Identification of Genes Causing Syndromic and Nonsyndromic Hearing Impairment

导致综合征性和非综合征性听力损伤的基因的鉴定

基本信息

批准号：
8745651
负责人：
Thomas Friedman
金额：
$ 350.22万
依托单位：
NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
依托单位国家：
美国
项目类别：
财政年份：
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/8745651
关键词：
15q21 16p 19p 3&apos Splice Site AIDS clinical trial group Adult Affect Alleles Alternative Splicing Amino Acids Antibodies Auditory system Boston Calcium-Binding Proteins Cell Line Cellular biology ChIP-seq Chromosomes Clinical Cochlea Collaborations Data Development Doctor of Philosophy Engineering Exons Family Female Gene Structure Genes Genetic Genetic Markers Genetic Variation Goals Gonadal Dysgenesis Growth Hearing Hepatocyte Growth Factor Human Human Genetics Image Inherited Knock-out Laboratories Labyrinth Length Life Link Lung Malignant Neoplasms Maps Medicine Mitochondria Molecular Molecular Biology Mus Mutant Strains Mice Mutate Mutation National Institute on Deafness and Other Communication Disorders Nature Paper Pathway interactions Perrault syndrome Phenotype Protein Isoforms Proteins Publications Publishing Pulmonary alveolar structure Regulation Reporting Research Reverse Transcriptase Polymerase Chain Reaction Signal Transduction Somatic Mutation Stereocilium Structure Surveys Technology Tissues Transcript Transgenes Type II Epithelial Receptor Cell United States National Institutes of Health Universities Untranslated Regions Usher Syndrome Work Wound Healing beta Actin cell type college deafness gamma Actin gene function genome wide association study hearing impairment interest lung development medical schools mouse model next generation sequencing novel polypeptide recessive genetic trait research study tool transcription factor

