Germline DNA was extracted from peripheral blood mononuclear cells at the Human Cancer Genetics sample bank at The Ohio State University using a simple salting out procedure.14 (link) Tumour DNA was extracted from archival material using Qiagen DNeasy kit (Qiagen, Valencia, California, USA). Primers and PCR conditions for sequencing of all exons of the BAP1 gene and the adjacent intronic sequences are listed in supplemental table 2. Mutational screening was carried out by direct sequencing of fragments obtained by PCR using an Applied Biosystems 3730 DNA sequencer (Applied Biosystems, Foster City, California, USA). Mutational screening for the hereditary melanoma candidate genes (CDKN2A, p14/ARF, and exon 2 of CDK4) were carried out, as previously described, in probands of families that were not included in the previous study.5 (link) For BAP1 the sequence results were read by aligning with the reference sequence provided by Genebank accession number NM_004656.2, utilising the Sequencher software (Version 4.8, Gene Codes Corp, Ann Arbor, Michigan, USA). All identified sequence variations were confirmed at least once in an independent PCR experiment.
Genotyping was carried out on the tumour tissues of three individuals from family FUM036, one lung adenocarcinoma (individual III.1, figure 1), one meningioma (individual III.2, figure 1), and one UM (individual III.6, figure 1). The three patients had germline mutation in BAP1. A total of 15 micro-satellite markers on chromosome 3 were used for genotyping, including three markers (D3S3026, D3S3561, and D3S1578) flanking the BAP1 gene (figure 2).