Utilization of Advanced Technologies for the Understanding of Human Structural Birth Defects

PROJECT SUMMARY The unifying theme of this proposal is the aim to use state-of-the-art technologies to investigate the basic biology of mammalian organ development and human structural birth defects. Our approach is wide-ranging, and aims to demonstrate how utilization of powerful technologies can inform many disorders. Importantly, this proposal marries a number of strengths of investigators at Seattle Children’s Research Institute and the University of Washington Department of Genome Sciences; specifically, expertise in the diagnosis and understanding of human congenital malformation syndromes and mammalian developmental biology, and the application of powerful new techniques for biological investigation. In Project 1, we propose to use single-cell RNA sequencing (sci-RNA-seq) technology to characterize mid- gestation embryos of mice carrying mutations relevant to human structural birth defects. Essentially, we are proposing to utilize sci-RNA-seq as a phenotype, with which one can annotate changes in expression and cell- type representation during abnormal organogenesis. Ideally, these profiles will be comparable to each other, and can potentially provide insight into fundamental biological pathways that are perturbed when developmentally important genes are lost. In Project 2, we will leverage recent advances in 3D imaging, computer vision and machine-learning to make the morphological characterization of mouse mutants more accurate, quantitative, reproducible and accessible. Progeny from the same lines studied in Project 1 will be harvested at E15.5 and imaged using microCT scanning. We will then employ several different data analysis techniques to identify differences in the tissue volume and shapes in the mutant mice compared to synthetic image constructed from a pool of ‘normative’ samples. The goal of Project 3 is to use novel technologies in prospective cohorts of children with structural birth defects to identify genetic variation not ascertained by current methods. These “hidden” variants include structural rearrangements, as well as DNA mutations that arise post-zygotically and are not present in blood-derived DNA. We will use long-read based DNA and RNA sequencing methods, or deep short-read based DNA sequencing of multiple, non-blood derived tissues, on patients with structural birth defects whose clinical workup has been non-diagnostic.