项目摘要

The goal of the Section on Human Genetics is to identify and study the function of mutated genes for human hereditary deafness. This work begins with the ascertainment of large families in which deafness appears to be inherited either as a dominant or a recessive trait. We then search for linkage of the deafness to genetic markers for the known (already reported) syndromic, DFNA (dominant) and DFNB (recessive) loci. If linkage to the known deafness loci can be excluded, we initiate a genome-wide screen to search for novel deafness loci followed by work to identify the causative gene. During the past year we ascertained several large families segregating deafness, mapped a novel deafness locus, and identified a novel gene for nonsyndromic deafness. Sections of the following projects were completed in the past year and have been published or are in press and likely to be published in 2013. 1. We will determine how noncoding mutations (del3 and del10 mutations) of HGF cause deafness DFNB39, which is the subject of ongoing work by Drs. Julie Schultz and Rob Morell. Hepatocyte growth factor is the 728 amino acid polypeptide product of the longest transcript arising from HGF (isoform 1). HGF activates downstream signaling only after proteolytic cleavage of the pro-HGFpolypeptide to form alpha and beta chains. HGF has indispensible functions involving growth and wound repair, and somatic mutations of HGF have been implicated in some cancers. There are also naturally occurring shorter isoforms of HGF that arise due to alternate splicing. Isoforms 3 and 4 use an alternate splice acceptor site in exon 5, resulting in the omission of five amino acids. Although not annotated as such in RefSeq, our own data demonstrates that isoform 5 also occurs in both an exon 5a and 5b configuration. In addition, we discovered a shorter HGF transcript designated isoform 6, which can be RT-PCR amplified from many tissues, including cochlea. The 3'-UTR of isoform 6 transcripts include the deleted sequence of DFNB39 mutations. Despite a wealth of research on HGF, no comprehensive study of the temporal and spatial regulation of HGF isoforms has been made. Previously we described two different Hgf mouse models and demonstrated that both have hearing loss (Schultz et al., 2009). An Hgf transgene ubiquitously over-expresses full length HGF, and a conditional knockout of Hgf in which exon 5 was flanked by flox sequences. Taken together, these data indicate that dysregulation (too much or too little) of HGF causes hearing loss. To recapitulate deafness due to the del3 and del10 mutations in the 3-UTR of human HGF isoform 6, we have engineered an orthologous del10 deletion of the 3-UTR of Hgf isoform 6 in mouse. This project promises to provide details of the necessary function of HGF in the auditory system. 2. We recently mapped a novel nonsyndromic deafness locus (DFNB81) to chromosome 19p, which is distinct from the closely linked DFNB72 locus (Rehman et al., 2011 EJHG). Mutations of GIPC3 are responsible for DFNB72 deafness. Using next-generation sequencing technology Atteeq rehman, PhD, a fellow in the LMG, identified mutations in CLPP encoding a mitochondrial chambered proteas. DFNB81 deafness was initially presumed to be nonsyndromic. However, further clinical chactareization of the affected subjects indicates Perrault syndrome characterized by hearing loss and female gonadal dysgenesis. This paper was published earlier in 2013 (Jenkinson, rehman et al., AJHG 2013). Atteeq is the co-first author and Friedman is the co-communicating author with William Newman, MD, PhD. Meghan Drummond, PhD, a fellow in the LMG/NIDCD is continuing to work on the function of CLPP in the auditory system. 3. In 2011 we mapped a novel locus for nonsyndromic deafness to chromosome 16p. The locus is designated DFNB86 (Ali et al., 2011). recently, Atteeq Rehman, a fellow in the LMG, identified mutations in a novel deafness gene. A publication is being prepared as a acollaboration with Suzanne Leal, PhD at baylor College of Medicine and Andrew J. Griffith, MD, PhD, NIDCD/NIH. 4. Grhl2-TMinsC/+ is a mouse model of DFNA28 human progressive hearing loss, which we reported ten years ago (Peters et al., 2002). A goal of our present study is to understand the function of the GRHL2 transcription factor in the auditory system. To that end, we have performed ChIP-Seq experiments using our own and commercially available antibodies to GRHL2, in a variety of tissues and cell types. In collaboration with Drs. Maria Ramirez and Saaket Varma at Boston University Medical School, and as a way to validate this technology in our laboratory, we extended our ChIP and expression analyses to include developing lung buds and the MLE15 cell line derived from adult mouse lung. GRHL2 is required for normal lung development. Varma et al. 2012, (Morell and Friedman are co-authors) showed that GRHL2 and NKX2-1 transactive each other and form a regulatory loop defining the critical transition from a type II cell type (cuboidal) to type I (squamous) in the lung alveolus. We are making a more comphrehensive survey of GRHL2 regulatory targets in the lung as compared to the auditory system using ChIP-Seq and our AB5500 sequencer. 5. Nonsyndromic deafness DFNB48 and a novel type 1 Usher syndrome were previously genetically mapped by us to an overlapping interval on chromosome 15q21-q23 (Ahmed et al., 2009). We recently demonstrated that mutations of CIB2 are associated with DFNB48 and USH1J are allelic and that the causative gene encodes a calcium binding protein (Riazuddin et al., November 2012, Nature Genetics). 6. Meghan Drummond PhD is working on the structure and dynamics of the gamma-actin and beta-actin in stereocilia using live-imaging and mouse mutants of gamma-actin some of which are equivalent to those alleles associated with dominantly inherited deafness DFNA20, a locus genetically mapped and reported by staff of the LMG several years ago and positionally cloned and identified as ACTG.

