BAP1 UCH ‐domain enzymatic activity

Purpose: Pathogenic variants in BRCA‐associated protein 1 ( BAP1 ) cause BAP1 tumour predisposition syndrome (BAP1‐TPDS) with an elevated risk of several cancers including uveal melanoma (UM). BAP1 is a deubiquitinating enzyme that needs to retain its enzymatic activity and be able to locate in the nucleus to function as a tumour suppressor. BAP1 loss of function variants are easily interpreted as pathogenic, but many missense variants remain as of unknown significance (VUS). We studied the effect of UCH domain missense variants on the enzymatic activity of BAP1. Methods: We selected 22 rare missense variants within or near the UCH domain (aa1‐240). Nineteen have been identified in patients with UM (Walpole et al.) and three in the general population (according to gnomAD). The variants were cloned to a bacterial vector and expressed as a GST‐fusion protein. Finally, we assayed the purified fusion proteins for their ability to cleave ubiquitin. Results: Nine variants significantly reduced the deubiquitinating activity of BAP1. Seven mutants with variants found in patients with familial BAP1‐TPDS, retained <20% of their activity. Three variants retained about 50% of activity, and the function of 12 variants was interpreted as normal (70%–120%). Conclusions: Functional studies are needed for accurate classification of BAP1 missense variants. Although BAP1 variants are dominant, penetrance might be affected by variants effect on enzymatic activity and patients with pathogenic variants might not exhibit familial BAP1 ‐TPDS. Some pathogenic variants that have been suggested to be pathogenic retained enzymatic activity, casting doubt on their classification. If enzymatic activity is retained, re‐classification should be considered or, when found in a family with clear BAP1‐TPDS, further studies need to be conducted. References. 1. Walpole S, Pritchard AL, Cebulla CM, et al. Comprehensive Study of the Clinical Phenotype of Germline BAP1 Variant‐Carrying Families Worldwide. JNCI: Journal of the National Cancer Institute 2018; 110(12): 1328–1341.