We report a type of melanocytic neoplasm that was inherited in an autosomal dominant pattern in two unrelated families and was clinically, histopathologically, and genetically distinct from common acquired nevi (Fig. 1a). Beginning in the second decade of life, affected family members progressively developed skin-colored to reddish-brown, dome-shaped to pedunculated, well-circumscribed papules with an average size of 5mm (Fig. 1b; Supplementary Fig. 1–4). The number of tumors per patient varied markedly, ranging from 5 to over 50. No intellectual disabilities or dysmorphic features were identified in affected individuals.

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Figure 1

BAP1 germline mutations in two families; left panel: family 1; right panel: family 2. (a) Pedigrees. Ages of onset of the first melanoma or ‘melanocytic tumor of uncertain malignant potential’ are written below the symbols. Only subjects older than 18 years were tested for mutations. An extended pedigree of family 1 is shown in Supplementary Fig. 15. (b) Representative melanocytic neoplasms from affected individuals (II-4 and II-7 from family 1; III-1 and III-3 from family 2). Note the characteristic inconspicuous, skin-colored to reddish-brown, dome-shaped appearance. Scale bars, 1 mm. (c) Histopathology of representative melanocytic tumors from both families showing relatively symmetrical intradermal proliferations of large epithelioid melanocytes with abundant cytoplasm and enlarged, irregularly-shaped, vesicular nuclei, some with prominent nucleoli. Scale bars, 50 μm. Scale bars inset, 1 mm.

Histopathological examination revealed primarily dermal tumors composed entirely or predominantly of epithelioid melanocytes with abundant amphophilic cytoplasm and prominent nucleoli. The melanocytes often contained large, vesicular nuclei that varied significantly in size and shape (Fig. 1c; Supplementary Fig. 5–7). The cytological features of some of the cells were reminiscent of Spitz nevi; however, characteristic features (such as epidermal hyperplasia, hypergranulosis, Kamino bodies, clefting around junctional melanocytic nests, and spindle-shaped melanocytes) frequently seen in Spitz nevi were consistently absent. In addition, 37 of 42 (88%) tumors in the families showed BRAF mutations, which are typically absent in Spitz nevi.¹

Some of the neoplasms exhibited one or more atypical features, such as high cellularity, considerable nuclear pleomorphism, and several chromosomal aberrations. These tumors were classified as ‘neoplasms of uncertain malignant potential’ and the patients were managed as if they had melanoma (Supplementary Fig. 8). Both families were identified because of the occurrence of multiple epithelioid melanocytic tumors, but in each family, one affected individual had uveal melanoma, and three members of family 2 had been diagnosed with cutaneous melanoma (Fig. 1a, Supplementary Table 1).

Twenty-two melanocytic neoplasms from three affected individuals (II-1, II-4, II-7) in family 1 were analyzed with array-based comparative genomic hybridization (aCGH). Losses affecting the entire chromosome 3 or portions of the short arm of chromosome 3 were found in 50% of tumors. The smallest overlap of the deleted regions encompassed 5.8 megabases, extending from base-pair position 47,976,758 to 53,848,761 (hg 18 assembly) and encoded at least 150 known genes (Fig. 2a).

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Figure 2

Identification of the syndrome-causing gene. (a) Recurrent deletions affecting chromosome 3 in the melanocytic neoplasms of family 1. The two uppermost array-CGH profiles illustrate deletions of the entire chromosome 3, whereas the other profiles show focal deletions in 3p21. The minimally deleted region (black arrows) encompassed approximately 6 megabases. The next to last profile shows in addition a loss of chromosome 11 and the last profile a gain of chromosome 15. (b) Gene structure of BAP1 and germline mutations observed in affected individuals from family 1 and 2.

The frequent loss of the 3p21 region suggested a second hit², resulting in the elimination of the remaining wild-type allele of a mutated tumor suppressor gene in this region. To support this hypothesis, the haplotypes of 6 members of family 1 were reconstructed with SNP-arrays. Affected siblings in the second generation (II-1, II-4, II-7) inherited the same maternal copy of the 3p21 region, whereas the non-affected brother (II-5) received the other maternal 3p21 copy (Supplementary Fig. 9a). SNP-arrays showed that in all tumors with chromosome 3 loss, the paternal copy of chromosome 3 was lost, while the maternal copy was retained (Supplementary Fig. 9b). In conclusion, these data strongly suggested that a mutated gene in the 3p21 region was inherited from the maternal side of the family.

