Molecular Analysis Of Human Hereditary Deafness

TMC基因 我们已经产生了TMC1和TMC2基因敲除(空)的小鼠。我们正在研究它们的听力和平衡功能。TMC1基因敲除等位基因纯合的小鼠是聋子。TMC2基因敲除等位基因纯合的小鼠听力和平衡能力正常。两种基因敲除等位基因纯合的小鼠是聋人，平衡功能不正常。这些结果表明，正常的平衡功能需要TMC1和TMC2，而听力只需要TMC1。我们目前正在努力确定TMC1和TMC2蛋白在听力和平衡器官的感觉毛细胞中的位置和功能。 为了更好地了解TMC基因和蛋白质的功能(S)，我们制造了Tmc6和Tmc8基因敲除小鼠。人类TMC6或TMC8基因的突变会导致疣状表皮发育不良，这是一种隐性疾病，会导致慢性皮肤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。我们已经做了大量的RNA表达分析，表明Tmc6和Tmc8主要在淋巴样细胞和组织以及肺和皮肤中表达，而且主要在发育过程中表达。纯合基因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了变化。 旋转式鼠标 我们在18号染色体上发现了ZEB1基因第一内含子的twirler突变。带有该突变的转基因小鼠的表型证实了它的致病性。得到的菌株具有Twirler的所有表型特征，从而证明了候选突变的致病作用。该突变改变了C-Myb蛋白与被该突变破坏的共识Myb结合位点的结合。突变小鼠表现出ZEB1RNA和蛋白质表达水平的增加。我们的合作者Ronna Hertzano博士和她的同事表明，ZEB1是其他基因的表达调节器，这些基因对于维持小鼠发育中的间充质细胞与上皮细胞的特性至关重要。它通过抑制通常在上皮细胞中表达的基因的间质表达来实现这一点。ZEB1在旋转耳中的表达变化导致其靶基因表达异常，破坏内耳结构的发育，导致内耳畸形和听力和平衡的丧失。 扩大前庭导水管(EVA) 我们确定有多个成员的非综合征性EVA家族与可检测到的SLC26A4突变或Pendred综合征无关。我们正在利用这些家系以遗传连锁为基础的策略来确定EVA的其他遗传原因。我们正在评估几个潜在连锁的候选区域。 我们建立了多西环素可诱导表达SLC26A4的小鼠系。这个转基因小鼠品系允许我们通过在饮用水中注射多西环素来操纵SLC26A4的表达(在SLC26A4基因敲除的背景下)。我们定义了胚胎16.5天到出生后2天的时间窗口，在此期间SLC26A4是听觉发育和功能所必需的。我们可以操纵多西环素给药来产生小鼠，在其中有显著的剩余听力和孤立的EVA，这是一种比现有的基因敲除小鼠更接近于模拟人类EVA的表型。我们与Philine Wangemann博士合作，展示了这些小鼠的耳蜗具酸性内淋巴(充满耳蜗管的液体)和耳蜗内电位降低(正常内耳感觉细胞功能所需的电化学梯度)。我们目前正在评估这些小鼠的长期听力状况，因为患有EVA的人类患者有渐进性或波动性听力损失。 协作项目 我们与Thomas Friedman博士合作，在由FGF3突变引起的巴基斯坦Lamm综合征患者中，表征了与耳聋相关的内耳结构表型。 我们与中岛津博士合作，在Pendred综合征SLC26A4基因敲除小鼠模型中评估了膳食碘缺乏对甲状腺肿发展的影响。

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.