Genomic analysis of microphthalmia, anophthalmia and coloboma

小眼症、无眼症和缺损的基因组分析

• 批准号: 10746478
• 负责人: ANNE M. SLAVOTINEK
• 金额: $ 46.24万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10746478
• 关键词: Genomic; analysis; microphthalmia; anophthalmia; coloboma

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 therapies, and providing information to support 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 variants, in a minimum of 200 patients with MAC and their biological parents. We will prioritize patients with MAC who have had negative prior genetic testing, multiple affected family members, consanguineous parents and bilateral eye involvement to increase our changes of identifying novel variants and genes. This aim will enable us to investigate the full range of genomic variation in MAC and to determine the contribution of non-coding variation and structural cytogenetic variation to the genetic etiology of these birth defects. In our second Aim, we will use zebrafish with gene editing and CRISPR/Cas9 to investigate the effects of loss and gain of function for novel candidate genes for MAC and to determine the effects of specific sequence variants on eye development. We will comprehensively phenotype crispant and control larvae and perform RNA-Seq to study alterations in downstream gene expression. We will also study non-coding variation by performing bioinformatic studies, minigene assays, RT-PCR in patient cells as appropriate, and dual-color 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，促进未来疗法，并提供信息以支持研究 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）和体外 和体内功能研究，以促进我们对MAC基因组结构的理解。我们的第一个目标 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， 拷贝数变异和结构细胞遗传学变异，至少有200例MAC患者 亲生父母。我们将优先考虑先前基因检测负阴性的MAC患者，多个 受影响的家庭成员，亲戚父母和双边眼睛参与，以增加我们的变化 识别新颖的变体和基因。这个目标将使我们能够研究基因组的全部差异 MAC并确定非编码变化和结构细胞遗传学变异对 这些先天缺陷的遗传病因。在第二个目标中，我们将使用斑马鱼与基因编辑和 CRISPR/CAS9研究MAC的新型候选基因的损失和功能增长的影响 确定特定序列变体对眼睛发育的影响。我们将全面表型 清晰并控制幼虫并执行RNA-Seq以研究下游基因表达的改变。我们将 还通过进行生物信息学研究，微基因测定，患者细胞中的RT-PCR研究非编码变化 适当的，在斑马鱼中的双色记者转基因测定法。我们的结果将产生全面 有关可能导致MAC的基因组变异的数据，并将提供新基因的功能验证和 变体。该建议还将大大提高我们对眼睛发育的理解，此外 为未来的调查和临床医生之间的合作创造丰富的资源，发展 生物学家和眼科研究人员。