Functional analysis of MAB21L2 mutations in MAC spectrum disorders

MAC 谱系疾病中 MAB21L2 突变的功能分析

• 批准号: 9129744
• 负责人: Jeffrey Gross
• 金额: $ 15.43万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9129744
• 关键词: Affect; Alleles; Anophthalmos; Aphakia; Biochemical; Biological Assay; Biological Models; Bromodeoxyuridine; Caenorhabditis elegans; Cell Nucleus; Cell Transplantation; Cell physiology; Cells; Choroid; Chromatin; Coloboma; Complex; Congenital Disorders; DNA; Data; Defect; Developmental Biology; Disease; Dominant-Negative Mutation; Event; Eye; Eye Development; Eye diseases; Gene Expression; Genes; Genetic; Genetic Research; Health; Hereditary Disease; Human; Human Genetics; Incidence; Knock-out; Laboratories; Lateral; Lens Placodes; Link; Liquid Chromatography; Live Birth; Mediating; Microphthalmos; Mission; Modeling; Molecular; Mus; Mutate; Mutation; Nucleotides; Optics; Orthologous Gene; Pathogenicity; Patients; Phenotype; Physiologic pulse; Play; Proteins; Proteomics; Publishing; RNA; Regulator Genes; Reporting; Research; Role; Series; Single Nucleotide Polymorphism Map; Structural defect; Testing; Time; Transferase; United States National Institutes of Health; Work; Zebrafish; base; chromatin remodeling; cost; exome; exome sequencing; eye formation; gene repression; genome sequencing; in vitro activity; in vivo; lens; loss of function; malformation; mutant; next generation sequencing; nucleotidyltransferase; optic cup; personalized medicine; prevent; protein folding; protein function; research study; scaffold; tandem mass spectrometry; transcriptome sequencing; whole genome

 DESCRIPTION (provided by applicant): Microphthalmia-Anophthalmia-Coloboma (MAC) disorders are a heterogeneous spectrum of congenital ocular defects that result in severe structural malformations of the eye. MAC phenotypes can be isolated, or they can be components of a variety of syndromic disorders. Reported incidences vary widely across the globe but approximate ranges are 2.13 per 10,000 live births for anophthalmia and microphthalmia, to 2.6 to 7.5 per 10,000 for colobomata. Colobomata are also a component of over 50 human genetic disorders, where they are often associated with microphthalmia. Despite a significant amount of genetic research to identify MAC loci, causative mutations have been identified in very few cases, and for those that have, little is known about how the affected proteins facilitate normal eye development. Recently, several different mutations were identified in the MAB21L2 gene in human MAC patients [Rainger et al., 2014]. MAB21L2 has not been previously linked to MAC spectrum disorders or to any human congenital disorder. MAB21L2 encodes a protein of unknown biochemical function and it is unknown how the mutations affect MAB21L2 activity. Previous work in our laboratory identified a zebrafish mab21l2 mutant, and ocular defects in the mutant resemble those in human MAC patients. Research in this proposal utilizes zebrafish mab21l2 mutants as a translational model system through which we can identify the molecular underpinnings of human MAB21L2 mutations and determine the cellular function of MAB21L2 during normal eye development. We test the hypothesis that MAB21L2 activity is required for normal proliferation within the optic cup and that Mab21l2 deficiencies result in microphthalmia which prevents apposition of the lateral edges of the choroid fissure, thereby contributing to colobomata. We combine RNA-Seq, chromatin-association assays and LC-MS/MS proteomics to determine the in vivo function of MAB21L2 and elucidate the MAB21L2-dependent gene regulatory network and protein interactome underlying normal eye formation. These experiments fit the mission of the NIH and the NEI because they have direct relevance to furthering our understanding of early eye development and MAC spectrum disorders. Furthermore, they develop a zebrafish mutant model as an in vivo translational platform for determining the pathogenicity of human mutations. Given the prospects of personalized medicine, and the ease/low-cost of exome and whole-genome sequencing, mutations in genes encoding proteins for which little functional information exists will be increasingly identified. The pipeline established in this proposal will serve as a useful in vivo paradigm for elucidating the molecular underpinnings of these human mutations, and for rapidly determining the functions of the proteins they encode.

 描述（由申请方提供）：小眼-无眼-缺损（MAC）疾病是一种异质性的先天性眼部缺陷，可导致严重的眼部结构畸形。MAC表型可以是孤立的，或者它们可以是各种综合征性疾病的组成部分。地球仪报告的发病率差异很大，但无眼症和小眼症的近似范围为每10，000例活产2.13例，缺损为每10，000例2.6 - 7.5例。缺损也是50多种人类遗传疾病的组成部分，它们通常与小眼症有关。尽管有大量的遗传研究来确定MAC基因座，但在极少数情况下已经确定了致病突变，并且对于那些已经确定的突变，人们对受影响的蛋白质如何促进正常的眼睛发育知之甚少。最近，在人MAC患者的MAB 21 L2基因中鉴定了几种不同的突变[Rainger等人，2014年]。MAB 21 L2以前没有与MAC谱系障碍或任何人类先天性疾病相关。MAB 21 L2编码一种生物化学功能未知的蛋白质，突变如何影响MAB 21 L2活性尚不清楚。我们实验室先前的工作鉴定了斑马鱼mab 21 l2突变体，突变体中的眼部缺陷类似于人类MAC患者中的眼部缺陷。这项研究利用斑马鱼mab 21 l2突变体作为翻译模型系统，通过该系统，我们可以鉴定人MAB 21 L2突变的分子基础，并确定MAB 21 L2在正常眼睛发育过程中的细胞功能。我们测试的假设，MAB 21 L2活性是所需的视杯内的正常增殖和Mab 21 L2的缺陷导致小眼，防止脉络膜裂的侧缘的并置，从而有助于缺损。我们结合联合收割机RNA-Seq、染色质缔合分析和LC-MS/MS蛋白质组学来确定MAB 21 L2的体内功能，并阐明MAB 21 L2依赖的基因调控网络和正常眼形成的蛋白质相互作用组。这些实验符合NIH和NEI的使命，因为它们与我们进一步理解早期眼发育和MAC谱系障碍有直接关系。此外，他们开发了一种斑马鱼突变模型作为体内翻译平台，用于确定人类突变的致病性。考虑到个性化医疗的前景，以及外显子组和全基因组测序的简便/低成本，编码蛋白质的基因中几乎不存在功能信息的突变将越来越多地被识别出来。在这个提议中建立的管道将作为一个有用的体内范例，用于阐明这些人类突变的分子基础，并快速确定它们编码的蛋白质的功能。