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：
10250947
负责人：
Thomas Friedman
金额：
$ 251.29万
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/10250947
关键词：
Alleles Alternative Splicing Amino Acids Apical Atomic Force Microscopy Auditory Auditory system Basic Science Biochemistry Brain CRISPR/Cas technology Cell physiology Cells Cellular biology Chromosome 1 Clinical Clinical Research Collaborations Complement DOOR (deafness-onychodystrophy-osteodystrophy-retardation) syndrome Data Development Doctor of Philosophy Dystonia Embryo Engineering Enrollment Exercise Exons F-Actin Family Female infertility Female sterility Fertility Functional disorder Future Genes Genetic Genetic Variation Genomic DNA Genotype Goals Hair Cells Hearing Heterozygote Human Human Genetics Human Genome Individual Inherited Inner Supporting Cell Knockout Mice Laboratories Labyrinth Link Manuscripts Maps Measures Missense Mutation Modeling Molecular Molecular Genetics Mus Mutate Mutation National Institute on Deafness and Other Communication Disorders Natural History Nigeria Pathogenicity Perrault syndrome Phenotype Phosphoric Monoester Hydrolases Preparation Protein Isoforms Proteins Publications Publishing RNA Splicing Reporting Research Research Personnel Research Project Grants Rolandic Epilepsy SNP array Seizures Site Splice-Site Mutation Supporting Cell Syndrome Time Transcript Tunisia United States National Institutes of Health Universities Usher Syndrome Variant Visual Zebrafish Zein auditory pathway base clinical center conditional knockout consanguineous family deaf deafness gene function genetic approach hearing impairment human disease inner ear diseases interest male male fertility mouse model novel proband recessive genetic trait skills structural biology tool

项目摘要

The goal of the Laboratory of Molecular Genetics (LMG), Section on Human Genetics is to identify and study the functions of mutated genes associated with human syndromic and nonsyndromic deafness. A new study begins with the ascertainment of large families in which deafness appears to be inherited either as a monogenic dominant or as monogenic recessive trait. We then search for linkage of the deafness to 950,000 SNP markers distributed across the human genome. Staff in the LMG have been working on the following projects, some of which were completed in the past year, have been published recently, are in press or likely to be submitted for publication in the near future. 1. DFNB32/CDC14A: Eleven years ago, DFNB32 was mapped to chromosome 1 by by a research group in Tunisia. The underlying gene for DFNA32 deafness was never reported. In eight of our consanguineous families segregating recessively inherited nonsyndromic deafness linked to markers for the DFNB32 locus, we identified several truncating mutations, a splice site mutation and a missense mutation in CDC14A, a gene located in our refined DFNB32 interval. CRISPR-Cas9 edited alleles of mouse Cdc14a, when homozygous or in compound heterozygosity, result in deafness. Surprisingly, males are also sterile but females have normal fertility but are also deaf. Thus, CDC14A is essential for hearing and for male fertility. As a collaboration with Dr. Katie Kindt, we also constructed zebrafish models of the DFNB32 gene to probe its function. These data were published in Human Molecular Genetics (Imtiaz et al., 2018). CDC14A is a phosphatase. In hair cells, CDC14A is located in the kinocilia and in stereocilia (Imtiaz et al., 2018) The protein substrates of CDC14A phosphatase are not known. Y2H screens and mass-spec analyses are underway to identify substrates of CDC14A as part of a larger project to understand the function of this essential phosphatase in the auditory system. 