Functional assay for assessment of pathogenicity of BAP1 variants.

BACKGROUND: Pathogenic germline variants in BRCA1-Associated Protein 1 (BAP1) cause BAP1 tumor predisposition syndrome (BAP1-TPDS). Carriers run especially a risk of uveal (UM) and cutaneous melanoma, malignant mesothelioma, and clear cell renal carcinoma. Approximately half of increasingly reported BAP1 variants lack accurate classification. Correct interpretation of pathogenicity can improve prognosis of the patients through tumor screening with better understanding of BAP1-TPDS. METHODS: We edited five rare BAP1 variants with differing functional characteristics identified from patients with UM in HAP1 cells using CRISPR-Cas9 and assayed their effect on cell adhesion/spreading (at 4 h) and proliferation (at 48 h), measured as cell index (CI), using xCELLigence real-time analysis system. RESULTS: In BAP1 knockout HAP1 cultures, cell number was half of wild type (WT) cultures at 48 h (p = 0.00021), reaching confluence later, and CI was 78% reduced (p < 0.0001). BAP1-TPDS-associated null variants c.67+1G>T and c.1780_1781insT, and a likely pathogenic missense variant c.281A>G reduced adhesion (all p ≤ 0.015) and proliferation by 74%-83% (all p ≤ 0.032). Another likely pathogenic missense variant c.680G>A reduced both by at least 50% (all p ≤ 0.032), whereas cells edited with likely benign one c.1526C>T grew similarly to WT. CONCLUSIONS: BAP1 is essential for optimal fitness of HAP1 cells. Pathogenic and likely pathogenic BAP1 variants reduced cell fitness, reflected in adhesion/spreading and proliferation properties. Further, moderate effects were quantifiable. Variant modelling in HAP1 with CRISPR-Cas9 enabled functional analysis of coding and non-coding region variants in an endogenous expression system.

Pathogenic Germline Variants in Uveal Melanoma Driver and BAP1-Associated Genes in Finnish Patients with Uveal Melanoma.

Uveal melanoma (UM) is a rare yet aggressive eye cancer causing over 50% mortality from metastasis. Familial UM, amounting to 1%-6% of patients in Finland and the United States, mostly lack identified genetic cause, while 8% show associations with other cancer syndromes. We searched novel genetic associations for predisposition to UM, additional to already studied BAP1 and MBD4, by using targeted amplicon sequencing of 19 genes associated with UM, BAP1, or renal cell carcinoma in 270 consecutively enrolled Finnish patients with UM. Key UM drivers GNAQ, GNA11, CYSLTR2, PLCB4, EIF1AX, and SF3B1 lacked pathogenic germline variants. One patient carried the pathogenic BRCA1 variant c.3626del p.(Leu1209*), and one harbored a novel truncating MET variant c.252C > G p.(Tyr84*), classified as likely pathogenic. FLCN and BRCA2, previously identified with pathogenic variants in patients with UM, did not have such variants in our cohort. Two patients were heterozygous for a pathogenic recessive BLM variant c.2824-2A > T. None of the carriers of identified variants had familial UM. We identified BRCA1 and MET as genes with pathogenic germline variants in Finnish UM patients, each with a frequency of 0.4% (95% confidence interval, 0-2).