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Published on: August 15, 2019
Exome sequencing and functional studies in zebrafish identify WDR8 as the causative gene for isolated
M Madhangi1, Debanjan Dutta1, Sautan Show1
1Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Abstract:
Isolated Microspherophakia (MSP) is an autosomal recessive disorder characterized by a smaller than normal spherical lens. Till date, LTBP2 is the only gene shown to cause MSP. We used homozygosity mapping and whole-exome sequencing and identified a homozygous mutation, c.1148C > T (p.Pro383Leu), in the WDR8 (or WRAP73) gene in two Indian MSP families. In vitro experiments showed that the missense mutation renders the protein unstable. WDR8 is a centriolar protein that has important roles in centrosomal assembly, spindle pole formation and ciliogenesis. Co-immunoprecipitation experiments from HeLa cells indicated that the mutation interferes with the interaction of WDR8 with its binding partners. In zebrafish, both morpholino-mediated knockdown and CRISPR/Cas knockout of wdr8 resulted in decreased eye and lens size. The lack of wdr8 affected cell cycle progression in the retinal cells, causing a reduction in cell numbers in the retina and lens. The reduction in eye size and the cell cycle defects were rescued by exogenous expression of the human wild-type WDR8. However, the human mutant WDR8 (p.Pro383Leu) was unable to rescue the eye defects, indicating that the missense mutation abrogates WDR8 protein function. Thus, our zebrafish results suggested that WDR8 is the causative gene for MSP in these Indian families.
Insights
A mutation in the WDR8 gene causes isolated microspherophakia (MSP), a rare eye disorder. This study identified WDR8 as the causative gene, revealing its critical role in lens development and eye size.
Area of Science:
- Genetics
- Ophthalmology
- Cell Biology
Background:
- Isolated microspherophakia (MSP) is a rare autosomal recessive disorder affecting lens size.
- Mutations in the LTBP2 gene were previously the only known cause of MSP.
- The genetic basis for MSP in many families remained unidentified.
Purpose of the Study:
- To identify the causative gene for isolated microspherophakia in two Indian families.
- To investigate the functional impact of identified mutations on WDR8 protein function.
- To elucidate the role of WDR8 in eye development using a zebrafish model.
Main Methods:
- Homozygosity mapping and whole-exome sequencing were employed to identify genetic mutations.
- In vitro experiments (protein stability, co-immunoprecipitation) assessed the functional consequences of the mutation.
- Zebrafish models (morpholino knockdown, CRISPR/Cas9 knockout) were used to study gene function in vivo, including rescue experiments.
Main Results:
- A homozygous mutation (c.1148C>T, p.Pro383Leu) in the WDR8 gene was identified in both families.
- The WDR8 mutation led to protein instability and impaired interactions with binding partners.
- WDR8 deficiency in zebrafish caused reduced eye and lens size, retinal cell cycle defects, and decreased cell numbers.
Conclusions:
- The WDR8 gene is a novel causative gene for isolated microspherophakia.
- The identified missense mutation in WDR8 disrupts protein function, leading to developmental eye abnormalities.
- WDR8 plays a crucial role in centrosome function, cell cycle progression, and vertebrate eye development.
