Germline BAP1 mutations predispose to malignant mesothelioma

Abstract

Because only a small fraction of asbestos-exposed individuals develop malignant mesothelioma, and because mesothelioma clustering is observed in some families, we searched for genetic predisposing factors. We discovered germline mutations in the gene encoding BRCA1 associated protein-1 (BAP1) in two families with a high incidence of mesothelioma, and we observed somatic alterations affecting BAP1 in familial mesotheliomas, indicating biallelic inactivation. In addition to mesothelioma, some BAP1 mutation carriers developed uveal melanoma. We also found germline BAP1 mutations in 2 of 26 sporadic mesotheliomas; both individuals with mutant BAP1 were previously diagnosed with uveal melanoma. We also observed somatic truncating BAP1 mutations and aberrant BAP1 expression in sporadic mesotheliomas without germline mutations. These results identify a BAP1-related cancer syndrome that is characterized by mesothelioma and uveal melanoma. We hypothesize that other cancers may also be involved and that mesothelioma predominates upon asbestos exposure. These findings will help to identify individuals at high risk of mesothelioma who could be targeted for early intervention.

Figure 1

Pedigrees of two U.S. mesothelioma families. (a, b) Pedigrees showing family members with a germline mutation in BAP1, as confirmed by both sequencing and linkage analyses (orange) or by linkage analysis alone (yellow, i.e., no DNA was available for sequencing); individuals without the mutation (green) and individuals for whom DNA was unavailable (blue) are also shown. Presence or absence of germline BAP1 mutation is also indicated with + or − symbols, respectively. (a) Pedigree of family W showing presence or absence of germline mutation at BAP1 consensus splice acceptor site. (b) Pedigree of family L showing presence or absence of germline nonsense mutation. The development of other tumor types (Supplementary Table 1) in these families may also be related to BAP1 germline mutations. In family W, the presence of a breast cancer before age 45 and an ovarian cancer suggests that the BAP1 mutation is associated with a hereditary form of breast/ovarian cancer, as might be expected given BAP1’s relationship with the breast/ovarian cancer susceptibility gene product, BRCA1. In family L, the skin cancers shown were squamous cell carcinomas. Available mesothelioma tumor specimens with germline splice site mutation and either somatic 25-bp deletion (W-III-04T), genomic alteration (W-III-06T), or loss of wild-type BAP1 allele (W-III-08T) are indicated in Supplementary Table 1, as is the homozygous deletion of BAP1 seen in mesothelioma specimen L-III-18T.

Figure 2

Array-CGH analysis of two mesothelioma family members and schematic diagrams of predicted mutant BAP1 proteins. (a) Array-CGH showing focal deletion encompassing BAP1 within a larger 3p deletion (tumor L-III-18T) and amplicon adjacent to BAP1 locus (tumor W-III-06T). The BAP1 gene resides at chr3:52,435,027-52,444,009, and the Agilent Human 244K chip contains two probes within the BAP1 locus: A_16_P00704764 (chr3:52,438,014-52,438,066) and A_14_P128339 (chr3:52,443,209-52,443,268). In W-III-06T, the two BAP1 probes displayed log₂ ratios indicative of normal diploid DNA copy numbers, whereas the log₂ ratios of two probes immediately centromeric of BAP1 showed a transition to a higher copy number, indicating the start of an amplified region at or very near the BAP1 promoter (zoomed-in image and further details shown in Supplementary Fig. 6). In L-III-18T, the focal homozygous deletion encompassed the entire BAP1 locus and was ~218 kb in size. (b) Schematic diagram of predicted truncations of BAP1 resulting from germline mutations observed in two mesothelioma families (W, L) as well as in two sporadic mesothelioma patients who previously had developed uveal melanomas (SP-002; SP-008).

Figure 3

Immunohistochemistry on mesotheliomas from L and W families reveals lack of BAP1 nuclear expression and only weak, focal cytoplasmic BAP1 staining. (a) SP-024, sporadic mesothelioma with wild-type BAP1; note the normal nuclear expression of BAP1. (b) W-III-04, (c) L-III-18, and (d) W-III-06 represent mesotheliomas from patients with germline BAP1 mutations: note lack of nuclear expression and weak cytoplasmic staining. All magnifications 400X. Bar = 100 μm.

Figure 4

BAP1 truncating mutations and aberrant protein expression in sporadic mesothelioma tumor biopsies. (a) Schematic diagram of predicted truncations of BAP1 in four sporadic mesotheliomas harboring BAP1 mutations. Bracket at left indicates mutations in two different BAP1 alleles in tumor sample SP-015. NLS, nuclear localization signal at carboxy-terminus of BAP1. Frameshift sequences are shown as thinner gray bars. (b) Immunoblot analysis on whole tumor cell lysates of the same four sporadic mesotheliomas with somatic BAP1 mutations (lanes 2-5) and sporadic tumor lacking a BAP1 mutation (lane 1). Sporadic mesotheliomas with somatic BAP1 mutations show decreased expression of BAP1 compared to that seen in tumor without BAP1 mutation. Note that in mesotheliomas, whole tumor cell lysates inevitably contain some normal stromal cells that may be responsible for the faint BAP1 signal detected. Also note the presence of additional, faster-migrating BAP1 band in sample shown in lane 4 (SP-013), suggesting the presence of a truncated form of BAP1. The BAP1 protein products predicted in tumors SP-001 and SP-015 were not observed, suggesting nonsense-mediated mRNA decay. The mutation in tumor SP-018 results in a predicted protein product only 15 amino acids smaller, which presumably precludes detection of a small change in molecular weight compared to wild-type BAP1. GAPDH was used as a loading control.