RESUMO
OBJECTIVES: Auditory neuropathy (AN) is a clinical disorder characterized by the absence of auditory brainstem response and presence of otoacoustic emissions. A gradual loss of otoacoustic emissions has been reported for some cases of AN. Such cases could be diagnosed as cochlear hearing loss and lead to misunderstanding of the pathology when patients first visit clinics after the loss of otoacoustic emissions. The purpose of this study was to investigate the time course of changes in distortion product otoacoustic emissions (DPOAEs) in association with patients' genetic and clinical backgrounds, including the use of hearing aids. DESIGN: DPOAE measurements from 31 patients with AN were assessed. Genetic analyses for GJB2, OTOF, and mitochondrial m.1555A> G and m.3243A> G mutations were conducted for all cases, and the analyses for CDH23 and OPA1 were conducted for the selected cases. Patients who were younger than 10 years of age at the time of AN diagnosis were designated as the pediatric AN group (22 cases), and those who were 18 years of age or older were designated as the adult AN group (9 cases). DPOAE was measured at least twice in all patients. The response rate for DPOAEs was defined and analyzed. RESULTS: The pediatric AN group comprised 10 patients with OTOF mutations, 1 with GJB2 mutations, 1 with OPA1 mutation, and 10 with indefinite causes. Twelve ears (27%) showed no change in DPOAE, 20 ears (46%) showed a decrease in DPOAE, and 12 ears (27%) lost DPOAE. Loss of DPOAE occurred in one ear (2%) at 0 years of age and four ears (9%) at 1 year of age. The time courses of DPOAEs in patients with OTOF mutations were divided into those with early loss and those with no change, indicating that the mechanism for deterioration of DPOAEs includes not only the OTOF mutations but also other common modifier factors. Most, but not all, AN patients who used hearing aids showed deterioration of DPOAEs after the start of using hearing aids. A few AN patients also showed deterioration of DPOAEs before using hearing aids. The adult AN group comprised 2 patients with OPA1 mutations, 2 with OTOF mutations, and 5 with indefinite causes. Four ears (22%) showed no change in DPOAE, 13 ears (72%) showed a decrease, and one ear (6%) showed a loss of DPOAE. Although the ratio of DPOAE decrease was higher in the adult AN group than in the pediatric AN group, the ratio of DPOAE loss was lower in the adult AN group. DPOAE was not lost in all four ears with OPA1 mutations and in all four ears with OTOF mutations in the adult group. CONCLUSIONS: DPOAE was decreased or lost in approximately 70% of pediatric and about 80% of adult AN patients. Eleven percent of pediatric AN patients lost DPOAEs by 1 year of age. Genetic factors were thought to have influenced the time course of DPOAEs in the pediatric AN group. In most adult AN patients, DPOAE was rarely lost regardless of the genetic cause.
Assuntos
Perda Auditiva Central/fisiopatologia , Emissões Otoacústicas Espontâneas/fisiologia , Adolescente , Adulto , Idoso , Proteínas Relacionadas a Caderinas , Caderinas/genética , Criança , Pré-Escolar , Conexina 26 , Conexinas/genética , Erros de Diagnóstico , Progressão da Doença , Feminino , GTP Fosfo-Hidrolases/genética , Genes Mitocondriais/genética , Perda Auditiva Central/diagnóstico , Perda Auditiva Central/genética , Perda Auditiva Neurossensorial/diagnóstico , Humanos , Lactente , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
The access of bone morphogenetic protein (BMP) to the BMP receptors on the cell surface is regulated by its antagonist noggin, which binds to heparan-sulfate proteoglycans on the cell surface. Noggin is encoded by NOG and mutations in the gene are associated with aberrant skeletal formation, such as in the autosomal dominant disorders proximal symphalangism (SYM1), multiple synostoses syndrome, Teunissen-Cremers syndrome, and tarsal-carpal coalition syndrome. NOG mutations affecting a specific function may produce a distinct phenotype. In this study, we investigated a Japanese pedigree with SYM1 and conductive hearing loss and found that it carried a novel heterozygous missense mutation of NOG (c.406C>T; p.R136C) affecting the heparin-binding site of noggin. As no mutations of the heparin-binding site of noggin have previously been reported, we investigated the crystal structure of wild-type noggin to investigate molecular mechanism of the p.R136C mutation. We found that the positively charged arginine at position 136 was predicted to be important for binding to the negatively charged heparan-sulfate proteoglycan (HSPG). An in silico docking analysis showed that one of the salt bridges between noggin and heparin disappeared following the replacement of the arginine with a non-charged cysteine. We propose that the decreased binding affinity of NOG with the p.R136C mutation to HSPG leads to an excess of BMP signaling and underlies the SYM1 and conductive hearing loss phenotype of carriers.