2. DFNB28/TRIOBP: The gene responsible for human deafness DFNB28 human deafness was identified as TRIOBP (Kitajiri et al., Cell, 2010). TRIOBP encodes three distinct proteins that arise from alternative splicing of TRIOBP transcripts. TRIOBP isoforms are referred to as TRIOBP-1, TRIOBP-4 and TRIOBP-5. Loss of TRIOBP-1 causes embryonic lethality in mouse. The function, if any, of TRIOBP-1 in the inner ear is not known. Simultaneous loss of TRIOBP4 and TRIOBP-5 causes deafness as a result of the inabilityof hair cells to develop stereocilia rootlets. Purified TRIOBP-4 tightly bundles F-actin typical of stereocilia rootlets. At that time, the individual function(s) of TRIOBP-5 is not known. We engineered mice that do not express functional TRIOBP-5 and have wild type expression of TRIOBP-1 and TRIOBP-4. TRIOBP-5 deficient mice develop rootlets. However, the rootlets are unusually dysmorphic, stereocilia are floppy and the reticular lamina, as measured by Atomic Force Microscopy, is significantly less rigid due to the apical loss of TRIOBP-5 in supporting cells surrounding hair cells (Katsuno and Belyantseva et al., 2019). Studies are continuing to understand the function of TRIOBP-4 and TRIOBP-5 in supporting cells and the functions of each of the TRIOBP isoform function elsewhere in hair cells. For example, we are studying a TRIOBP-1 conditional knock out mouse which will us to delete TRIOBP-1 in hair cells or only in supporting cells of the inner ear and determine the phenotype. 3. The LMG is ascertaining Pakistani families segregating Perrault Syndrome, which is characterized by deafness and female infertility. This project is an ongoing collaboration with William Newman, MD, PhD in Manchester, UK. We are also engineering mouse models of the human genes responsible for Perrault syndrome in order to understand their function in the auditory system. 4. DFNB86/TBC1D24: In 2014, we reported that Variants of TBC1D24 are associated with nonsyndromic deafness DFNB86. Variants of TBC1D24 have also been associated with seizures, seizures and deafness and DOORS syndrome. Other variants of TBC1D24 have been associated with Rolandic epilepsy and exercise-induced dystonia (Luthy et al., 2019), expanding the genotype-phenotype range even further. Using CRISPR-Cas9, we have engineered mice with variants of Tbc1d2, one of which abruptly begins having seizures at P15. The abrupt onset of seizures in mouse is correlated with inclusion of a perfectly conserved alternatively spliced micro-exon encoding 8 amino acid residues and harboring a mutation of Tbc1d24 (Tona et al., 2019). The function of the micro-exon is being explored using a conditional knockout variant in which loxP sites were engineered surrounding the micro-exon. As a collaboration with Michelle Hastings, we are also developing a potential ASO-based therapy in mouse to circumvent splicing to include the micro-exon. Additionally, we are working on identifying the protein partners of TBC1D24 in the brain and inner ear and human disease-causing missense mutations of TBC1D24 that are predicted to disrupt such partnerships. 