Molecular Analysis Of Human Hereditary Deafness

人类遗传性耳聋的分子分析

• 批准号: 8349627
• 负责人: Andrew J Griffith
• 金额: $ 166.96万
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349627
• 关键词: Affect; Alleles; Auditory; Bilateral; Binding; Binding Sites; Birth; Cell physiology; Chromosomes, Human, Pair 18; Chronic; Cleft Palate; Cochlea; Cochlear duct; Consensus; Cutaneous; Defect; Development; Dietary Iodine; Disease; Doxycycline; Ear; Embryo; Endolymph; Epidermodysplasia Verruciformis; Epithelial Cells; Equilibrium; FGF3 gene; Family; Gene Expression; Gene Proteins; Gene Targeting; Genes; Genetic Transcription; Goals; Goiter; Hair Cells; Hearing; Hearing Impaired Persons; Hereditary Disease; Human; Human Papillomavirus; Individual; Infection; Inherited; Introns; Iodine; Knock-in Mouse; Knock-out; Knockout Mice; Laboratories; Labyrinth; Liquid substance; Location; Lung; Lymphoid Cell; Lymphoid Tissue; Maps; Mesenchymal; Modeling; Molecular; Molecular Analysis; Mus; Mutant Strains Mice; Mutation; Obesity; Organ; Other Genetics; Papilloma; Papillomavirus Infections; Pathogenicity; Patients; Pendred Syndrome; Phenotype; Predisposition; Proteins; Reporting; Residual state; Role; Sensorineural Hearing Loss; Sensory; Sensory Hair; Skin; Skin Carcinoma; Structure; Syndrome; Temporal bone structure; Thyroid Gland; Time; Transgenic Mice; Vestibular Aqueduct; Work; base; deafness; drinking water; gene function; genetic linkage; hearing impairment; iodine deficiency syndrome; malformation; member; mouse model; postnatal; protein expression; response

TMC GENES We have generated mice with knockout (null) alleles of Tmc1 and Tmc2. We are characterizing their hearing and balance function. Mice that are homozygous for the Tmc1 knockout allele are deaf. Mice that are homozygous for the Tmc2 knockout allele have normal hearing and balance. Mice that are homozygous for knockout alleles of both genes are deaf and have abnormal balance function. These results indicate that both Tmc1 and Tmc2 are required for normal balance function, whereas only Tmc1 is required for hearing. We are currently working to identify the location and function of TMC1 and TMC2 proteins in sensory hair cells of the hearing and balance organs. We generated knockout mice for Tmc6 and Tmc8 to better understand the function(s) of Tmc genes and proteins. Mutations in human TMC6 or TMC8 genes cause epidermodysplasia verruciformis, a recessive disease resulting in chronic cutaneous HPV infections (papillomas or warts) with increased susceptibility to non-melanoma skin cancers. We have done extensive RNA expression analyses to show that Tmc6 and Tmc8 are primarily expressed in lymphoid cells and tissues and lung and skin, and primarily during development. The homozygous knockout mice have no obvious phenotypic abnormalities, so we are collaborating with Dr. Paul Lambert to determine if these mice have alterations in their susceptibility or response to papillomavirus infection. TWIRLER MOUSE We have identified the Twirler mutation in the first intron of the Zeb1 gene on chromosome 18. The phenotype of a knock-in mouse line with this mutation confirmed its pathogenicity. The resulting line has all of the phenotypic features of Twirler, thus proving the pathogenic role of the candidate mutation. The mutation alters the binding of C-Myb protein to a consensus Myb binding site that is disrupted by the mutation. The mutant mice show increased levels of Zeb1 RNA and protein expression. Our collaborators Dr. Ronna Hertzano and her colleagues showed that Zeb1 is a regulator of expression of other genes that are important for maintaining mesenchymal versus epithelial cell identity in the developing mouse ear. It does this by repressing the mesenchymal expression of genes that are normally expressed in epithelial cells. The alteration of Zeb1 expression in Twirler ears results in abnormal expression of its target genes with disruption of the development of the inner ear structures, leading to inner ear malformations and loss of hearing and balance. ENLARGED VESTIBULAR AQUEDUCTS (EVA) We ascertain families with multiple members with nonsyndromic EVA that is not associated with detectable SLC26A4 mutations or Pendred syndrome. We are using those families in a genetic linkage-based strategy to identify other genetic causes of EVA. We are evaluating several candidate regions of potential linkage. We generated a doxycycline-inducible Slc26a4-expression mouse line. This transgenic mouse line allows us to manipulate Slc26a4 expression (on an Slc26a4-knockout background) by the administration of doxycycline in drinking water. We defined a time window of embryonic day 16.5 to postnatal day 2 during which Slc26a4 is required for auditory development and function. We can manipulate doxycycline administration to generate mice in which there is significant residual hearing and isolated EVA, a phenotype which models human EVA more closely than the existing knockout mouse. We collaborated with Dr. Philine Wangemann to show the cochleae of these mice have acidic endolymph (the fluid filling the cochlear duct) and a reduction of the endocochlear potential (the electrochemical gradient required for normal inner ear sensory cell function). We are currently evaluating the long-term hearing status of these mice since human patients with EVA have incrementally progressive or fluctuating hearing loss. COLLABORATIVE PROJECTS We collaborated with Dr. Thomas Friedman to characterize the inner ear structural phenotypes associated with deafness in Pakistani patients with LAMM syndrome caused by FGF3 mutations. We collaborated with Dr. Tsutomu Nakashima to evaluate the effect of dietary iodine deficiency on the development of goiter in the Slc26a4-knockout mouse model of Pendred syndrome.