人类遗传学部分的目标是识别和研究人类遗传性耳聋突变基因的功能。这项工作首先确定大家庭中耳聋似乎是作为显性或隐性特征遗传的。然后，我们寻找耳聋与已知（已报道）综合征、DFNA（显性）和 DFNB（隐性）基因座的遗传标记的联系。如果可以排除与已知耳聋基因座的连锁，我们将启动全基因组筛选来寻找新的耳聋基因座，然后确定致病基因。在过去的一年里，我们确定了几个分离耳聋的大家族，绘制了一个新的耳聋基因座，并鉴定了一个新的非综合征性耳聋基因。以下项目的部分内容已于去年完成，并已出版或正在出版，并可能于 2013 年出版。 1. 我们将确定 HGF 的非编码突变（del3 和 del10 突变）如何导致耳聋 DFNB39，这是 Drs. 正在进行的工作的主题。朱莉·舒尔茨和罗布·莫雷尔。肝细胞生长因子是由 HGF（亚型 1）产生的最长转录物的 728 个氨基酸的多肽产物。 HGF 仅在 HGF 多肽原蛋白水解裂解形成 α 链和 β 链后才激活下游信号传导。 HGF 具有涉及生长和伤口修复的不可或缺的功能，并且 HGF 的体细胞突变与某些癌症有关。还存在因交替剪接而产生的天然存在的较短 HGF 亚型。同种型 3 和 4 使用外显子 5 中的替代剪接受体位点，导致省略了 5 个氨基酸。尽管在 RefSeq 中没有这样注释，但我们自己的数据表明同工型 5 也出现在外显子 5a 和 5b 配置中。此外，我们还发现了一个较短的 HGF 转录物，称为亚型 6，可以从包括耳蜗在内的许多组织中进行 RT-PCR 扩增。同种型 6 转录物的 3'-UTR 包括 DFNB39 突变的删除序列。尽管对 HGF 进行了大量研究，但尚未对 HGF 亚型的时间和空间调控进行全面的研究。之前我们描述了两种不同的 Hgf 小鼠模型，并证明它们都有听力损失（Schultz 等人，2009）。 Hgf 转基因普遍过表达全长 HGF，并且条件性敲除 Hgf，其中外显子 5 两侧为 flox 序列。总而言之，这些数据表明 HGF 失调（过多或过少）会导致听力损失。为了重现由于人 HGF 亚型 6 的 3-UTR 中的 del3 和 del10 突变导致的耳聋，我们在小鼠中设计了 Hgf 亚型 6 3-UTR 的直系同源 del10 缺失。该项目承诺提供 HGF 在听觉系统中必要功能的详细信息。 2. 我们最近将一个新的非综合征性耳聋基因座 (DFNB81) 定位到染色体 19p，该基因座不同于紧密连锁的 DFNB72 基因座 (Rehman et al., 2011 EJHG)。 GIPC3 突变导致 DFNB72 耳聋。 LMG 研究员 Atteeq rehman 博士利用下一代测序技术，鉴定出了编码线粒体腔蛋白酶的 CLPP 中的突变。 DFNB81 耳聋最初被认为是非综合征性的。然而，受影响受试者的进一步临床特征表明佩罗综合征，其特征是听力损失和女性性腺发育不全。本文于 2013 年早些时候发表（Jenkinson、rehman 等人，AJHG 2013）。 Atteeq 是共同第一作者，Friedman 是医学博士、博士 William Newman 的共同通讯作者。 LMG/NIDCD 研究员 Meghan Drummond 博士正在继续研究 CLPP 在听觉系统中的功能。 3. 2011 年，我们将非综合征性耳聋的一个新基因座定位到染色体 16p。该基因座被指定为 DFNB86（Ali 等人，2011）。最近，LMG 研究员 Atteeq Rehman 发现了一种新型耳聋基因的突变。贝勒医学院的 Suzanne Leal 博士和 NIDCD/NIH 医学博士、哲学博士 Andrew J. Griffith 正在合作编写一份出版物。 4. Grhl2-TMinsC/+ 是 DFNA28 人类渐进性听力损失的小鼠模型，我们十年前报道过（Peters 等，2002）。我们目前研究的目标是了解 GRHL2 转录因子在听觉系统中的功能。为此，我们使用我们自己的和市售的 GRHL2 抗体在多种组织和细胞类型中进行了 ChIP-Seq 实验。与博士合作。波士顿大学医学院的 Maria Ramirez 和 Saaket Varma，作为在我们实验室验证这项技术的一种方法，我们扩展了 ChIP 和表达分析，以包括发育中的肺芽和源自成年小鼠肺的 MLE15 细胞系。 GRHL2 是正常肺部发育所必需的。瓦尔玛等人。 2012 年，（Morell 和 Friedman 为共同作者）表明 GRHL2 和 NKX2-1 相互交互并形成一个调节环路，定义肺泡中从 II 型细胞类型（立方形）到 I 型（鳞状）细胞的关键转变。与听觉系统相比，我们正在使用 ChIP-Seq 和我们的 AB5500 测序仪对肺部 GRHL2 调控靶点进行更全面的调查。 5. 非综合征性耳聋 DFNB48 和一种新型 1 型 Usher 综合征先前被我们通过基因映射到染色体 15q21-q23 上的重叠区间（Ahmed 等，2009）。我们最近证明 CIB2 的突变与 DFNB48 和 USH1J 相关，是等位基因，并且致病基因编码钙结合蛋白（Riazuddin 等人，2012 年 11 月，Nature Genetics）。 6. Meghan Drummond 博士正在利用实时成像和小鼠伽马肌动蛋白突变体研究静纤毛中伽马肌动蛋白和β-肌动蛋白的结构和动力学，其中一些突变体相当于与显性遗传性耳聋相关的等位基因 DFNA20， LMG 工作人员几年前对基因座进行了基因定位和报告，并进行了位置克隆并鉴定为 ACTG。