To identify the mutated gene, we sequenced the minimally deleted region of chromosome 3 in 2 affected (I-2, II-4) and 2 unaffected (I-1, II-5) subjects from family 1 using an in-solution hybrid capture technique followed by massively parallel sequencing.³ This analysis revealed a frameshift mutation in the BAP1 gene (c.1305delG, p.Gln436Asnfs*135) that was subsequently found to segregate with the phenotype (Fig. 2b, Supplementary Fig. 10). To rule out BAP1 germline mutations in the general population, we reviewed sequence data from 629 individuals in the 1000genomes database (http://www.1000genomes.org/home). No truncating mutations were found indicating that BAP1 germline mutations are infrequent in the general population.

The status of the second BAP1 allele was assessed in 29 skin tumors and in the uveal melanoma (I-2) from family 1. All tumors in which a loss of 3p21 was detected previously by aCGH, also showed a loss of the wild-type BAP1 allele in the sequencing electropherogram. In five additional cases without loss of the 3p21 region, the electropherogram showed markedly suppressed residual wild-type sequences, indicating that the neoplastic cells had lost the wild-type BAP1 allele through copy number neutral mechanisms, resulting in maternal uniparental disomy. In four other neoplasms without 3p21 deletions, we found additional acquired somatic nonsense (2 cases), frameshift (1 case) and missense (1 case) mutations in BAP1. Immunohistochemistry for BAP1 showed loss of nuclear expression in all melanocytic neoplasms, including the tumors without detectable alteration of the wild-type BAP1 allele. In summary, these data suggest that the remaining wild-type allele of BAP1 is lost by various somatic alterations in the melanocytic tumors (Supplementary Table 2, Supplementary Fig. 11).

In family 2, we found a different germline mutation in BAP1 that segregated with the phenotype and removed the acceptor splice site at the last exon (c.2057-2A>G, p.Met687Glufs*28, Fig. 2b). Analysis of cDNA from 2 affected family members confirmed that the last intron was not removed by splicing (Supplementary Fig. 12). In family 2, inactivation of the remaining wild-type BAP1 allele was found in 9 of 13 skin tumors, in the uveal melanoma (II-1), and in the cutaneous melanoma from patient II-3 (Fig. 3a–d, Supplementary Table 3). The metastatic melanoma of individual II-6 did not show loss of heterozygosity of BAP1, but no additional tissue was available to investigate alternative mechanisms of BAP1 inactivation. Besides the elevated, skin-colored melanocytic neoplasms, common acquired nevi (flat, brown maculae) were also excised from 4 patients (II-3, III-3, III-4, III-5). Histopathologically these nevi were composed of small uniform melanocytes and showed strong nuclear expression of BAP1 by immunohistochemistry (Supplementary Figure 13).

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Figure 3

Bi-allelic BAP1 loss is associated with characteristic histological features in familial melanocytic neoplasms. (a) A melanocytic neoplasm from patient III-3, family 2, presents as a combined lesion with (b) an area of small cells (common acquired nevus) on the left, and (c) an area of large epithelioid cells on the right. Scale bars, 50 μm. Fluorescence in-situ hybridization (red signal: chr. 3p21 [BAP1]; orange: chr. 3p25 [control]; green: chr. 4p12 [control]) shows loss of BAP1 in the area with large epithelioid melanocytes (insert in c: one red BAP1 signal, but 2 orange and green control signals per nucleus. Scale bar, 10 μm), but no loss in the region of the common nevus (insert in b: two signals of each probe. Scale bar, 10 μm.). (d) BAP1 immunohistochemistry at the transition area between common nevus and epithelioid cell area shows a strong nuclear staining in the common nevus component (left) and loss of nuclear staining in the epithelioid component (right). Scale bar, 100 μm.

