Genomic analysis of microphthalmia, anophthalmia and coloboma

摘要 小眼症、无眼症和缺陷症(MAC)是非常重要的出生缺陷，因为 视力严重减退的终生影响。Mac可由致病序列变异和拷贝数引起 影响转录因子和其他与眼睛发育有关的基因的变异。患有MAC的患者通常 通过微阵列、基因面板和外显子组测序进行基因测试，但基因病因学不是 为许多患者所确认。一份完整的MAC下基因组变异目录对于 优化患者护理，促进未来的治疗，并提供信息以支持对 眼睛缺陷的发育生物学。在这项提案中，我们将使用全基因组测序(WGS)和体外 以及体内功能研究，以促进我们对MAC基因组结构的理解。我们的首要目标是 涉及使用WGS来获得完整的基因组数据，包括编码和非编码序列变体， 至少200例MAC及其患者的拷贝数变异和结构细胞遗传学变异 亲生父母。我们将优先考虑先前基因检测为阴性的MAC患者，多次 受影响的家庭成员、血缘关系密切的父母和双眼参与增加我们的变化 识别新的变种和基因。这一目标将使我们能够研究人类基因组的全部变异。 并确定非编码变异和结构细胞遗传学变异对 这些出生缺陷的遗传病因学。在我们的第二个目标中，我们将使用斑马鱼进行基因编辑和 CRISPR/Cas9研究MAC和TO新候选基因功能丧失和获得的影响 确定特定序列变异对眼睛发育的影响。我们将全面表型 并对幼虫进行RNA-Seq以研究下游基因表达的变化。我们会 还通过在患者细胞中进行生物信息学研究、微型基因分析、RT-PCR来研究非编码变异 适当的，和双色报告斑马鱼转基因检测。我们的结果将产生全面的 关于可导致MAC的基因组变异的数据，并将提供新基因和 变种。这项建议将极大地提高我们对眼睛发育的理解，除了 为未来的研究和临床医生之间的合作产生丰富的资源，开发 生物学家和眼科研究人员。

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.