3D Genome Architecture in Congenital Disease

在过去的十年中，基因组学的一个重大科学突破是发现基因组在细胞核的三维空间中折叠，这与基因调控直接相关。在这个提案中，我建议通过将研究3D基因组架构与全面的基因组分析相结合，将疾病导向的基因组分析提升到另一个层次，从而解决我们目前对罕见疾病的理解中的一个主要问题。我推测，许多迄今未确诊的病例实际上是由异常基因调控引起的，由于突变改变了基因组结构，从而改变了调节增强子-启动子的相互作用。我建议检测和研究这种新的突变机制基因组范围内使用染色体构象捕获（HiC）从先天性肢体畸形患者的细胞。HiC将用于鉴定可能影响发育基因表达的异常增强子启动子接触。这些数据将与使用短（Illumina）和长（PacBio）读取技术的全基因组分析进行比较，以识别相应的变化，包括序列和结构的变体以及可能改变3D基因组结构的重复序列。将使用适应的Crispr/Cas9方案在小鼠中体内或在由这些细胞产生的成纤维细胞或iPSC中体外研究潜在致病性的变化。

One of the major scientific breakthoughs in genomics of the last decade has been the discovery that the genome folds in the threedimensional space in the nucleus and that this is directly related to gene regulation. In this proposal I propose to take disease oriented genome analysis to another level by combining the study 3D genome architecture with comprehensive genome analysis, thereby addressing one of the major problems in our current understanding of rare diseases. I hypothesize that many so far undiagnosed cases are in fact caused by abnormal gene regulation due to mutations that alter genomic architecture thereby changing regulatory enhancer-promoter interactions. I propose to detect and investigate such novel mutational mechanisms genome wide using a chromosome conformation capture (HiC) in cells from patients with congenital limb malformations. HiC will be used to identify aberrant enhancerpromoter contacts that might influence developmental gene expression. These data will be compared to whole genome analysis with short (Illumina) and long (PacBio) read technology to identify corresponding changes including variants of sequence and structure as well as repetitive sequences that might change 3D genome architecture. Changes with potential pathogenicity will be investigated in vivo in mice using an adapted Crispr/Cas9 protocol or in vitro in fibroblasts or iPSCs generated from these cells.