To address the role of BAP1 mutations in sporadic melanocytic neoplasms, we sequenced BAP1 in 156 randomly selected tumors without family history: common nevi with uniform small melanocytes (n=28); Spitz nevi (n=17); neoplasms with overlapping features between Spitz nevus and melanoma (so-called “atypical Spitz tumors”, n=18); primary melanomas originating from acral skin (n=15), mucosa (n=15), or skin with (n=15) or without (n=15) chronic sun-induced damage, and uvea (n=33). Thirteen (40%) uveal melanomas, two (11%) atypical Spitz tumors and three (5%) of the melanomas (2 melanoma on skin without chronic sun-induced damage and 1 acral melanoma) had somatic BAP1 mutations. No mutations were found in the other categories (Supplementary Table 4–8). The mutation frequency seen in uveal melanoma is similar to a recent report.⁴ Two of five sporadic cutaneous melanomas that arose in nevi harbored BAP1 mutations. In both cases the BAP1 mutations were absent in the nevus portion, suggesting that loss of function of BAP1 may play a role in progression from nevus to melanoma in some cases (Fig. 4a–g).

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Figure 4

Progression of a nevus to melanoma is associated with loss of BAP1. (a) Scanning magnification of a sporadic melanoma (M) arising within a nevus (N) (hematoxylin-eosin stain). (b) Nevus component with bland melanocytes containing monomorphous round and oval-shaped nuclei. Scale bar, 50 μm. (c) Melanoma component showing melanocytes with large vesicular nuclei and prominent nucleoli. Scale bar, 50 μm. (d) MIB-1 immunohistochemistry shows a high proliferation rate in the melanoma compared to the nevus component. Scale bar, 100 μm. (e) BAP1 immunohistochemistry displays a conspicuous nuclear staining in the nevus contrasted with absent nuclear staining in the melanoma. Scale bar 100 μm. (f) The nevus component shows a BRAF^V600E mutation, but no BAP1 mutation or chromosomal aberrations in aCGH. (g) The melanoma component shows the same BRAF mutation and in addition a focal loss of chromosome region 3p21 spanning the BAP1-locus in aCGH. The electropherogram of BAP1 demonstrates a frameshift mutation of the second BAP1 allele.

The two atypical Spitz tumors with somatic BAP1 mutations had similar morphologic features to the melanocytic neoplasms seen in both families, lacked immunohistochemical expression of BAP1, and harbored BRAF mutations (Supplementary Figure 14). This finding suggests that bi-allelic inactivation of BAP1 is associated with a clinically and morphologically distinct type of melanocytic neoplasm. Histopathologically, the tumors range from intradermal nevi composed of bland epithelioid melanocytes to atypical proliferations of epithelioid melanocytes with morphological and cytogenetic features overlapping with melanoma. These tumors have been previously subsumed under the category of “spitzoid” melanocytic neoplasms because they share cytologic features with Spitz nevi.

As illustrated by the families, inheriting one mutant copy of BAP1 results in a markedly increased number of these neoplasms. In aggregate, several hundred papular melanocytic tumors were present in affected family members, while the number of melanomas was substantially lower. This indicates that the risk of malignant progression in individual tumors is low, and that bi-allelic loss of BAP1 in conjunction with mutations in BRAF is not sufficient for melanoma formation in the skin. Harbour et al.⁴ proposed that bi-allelic loss of BAP1 in uveal melanoma, which carry mutations in GNAQ⁵ or GNA11⁶ but not in BRAF, marks the transition to metastatic disease. Our findings may indicate that the role of BAP1 in melanocytic neoplasia depends on the associated oncogene and/or the cell of origin.

BAP1 was originally discovered as a binding partner of BRCA1 and has been functionally implicated in DNA damage response^7,8, as well as in regulation of apoptosis, senescence, and the cell cycle.⁹ Its Drosophila counterpart Calypso is involved in chromatin remodeling, which was shown to oppose the mono-ubiquitination activity of the polycomb repressive complex 1, a critical component of transcriptional silencing.¹⁰ In cancer, mutations and deletions in BAP1 have been reported in breast and lung cancers^11–13, but none of the individuals in our study developed breast or lung cancers.

In summary, we describe a novel autosomal dominant syndrome that is caused by germline mutations of BAP1, characterized by a high penetrance of melanocytic neoplasms with distinctive clinical and histopathological features, and possibly associated with an increased risk for uveal and cutaneous melanoma.