Assuntos
Proteínas de Transporte/metabolismo , Articulações dos Dedos/anormalidades , Perda Auditiva Condutiva/genética , Heparina/metabolismo , Artropatias/congênito , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Arginina/química , Arginina/genética , Povo Asiático/genética , Sítios de Ligação/genética , Proteína Morfogenética Óssea 7/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Criança , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Feminino , Heterozigoto , Humanos , Japão , Artropatias/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , Transdução de SinaisRESUMO
OBJECTIVES: The crystal structure of the six protomers of gap junction protein beta 2 (GJB2) enables prediction of the effect(s) of an amino acid substitution, thereby facilitating investigation of molecular pathogenesis of missense variants of GJB2. This study mainly focused on R143W variant that causes hearing loss, and investigated the relationship between amino acid substitution and 3-D structural changes in GJB2. METHODS: Patients with nonsyndromic hearing loss who appeared to have two GJB2 pathogenic variants, including the R143W variant, were investigated. Because the X-ray crystal structure of the six protomers of the GJB2 protein is known, R143W and structurally related variants of GJB2 were modeled using this crystal structure as a template. The wild-type crystal structure and the variant computer-aided model were observed and the differences in molecular interactions within the two were analyzed. RESULTS: The predicted structure demonstrated that the hydrogen bond between R143 and N206 was important for the stability of the protomer structure. From this prediction, R143W related N206S and N206T variants showed loss of the hydrogen bond. CONCLUSION: Investigation of the genotypes and clinical data in patients carrying the R143W variant on an allele indicated that severity of hearing loss depends largely on the levels of dysfunction of the pathogenic variant on the allele, whereas a patient with the homozygous R143W variant demonstrated profound hearing loss. We concluded that these hearing impairments may be due to destabilization of the protomer structure of GJB2 caused by the R143W variant.
Assuntos
Conexina 26 , Conexinas , Perda Auditiva , Humanos , Conexina 26/genética , Conexinas/genética , Conexinas/química , Perda Auditiva/genética , Feminino , Masculino , Criança , Modelos Moleculares , Pré-Escolar , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Ligação de Hidrogênio , Cristalografia por Raios X , Adolescente , AdultoRESUMO
Genetic mutation is one of the causative factors for idiopathic progressive hearing loss. A patient with late-onset, moderate, and high-frequency hearing loss was found to have a novel, heterozygous KCNQ4 mutation, c.806_808delCCT, which led to a p.Ser260del located between S5 and the pore helix (PH). Molecular modeling analysis suggested that the p.Ser269del mutation could cause structural distortion and change in the electrostatic surface potential of the KCNQ4 channel protein, which may impede K+ transport. The present study supports the idea that a non-truncating mutation around the N-terminus of PH may be related to moderate hearing loss.
Assuntos
Perda Auditiva de Alta Frequência/genética , Canais de Potássio KCNQ/química , Canais de Potássio KCNQ/genética , Adulto , Sequência de Aminoácidos , Feminino , Heterozigoto , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Estrutura Secundária de Proteína/genética , Deleção de Sequência , Serina/química , Serina/genéticaRESUMO
Otof, which encodes otoferlin, knockout mice are considered model mice for auditory neuropathy spectrum disorder, which is characterized by an absent auditory brainstem response (ABR) despite preserved distortion product otoacoustic emission (DPOAE). Although otoferlin-deficient mice lack neurotransmitter release at the inner hair cell (IHC) synapse, it remains unclear how the Otof mutation affects spiral ganglions. Thus, we used Otof-mutant mice carrying the Otoftm1a(KOMP)Wtsi allele (Otoftm1a) and analyzed spiral ganglion neurons (SGNs) in Otoftm1a/tm1a mice by immunolabeling type â SGNs (SGN-â ) and type II SGNs (SGN-II). We also examined apoptotic cells in SGNs. Four-week-old Otoftm1a/tm1a mice had an absent ABR but normal DPOAEs. The number of SGNs was significantly lower in Otoftm1a/tm1a mice on postnatal day 7 (P7), P14, and P28 compared with that of wild-type mice. Moreover, significantly more apoptotic SGNs were observed in Otoftm1a/tm1a mice than in wild-type mice on P7, P14, and P28. SGN-IIs were not significantly reduced in Otoftm1a/tm1a mice on P7, P14, and P28. No apoptotic SGN-IIs were observed under our experimental conditions. In summary, Otoftm1a/tm1a mice showed a reduction in SGNs accompanied by apoptosis of SGN-â s even before the onset of hearing. We speculate that the reduction in SGNs with apoptosis is a secondary defect caused by a lack of otoferlin in IHCs. Appropriate glutamatergic synaptic inputs may be important for the survival of SGNs.
