Molecular Analysis Of Human Hereditary Deafness

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。我们的假设是，这些家庭分离隐性等位基因在一个或多个其他遗传基因座，导致非综合征型伊娃。我们正在使用这些家庭在连锁为基础的外显子组测序策略，以确定其他遗传原因的伊娃。我们还使用重组断裂点定位和外显子组测序来寻找SLC26 A4的隐藏的（未鉴定的）突变，在分离的非综合征性伊娃中，只有一个可检测的突变等位基因的SLC26 A4。我们的假设是，这些家庭隔离的第二个，身份不明，突变SLC26A4。 我们产生了强力霉素诱导的Slc26a4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Slc26a4表达（在Slc26a4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，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。由于伊娃患者的听力损失呈递增性进展或波动性，我们目前正在评估这些小鼠听力损失的长期自然史和分子细胞生理学发病机制。 TMC基因 我们产生了敲除（无效）等位基因Tmc 1和Tmc 2的小鼠。我们描述了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。 我们发现Tmc 1和Tmc 2在功能上是冗余的，并且需要在出生后耳蜗和前庭感觉毛细胞的静纤毛中进行机械转导。荧光团标记的TMC 1或TMC 2定位在静纤毛的尖端处或附近，表明这些蛋白质或者包含毛细胞机电转导通道的组成部分，或者密切参与其发育和/或功能。我们观察到Tmc 1和Tmc 2 mRNA在发育和成熟的前庭毛细胞中表达，而Tmc 2仅在发育中的耳蜗毛细胞中短暂表达，而Tmc 1表达在成熟的耳蜗毛细胞中持续存在。这些结果表明，为什么人类和小鼠与Tmc 1突变等位基因耳聋，但前庭功能障碍的模型：Tmc 2是可用于补偿Tmc 1功能的前庭，但不是耳蜗毛细胞的损失。目前，我们正在使用转基因小鼠来验证这一假设。我们还在进行进一步的研究，以明确定位TMC1和TMC2蛋白在听觉系统中，以及测试的假设，它们是毛细胞机电转导通道的组成部分。 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。 合作项目 我们与托马斯·弗里德曼（Thomas Friedman）博士合作，对非综合征型DFNB 12耳聋或I型亚瑟综合征患者的CDH 23基因型以及听觉和眼部表型进行了研究。结果支持非综合征性耳聋表型（即视力正常）与保留残余钙粘蛋白-23功能的突变之间的相关性。

ENLARGED VESTIBULAR AQUEDUCTS (EVA) We ascertain families with multiple members with nonsyndromic EVA that is not associated with detectable SLC26A4 mutations or Pendred syndrome. Our hypothesis is that these families segregate recessive alleles at one or more other genetic loci that cause nonsyndromic EVA. We are using those families in a linkage-based exome sequencing strategy to identify other genetic causes of EVA. We are also using recombination breakpoint mapping and exome sequencing to search for occult (unidentified) mutations of SLC26A4 in families segregating nonsyndromic EVA with only one detectable mutant allele of SLC26A4. Our hypothesis is that these families segregate a second, unidentified, mutation of SLC26A4. 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 natural history and molecular-cellular-physiologic pathogenesis of hearing loss in these mice since human patients with EVA have incrementally progressive or fluctuating hearing loss. TMC GENES We generated mice with knockout (null) alleles of Tmc1 and Tmc2. We characterized 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. We showed that Tmc1 and Tmc2 are functionally redundant and required for mechanotransduction in the stereocilia of postnatal cochlear and vestibular sensory hair cells. Fluorophore-tagged TMC1 or TMC2 localized at or near tips of stereocilia, suggesting these proteins either comprise and integral component of the hair cell mechanoelectrical transduction channel, or are intimately involved in its development and/or function. We observed that both Tmc1 and Tmc2 mRNA is expressed in both developing and mature vestibular hair cells, whereas Tmc2 is expressed only transiently in developing cochlear hair cells whereas Tmc1 expression persists in mature cochlear hair cells. These results suggest a model for why humans and mice with Tmc1 mutant alleles have deafness but not vestibular dysfunction: Tmc2 is available to compensate for loss of Tmc1 function in vestibular but not cochlear hair cells. We are currently testing this hypothesis using transgenic mice. We are also pursuing further studies to definitively localize TMC1 and TMC2 proteins in the auditory system, as well as to test the hypothesis that they are integral components of the hair cell mechanoelectrical transduction channel. 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. COLLABORATIVE PROJECTS We collaborated with Dr. Thomas Friedman to CDH23 genotypes and auditory and ocular phenotypes in patients with nonsyndromic DFNB12 deafness or type I Usher syndrome. The results support a correlation of a nonsyndromic deafness phenotype (i.e. with normal vision) with mutations that preserve residual cadherin-23 function.