Genomic analysis of microphthalmia, anophthalmia and coloboma

小眼症、无眼症和缺损的基因组分析

基本信息

批准号：
10280960
负责人：
ANNE M. SLAVOTINEK
金额：
$ 47.67万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-30 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10280960
关键词：
Affect Animal Model Anophthalmos Architecture Bilateral Biological Biological Assay Biological Models Biology CRISPR/Cas technology Candidate Disease Gene Catalogs Categories Cells Clinical Management Clustered Regularly Interspaced Short Palindromic Repeats Code Collaborations Coloboma Color Congenital Abnormality Copy Number Polymorphism Cytogenetics Data Data Set Defect Development Developmental Biology Diagnosis Dideoxy Chain Termination DNA Sequencing Dominant-Negative Mutation Evaluation Exons Eye Eye Development Eye diseases Failure Family member Fissural Future Gene Expression Gene Transfer Techniques Genes Genetic Genetic Heterogeneity Genetic Predisposition to Disease Genomics Genotype Immunohistochemistry Impairment In Vitro Injections Investigation Karyotype Knowledge Larva Medical Genetics Methodology Microphthalmos Modeling Morphogenesis Morphology Nucleic Acid Regulatory Sequences Nucleotides Oligonucleotides Optics Parents Pathogenesis Pathogenicity Patient Care Patients Phenotype Prognosis Quality of life RNA RNA Splicing Regulatory Element Reporter Research Personnel Research Support Resources Reverse Transcriptase Polymerase Chain Reaction Risk Role Site Spliced Genes Structure Testing Therapeutic Trials Time Untranslated RNA Validation Variant Vision Visual impairment Work Zebrafish causal variant exome sequencing experience gain of function genetic testing genetic variant genome sequencing genomic data genomic variation improved in silico in vivo insertion/deletion mutation loss of function malformation medical complication mutant novel programs segregation spatiotemporal therapy development transcription factor transcriptome sequencing whole genome

项目摘要

SUMMARY Microphthalmia, anophthalmia and coloboma (MAC) are highly important birth defects because of the lifelong effects of severely reduced vision. MAC can be caused by pathogenic sequence variants and copy number variants affecting transcription factors and other genes involved in eye development. Patients with MAC typically undergo genetic testing with microarrays, gene panels and exome sequencing, but a genetic etiology is not identified for many patients. A complete catalogue of the genomic variation underlying MAC is critical for optimizing patient care, facilitating future therapeutic trials and to provide novel information supporting research into the developmental biology of eye defects. In this proposal, we will use whole genome sequencing (WGS) and in vitro and in vivo functional studies to advance our understanding of the genomic architecture of MAC. Our first Aim involves the use of WGS to obtain complete genomic data, including coding and non-coding sequence variants, copy number variants and structural cytogenetic variation, in a minimum of 200 patients with MAC. We will prioritize patients with MAC who have multiple affected family members, consanguineous parents, bilateral eye involvement, and negative prior genetic testing. This aim will enable us to investigate the full range of genomic variation in MAC and to determine whether non-coding variation and structural cytogenetic variation contribute to the genetic etiology of these birth defects. In our second Aim, we will use zebrafish with CRISPR to investigate the effects of loss and gain of function for novel candidate genes for MAC and to determine the effects of specific, coding sequence variants on eye development. We will comprehensively phenotype control and mutant larvae and perform RNA-Seq to study alterations in downstream gene expression. We will also study non-coding variation by performing RT-PCR in patient cells, minigene assays and CRISPR/Cas9 with dual reporter transgenesis assays in zebrafish. Our results will generate comprehensive data on the genomic variation that can cause MAC and will provide functional validation of novel genes and variants. This proposal will substantially improve our understanding of eye development, in addition to generating rich resources for future investigations and collaborations between clinicians, developmental biologists and eye researchers.

概括微观噬菌体，性心疗和造口症（MAC）是非常重要的先天缺陷视力严重降低的终身影响。 MAC可能是由致病序列变体和拷贝数引起的影响转录因子和其他基因参与眼睛发育的变体。 MAC患者通常通过微阵列，基因面板和外显子组测序进行基因检测，但遗传病因不是为许多患者确定。 MAC基因组变异的完整目录对于优化患者护理，促进未来的治疗试验并提供新的信息支持研究眼缺陷的发育生物学。在此提案中，我们将使用整个基因组测序（WGS）以及体外和体内功能研究，以促进我们对苹果。我们的第一个目的涉及使用WGS获得完整的基因组数据，包括编码和非编码序列变体，拷贝数变体和结构细胞遗传学变异，至少有200例苹果。我们将优先考虑具有多个受影响家庭成员的MAC患者，亲密的父母，双侧眼睛受累和先前的基因检测。这个目标将使我们能够调查整个范围 MAC中基因组变异并确定非编码变化和结构细胞遗传学变异是否有助于这些先天缺陷的遗传病因。在我们的第二个目标中，我们将使用斑马鱼和crispr 研究新型候选基因的MAC的损失和功能增长的影响，并确定效果特定的编码序列变体在眼睛发育中。我们将全面地表型控制和突变幼虫并执行RNA-Seq研究下游基因表达的改变。我们还将学习通过在患者细胞，小型分析和CRISPR/CAS9中执行RT-PCR的非编码变化斑马鱼中的记者转基因测定法。我们的结果将产生有关基因组变异的全面数据这可能会导致MAC，并将提供新型基因和变体的功能验证。该提议将除了为未来创造丰富的资源外，还要大大提高我们对眼睛发育的理解临床医生，发育生物学家和眼科研究人员之间的调查和合作。