Functional analysis of MAB21L2 mutations in MAC spectrum disorders

MAC 谱系疾病中 MAB21L2 突变的功能分析

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：摩根氏菌 - 恐怖分子 - 核瘤（MAC）疾病是先天性眼缺陷的异质谱，导致眼睛严重的结构性畸形。 MAC表型可以分离，也可以是多种综合症的组成部分。据报道，全球范围内的发明差异很大，但近10,000个生物症和微观恐怖分子的近似范围为每10,000个活产，山脉的射发射范围为每10,000次，至2.6至7.5 / 10,000。 colobomata也是50多种人遗传疾病的组成部分，它们通常与微观恋爱相关。尽管大量的遗传研究以鉴定MAC位置，但在很少的情况下已经确定了真正的突变，并且对于那些对受影响蛋白如何偏爱正常眼睛发育的人几乎不了解。最近，在人类MAC患者的MAB21L2基因中发现了几种不同的突变[Rainger等，2014]。 MAB21L2以前尚未与MAC谱系障碍或任何人类先天性疾病有关。 MAB21L2编码一种未知生化功能的蛋白质，未知突变如何影响MAB21L2活性。我们实验室中的先前工作确定了斑马鱼MAB21L2突变体，突变体中的眼部缺陷类似于人类MAC患者中的缺陷。该提案中的研究利用斑马鱼MAB21L2突变体作为翻译模型系统，通过该模型，我们可以鉴定人MAB21L2突变的分子基础，并确定正常眼发育过程中MAB21L2的细胞功能。我们测试了视力杯内正常增殖需要MAB21L2活性的假设，并且MAB21L2缺陷导致微观摩托感染，从而阻止了脉络膜裂缝的侧向边缘的占用，从而导致了果酱。我们结合了RNA-SEQ，染色质关联测定和LC-MS/MS蛋白质组学，以确定MAB21L2的体内功能，并阐明MAB21L2依赖性基因调节网络和蛋白质相互作用组的基础正常眼睛形成。这些实验符合NIH和NEI的使命，因为它们具有直接的意义，可以进一步了解我们对早期眼睛发育和MAC谱系障碍的理解。此外，他们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。鉴于个性化医学的前景，以及外显子和全基因组测序的易于/低成本，将增加编码很少有功能信息的蛋白质的突变。在本提案中建立的管道将是阐明这些人类突变的分子基础的体内范式中有用的，并迅速确定它们编码的蛋白质的功能。