Assuntos
Neurônios , Gânglio Espiral da Cóclea , Animais , Camundongos , Gânglio Espiral da Cóclea/metabolismo , Neurônios/metabolismo , Apoptose/fisiologia , Transmissão Sináptica/fisiologia , Camundongos Knockout , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
It is well accepted that G protein-coupled receptors (GPCRs) arrange into dimers or higher-order oligomers that may modify various functions of GPCRs. GPCR-type purinergic receptors (i.e. adenosine and P2Y receptors) tend to form heterodimers with GPCRs not only of the different families but also of the same purinergic receptor families, leading to alterations in functional properties. In the present review, we focus on current knowledge of the formation of heterodimers between metabotropic purinergic receptors that activate novel functions in response to extracellular nucleosides/nucleotides, revealing that the dimerization seems to be employed for 'fine-tuning' of purinergic signaling. Thus, the relationship between adenosine and adenosine triphosphate is likely to be more and more intimate than simply being a metabolite of the other.
Assuntos
Dimerização , Estrutura Quaternária de Proteína , Receptores Acoplados a Proteínas G , Receptores Purinérgicos , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Plaquetas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos/química , Receptores Purinérgicos/metabolismoRESUMO
HYPOTHESIS: The phenotype of DFNA11 consists of specific features at diverse developmental and age stages. BACKGROUND: Only eight mutations have been identified for autosomal dominant non-syndromic hearing loss related to MYO7A (DFNA11), and the onset and progression of DFNA11 are poorly understood. METHODS: After linkage analysis and following Sanger sequencing in a family suspected to have autosomal dominant hereditary hearing loss, we analyzed the audiometric and vestibular functions and their long-term changes in the subjects carrying the variant. RESULTS: A reported variant of uncertain significance, NP_000251.3:p.Arg853His, in MYO7A was detected and cosegregation data of this large family provided evidence that the variant was likely pathogenic for DFNA11. Family members with the variant had no other symptoms associated with hearing loss and were confirmed to have autosomal dominant non-syndromic sensorineural hearing loss. Audiograms tended to show gently sloping configuration in childhood and flat configuration after the age of 30 years. Hearing loss at high frequencies progressed slowly, while hearing at low frequencies started to deteriorate later but progressed more rapidly. Some subjects showed partly abnormal results in the distortion products of otoacoustic emissions before the elevation of hearing thresholds. Vestibular function was within the normal range in all the subjects tested. CONCLUSION: We revealed that hearing loss at high frequencies was mainly noted in early developmental stages and that thresholds increased more rapidly in the low frequency range, resulting in changes in audiometric configuration. Deterioration of distortion product otoacoustic emissions (DPOAE) before the elevation of hearing thresholds was considered as a clinical feature of DFNA11.
