July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Mutations in proteasome 26S subunit, non-ATPase 5 gene (psmd5) cause ocular coloboma and vertebral defects
Author Affiliations & Notes
  • VIJAY Kumar KALASKAR
    Pediatric, Developmental & Genetic Ophthalmology Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Natalia Diaz Torres
    University of Puerto Rico School of Medicine, Puerto Rico, United States
  • Aman George
    Pediatric, Developmental & Genetic Ophthalmology Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Tiziana Cogliati
    Pediatric, Developmental & Genetic Ophthalmology Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Brian Patrick Brooks
    Pediatric, Developmental & Genetic Ophthalmology Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   VIJAY KALASKAR, None; Natalia Diaz Torres, None; Aman George, None; Tiziana Cogliati, None; Brian Brooks, None
  • Footnotes
    Support  Intramural Research Program of the National Eye Institute, National Institutes of Health
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5431. doi:https://doi.org/
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      VIJAY Kumar KALASKAR, Natalia Diaz Torres, Aman George, Tiziana Cogliati, Brian Patrick Brooks; Mutations in proteasome 26S subunit, non-ATPase 5 gene (psmd5) cause ocular coloboma and vertebral defects. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5431. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : psmd5 encodes a non-ATPase subunit of the 19S regulator base of 26S proteasome complex and functions as a chaperone protein during 26S proteasomal assembly. Mutations in genes encoding the non-ATPase subunits of the 19S regulator base of 26S proteasome complex have been associated with ophthalmological and vertebral defects in humans. The purpose of this study was to evaluate the role of psmd5 during optic fissure closure and vertebral development in zebrafish.

Methods : Zebrafish psmd5 CRISPR mutants were created by co-injection of Cas9 protein with sgRNAs targeting specific regions of psmd5 into 1-2 cell stage ABTL zebrafish embryos. Sanger sequencing was used to confirm the mutations induced in the psmd5 gene. qRT-PCR, in situ hybridization (ISH) and H&E staining were used for morphological and expression analysis. Cartilage and bone defects were determined by Alcian blue and Alizarin red staining, respectively. CRISPR induced phenotypes were verified by injecting two different morpholinos targeting psmd5.

Results : ISH revealed maternal expression of psmd5 with continued expression during the early stages of the developing zebrafish embryo. At 26 hours post fertilization, expression was observed in the entire eye and the brain with less intense expression in other parts of the body. CRISPR targeted disruption of psmd5 gene resulted in different mutations including, single base pair (bp) changes and indels carrying 23 or 25 bp deletions. A significant reduction up to 50 - 60 % of psmd5 expression was detected by qRT-PCR in the mutant embryos. F1 embryos obtained by intercrossing F0 mutant fish showed ocular coloboma and vertebral defects. Coloboma was observed in the ventral retina/retinal pigment epithelium (RPE) and in the optic stalk. Vertebral defects including bone fusions, multiple transverse processes on vertebral bodies, and missing vertebrae were detected in psmd5 mutants. No coloboma nor vertebral defects were observed in control ABTL zebrafish adults. Preliminary experiments with two different splice blocking morpholinos targeting the 2nd and 3rd exons of the psmd5 gene showed dose-dependent severity of the coloboma phenotype along with other body defects in zebrafish embryos.

Conclusions : Mutations in psmd5 gene result in ocular coloboma and vertebral defects in zebrafish, suggesting an important role for psmd5 during optic fissure closure and vertebral development.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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