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Epigenetic Regulation Identifies RASEF as a Tumor-Suppressor Gene in Uveal Melanoma

¹From the Departments of Ophthalmology and ³Dermatology, Leiden University Medical Center (LUMC), Leiden, The Netherlands; and ²Hogeschool Leiden, Leiden, The Netherlands.

PURPOSE. Recently, a segregation study in families with uveal and cutaneous melanoma identified 9q21 as a potential locus harboring a tumor-suppressor gene (TSG). One of the genes in this area, RASEF, was then analyzed as a candidate TSG, but lack of point mutations and copy number changes could not confirm this. In this study, the RASEF gene was investigated for potential mutations and gene silencing by promoter methylation in uveal melanoma.

METHODS. Eleven uveal melanoma cell lines and 35 primary uveal melanoma samples were screened for mutations in the RASEF gene by high-resolution melting-curve and digestion analysis. Expression of RASEF was determined by real-time RT-PCR in all cell lines and 16 primary uveal melanoma samples, and the methylation status of the promoter of the RASEF gene was analyzed and confirmed by direct sequencing.

RESULTS. Mutation screening revealed a known polymorphism (R262C; CT) in exon 5 of the RASEF gene that displayed a normal frequency (54%). Of the primary uveal melanomas, 46% presented a heterozygous genotype, and 10 (91%) of 11 cell lines showed a homozygous genotype. Melting-curve analysis indicated loss of heterozygosity in at least two primary tumors. Low RASEF expression in the cell lines and primary tumors correlated with methylation of the RASEF promoter region. Homozygosity and methylation of the RASEF gene in primary tumors were associated with decreased survival (P = 0.019).

CONCLUSIONS. Homozygosity, in combination with methylation, appears to be the mechanism targeting RASEF in uveal melanoma, and allelic imbalance at this locus supports a TSG role for RASEF.