HAP1 cell utilization for accurate interpretation of germline BAP1 variants

Abstract

Aims/Purpose: Pathogenic germline variants in BRCA1‐Associated Protein 1 (BAP1) gene cause a rare, dominant, life‐threatening tumour predisposition syndrome (BAP1‐TPDS). It increases risk for atypical cutaneous Spitz tumours and for several index cancers: uveal melanoma, malignant mesothelioma, cutaneous melanoma, clear cell renal cell carcinoma, and basal cell carcinoma. Because about 50% of variants in the public database CLINVAR are of unknown significance (VUS) and accurate interpretation of variants is crucial for screening cancer in carriers, improved tools for genomic BAP1 variant interpretation are needed. We aimed to create a mammalian cell based tool for this purpose.Methods: Four pathogenic BAP1 variants (c.67 + 1G > T, c.281A > G, c.680G > A, and c.1780_1781insT) and one VUS (c.1526C > T) were edited into the genome of near haploid HAP1 cells with CRISPR‐Cas9 and assayed for BAP1 expression and cell fitness. Proliferation and cell adhesion was quantified using xCelligence real‐time cell analysis system.Results: BAP1 loss reduces growth of HAP1 cells, which we exploited as phenotypic readout for pathogenicity of inserted variants. Three BAP1‐TPDS history‐associated variants c.67 + 1G > T, c.281A > G, and c.1780_1781insT reduced HAP1 adhesion and proliferation roughly by 70–80% whereas the c.680G > A missense variant, identified in a patient with UM without BAP1‐TPDS family history, reduced proliferation by 50%. VUS c.1526C > T behaved similarly to wild type.Conclusions: Qualitatively, the results of our in vitro tool for BAP1 variant interpretation agree with previous studies, validating the tool. The validated system has potential for scaling up for simultaneous analysis of multiple variants. Quantitatively, it might have potential for predicting higher or lower risk in family members.