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

Area of Science:

• Genetics
• Developmental Biology
• Ophthalmology

Background:

• Ocular congenital cranial dysinnervation disorders (oCCDDs) are a group of rare genetic conditions affecting eye movement.
• Genetic causes for many oCCDDs remain unknown, necessitating functional validation of candidate genes.
• Zebrafish models offer a powerful platform for rapid genetic screening and functional gene assessment.

Purpose of the Study:

• To functionally evaluate novel human sequence-derived candidate genes and variants implicated in unsolved oCCDDs.
• To prioritize candidate genes and variants identified through exome and genome sequencing in a human oCCDD cohort.
• To investigate the functional impact of variants of uncertain significance in transcription factor genes.

Main Methods:

• Utilized a G0 CRISPR/Cas9 knockout assay in zebrafish to screen 43 human and 57 zebrafish candidate genes.
• Generated F2 germline mutants for 17 prioritized genes for further analysis.
• Employed protein binding microarrays to assess the DNA binding capacity of human variants in transcription factors.

Main Results:

• Demonstrated the efficacy of the G0 zebrafish screen, with 70-90% recapitulating null-equivalent phenotypes for known oCCDD genes.
• Identified SEMA3F, OLIG2, and FRMD4B as three novel candidate genes contributing to cranial motor development.
• Confirmed reduced or abolished DNA binding for variants in PHOX2A, MAFB, and OLIG2 using protein binding microarrays.

Conclusions:

• Nominated SEMA3F, OLIG2, and FRMD4B as strong novel candidate genes for oCCDDs.
• Supported the pathogenicity of specific variants in PHOX2A, MAFB, and OLIG2 transcription factors.
• Validated the combined approach of zebrafish G0 screening, human sequence analysis, and protein binding assays for oCCDD gene/variant prioritization.