RESUMO
Mutation in the gene encoding microphthalmia-associated transcription factor (MITF) lead to Waardenburg syndrome 2 (WS2), an autosomal dominantly inherited syndrome with auditory-pigmentary abnormalities, which is clinically and genetically heterogeneous. Haploinsufficiency may be the underlying mechanism for WS2. However, the mechanisms explaining the genotypic and phenotypic variations in WS2 caused by MITF mutations are unclear. A previous study revealed that MITF interacts with LEF-1, an important factor in the Wnt signaling pathway, to regulate its own transcription through LEF-1-binding sites on the MITF promoter. In this study, four different WS2-associated MITF mutations (p.R217I, p.R217G, p.R255X, p.R217del) that are associated with highly variable clinical features were chosen. According to the results, LEF-1 can activate the expression of MITF on its own, but MITF proteins inhibited the activation. This inhibition weakens when the dosage of MITF is reduced. Except for p.R217I, p.R255X, p.R217G, and p.R217del lose the ability to activate TYR completely and do not inhibit the LEF-1-mediated activation of the MITF-M promoter, and the haploinsufficiency created by mutant MITF can be overcome; correspondingly, the mutants' associated phenotypes are less severe than that of p.R217I. The dominant negative of p.R217del made it have a second-most severe phenotype. This study's data imply that MITF has a negative feedback loop of regulation to stabilize MITF gene dosage that involves the Wnt signaling pathway and that the interaction of MITF mutants with this pathway drives the genotypic and phenotypic differences observed in Waardenburg syndrome type 2 associated with MITF mutations.
Assuntos
Genótipo , Fator de Transcrição Associado à Microftalmia/genética , Mutação , Fenótipo , Síndrome de Waardenburg/genética , Síndrome de Waardenburg/metabolismo , Via de Sinalização Wnt , Linhagem Celular , Epistasia Genética , Genes Reporter , Estudos de Associação Genética , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Waardenburg syndrome (WS) is an autosomal dominant inherited non-syndromic type of hereditary hearing loss characterized by varying combinations of sensorineural hearing loss and abnormal pigmentation of the hair, skin, and inner ear. WS is classified into four subtypes (WS1-WS4) based on additional symptoms. WS2 is characterized by the absence of additional symptoms. Recently, we identified a SOX10 missense mutation c.422T > C (p.L141P) associated with WS2. We performed functional assays and found the mutant loses DNA-binding capacity, shows aberrant cytoplasmic and nuclear localization, and fails to interact with PAX3. Therefore, the mutant cannot transactivate the MITF promoter effectively, inhibiting melanin synthesis and leading to WS2. Our study confirmed haploinsufficiency as the underlying pathogenesis for WS2.
Assuntos
Haplótipos/genética , Fator de Transcrição PAX3/genética , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição SOXE/genética , Síndrome de Waardenburg/genética , Adolescente , Humanos , Masculino , Mutação/genéticaRESUMO
Hearing impairment, or deafness (in its most severe form), is one of the most common human sensory disorders. There have been several reports of autosomal dominant mutations in the POU4F3 gene, which is associated with non-syndromic hearing loss. In this study, we identified a novel heterozygous mutation (c.602delT, p.L201fs) in the gene POU4F3 by taking advantage of whole-exome sequencing, which was validated by Sanger sequencing and completely co-segregated within a large hearing impaired Chinese family. We have focused on this pedigree since 2002, and we have mapped a deafness locus named DFNA42 (which has been renamed DFNA52, OMIM entry 607683) via a genome-wide scan. Furthermore, we analyzed this mutational variant and found that it was located at the beginning of the first functional domain of POU4F3, which could theoretically impair the function of POU4F3. We have identified a novel frameshift mutation in the POU4F3 gene. Further functional studies of variants of this specific gene are needed to illustrate the pathogenic mechanism(s) that underlie hearing impairment.
