Gene identification for ocular congenital cranial motor neuron disorders using human sequencing, zebrafish screening, and protein binding microarrays.

PURPOSE

To functionally evaluate novel human sequence-derived candidate genes and variants for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs).

METHODS

Through exome and genome sequencing of a genetically unsolved human oCCDD cohort, we previously identified variants in 80 strong candidate genes. Here, we further prioritized a subset of these (43 human genes, 57 zebrafish genes) using a G0 CRISPR/Cas9-based knockout assay in zebrafish and generated F2 germline mutants for seventeen. We tested the functionality of variants of uncertain significance in known and novel candidate transcription factor-encoding genes through protein binding microarrays.

RESULTS

We first demonstrated the feasibility of the G0 screen by targeting known oCCDD genes and . 70-90% of gene-targeted G0 zebrafish embryos recapitulated germline homozygous null-equivalent phenotypes. Using this approach, we then identified three novel candidate oCCDD genes (, and ) with putative contributions to human and zebrafish cranial motor development. In addition, protein binding microarrays demonstrated reduced or abolished DNA binding of human variants of uncertain significance in known and novel sequence-derived transcription factors (p.(Trp137Cys)), (p.(Glu223Lys)), and (p.(Arg156Leu)).

CONCLUSIONS

This study nominates three strong novel candidate oCCDD genes (, and ) and supports the functionality and putative pathogenicity of transcription factor candidate variants p.(Trp137Cys), p.(Glu223Lys), and p.(Arg156Leu). Our findings support that G0 loss-of-function screening in zebrafish can be coupled with human sequence analysis and protein binding microarrays to aid in prioritizing oCCDD candidate genes/variants.