PUBLICATION
Gene Identification for Ocular Congenital Cranial Motor Neuron Disorders Using Human Sequencing, Zebrafish Screening, and Protein Binding Microarrays
- Authors
- Jurgens, J.A., Matos Ruiz, P.M., King, J., Foster, E.E., Berube, L., Chan, W.M., Barry, B.J., Jeong, R., Rothman, E., Whitman, M.C., MacKinnon, S., Rivera-Quiles, C., Pratt, B.M., Easterbrooks, T., Mensching, F.M., Di Gioia, S.A., Pais, L., England, E.M., de Berardinis, T., Magli, A., Koc, F., Asakawa, K., Kawakami, K., O'Donnell-Luria, A., Hunter, D.G., Robson, C.D., Bulyk, M.L., Engle, E.C.
- ID
- ZDB-PUB-250401-24
- Date
- 2025
- Source
- Investigative ophthalmology & visual science 66: 6262 (Journal)
- Registered Authors
- Asakawa, Kazuhide, Chan, Wai-Man, Di Gioia, Silvio Alessandro, Easterbrooks, Teresa, Jurgens, Julie, Kawakami, Koichi
- Keywords
- none
- MeSH Terms
-
- Disease Models, Animal
- Exome Sequencing
- Zebrafish*
- CRISPR-Cas Systems
- Animals
- Homeodomain Proteins/genetics
- Homeodomain Proteins/metabolism
- Humans
- Transcription Factors/genetics
- Transcription Factors/metabolism
- Zebrafish Proteins/genetics
- Zebrafish Proteins/metabolism
- PubMed
- 40162949 Full text @ Invest. Ophthalmol. Vis. Sci.
Citation
Jurgens, J.A., Matos Ruiz, P.M., King, J., Foster, E.E., Berube, L., Chan, W.M., Barry, B.J., Jeong, R., Rothman, E., Whitman, M.C., MacKinnon, S., Rivera-Quiles, C., Pratt, B.M., Easterbrooks, T., Mensching, F.M., Di Gioia, S.A., Pais, L., England, E.M., de Berardinis, T., Magli, A., Koc, F., Asakawa, K., Kawakami, K., O'Donnell-Luria, A., Hunter, D.G., Robson, C.D., Bulyk, M.L., Engle, E.C. (2025) Gene Identification for Ocular Congenital Cranial Motor Neuron Disorders Using Human Sequencing, Zebrafish Screening, and Protein Binding Microarrays. Investigative ophthalmology & visual science. 66:6262.
Abstract
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 reported the identification of variants in many candidate genes. Here, we describe a parallel study that prioritized a subset of these genes (43 human genes, 57 zebrafish genes) using a G0 CRISPR/Cas9-based knockout assay in zebrafish and generated F2 germline mutants for 17. 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 phox2a and mafba. Approximately 70% to 90% of gene-targeted G0 zebrafish embryos recapitulated germline homozygous null-equivalent phenotypes. Using this approach, we then identified three novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) 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 PHOX2A (p.(Trp137Cys)), MAFB (p.(Glu223Lys)), and OLIG2 (p.(Arg156Leu)).
Conclusions This study nominates three strong novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) and supports the functionality and putative pathogenicity of transcription factor candidate variants PHOX2A p.(Trp137Cys), MAFB p.(Glu223Lys), and OLIG2 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping