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The Phenotypic Effects of Loss-of-Function Variants in WDR45

Laboratory Genetics and Genomics
  • Primary Categories:
    • Laboratory Genetics
  • Secondary Categories:
    • Laboratory Genetics
Introduction:
WDR45 (WIPI4), a member of the WIPI family (WIPI1-4), is crucial for autophagosome biogenesis as part of the PROPPIN family. While the roles of WIPI1 and WIPI2 are well-characterized, the functions of WDR45 and WDR45B (WIPI3) in autophagy remain unclear. Mutations in WDR45 are associated with beta-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation (NBIA). WDR45 deficiency has been linked to selective ferritinophagy defects and iron accumulation, but the specific mechanisms remain elusive. In this study, we identified a de novo non-canonical splice site variant, c.344+5G>T, in the WDR45 gene of a female patient through whole exome sequencing and conducted a detailed analysis of its associated phenotypic effects.

Methods:
To investigate the pathogenicity of the c.344+5G>T variant, we generated a patient-derived induced pluripotent stem cell (iPSC) line (FDHPIi001-A) and engineered WDR45 gene knockin/knockout SH-SY5Y cells. RT-PCR was used to assess the impact of the variant on RNA splicing, revealing an insertion of 28 bp in intron 6. This defect was further analyzed by qPCR to measure mutant (mutWDR45) and wild-type (wtWDR45) transcript levels. We also overexpressed Flag-tagged wild-type and mutant WDR45 in 293T cells to evaluate functional consequences. A series of assays were performed to assess autophagy, ferritinophagy, iron homeostasis, and ROS levels to elucidate the cellular phenotypes associated with WDR45 deficiency.

Results:
Our findings demonstrated that the c.344+5G>T variant disrupted RNA splicing, leading to a 28 bp insertion and subsequent nonsense-mediated decay (NMD) of the aberrant transcript. In the SH-SY5Y cell model, WDR45 mutation impaired ferritinophagy, resulting in iron overload, elevated ROS levels, and insufficient intracellular bioavailable iron. This imbalance triggered compensatory activation of iron uptake pathways, exacerbating neurotoxic iron overload. LC3B analysis showed that WDR45 loss did not completely block autophagy but caused defects in selective autophagic processes. Treatment with Baf A1 further revealed autophagy pathway disruptions, confirming that WDR45 is essential for selective autophagy rather than general autophagic flux.This study highlights the pathogenicity of a non-canonical splice site variant in WDR45, revealing its crucial role in neuronal iron homeostasis through ferritinophagy. Loss of WDR45 leads to selective autophagy defects, iron dysregulation, and oxidative stress, offering novel insights into the molecular mechanisms underlying BPAN. These findings underscore the importance of WDR45 in maintaining neuronal health and provide a foundation for further research into targeted therapeutic strategies for BPAN and related disorders.

Conclusion:
This study highlights the pathogenicity of a non-canonical splice site variant in WDR45, revealing its crucial role in neuronal iron homeostasis through ferritinophagy. Loss of WDR45 leads to selective autophagy defects, iron dysregulation, and oxidative stress, offering novel insights into the molecular mechanisms underlying BPAN. These findings underscore the importance of WDR45 in maintaining neuronal health and provide a foundation for further research into targeted therapeutic strategies for BPAN and related disorders.

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