Assuntos
Perda Auditiva Neurossensorial , Miosina VIIa/genética , Adulto , Envelhecimento , Criança , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Humanos , Miosinas/genética , Emissões Otoacústicas Espontâneas , LinhagemRESUMO
BACKGROUND: Hereditary hearing loss is characterized by a very high genetic heterogeneity. The OTOF (Locus: DFNB9), encoding otoferlin, is reported to be one of the major causes of non-syndromic hearing loss, and is also reported to be the most common cause of non-syndromic recessive auditory neuropathy spectrum disorder. METHODS: In this study, whole exome sequencing was employed for detection of novel pathogenic variant that segregates with autosomal recessive nonsyndromic hearing loss in a tribal family from Rajouri, Jammu and Kashmir. Proband was a 9-year-old male born to first-cousin parents and presented with sensorineural hearing loss since birth. Family resides in an area with high consanguinity and lack of basic health care facilities including genetic counselling services. RESULTS: We report a novel OTOF pathogenic variant NM_194248.2:c.4249_4250insG (p.Ser1417CysfsTer4) co-segregating with hearing loss in this family and not present in any public databases. CONCLUSIONS: Our findings not only extend the geographical and mutational spectrum of autosomal recessive nonsyndromic hearing loss but also support the need for introducing genetic counselling services to rural and tribal areas in India with high consanguinity.
Assuntos
Surdez/genética , Proteínas de Membrana/genética , Mutação/genética , Criança , Consanguinidade , Feminino , Humanos , Índia , Masculino , Linhagem , Sequenciamento do ExomaRESUMO
Dimerization between G protein-coupled receptors (GPCRs) is a clearly established phenomenon. However, limited information is currently available on the interface essential for this process. Based on structural comparisons and sequence homology between rhodopsin and A(1) adenosine receptor (A(1)R), we initially hypothesized that four residues in transmembrane (TM) 4 and TM5 are involved in A(1)R homodimerization. Accordingly, these residues were substituted with Ala by site-directed mutagenesis. Interestingly, the mutant protein displayed no significant decrease in homodimer formation compared with wild-type A(1)R, as evident from coimmunoprecipitation and BRET(2) analyses (improved bioluminescence resonance energy transfer system offered by Perkin-Elmer Life Sciences), but lost ligand binding activity almost completely. Further studies disclosed that this effect was derived from the mutation of one particular residue, Trp132, which is highly conserved among many GPCRs. Confocal immunofluorescence and cell-surface biotinylation studies revealed that the mutant receptors localized normally at transfected cell membranes, signifying that loss of ligand binding was not because of defective cellular trafficking. Molecular modeling of the A(1)R-ligand complex disclosed that Trp132 interacted with several residues located in TM3 and TM5 that stabilized agonist binding. Thus, loss of interactions of Trp with these residues may, in turn, disrupt binding to agonists. Our study provides strong evidence of the essential role of the highly conserved Trp132 in TM4 of adenosine receptors.
Assuntos
Adenosina/metabolismo , Membrana Celular/metabolismo , Multimerização Proteica , Receptor A1 de Adenosina/química , Receptor A1 de Adenosina/metabolismo , Triptofano/metabolismo , Adenosina/agonistas , Adenosina/análogos & derivados , Agonistas do Receptor A1 de Adenosina , Sequência de Aminoácidos/fisiologia , Membrana Celular/química , Sequência Conservada/fisiologia , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Humanos , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Triptofano/químicaRESUMO
Pyrococcus furiosus is a hyperthermophilic archaeal microorganism found near deep-sea thermal vents and its optimal growth temperature of 100 degrees C. Recently, a 38.8-kDa protein from P. furiosus DSM 3638 was isolated and characterized. Electron microscopy revealed that this protein aggregated as spheres of approximately 30 nm in diameter, which we designated P. furiosus virus-like particles (PfVs). X-ray crystallographic analysis at 3.6-A resolution revealed that each PfV consisted of 180 copies of the 38.8-kDa protein and retained T=3 icosahedral symmetry, as is often the case in spherical viruses. The total molecular mass of each particle was approximately 7 MDa. An examination of capsid structures suggested strong evolutionary links among PfV, tailed double-stranded DNA bacteriophages, and herpes viruses. The similar three-dimensional structures of the various coat proteins indicate that these viral capsids might have originated and evolved from a common ancestor. The structure of PfV provides a previously undescribed example of viral relationships across the three domains of life (Eukarya, Bacteria, and Archaea).
