Mesothelioma, an aggressive cancer often caused by exposure to asbestos, is one of the malignancies currently seen with increasing frequency in younger patients. We have sequenced and analyzed the DNA of 61 patients who developed mesothelioma at young age (55< y old) and who had no evidence of asbestos exposure. We discovered that ~1.8% of all mesothelioma patients and 4.9% of those younger than 55, carry rare pathogenic germline variants of the BRCA1 associated RING domain 1 (BARD1) gene.

Cell cultures Human dermal skin fibroblasts from explants of skin biopsies were collected, as previously described (Bononi et al 2017) from BARD1WT and BARD1 mutant carriers. Fibroblasts were cultured in Dulbecco modified Eagle’s medium (DMEM) (Corning Cellgro, # 10-017-CV), supplemented with 10% fetal bovine serum (FBS) (Seradigm, #1500-500), and 1% penicillin–streptomycin (PS) (Life Technologies, # 15140-163). Primary human mesothelial cells (HM) were derived from pleural fluids accumulated in individuals with congestive heart failure and cultured in DMEM with 20% FBS and 1% penicillin–streptomycin (PS).PBMCs were collected from BARD1WT and BARD1 mutant carriers. Gene Silencing with siRNA siRNA oligonucleotides were obtained from Qiagen. GeneSoluton siRNAs targeting four different BARD1 mRNAs were as follows: Hs_BARD1_2, (cat.no.1027415, D: SI00010136) ; Hs_BARD1_3, (cat.no.1027415, ID: SI00010143); Hs_BAP1_9, (cat.no.1027415, ID: SI05103847); Hs_BAP1_10, (cat.no.1027415, ID: SI05103854). A nonspecific control siRNA was used as a negative control (cat. no. 1027280). Transfection was performed with HiPerfect (Qiagen) for 24 hours. Western Blot: Total cell lysates were prepared in M-PER (Thermo Scientific, cat.no. 78501) reagent supplemented with proteases and phosphatases inhibitors (2mM Na3VO4, 2mM NaF, 50nM okadaic acid, 1mM PMSF and protease inhibitor cocktail) and 1mM DTT. 10 μg of protein were loaded and separated on NuPAGE Novex 4–12% Bis-Tris Gel (Life Technologies), and electron-transferred to PVDF according to standard procedures.

We performed functional assays in primary fibroblasts obtained from a patient carrying a heterozygous BARD1 mutation. We found that these cells had genomic instability, reduced DNA repair, and impaired apoptosis.. Studying underlying signaling pathways, we found that BARD1 interacts with p53 and SERCA2 modulating calcium signaling and apoptosis.

Our study elucidated mechanisms of BARD1 activity and revealed that heterozygous germline BARD1 mutations favor mesothelioma development in young patients and increase the susceptibility to asbestos carcinogenesis. These mesotheliomas are significantly less aggressive compared to asbestos-caused mesotheliomas. These patients experience significantly prolonged survival up to 20+ y and they require tailored screening and personalized therapeutic approaches.