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: 8951973
• 负责人: Jeffrey Gross
• 金额: $ 19.25万
• 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 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8951973
• 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; 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; public health relevance; research study; scaffold; tandem mass spectrometry; transcriptome sequencing

 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(由申请人提供)：小眼球-无眼症-眼球缺乏症(MAC)是一种不同类型的先天性眼睛缺陷，会导致严重的眼睛结构畸形。Mac表型可以是孤立的，也可以是各种综合症疾病的组成部分。报告的发病率在全球范围内差异很大，但大致范围是每10,000名活产儿中有2.13人患有无眼症和小眼球症，到每10,000名活产儿中有2.6至7.5人患有错构症。蛇形虫也是50多种人类遗传性疾病的一个组成部分，在这些疾病中，它们经常与小眼球有关。尽管有大量的基因研究来确定MAC基因座，但在极少数情况下发现了致病突变，对于那些已经发生突变的人来说，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，在人类MAC患者中发现了MAB21L2基因的几种不同突变[Rainger等人，2014]。MAB21L2以前没有被认为与MAC谱系障碍或任何人类先天性疾病有关。MAB21L2编码一种生化功能未知的蛋白质，目前尚不清楚这些突变如何影响MAB21L2的活性。我们实验室以前的工作发现了一个斑马鱼mab21l2突变体，该突变体的眼睛缺陷与人类MAC患者的眼睛缺陷相似。这项研究利用斑马鱼mab21L2突变体作为翻译模型系统，通过它我们可以确定人类MAB21L2突变的分子基础，并确定MAB21L2在正常眼睛发育过程中的细胞功能。我们验证了这样的假设，即MAB21L2活性是视杯内正常增殖所必需的，并且Mab21L2缺乏会导致小眼球，从而阻止脉络膜裂外侧边缘的对接，从而导致错构症。我们结合RNA-Seq、染色质结合分析和LC-MS/MS蛋白质组学来确定MAB21L2的体内功能，并阐明MAB21L2依赖的基因调控网络和正常眼睛形成的蛋白质相互作用组。这些实验符合NIH和NEI的使命，因为它们与加深我们对早期眼睛发育和MAC光谱障碍的理解有直接关系。此外，他们还开发了斑马鱼突变模型，作为确定人类突变致病性的体内翻译平台。鉴于个性化医疗的前景，以及外显子组和全基因组测序的容易/低成本，编码蛋白质的基因突变将越来越多地被识别出来，这些基因的功能信息很少。这一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。