Assuntos
Evolução Biológica , Estrutura Quaternária de Proteína , Pyrococcus furiosus/virologia , Proteínas Virais , Vírus , Sequência de Aminoácidos , Bacteriófagos/química , Bacteriófagos/ultraestrutura , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Pyrococcus furiosus/química , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/classificação , Proteínas Virais/ultraestrutura , Vírus/química , Vírus/classificação , Vírus/ultraestruturaRESUMO
OBJECTIVE: To examine and expand the genetic spectrum of Waardenburg syndrome type 1 (WS1). METHODS: Clinical features related to Waardenburg syndrome (WS) were examined in a five-year old patient. Mutation analysis of genes related to WS was performed in the proband and her parents. Molecular modeling of EDNRB and the p.R319W mutant was conducted to predict the pathogenicity of the mutation. RESULTS: The proband showed sensorineural hearing loss, heterochromia iridis, and dystopia canthorum, fulfilling the clinical criteria of WS1. Genetic analyses revealed that the proband had no mutation in PAX3 which has been known as the cause of WS1, but had a homozygous missense mutation (p.R319W) in endothelin receptor type B (EDNRB) gene. The asymptomatic parents had the mutation in a heterozygote state. This mutation has been previously reported in a heterozygous state in a patient with Hirschsprung's disease unaccompanied by WS, but the patient and her parents did not show any symptoms in gastrointestinal tract. Molecular modeling of EDNRB with the p.R319W mutation demonstrated reduction of the positively charged surface area in this region, which might reduce binding ability of EDNRB to G protein and lead to abnormal signal transduction underlying the WS phenotype. CONCLUSIONS: Our findings suggested that autosomal recessive mutation in EDNRB may underlie a part of WS1 with the current diagnostic criteria, and supported that Hirschsprung's disease is a multifactorial genetic disease which requires additional factors. Further molecular analysis is necessary to elucidate the gene interaction and to reappraise the current WS classification.
Assuntos
Receptor de Endotelina B/genética , Síndrome de Waardenburg/genética , Pré-Escolar , Feminino , Homozigoto , Humanos , Modelos Moleculares , Mutação de Sentido Incorreto , Síndrome de Waardenburg/fisiopatologiaRESUMO
OBJECTIVES/HYPOTHESIS: To determine the frequency of the incomplete partition type III anomaly and the genetic and clinical features associated with POU3F4 mutations in children with hearing loss. STUDY DESIGN: Retrospective case series from 2000 to 2014 at the National Hospital Organization Tokyo Medical Center and collaborating hospitals. METHODS: A total of 1,004 patients (from 938 families) who had hearing loss by 10 years of age and had undergone computed tomography scanning of their temporal bones were enrolled in this genetic, clinical, and radiological study. RESULTS: The incomplete partition type III anomaly was identified in six patients (0.6%), each of whom had an enlargement of the vestibular aqueduct at the end close to the vestibule. The six patients also had POU3F4 variants, and a genetic analysis revealed frameshift deletions in three patients, a missense variant in two patients of the same family, and a large deletion in one patient. Three of the six patients with POU3F4 variants were sporadic cases, and in one patient the genetic mutation occurred de novo. CONCLUSIONS: It was indicated that POU3F4 mutations can be predicted by incomplete partition type III anomaly by radiological examination of the inner ear. All six of the patients showed mixed hearing loss, but none showed fluctuations in hearing, which may be related to the lack of vestibular aqueduct enlargement at the operculum. LEVEL OF EVIDENCE: 4 Laryngoscope, 127:1663-1669, 2017.
