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，促进未来的治疗试验，并提供新的信息支持 研究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)和体外和体内功能研究，以促进我们对基因组结构的理解 麦克。我们的第一个目标是使用WGS来获得完整的基因组数据，包括编码和非编码 至少200例患者的序列变异、拷贝数变异和结构细胞遗传学变异 麦克。我们将优先考虑有多个受影响家庭成员、近亲父母、 双眼受累，且先前基因检测呈阴性。这一目标将使我们能够全面调查 并确定非编码变异和结构细胞遗传学变异 有助于这些出生缺陷的遗传病因学。在我们的第二个目标中，我们将使用斑马鱼和CRISPR来 研究新的MAC候选基因的功能丧失和功能获得的影响并确定其影响 对眼睛发育的特定编码序列变体的研究。我们将全面控制表型和 并进行RNA-Seq以研究下游基因表达的变化。我们还将研究 在患者细胞中进行RT-PCR、微基因分析和CRISPR/Cas9的非编码变异 记者在斑马鱼中进行转基因检测。我们的结果将产生关于基因组变异的全面数据 这可能导致MAC，并将提供新基因和变种的功能验证。这项提议将 除了为未来提供丰富的资源外，还大大提高了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。

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.