TMC基因 我们已经产生了Tmc 1和Tmc 2的敲除（无效）等位基因的小鼠。我们正在描述他们的听觉和平衡功能。Tmc 1基因敲除的纯合子小鼠是耳聋的。 Tmc 2基因敲除等位基因纯合的小鼠具有正常的听力和平衡能力。两个基因的等位基因敲除纯合的小鼠是耳聋的，并且具有异常的平衡功能。 这些结果表明，Tmc 1和Tmc 2都是正常平衡功能所需的，而听力只需要Tmc 1。我们目前正在努力确定TMC 1和TMC 2蛋白在听觉和平衡器官的感觉毛细胞中的位置和功能。 我们产生了Tmc 6和Tmc 8的敲除小鼠，以更好地理解Tmc基因和蛋白质的功能。人类TMC 6或TMC 8基因突变导致疣状表皮发育不良，这是一种隐性疾病，导致慢性皮肤HPV感染（乳头状瘤或疣），并增加对非黑色素瘤皮肤癌的易感性。我们已经进行了广泛的RNA表达分析，表明Tmc 6和Tmc 8主要在淋巴细胞和组织以及肺和皮肤中表达，并且主要在发育期间表达。 纯合子敲除小鼠没有明显的表型异常，因此我们正在与Paul Lambert博士合作，以确定这些小鼠对乳头瘤病毒感染的易感性或反应是否发生改变。 扭鼠 我们已经在18号染色体上的Zeb 1基因的第一个内含子中鉴定了Twirler突变。具有该突变的敲入小鼠品系的表型证实了其致病性。所得品系具有Twirler的所有表型特征，从而证明候选突变的致病作用。突变改变了C-Myb蛋白与被突变破坏的共有Myb结合位点的结合。突变小鼠显示Zeb 1 RNA和蛋白质表达水平增加。我们的合作者Ronna Hertzano博士和她的同事表明，Zeb 1是其他基因表达的调节因子，这些基因对于维持发育中小鼠耳朵的间充质细胞与上皮细胞的身份非常重要。它通过抑制上皮细胞中正常表达的基因的间充质表达来实现这一点。Twirler耳中Zeb 1表达的改变导致其靶基因的异常表达，并破坏内耳结构的发育，导致内耳畸形以及听力和平衡的丧失。 前庭导水管扩大（伊娃） 我们确定了多个成员患有非综合征型伊娃的家族，该家族与可检测到的SLC 26 A4突变或Pendred综合征无关。我们正在以基于遗传联系的策略使用这些家庭来确定伊娃的其他遗传原因。我们正在评估几个可能存在关联的候选区域。 我们产生了强力霉素诱导的Slc 26 a4表达小鼠系。该转基因小鼠系允许我们通过在饮用水中施用强力霉素来操纵Slc 26 a4表达（在Slc 26 a4敲除背景上）。我们定义了一个时间窗口，胚胎16.5天出生后2天，在此期间，Slc 26 a4是听觉发育和功能所必需的。我们可以操纵多西环素给药产生小鼠，其中有显着的残余听力和孤立的伊娃，一种表型，模型人类伊娃更接近于现有的敲除小鼠。我们与Philine Wangemann博士合作，证明这些小鼠的耳蜗具有酸性内淋巴（填充耳蜗导管的液体）和耳蜗内电位（正常内耳感觉细胞功能所需的电化学梯度）的降低。我们目前正在评估这些小鼠的长期听力状况，因为伊娃人类患者的听力损失是渐进性或波动性的。 合作项目 我们与托马斯弗里德曼博士合作，在巴基斯坦患有由FGF 3突变引起的LAMM综合征的患者中描述与耳聋相关的内耳结构表型。 我们与Tsutomu Nakashima博士合作，在Pendred综合征的Slc 26 a4基因敲除小鼠模型中评估膳食碘缺乏对甲状腺肿发展的影响。