Assuntos
Cóclea/anormalidades , Análise Mutacional de DNA , Frequência do Gene , Perda Auditiva Condutiva-Neurossensorial Mista/diagnóstico por imagem , Perda Auditiva Condutiva-Neurossensorial Mista/genética , Fatores do Domínio POU/genética , Criança , Pré-Escolar , Cóclea/diagnóstico por imagem , Feminino , Humanos , Masculino , Linhagem , Estudos Retrospectivos , Tóquio , Tomografia Computadorizada por Raios X , Aqueduto Vestibular/anormalidades , Aqueduto Vestibular/diagnóstico por imagemRESUMO
BACKGROUND: To date, 102 genes have been reported as responsible for non-syndromic hearing loss, some of which are associated with specific audiogram features. Four genes have been reported as causative for mid-frequency sensorineural hearing loss (MFSNHL), among which TECTA is the most frequently reported; however, the prevalence of TECTA mutations is unknown. To elucidate the prevalence of TECTA mutation in MFSNHL and clarify genotype-phenotype correlations, we analyzed the genetic and clinical features of patients with MFSNHL. METHODS: Subjects with bilateral non-syndromic hearing loss were prescreened for GJB2 and m.1555A > G and m.3243A > G mitochondrial DNA mutations, and patients with inner ear malformations were excluded. We selected MFSNHL patients whose audiograms met the U-shaped criterion proposed by the GENDEAF study group, along with those with shallow U-shaped audiograms, for TECTA analysis. All TECTA exons were analyzed by Sanger sequencing. Novel missense variants were classified as possibly pathogenic, non-pathogenic, and variants of uncertain significance, based on genetic data. To evaluate novel possibly pathogenic variants, we predicted changes in protein structure by molecular modeling. RESULTS: Pathogenic and possibly pathogenic variants of TECTA were found in 4 (6.0%) of 67 patients with MFSNHL. In patients with U-shaped audiograms, none (0%) of 21 had pathogenic or possibly pathogenic variants. In patients with shallow U-shaped audiograms, four (8.7%) of 46 had pathogenic or possibly pathogenic variants. Two novel possibly pathogenic variants were identified and two previously reported mutations were considered as variant of unknown significance. The clinical features of patients with pathogenic and possibly pathogenic variants were consistent with those in previous studies. Pathogenic or possibly pathogenic variants were identified in 3 of 23 families (13.0%) which have the family histories compatible with autosomal dominant and 1 of 44 families (2.3%) which have the family histories compatible with sporadic or autosomal recessive. CONCLUSIONS: TECTA mutations were identified in 6.0% of MFSNHL. These mutations were more frequent in patients with shallow U-shaped audiograms than those with U-shaped audiograms, and in families which have the family histories compatible with autosomal dominant than those with the family histories compatible with sporadic or autosomal recessive.
Assuntos
Proteínas da Matriz Extracelular/genética , Perda Auditiva Neurossensorial/genética , DNA Mitocondrial/genética , Feminino , Proteínas Ligadas por GPI/genética , Humanos , Masculino , Mutação , LinhagemRESUMO
OBJECTIVE: To report two novel LOXHD1 mutations, including missense mutations and the clinical features of the patients. METHODS: We studied a three-generation Japanese family with hearing loss. Targeted next-generation sequencing was used for genetic analysis. Conditional orientation response audiometry and pure tone audiometry were used to assess hearing. SWISS-MODEL was used for molecular modeling of the PLAT domain in LOXHD1 protein. RESULTS: The two sisters, who had either mild or severe high-frequency hearing loss, were compound heterozygous for two novel mutations (c.5674G>T [p.V1892F] and c.4212+1G>A) in LOXHD1, which is responsible for autosomal-recessive nonsyndromic hearing loss DFNB77. These cases showed less severe hearing impairment than the previously reported cases carrying LOXHD1 mutations, but their hearing loss appeared to be progressive. Molecular modeling predicted that distorted structure of the PLAT domain in the p.V1892F mutant could lead to decreased affinity of the protein to lipid membrane resulting in hair cell dysfunction. CONCLUSION: We report a Japanese family carrying compound heterozygotes of truncating and nontruncating mutations in LOXHD1 identified by targeted NGS analysis. The fact of lower degree of hearing impairment in our cases than previously reported and the molecular modeling of the missense mutant provide insight to the genotype-phenotype correlation of DFNB77.
