Gene Identification for Ocular Congenital Cranial Motor Neuron Disorders Using Human Sequencing, Zebrafish Screening, and Protein Binding Microarrays.

• DOI: 10.1167/iovs.66.3.62
• Publication Date: 2025-03-03
• Authors: Julie A. Jurgens; Paola M. Matos Ruiz; Jessica M. King; Emma E. Foster; Lindsay Berube; Wai‐Man Chan; Brenda J. Barry; Raehoon Jeong; Elisabeth Rothman; Mary C. Whitman; Sarah MacKinnon; Cristina Rivera-Q...; Brandon M. Pratt; Teresa Easterbrooks; Fiona M. Mensching; Silvio Alessandro...; Lynn Pais; Eleina England; Teresa de Berardinis; Adriano Magli; Feray Koc; Kazuhide Asakawa; Koichi Kawakami; Anne O’Donnell‐Luria; David G. Hunter; Caroline D. Robson; Martha L. Bulyk; Elizabeth C. Engle
• Keywords: Identification

To functionally evaluate novel human sequence-derived candidate genes and variants for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Through exome genome sequencing of a genetically oCCDD cohort, we previously reported the identification in many genes. Here, describe parallel study that prioritized subset these (43 genes, 57 zebrafish genes) using G0 CRISPR/Cas9-based knockout assay generated F2 germline mutants 17. We tested functionality uncertain significance known transcription factor-encoding through protein binding microarrays. first demonstrated feasibility screen by targeting phox2a mafba. Approximately 70% to 90% gene-targeted embryos recapitulated homozygous null-equivalent phenotypes. Using this approach, then identified three (SEMA3F, OLIG2, FRMD4B) with putative contributions motor development. In addition, microarrays reduced or abolished DNA factors PHOX2A (p.(Trp137Cys)), MAFB (p.(Glu223Lys)), OLIG2 (p.(Arg156Leu)). This nominates strong supports pathogenicity factor p.(Trp137Cys), p.(Glu223Lys), p.(Arg156Leu). Our findings support loss-of-function screening can be coupled sequence analysis aid prioritizing genes/variants.