Targeted Next Generation Sequencing reveals previously unidentified TSC1 and TSC2 mutations.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 and TSC2. Conventional DNA diagnostic screens identify a TSC1 or TSC2 mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undetectable using conventional methods, or possibly a mutation in another as yet unidentified gene. METHODS: Here we apply a targeted Next Generation Sequencing (NGS) approach to screen the complete TSC1 and TSC2 genomic loci in 7 individuals fulfilling the clinical diagnostic criteria for definite TSC in whom no TSC1 or TSC2 mutations were identified using conventional screening methods. RESULTS: We identified and confirmed pathogenic mutations in 3 individuals. In the remaining individuals we identified variants of uncertain clinical significance. The identified variants included mosaic changes, changes located deep in intronic sequences and changes affecting promoter regions that would not have been identified using exon-only based analyses. CONCLUSIONS: Targeted NGS of the TSC1 and TSC2 loci is a suitable method to increase the yield of mutations identified in the TSC patient population.

Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma.

Uveal melanoma is a lethal cancer with a strong propensity to metastasize. Limited therapeutic options are available once the disease has disseminated. A strong predictor for metastasis is the loss of chromosome 3. Inactivating mutations in BAP1 encoding the BRCA1-associated protein 1 and located on chromosome 3p21.1, have been described in uveal melanoma and other types of cancer. In this study, we determined the prevalence of somatic BAP1 mutations and examined whether these mutations correlate with the functional expression of BAP1 in uveal melanoma tissue and with other clinical, histopathological and chromosomal parameters. We screened a cohort of 74 uveal melanomas for BAP1 mutations, using different deep sequencing methods. The frequency of BAP1 mutations in our study group was 47%. The expression of BAP1 protein was studied using immunohistochemistry. BAP1 staining was absent in 43% of the cases. BAP1 mutation status was strongly associated with BAP1 protein expression (P<0.001), loss of chromosome 3 (P<0.001), and other aggressive prognostic factors. Patients with a BAP1 mutation and absent BAP1 expression had an almost eightfold higher chance of developing metastases compared with those without these changes (P=0.002). We found a strong correlation between the immunohistochemical and sequencing data and therefore propose that, immunohistochemical screening for BAP1 should become routine in the histopathological work-up of uveal melanoma. Furthermore, our analysis indicates that loss of BAP1 may be particularly involved in the progression of uveal melanoma to an aggressive, metastatic phenotype.