Assuntos
Mutação da Fase de Leitura/genética , Perda Auditiva Neurossensorial/genética , Proteínas de Homeodomínio/genética , Fator de Transcrição Brn-3C/genética , Povo Asiático/genética , Sequência de Bases , Exoma/genética , Perda Auditiva Neurossensorial/patologia , Humanos , Linhagem , Análise de Sequência de DNARESUMO
OBJECTIVE: To study the therapeutic effect of ventilation tube insertion in the middle ear and the external auditory canal on chronic secretory otitis media in children. METHODS: A retrospective study on 30 patients (40 ears) with chronic secretory otitis media and who underwent the operation of middle ear exploration and ventilation tube insertion in the middle ear and the external auditory canal was performed. Poor tympanic membrane, even with adhesion, was seen in 23 ears. Ten patients had evidence of bilateral secretory otitis media. From this group one ear was first injected with drugs (dexamethasone, mucosolvin, etc) and then tube insertion into the auditory tube was performed; the other ear only received drug injections into the auditory tube. The remaining 20 patients who had evidence of unilateral secretory otitis media only received drug injections into the auditory tube. RESULTS: The tubes inserting into the auditory tube all dropped out 5-8 days after operation. None of the ventilation tubes into the middle ear dropped out and the patients' tympanum recovered after the ventilation tubes were removed (6-8 months after operation). The total cure rate was 87.5% (35/40) and the improvement rate was 12.5% (5/40). The operation of inserting tubes into the auditorytube did not improve the therapeutic effects. In the 0.5-2 years postoperative follow-up, middle ear effusions recurred in one ear, and three ears were transferred from type C to type A. CONCLUSIONS: The surgery of ventilation tube insertion in the middle ear and the external auditory canal for chronic secretory otitis media can prevent the tympanic membrane from damage and dropping out of the ventilation tube and reduce recurrence in children. It is a preferred selection for the patients with poor tympanic membrane or adhesive tympanic membrane. It is no use to insert the tube into the auditory tube for the improvement of therapeutic effects.
Assuntos
Ventilação da Orelha Média/métodos , Otite Média com Derrame/cirurgia , Adolescente , Criança , Doença Crônica , Meato Acústico Externo/cirurgia , Orelha Média/cirurgia , Feminino , Humanos , Masculino , Estudos RetrospectivosRESUMO
Bartter syndrome type IV, characterized by salt-losing nephropathies and sensorineural deafness, is caused by mutations of BSND or simultaneous mutations of both CLCNKA and CLCNKB. GJB2 is the primary causative gene for non-syndromic sensorineural deafness and associated with several syndromic sensorineural deafness. Owing to the rarity of Bartter syndrome, only a few mutations have been reported in the abovementioned causative genes. To investigate the underlying mutations in a Chinese patient with Bartter syndrome type IV, genetic analysis of BSND, CLCNKA, CLCNKB and GJB2 were performed by polymerase chain reaction and direct sequencing. Finally, double homozygous mutations c.22C > T (p.Arg8Trp) and c.127G > A (Val43Ile) were detected in exon 1 of BSND. Intriguingly, compound heterozygous mutations c.235delC (p.Leu79CysfsX3) and c.109G > A (p.Val37Ile) were also revealed in exon 2 of GJB2 in the same patient. No pathogenic mutations were found in CLCNKA and CLCNKB. Our results indicated that the homozygous mutation c.22C > T was the key genetic reason for the proband, and a digenic effect of BSND and GJB2 might contributed to sensorineural deafness. To our knowledge, it was the first report showing that the GJB2 gene mutations were detected in Bartter syndrome.
Assuntos
Síndrome de Bartter/genética , Canais de Cloreto/genética , Conexinas/genética , Perda Auditiva Neurossensorial/genética , Mutação , Síndrome de Bartter/complicações , Pré-Escolar , Conexina 26 , Éxons , Feminino , Homozigoto , Humanos , Lactente , Recém-NascidoRESUMO
Waardenburg syndrome type IV (WS4) is a rare genetic disorder, characterized by auditory-pigmentary abnormalities and Hirschsprung disease. Mutations of the EDNRB gene, EDN3 gene, or SOX10 gene are responsible for WS4. In the present study, we reported a case of a Chinese patient with clinical features of WS4. In addition, the three genes mentioned above were sequenced in order to identify whether mutations are responsible for the case. We revealed a novel nonsense mutation, c.1063C>T (p.Q355*), in the last coding exon of SOX10. The same mutation was not found in three unaffected family members or 100 unrelated controls. Then, the function and mechanism of the mutation were investigated in vitro. We found both wild-type (WT) and mutant SOX10 p.Q355* were detected at the expected size and their expression levels are equivalent. The mutant protein also localized in the nucleus and retained the DNA-binding activity as WT counterpart; however, it lost its transactivation capability on the MITF promoter and acted as a dominant-negative repressor impairing function of the WT SOX10.
