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）：小眼症-无眼症-缺损（MAC）疾病是一系列异质性先天性眼部缺陷，可导致严重的眼睛结构畸形。 MAC 表型可以是分离的，也可以是多种综合征性疾病的组成部分。全球范围内报告的发病率差异很大，但无眼症和小眼症的大致范围为每 10,000 名活产儿中 2.13 例，而缺眼症为每 10,000 名活产儿 2.6 至 7.5 例。缺损也是 50 多种人类遗传性疾病的组成部分，通常与小眼症有关。尽管进行了大量的基因研究来确定 MAC 位点，但在极少数情况下发现了致病突变，而对于那些已发现的突变，人们对受影响的蛋白质如何促进正常眼睛发育知之甚少。最近，在人类 MAC 患者的 MAB21L2 基因中发现了几种不同的突变 [Rainger et al., 2014]。此前尚未发现 MAB21L2 与 MAC 谱系疾病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，并且突变如何影响 MAB21L2 活性尚不清楚。我们实验室之前的工作发现了一种斑马鱼 mab21l2 突变体，该突变体的眼部缺陷与人类 MAC 患者的眼部缺陷相似。该提案中的研究利用斑马鱼 mab21l2 突变体作为翻译模型系统，通过该系统我们可以识别人类 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。我们测试了这样的假设：MAB21L2 活性是视杯内正常增殖所必需的，并且 Mab21L2 缺陷会导致小眼球，从而阻止脉络膜裂侧缘的并置，从而导致缺损。我们结合 RNA-Seq、染色质关联测定和 LC-MS/MS 蛋白质组学来确定 MAB21L2 的体内功能，并阐明正常眼睛形成背后的 MAB21L2 依赖性基因调控网络和蛋白质相互作用组。这些实验符合 NIH 和 NEI 的使命，因为它们与进一步了解早期眼睛发育和 MAC 谱系疾病有直接关系。此外，他们开发了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，编码蛋白质的基因突变将越来越多地被识别，而这些蛋白质的功能信息很少。该提案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。