Molecular Analysis Of Human Hereditary Deafness

TMC基因 我们已经产生了分离敲除（无效）等位基因的Tmc 1和Tmc 2的小鼠。我们正在描述他们的突变听觉和前庭（平衡）表型。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。敲除两个基因的等位基因的纯合子小鼠是耳聋的，并且具有异常的前庭功能。 这些结果表明，Tmc 1和Tmc 2都是正常前庭功能（平衡）所必需的，而只有Tmc 1是听力所必需的。我们正在与Charley Della Santina博士合作，他评估了小鼠的前庭功能，以证明它们的异常平衡和步态是由于前庭系统的半规管（嵴）耳石器官的功能异常。我们目前正在努力确定毛细胞中TMC 1和TMC 2蛋白的细胞内位置和功能。 我们使用酵母双杂交筛选来分离编码可能与TMC 1相互作用的蛋白质的基因。我们将名单缩小到几个与囊泡运输、抗凋亡和细胞存活有关的候选基因。我们正在使用的方法来确定哪些相互作用发生在毛细胞原位组合。 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），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 我们已经确定了一个候选突变，并产生了一个敲入小鼠系与此突变，以确认其致病性。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。 前庭导水管扩大（伊娃） 我们正在确定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。我们正在使用这些家庭在一个遗传连锁为基础的战略，以确定其他遗传原因的伊娃。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）。我们已经定义了一个时间窗口，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。 基于其他实验室的结果表明野生型Slc 26 a4有助于小鼠哮喘和高血压的发病机制，我们假设SLC 26 A4突变可能对人类高血压或哮喘的发展具有保护作用。我们分析了伊娃先证者及其家庭成员的医疗记录来解决这个问题。21个SLC 26 A4有两个突变等位基因的个体中没有一个患有高血压或哮喘，但这一观察结果没有统计学意义。需要进行更大规模的研究来验证这一重要假设。 我们与Sheikh Riazuddin和托马斯·弗里德曼博士合作，完成了巴基斯坦SLC 26 A4突变引起的耳聋的基因型调查。该研究发现了8种新的突变，研究表明，SLC 26 A4突变占巴基斯坦严重至极深的舌前耳聋的约7.2%。因此，SLC 26 A4突变是该人群中最常见的已知耳聋原因。 我们与NIDCD的Byung Choi博士和首尔国立大学的Seung-Ha Oh博士合作，确定了韩国伊娃患者队列中SLC 26 A4突变的患病率和谱。基于这些结果，我们开发了一种包括变性高效液相色谱和直接测序的分级突变筛选策略。我们用已发表的东亚伊娃队列在计算机上验证了这一策略，以表明这是东亚人群中伊娃分子遗传学诊断的有效且具有成本效益的策略。 合作项目 我们与NIDCD的托马斯弗里德曼博士合作，在巴基斯坦完成了一项由OTOF突变引起的DFNB 9耳聋的基因型调查。我们确定了10个新的突变，并表明OTOF突变约占巴基斯坦严重至深度，舌前发作性耳聋的2.3%。 我们与NIDCD的托马斯弗里德曼博士合作，将常染色体显性耳聋基因座DFNA 27定位到染色体4 q12 -13.1。 我们与NIDCD的托马斯弗里德曼博士合作，将常染色体隐性耳聋基因座DFNB 79定位到染色体9q34.3。 我们与NIDCD的托马斯弗里德曼博士合作，鉴定了一个常染色体隐性耳聋基因LRTOMT，这是一个具有替代阅读框架的融合基因。 我们与NHGRI的Leslie Biesecker博士和NIDCD的Matthew Kelley博士合作，将低频感音神经性听力损失确定为GLI 3基因突变引起的Pallister-Hall综合征患者的一种新的不完全的外显特征。这项研究证明了GLI 3在音速刺猬信号传导中的作用，以及突变如何影响内耳的这一发育途径。

TMC GENES We have generated mice segregating knockout (null) alleles of Tmc1 and Tmc2. We are characterizing their mutant auditory and vestibular (balance) phenotypes. 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 vestibular function. These results indicate that both Tmc1 and Tmc2 are required for normal vestibular function (balance), whereas only Tmc1 is required for hearing. We are collaborating with Dr. Charley Della Santina who has evaluated vestibular function in the mice to demonstrate that their abnormal balance and gait is due to abnormal function of the semicircular canals (cristae) otolithic organs of the vestibular system. We are currently working to identify the intracellular location and function of TMC1 and TMC2 proteins in hair cells. We used a yeast two-hybrid screen to isolate genes encoding proteins that potentially interact with TMC1. We narrowed the list to a few candidate genes implicated in vesicular trafficking and in antiapoptosis and cell survival. We are using a combination of approaches to determine which interactions occur in situ in hair cells. 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) 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 a candidate mutation and generated a knock-in mouse line with this mutation to confirm its pathogenicity. The resulting line has all of the phenotypic features of Twirler, thus proving the pathogenic role of the candidate mutation. ENLARGED VESTIBULAR AQUEDUCTS (EVA) We are ascertaining 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 have defined a time window 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. Based upon results from other laboraties demonstrating that wild type Slc26a4 contributes to the pathogenesis of asthma and hypertension in mice, we postulated that SLC26A4 mutations may confer a protective effect against the development of hypertension or asthma in humans. We analyzed medical records for our cohort of EVA probands and their family members to address this question. None of 21 individuals with two mutant alleles of SLC26A4 had hypertension or asthma, but this observation was not statistically significant. A larger study is required to test this important hypothesis. We collaborated with Drs. Sheikh Riazuddin and Thomas Friedman to complete a genotypic survey for deafness caused by SLC26A4 mutations in Pakistan. Eight novel mutations were identified and the study showed that SLC26A4 mutations account for approximately 7.2% of severe to profound, prelingual-onset deafness in Pakistan. SLC26A4 mutations are thus the most common known cause of deafness in that population. We collaborated with Dr. Byung Choi of the NIDCD and Dr. Seung-Ha Oh of Seoul National University to define the prevalence and spectrum of SLC26A4 mutations among a cohort of Korean patients with EVA. Based upon those results, we developed a hierarchical mutation screening strategy involving denaturing high performance liquid chromatography and direct sequencing. We validated this strategy in silico with published east Asian EVA cohorts to show that it is an efficient and cost-effective strategy for the molecular genetic diagnosis of EVA in east Asian populations. COLLABORATIVE PROJECTS We collaborated with Dr. Thomas Friedman of the NIDCD to complete a genotypic survey for DFNB9 deafness caused by OTOF mutations in Pakistan. We identified 10 novel mutations and showed that OTOF mutations account for approximately 2.3% of severe to profound, prelingual-onset deafness in Pakistan. We collaborated with Dr. Thomas Friedman of the NIDCD to map the autosomal dominant deafness locus DFNA27 to chromosome 4q12-13.1. We collaborated with Dr. Thomas Friedman of the NIDCD to map the autosomal recessive deafness locus DFNB79 to chromosome 9q34.3. We collaborated with Dr. Thomas Friedman of the NIDCD to identify an autosomal recessive deafness gene as LRTOMT, a fusion gene with alternative reading frames. We collaborated with Drs. Leslie Biesecker of the NHGRI and Matthew Kelley of the NIDCD to identify low-frequency sensorineural hearing loss as a novel and incompletely penetrant feature of patients with Pallister-Hall syndrome caused by mutations of the GLI3 gene. This study demonstrated the role of GLI3 in sonic hedgehog signaling and how mutations affect this developmental pathway in the inner ear.