Assuntos
Proteínas de Transporte/genética , Perda Auditiva de Alta Frequência/genética , Perda Auditiva Neurossensorial/genética , Mutação de Sentido Incorreto , Irmãos , Povo Asiático/genética , Audiometria , Audiometria de Tons Puros , Criança , Análise Mutacional de DNA , Feminino , Genótipo , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Japão , Linhagem , FenótipoRESUMO
HYPOTHESIS: Different missense mutations of the optic atrophy 1 gene (OPA1) identified in optic atrophy patients with auditory neuropathy spectrum disorder (ANSD) induce functional impairment through different molecular mechanisms. BACKGROUND: OPA1 is the gene responsible for autosomal dominant optic atrophy (ADOA), but some of its mutations are also associated with ANSD. OPA1 is a member of the GTPase family of proteins and plays a key role in the maintenance of mitochondrial activities that are dependent on dimer formation of the protein. There are many reports of OPA1 mutations, but the molecular mechanisms of their functional impairments are unclear. METHODS: The sequences of coding regions in OPA1 were analyzed from blood samples of ADOA patients with ANSD. Molecular modeling of the protein's ability to form dimers and its GTP-binding ability were conducted to study the effects of structural changes in OPA1 caused by two identified mutations and their resultant effects on protein function. RESULTS: Two heterozygous mutations, p.T414P (c.1240A>C) and p.T540P (c.1618A>C), located in the GTPase and middle domains of OPA1, respectively, were identified in two patients. Molecular modeling indicated decreased dimer formation caused by destabilization of the association structure of the p.T414P mutant, and decreased GTP-binding caused by destabilization of the binding site structure in the p.T540P mutant. CONCLUSION: These two different conformational changes might result in decreased GTPase activities that trigger ADOA associated with ANSD, and are likely to be associated with mild clinical features. Molecular modeling would provide useful information in clinical practice.
Assuntos
GTP Fosfo-Hidrolases/genética , Perda Auditiva Central/genética , Atrofia Óptica Autossômica Dominante/genética , Adulto , Feminino , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Humanos , Masculino , Modelos Moleculares , Mutação de Sentido IncorretoRESUMO
Spherical particles (SPs) of approximately 30 nm in diameter were found in the hyperthermophilic archaeon Pyrococcus furiosus. The SPs contained no nucleic acid and were composed of a single 39-kDa protein. The amino acid sequences of the amino-terminal and internal fragments were identical to portions of the deduced amino acid sequence of the putative 38.7-kDa protein encoded by the genome of P. furiosus, suggesting that the protein was expressed from the genome of P. furiosus. This possibility was confirmed by the observation that the 38.7-kDa protein expressed in Escherichia coli reacted specifically with the antibody against purified SPs, and it also formed SPs similar to those found in P. furiosus. Of the 345 amino acid residues in the 38.7-kDa protein, the amino-terminal 100 amino acids exhibited strong homology to putative proteins from other species of Pyrococcus, while the remaining 245 carboxy-terminal residues were not significantly homologous to putative proteins from other members of archaea. Thus, the carboxy-terminal region might be the product of a foreign gene that was incorporated relatively recently into the genome of P. furiosus.
Assuntos
Proteínas Arqueais/química , Pyrococcus furiosus/química , Sequência de Aminoácidos , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/ultraestrutura , Sequência de Bases , DNA Arqueal/química , Dados de Sequência Molecular , Pyrococcus furiosus/genética , Proteínas Recombinantes/químicaRESUMO
BACKGROUND: Mutations in CDH23 are responsible for Usher syndrome 1D and recessive non-syndromic hearing loss. In this study, we revealed the prevalence of CDH23 mutations among patients with specific clinical characteristics. METHODS: After excluding patients with GJB2 mutations and mitochondrial m.1555A > G and m.3243A > G mutations, subjects for CDH23 mutation analysis were selected according to the following criteria: 1) Sporadic or recessively inherited hearing loss 2) bilateral non-syndromic congenital hearing loss, 3) no cochlear malformation, 4) a poorer hearing level at high frequencies than at low frequencies, and 5) severe or profound hearing loss at higher frequencies. RESULTS: Seventy-two subjects were selected from 621 consecutive probands who did not have environmental causes for their hearing loss. After direct sequencing, 13 of the 72 probands (18.1%) had homozygous or compound heterozygous CDH23 mutations. In total, we identified 16 CDH23 mutations, including five novel mutations. The 16 mutations included 12 missense, two frameshift, and two splice-site mutations. CONCLUSIONS: These results revealed that CDH23 mutations are highly prevalent in patients with congenital high-frequency sporadic or recessively inherited hearing loss and that the mutation spectrum was diverse, indicating that patients with these clinical features merit genetic analysis.
