Developing a molecular assay to predict hemangioblastoma phenotype severity in VHL disease

Abstract

Purpose : Von Hippel-Lindau (VHL) disease is an inherited cancer predisposition syndrome that commonly causes retinal hemangioblastomas. Genetically related patients carrying the same mutation can exhibit vast differences in ocular phenotype severity. Currently, no method exists to determine an individual’s prognosis. We used patient-derived fibroblasts to test the hypothesis that a simple molecular assay can provide prognostic information.

Methods : We studied fibroblasts from four genetically related female patients with molecularly confirmed VHL disease (heterozygous for Tyr98His) and age-matched non-disease controls. Notably, two of the VHL patients have severe ocular phenotypes while the other two have mild disease. We performed siRNA-mediated knockdown of VHL to mimic loss of heterozygosity, which is essential to inducing VHL disease phenotypes. We also exposed these treated cells to hypoxia (1%), which we hypothesized would further enhance any differences observed between patients since VHL regulates HIF signaling. We performed qPCR to compare differences in expression of the VHL gene and its downstream targets (HIF1a and HIF2a).

Results : In the control lines, all three genes (VHL, HIF1a and HIF2a) were stably expressed across treatment conditions and oxygen tensions. As expected, VHL was decreased following siRNA treatment (Control: mean 72% decrease in gene expression in both oxygen tensions; disease at normoxia: 56% - 74% decrease; disease at hypoxia: 28% - 67% decrease). Except for one patient with severe disease, all disease cell lines displayed increased VHL gene expression relative to the control at baseline conditions. Interestingly, the cells that did not follow this trend were obtained from a patient that was over 3 decades older at biopsy acquisition than the other individual with severe disease. In this cell line VHL gene expression was similar to controls in all treatment conditions and oxygen tensions. Furthermore, these cells showed the lowest levels of HIF1a and HIF2a in hypoxic conditions relative to all other disease cell lines.

Conclusions : In conclusion, these findings suggest that mRNA expression in patient derived dermal fibroblasts does not predict disease severity regardless of oxygen condition or loss of heterozygosity.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.