Abstract
Vacuolar protein sorting-associated protein 52 (VPS52) is a crucial subunit of both the Golgi- associated retrograde protein (GARP) and the endosome-associated recycling protein (EARP) complexes. These heterotetramers play a vital role in the intracellular vesicle trafficking network. Recently, the Bicknell lab discovered biallelic variants in the VPS52 gene of a New Zealand family, with an additional seven families since added to the cohort. These patients have either homozygous recessive missense variants or compound heterozygous variants - a missense variant combined with a truncating variant.
This project aimed to investigate the pathogenicity of the identified missense and NZ biallelic variants for their impact on cellular function. Previous reports on the pathogenicity of variants in the other subunits support VPS52 as a probable disease gene requiring further investigation. Therefore, these novel variants were hypothesised to negatively affect cell function. To explore this hypothesis, plasmid-transfected reporter cells and patient-derived fibroblasts were utilised to investigate missense and biallelic variants in the VPS52 gene, respectively.
The coding sequence of WT VPS52 was inserted into a plasmid vector, which was then subjected to site-directed mutagenesis to introduce targeted missense variants. These plasmids were transfected into HEK293 cells, where protein levels were measured through western blotting. The VPS52 mRNA and protein levels were also measured in the patient's primary fibroblasts using RT-qPCR and western blot, respectively. Finally, Lysotracker stain was used to compare lysosome states in the patient and control cells.
The results showed that cells expressing VPS52 missense variants Arg479Trp, Cys446Tyr, Glu396Lys, and Arg301Cys had reduced VPS52 protein levels. Patient fibroblasts with biallelic VPS52 variants also displayed decreased mRNA and protein levels. This suggests that the missense variants may induce structural alterations in the protein's conformation, leading to reduced protein expression due to instability. However, further investigation of this theory is required.
Patient and control fibroblasts were stained with Lysotracker and imaged to visualise the cellular lysosome quantity and appearance. Results were highly variable, revealing no consistent pattern between cell types and confirming the need for further research.
These findings offer preliminary evidence for classifying the VPS52 variants on their pathogenicity in accordance with ACMG guidelines. This classification would provide the families with confirmation regarding the aetiology of their disease. Conclusions drawn from this study will establish foundational knowledge for future research aimed at developing diagnostic tools, treatment strategies, and a deeper comprehension of the GARP/EARP subunits for their broader roles in brain development.