Assuntos
Códon sem Sentido , Fatores de Transcrição SOXE/genética , Síndrome de Waardenburg/genética , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Pré-Escolar , Éxons , Doença de Hirschsprung , Humanos , Masculino , Linhagem , Ligação Proteica , Fatores de Transcrição SOXE/metabolismo , Ativação Transcricional , Síndrome de Waardenburg/diagnóstico , Síndrome de Waardenburg/metabolismoRESUMO
OBJECTIVE: To explore interaction proteins affect functions of connexin 30 (Cx30) by screening and identification interaction proteins of Cx30. METHODS: The fusion expression vecto of CX30-C-terminal functional domain-pGEX-4T-2-GST was constructed, and then, fusion protein and GST were purified. They were incubated with the proteins of the foetus brain tissue disruption to pull down interaction proteins. The interaction proteins were separated by SDS-PAGE. Differential straps were cut to enzymolysis to prepare for mass chromatographic analysis, and then to index and screen interaction proteins in NCBInr database. The interaction proteins were identified by immunolocalization. RESULTS: The four interaction proteins of Cx30 were screened in the foetus brain tissue, as follow, Keratin 16, Camk2b, Tubulin beta-3 and alpha-tubulin. Cx30 was proved to coexist with Keratin 16 and Tubulin beta-3. CONCLUSIONS: Keratin 16, Camk2b, Tubulin beta-3 and alpha-tubulin are the interaction proteins of Cx30. The interaction proteins affect the assembly, intracellular transport, and channel switch of Cx30.
Assuntos
Conexinas/genética , Vetores Genéticos , Conexina 30 , Conexinas/metabolismo , Glutationa Transferase , Humanos , Testes de Mutagenicidade , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
OBJECTIVE: To undergo tinnitus evaluation test and masking therapy of subject tinnitus associated with sensorineural hearing loss as a reference for diagnosis and guiding masking therapy. METHODS: The 66 patients with subject tinnitus were diagnosed as sensorineural hearing loss. Sixty-six patients divide into three groups according to the results of pure tone audiometry, including steep drop type in 28 patients, slow drop type in 20 patients, and flat type in 18 patients. All the patients underwent tinnitus evaluation tests (pitch matching, intensity matching, tinnitus masking curves, residual inhibition) and masking therapy. RESULTS: Tinnitus with steep drop type manifest was as low intensity [average intensity (5.7 +/- 2.9) dB (x(-) +/- s)] and high frequency (median with 4750 Hz). Residual inhibition was almost positive, but was usually consistent with convergence and congruence tinnitus masking curves. Masking therapy had better effect in the treatment of this type of tinnitus (effective rate 89.3%). Tinnitus with slow drop type manifest was as low intensity [average intensity: (6.2 +/- 4.8) dB] and high frequency (median: 4050 Hz). The distribution of residual inhibition and tinnitus masking curves had no obviously characteristics. The effective rate of masking therapy of this type of tinnitus (55.0%) was higher than tinnitus with flat type but low than that of tinnitus with steep drop type. The intensity of tinnitus with flat type [average intensity: (9.2 +/- 5.0) dB] was higher than that of the previous groups. The distribution of frequency of this type had no obviously characteristics. The residual inhibition was almost negative, and was usually consistent with divergence and persistence tinnitus masking curves. Masking therapy had unsatisfactory curative effect in the treatment of this type of tinnitus (effective rate 11.1%). The effective rate of masking therapy was significant differences among the three groups (chi(2) = 9.127, P < 0.05). CONCLUSIONS: For the tinnitus patients with steep drop type audiometric curve, they are recommended masking therapy. For the tinnitus patients with slow drop type audiometric curve, masking therapy may be attempted to perform. For the tinnitus patients with flat type audiometric curve, they do not recommend the masking therapy.