Cohorts and pedigrees.

<p>(<b>A</b>). Ocular melanoma and cutaneous melanoma cohorts used in this study. (<b>B</b>). Fam-562 pedigree and clinical images of two nevoid melanoma-like melanocytic proliferations (NEMMPs) diagnosed in Fam729. (<b>C</b>). Fam-729 pedigree. One carrier had a Spitz nevus, which has been reported to harbor somatic <i>BAP1</i> mutations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035295#pone.0035295-Wiesner1" target="_blank">[4]</a>. Abbreviations: M, mutation carrier; wt, wildtype germline sequence; Ob, obligate carrier; OCMEL, ocular melanoma; MEL, cutaneous melanoma; NEMMPs, nevoid melanoma-like melanocytic proliferations; BR, breast cancer; CHL, cholangiocarcinoma; LG, lung cancer; KID, kidney cancer; UNP, melanoma of unknown primary site; CNS, central nervous system tumor; LK, leukemia. Crosses indicate CM with a nevoid pattern. The numbers next to the “MEL” indicate ages of diagnosis. <i>For the sake of confidentiality, the pedigrees have been masked for some non-affected individuals and siblings. Nonessential gender information has also been disguised by a diamond; the number of individuals collapsed into the diamond is indicated.</i></p

Distribution of <i>BAP1</i> mutations.

<p>Inactivating germline mutations identified in this study are indicated by the red arrows. Reported somatic missense mutations (blue bars) and indels (blue arrows) from ocular melanoma specimens (COSMIC database; <a href="http://www.sanger.ac.uk/genetics/CGP/cosmic/" target="_blank">http://www.sanger.ac.uk/genetics/CGP/cosmic/</a>) and germline variants (blue lines) from other families are also shown. Half of the germline variants occur in the terminal 150 amino acids while the somatic changes are more scattered.</p

BAP1 Mutations Identified In Study.

<p>Oc mel = ocular melanoma, Cut mel = cutaneous melanoma, NEMMP = nevoid melanoma-like melanocytic proliferation, SSM = superficial spreading melanoma; DCIS = breast ductal carcinoma-in-situ; DOD = died of disease; mat = maternal; pat = paternal; GM = grandmother.</p>*<p>found to be part of the same kindred.</p>#<p>see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035295#pone-0035295-g001" target="_blank">Figure 1</a>.</p

Histologic and molecular analyses of tumors from Fam-562.

<p>(<b>A</b>)–(<b>F</b>): Histology of 2 distinct NEMMPs. (<b>A</b>) Scanning view of the first lesion showing two expansile dermal nodules (H&E, 2×,) with a (<b>B</b>) benign nevoid appearance (H&E, 10×). (<b>C</b>) Atypical cytological features including nuclear pleomorphism and prominent nucleoli and a dermal mitotic figure (arrow) (H&E, 40×) along with focal increases in Ki67 staining (inset). (<b>D</b>) In the second lesion, there is an expansile dermal proliferation (H&E, 4×). (<b>E</b>) Detail of a field populated by dermal nevic cells with bland nuclear features (H&E,20×). (<b>F</b>) A proliferative area showing marked nuclear atypia and hyperchromasia along with elevated Ki 67 staining (inset). Biallelic inactivation of <i>BAP1</i> in two tumors through (<b>G</b>) loss of the wildtype allele in a nevoid melanoma (ie. LOH; arrow) or (<b>H</b>) a secondary mutation (p.Ser123Lysfs*3) in a NEMMP that did not exhibit LOH.</p

EIF1AX-regulated growth and translation in uveal melanoma.

<p><b>(A)</b> Distribution of <i>EIF1AX</i> mutations observed in cohort of 52 uveal melanomas in comparison to other cancer types (as reported by <a href="http://www.tumorportal.org" target="_blank">http://www.tumorportal.org</a>). <b>(B)</b> <i>EIF1AX</i> wild type (WT) or mutant (MUT) uveal melanoma cells were infected with <i>EIF1AX</i> or control shRNAs and cell viability was determined after 6 days using MTS. Percent growth is relative to shLuc-expressing cells. Error bars represent SD of mean from 3 independent experiments. <b>(C)</b> Immunoblot analysis of EIF1AX protein levels in shRNA-expressing cells. <b>(D)</b> Polysome profiles of cell lines expressing shRNAs against <i>EIF1AX</i> and <i>Luciferase</i>.</p

Somatic mutations in primary and metastatic uveal melanoma.

<p><b>(A)</b> The number of synonymous and nonsynonymous mutations per megabase of DNA sequence for 52 samples, arranged in columns. <b>(B)</b> Mutations in recurrently mutated genes are color-coded and ordered by significance. <b>(C)</b> Boxplots represent the distributions of allelic fractions observed per sample where the thick line represents 25-75^th percentile, and thin line 5-95^th. <b>(D)</b> The percentage of tumor cells (CCF) harboring a given mutation in the primary tumor in comparison to a metastatic liver sample from the same patient (UM45). <b>(E)</b> As in (D), but comparing a pre-treatment metastatic tumor sample to a post-treatment metastasis (Trio 2).</p

Decreased EIF1AX expression impairs translation of protein synthesis machinery in wildtype, but not mutated setting.

<p><b>(A)</b> Principal component analysis depicts 4 color-coded clusters of 141 genes. <b>(B)</b> The trend in translational efficiency is depicted for each cluster in cells expressing control shRNAs (CN) or <i>EIF1AX</i> shRNAs (KD). Each line represents a different gene. Ribosomal protein genes are highlighted in red. Translational efficiency was calculated as polysome CPM / total CPM. <b>(C)</b> Boxplots demonstrate the distribution of the translational efficiencies of 78 ribosomal proteins in cells as in (B).</p