Metiltransferases/genética , Camundongos , Camundongos Knockout , Proto-Oncogene Mas , Células Receptoras Sensoriais/patologia , Peixe-ZebraRESUMO
MAP3K1 is a serine/threonine kinase that is activated by a diverse set of stimuli and exerts its effect through various downstream effecter molecules, including JNK, ERK1/2 and p38. In humans, mutant alleles of MAP3K1 are associated with 46,XY sex reversal. Until recently, the only phenotype observed in Map3k1(tm1Yxia) mutant mice was open eyelids at birth. Here, we report that homozygous Map3k1(tm1Yxia) mice have early-onset profound hearing loss accompanied by the progressive degeneration of cochlear outer hair cells. In the mouse inner ear, MAP3K1 has punctate localization at the apical surface of the supporting cells in close proximity to basal bodies. Although the cytoarchitecture, neuronal wiring and synaptic junctions in the organ of Corti are grossly preserved, Map3k1(tm1Yxia) mutant mice have supernumerary functional outer hair cells (OHCs) and Deiters' cells. Loss of MAP3K1 function resulted in the downregulation of Fgfr3, Fgf8, Fgf10 and Atf3 expression in the inner ear. Fgfr3, Fgf8 and Fgf10 have a role in induction of the otic placode or in otic epithelium development in mice, and their functional deficits cause defects in cochlear morphogenesis and hearing loss. Our studies suggest that MAP3K1 has an essential role in the regulation of these key cochlear morphogenesis genes. Collectively, our data highlight the crucial role of MAP3K1 in the development and function of the mouse inner ear and hearing.
Assuntos
Células Ciliadas Auditivas Externas/enzimologia , Células Ciliadas Auditivas Externas/patologia , MAP Quinase Quinase Quinase 1/metabolismo , Animais , Limiar Auditivo , Corpos Basais/metabolismo , Sobrevivência Celular , Regulação para Baixo/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Células Ciliadas Auditivas Externas/ultraestrutura , Perda Auditiva/metabolismo , Perda Auditiva/patologia , Perda Auditiva/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Transporte Proteico , Transdução de Sinais/genética , Gânglio Espiral da Cóclea/patologia , Núcleo Vestibular Lateral/patologiaRESUMO
Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor of the melanocyte's plasma membrane, is a major determinant of skin pigmentation and phototype. Upon activation by α-melanocyte stimulating hormone, MC1R triggers the cAMP cascade to stimulate eumelanogenesis. We used whole-exome sequencing to identify causative alleles in Pakistani families with skin and hair hypopigmentation. Six MC1R mutations segregated with the phenotype in seven families, including a p.Val174del in-frame deletion and a p.Tyr298* nonsense mutation, that were analyzed for function in heterologous HEK293 cells. p.Tyr298* MC1R showed no agonist-induced signaling to the cAMP or ERK pathways, nor detectable agonist binding. Conversely, signaling was comparable for p.Val174del and wild-type in HEK cells overexpressing the proteins, but binding analysis suggested impaired cell surface expression. Flow cytometry and confocal imaging studies revealed reduced plasma membrane expression of p.Val174del and p.Tyr298*. Therefore, p.Tyr298* was a total loss-of-function (LOF) allele, while p.Val174del displayed a partial LOF attribute.
Assuntos
Alelos , Mutação/genética , Receptor Tipo 1 de Melanocortina/genética , Família , Feminino , Humanos , Hipopigmentação/genética , Masculino , Paquistão , Linhagem , FenótipoRESUMO
Neurofibromatosis type-1 (NF-1) is a rare genetic disorder with an extremely variable phenotype. A broad spectrum of associations have also been reported with it. We present a florid case of NF, presenting with unusual symptoms, which was found to have an associated Caroli's disease, a rare congenital disorder of the intrahepatic bile ducts. The case is reported along with a brief review of both the disorders.
Assuntos
Doença de Caroli/epidemiologia , Neurofibromatoses/epidemiologia , Adulto , Comorbidade , Feminino , HumanosRESUMO
The two compositionally distinct extracellular cochlear fluids, endolymph and perilymph, are separated by tight junctions that outline the scala media and reticular lamina. Mutations in TRIC (also known as MARVELD2), which encodes a tricellular tight junction protein known as tricellulin, lead to nonsyndromic hearing loss (DFNB49). We generated a knockin mouse that carries a mutation orthologous to the TRIC coding mutation linked to DFNB49 hearing loss in humans. Tricellulin was absent from the tricellular junctions in the inner ear epithelia of the mutant animals, which developed rapidly progressing hearing loss accompanied by loss of mechanosensory cochlear hair cells, while the endocochlear potential and paracellular permeability of a biotin-based tracer in the stria vascularis were unaltered. Freeze-fracture electron microscopy revealed disruption of the strands of intramembrane particles connecting bicellular and tricellular junctions in the inner ear epithelia of tricellulin-deficient mice. These ultrastructural changes may selectively affect the paracellular permeability of ions or small molecules, resulting in a toxic microenvironment for cochlear hair cells. Consistent with this hypothesis, hair cell loss was rescued in tricellulin-deficient mice when generation of normal endolymph was inhibited by a concomitant deletion of the transcription factor, Pou3f4. Finally, comprehensive phenotypic screening showed a broader pathological phenotype in the mutant mice, which highlights the non-redundant roles played by tricellulin.
Assuntos
Células Ciliadas Auditivas Externas/metabolismo , Perda Auditiva/metabolismo , Proteína 2 com Domínio MARVEL/deficiência , Junções Íntimas/metabolismo , Animais , Feminino , Perda Auditiva/patologia , Proteína 2 com Domínio MARVEL/genética , Masculino , Potenciais da Membrana , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Mutação de Sentido Incorreto , Órgão Espiral/patologia , Estria Vascular/metabolismo , Estria Vascular/patologia , Vestíbulo do Labirinto/metabolismo , Vestíbulo do Labirinto/patologiaRESUMO
Sensorineural hearing loss is genetically heterogeneous. Here, we report that mutations in CIB2, which encodes a calcium- and integrin-binding protein, are associated with nonsyndromic deafness (DFNB48) and Usher syndrome type 1J (USH1J). One mutation in CIB2 is a prevalent cause of deafness DFNB48 in Pakistan; other CIB2 mutations contribute to deafness elsewhere in the world. In mice, CIB2 is localized to the mechanosensory stereocilia of inner ear hair cells and to retinal photoreceptor and pigmented epithelium cells. Consistent with molecular modeling predictions of calcium binding, CIB2 significantly decreased the ATP-induced calcium responses in heterologous cells, whereas mutations in deafness DFNB48 altered CIB2 effects on calcium responses. Furthermore, in zebrafish and Drosophila melanogaster, CIB2 is essential for the function and proper development of hair cells and retinal photoreceptor cells. We also show that CIB2 is a new member of the vertebrate Usher interactome.