Multi-organism platform for functional assessment of human birth defect associated genomic variants

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Birth defects, which occur in 3 - 5% of US-born infants per year, are a leading cause of childhood mortality and repeated hospitalization and are a large burden to families and society. Birth defects typically result from rare genetic changes, but determining which gene-variant causes a phenotype and disease remains challenging, despite significant advances in next-generation sequencing and analysis. Whereas ~4,000 human genes have been linked to monogenic, rare diseases, it has been estimated that 6,000-13,000 additional rare disease genes remain to be identified, many of which are likely causal for birth defects; this underscores a need for effective strategies to assess the functional effects of associated variants. In this application, we propose a multi-organism approach to connect patient presentation with genes not previously associated with disease (genes of uncertain significance, GUS). Focusing on intellectual and developmental disorders and structural birth defects, we will first screen patient birth-defect associated GUS leveraging experimental tractability of C. elegans, Drosophila, or zebrafish, followed by assessment of patient- related phenotypes in vertebrate organisms (zebrafish, mouse, or established human embryonic stem cell lines (hESCs)) for a subset of these genes. In Aim 1, bioinformatic analysis of family based clinical exome sequence will identify high probability candidate gene-variants that may be causal for the patient’s symptom(s) for further study. In Aim 2, nominated candidate gene-variants will be screened in worm, fly or fish to obtain in vivo functional data in support of variant causality. Functional information will help determine whether the gene-variant is damaging, if the gene is required in a specific tissue, and whether the observed genetic mechanism (e.g., hypomorph, dominant negative, etc.) is consistent with patient genetics. Additionally, these experiments may illuminate the molecular or cell biological mechanism disrupted by the gene-variant (e.g., disruption of cytoskeleton, etc.). We will take advantage of the strengths of the different model organisms—CRISPR editing for C. elegans, tissue-specific RNAi in Drosophila, and mRNA and CRISPR embryo injections for zebrafish—in the genetic screening approach. A total of 84 candidate birth defect GUS will be screened in Aim 2 over the grant period. Often, the phenotypic effects of the corresponding disruption of the orthologous gene in worm or fly are not obviously related to the phenotype observed in humans. Therefore, in Aim 3, for a subset of genes (23) identified from the screen as likely disease-causing in Aim 2, we will examine phenotypes in zebrafish, mouse, or hESC systems, to advance our understanding of disease phenotype and progression that is not possible in simple model organisms or with the patient. These studies will leverage our experience in disease gene modeling and our work with clinical collaborators to understand the phenotypic, genetic, molecular, and cell biological basis of each patient’s disease. This innovative multi-organism experimental platform will significantly accelerate identification of birth defect-causing genes and will open avenues to diagnosis, prevention, and therapies.