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.