5. Usher syndrome is genetically and clinically heterogeneous. In collaboration with Carmen Brewer, PhD, Andrew Griffith MD, PhD and Wadih Zein MD (NEI) we are studying the natural history of the visual, auditory and vestibular phenotypes of Usher syndrome subjects enrolled at the NIH Clinical Center. All of these Usher subjects have biallelic molecular genetic likely pathogenic variants of the reported Usher genes from sequencing analyses conducted by staff in the LMG. A manuscript is in preparation (Wafa et al., 2019). 6. In collaboration with Adebolajo Adeyemo, MD at the University of Ibadan and Andrew J. Griffith, NIDCD/NIH, staff in the LMG are identifying the pathogenic variants associated with deafness in Nigeria. Approximately 50 small families with a deaf probing were ascertained in Nigeria. The deaf proband's gDNA is being whole exam sequenced and evaluated for putative pathogenic variants.

分子遗传学实验室 (LMG) 人类遗传学部分的目标是识别和研究与人类综合征性和非综合征性耳聋相关的突变基因的功能。一项新的研究首先确定了一些大家族，其中耳聋似乎是作为单基因显性性状或单基因隐性性状遗传的。然后，我们寻找耳聋与分布在人类基因组中的 950,000 个 SNP 标记之间的联系。 LMG 的工作人员一直致力于以下项目，其中一些项目在过去一年完成，最近已出版，正在出版或可能在不久的将来提交出版。 1. DFNB32/CDC14A：11年前，突尼斯的一个研究小组将DFNB32定位到1号染色体。 DFNA32 耳聋的潜在基因从未被报道过。在我们分离出与 DFNB32 基因座标记相关的隐性遗传性非综合征性耳聋的 8 个近亲家庭中，我们在 CDC14A（位于我们精制的 DFNB32 间隔中的基因）中鉴定了几个截短突变、一个剪接位点突变和一个错义突变。 CRISPR-Cas9 编辑的小鼠 Cdc14a 等位基因，当纯合或复合杂合时，会导致耳聋。令人惊讶的是，雄性也是不育的，而雌性则具有正常的生育能力，但也是聋子。因此，CDC14A 对于听力和男性生育能力至关重要。作为与 Katie Kindt 博士的合作，我们还构建了 DFNB32 基因的斑马鱼模型来探测其功能。这些数据发表在《人类分子遗传学》上（Imtiaz 等人，2018）。 CDC14A 是一种磷酸酶。在毛细胞中，CDC14A 位于动纤毛和静纤毛中 (Imtiaz et al., 2018) CDC14A 磷酸酶的蛋白质底物尚不清楚。 Y2H 筛选和质谱分析正在进行中，以确定 CDC14A 的底物，作为了解这种重要磷酸酶在听觉系统中的功能的更大项目的一部分。 2. DFNB28/TRIOBP：导致人类耳聋的基因DFNB28人类耳聋被鉴定为TRIOBP（Kitajiri等人，Cell，2010）。 TRIOBP 编码三种不同的蛋白质，这些蛋白质由 TRIOBP 转录物的选择性剪接产生。 TRIOBP 同工型称为 TRIOBP-1、TRIOBP-4 和 TRIOBP-5。 TRIOBP-1 缺失会导致小鼠胚胎死亡。 TRIOBP-1 在内耳中的功能（如果有的话）尚不清楚。 TRIOBP4 和 TRIOBP-5 同时缺失会导致耳聋，因为毛细胞无法发育出静纤毛细根。纯化的 TRIOBP-4 紧密捆绑静纤毛细根典型的 F-肌动蛋白。当时，TRIOBP-5 的各个功能尚不清楚。我们设计了不表达功能性 TRIOBP-5 且具有野生型表达 TRIOBP-1 和 TRIOBP-4 的小鼠。 TRIOBP-5 缺陷小鼠会长出细根。然而，根部异常畸形，立体纤毛松软，并且通过原子力显微镜测量，由于毛细胞周围的支持细胞中 TRIOBP-5 的顶端缺失，网状层的刚性明显降低（Katsuno 和 Belyantseva 等人， 2019）。研究正在继续了解 TRIOBP-4 和 TRIOBP-5 在支持细胞中的功能以及每种 TRIOBP 同工型在毛细胞其他部位的功能。例如，我们正在研究TRIOBP-1条件敲除小鼠，该小鼠将删除毛细胞中的TRIOBP-1或仅删除内耳支持细胞中的TRIOBP-1并确定表型。 3. LMG 正在查明患有佩罗综合症的巴基斯坦家庭，该综合症的特点是耳聋和女性不孕。该项目是与英国曼彻斯特医学博士 William Newman 持续合作的项目。我们还在设计导致佩罗综合征的人类基因的小鼠模型，以了解它们在听觉系统中的功能。 4. DFNB86/TBC1D24：2014年，我们报道TBC1D24的变异体与非综合征性耳聋DFNB86相关。 TBC1D24 的变异体也与癫痫发作、癫痫发作和耳聋以及 DOORS 综合征有关。 TBC1D24 的其他变体与 Rolandic 癫痫和运动诱发的肌张力障碍有关 (Luthy et al., 2019)，进一步扩大了基因型-表型范围。使用 CRISPR-Cas9，我们设计了带有 Tbc1d2 变体的小鼠，其中一只在 P15 时突然开始癫痫发作。小鼠癫痫发作的突然发作与包含一个完全保守的可变剪接微外显子相关，该微外显子编码 8 个氨基酸残基并含有 Tbc1d24 突变（Tona 等人，2019）。微外显子的功能正在使用条件敲除变体进行探索，其中loxP位点被设计在微外显子周围。作为与 Michelle Hastings 的合作，我们还在小鼠身上开发一种潜在的基于 ASO 的疗法，以规避包含微外显子的剪接。此外，我们正在努力鉴定大脑和内耳中 TBC1D24 的蛋白质伙伴关系以及导致人类疾病的 TBC1D24 错义突变，预计这些突变会破坏这种伙伴关系。 5. Usher综合征在遗传和临床上具有异质性。我们与 Carmen Brewer 博士、Andrew Griffith 博士和 Wadih Zein 博士 (NEI) 合作，研究 NIH 临床中心登记的 Usher 综合征受试者的视觉、听觉和前庭表型的自然史。根据 LMG 工作人员进行的测序分析，所有这些 Usher 受试者都具有所报道的 Usher 基因的双等位分子遗传可能致病变异。手稿正在准备中（Wafa 等人，2019）。 6. LMG 的工作人员与伊巴丹大学医学博士 Adebolajo Adeyemo 和 NIDCD/NIH 的 Andrew J. Griffith 合作，正在鉴定与尼日利亚耳聋相关的致病变异。在尼日利亚，大约有 50 个小家庭患有耳聋探查。聋哑先证者的 gDNA 正在接受全面检查测序并评估推定的致病变异。