Assuntos
Caderinas/genética , Perda Auditiva/genética , Adolescente , Adulto , Proteínas Relacionadas a Caderinas , Criança , Pré-Escolar , Conexina 26 , Conexinas , Análise Mutacional de DNA , Feminino , Perda Auditiva Neurossensorial/genética , Humanos , Lactente , Recém-Nascido , Masculino , Mutação/genética , Fenótipo , Prevalência , Adulto JovemRESUMO
Waardenburg syndrome is characterized by hearing loss, pigmentation abnormalities, dysmorphologic features, and neurological phenotypes. Waardenburg syndrome consists of four distinct subtypes, and SOX10 mutations have been identified in type II and type IV. Type IV differs from type II owing to the presence of Hirschsprung disease. We identified a de novo nonsense mutation in SOX10 (p.G39X) in a female pediatric patient with Waardenburg syndrome with heterochromia iridis, profound bilateral sensorineural hearing loss, inner ear malformations, and overall hypopigmentation of the hair without dystopia canthorum. This patient has experienced chronic constipation since she was a neonate, but anorectal manometry showed a normal anorectal reflex. Chronic constipation in this patient was likely to be a consequence of a mild intestinal disorder owing to the SOX10 mutation, and this patient was considered to have a clinical phenotype intermediate between type II and type IV of the syndrome. Chronic constipation may be recognized as indicative of a SOX10 mutation in patients with Waardenburg syndrome.
Assuntos
Códon sem Sentido , Constipação Intestinal/diagnóstico , Fatores de Transcrição SOXE/genética , Síndrome de Waardenburg/diagnóstico por imagem , Sequência de Bases , Pré-Escolar , Doença Crônica , Constipação Intestinal/genética , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Humanos , Linhagem , Radiografia , Osso Temporal/diagnóstico por imagem , Síndrome de Waardenburg/genéticaRESUMO
CONCLUSION: PAX3 genetic analysis increased the diagnostic accuracy for Waardenburg syndrome type I (WS1). Analysis of the three-dimensional (3D) structure of PAX3 helped verify the pathogenicity of a missense mutation, and multiple ligation-dependent probe amplification (MLPA) analysis of PAX3 increased the sensitivity of genetic diagnosis in patients with WS1. OBJECTIVES: Clinical diagnosis of WS1 is often difficult in individual patients with isolated, mild, or non-specific symptoms. The objective of the present study was to facilitate the accurate diagnosis of WS1 through genetic analysis of PAX3 and to expand the spectrum of known PAX3 mutations. METHODS: In two Japanese families with WS1, we conducted a clinical evaluation of symptoms and genetic analysis, which involved direct sequencing, MLPA analysis, quantitative PCR of PAX3, and analysis of the predicted 3D structure of PAX3. The normal-hearing control group comprised 92 subjects who had normal hearing according to pure tone audiometry. RESULTS: In one family, direct sequencing of PAX3 identified a heterozygous mutation, p.I59F. Analysis of PAX3 3D structures indicated that this mutation distorted the DNA-binding site of PAX3. In the other family, MLPA analysis and subsequent quantitative PCR detected a large, heterozygous deletion spanning 1759-2554 kb that eliminated 12-18 genes including a whole PAX3 gene.
Assuntos
Predisposição Genética para Doença , Fatores de Transcrição Box Pareados/genética , Síndrome de Waardenburg/diagnóstico , Síndrome de Waardenburg/genética , Estudos de Casos e Controles , Pré-Escolar , Feminino , Testes Genéticos , Heterozigoto , Humanos , Lactente , Japão , Masculino , Mutação de Sentido Incorreto , Fator de Transcrição PAX3 , Linhagem , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
The formation of homo- and hetero-oligomers between various G protein-coupled receptors (GPCRs) has been demonstrated over the past decade. In most cases, GPCR heterodimerization increases the diversity of intracellular signaling. GPCR-type purinergic receptors (adenosine and P2Y receptors) are actively reported to form hetero-oligomers with each other, with GPCRs belonging to the same group (type 1, rhodopsin-like), and even with GPCRs from another group. This chapter describes common strategies to identify dimerization of purinergic receptors (coimmunoprecipitation, bioluminescence resonance energy transfer (BRET), and immunoelectron microscopy) and to assess the alteration of their pharmacology (ligand binding, intracellular cAMP, and intracellular Ca(2+) assays). We have reported dimerization of purinergic receptors using these strategies in transfected human embryonic kidney 293T cells and native brain tissue. Our data suggest that homo- and hetero-oligomerization between purinergic receptors exert unique pharmacology in this receptor group. According to these discoveries, heterodimerization is likely to be employed for the "fine-tuning" of purinergic receptor signaling.