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bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.12.612713
preprint
1
Article
Gene identification for ocular congenital cranial motor neuron disorders using human sequencing, zebrafish screening, and protein binding microarrays
Jurgens Julie A. http://orcid.org/0000-0003-4822-9384

Ruiz Paola M. Matos
King Jessica
Foster Emma E.
Berube Lindsay
Chan Wai-Man
Barry Brenda J.
Jeong Raehoon
Rothman Elisabeth
Whitman Mary C.
MacKinnon Sarah
Rivera-Quiles Cristina
Pratt Brandon M.
Easterbrooks Teresa
Mensching Fiona M.
Di Gioia Silvio Alessandro
Pais Lynn
England Eleina M.
de Berardinis Teresa
Magli Adriano
Koc Feray
Asakawa Kazuhide http://orcid.org/0000-0003-0628-1176

Kawakami Koichi
O’Donnell-Luria Anne http://orcid.org/0000-0001-6418-9592

Hunter David G. http://orcid.org/0000-0002-4587-4794

Robson Caroline D.
Bulyk Martha L.
Engle Elizabeth C.
15 9 2024
2024.09.12.612713https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
http://biorxiv.org/lookup/doi/10.1101/2024.09.12.612713
nihpp-2024.09.12.612713.pdf
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 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 phox2a and mafba . 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 ( 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.
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