Frequent loss of differential methylation at the imprinted DIRAS3 locus in uveal melanoma

Proc Amer Assoc Cancer Res, Volume 47, 2006 1574 Uveal melanoma is the most common primary ocular tumour with an annual incidence of about six cases per million people. Approximately 50% of the patients die of metastases predominantly in the liver. The metastases mainly arise from primary tumours that show loss of an entire chromosome 3 (monosomy 3), whereas tumours with disomy 3 rarely give rise to metastatic disease. Loss of the entire short arm of chromosome 1 is another frequent alteration only observed in tumors with monosomy 3. This indicates a potential role of genes located in this region in tumor progression. The imprinted putative tumour suppressor gene DIRAS3 (Ras homolog member I), which frequently shows loss of imprinting (LOI) in breast and ovarian cancer cells, is located within this region. To evaluate the role of DIRAS3 in uveal melanoma pathogenesis we determined the DIRAS3 expression by quantitative real time PCR. DIRAS3 expression was strongly reduced in 23 from 25 primary tumors compared to its expression level in cultured melanocytes derived from the uveal tract. Furthermore, we analyzed the DIRAS3 methylation pattern in DNA from 41 primary uveal melanomas, 13 retinoblastomas and 6 neuroblastomas. Among 32 uveal melanomas with retention of heterozygosity of 1p we found loss of differential methylation in 50% of tumours, which was independent of the chromosome 3 status. In five of nine tumors showing 1p LOH the remaining allele was methylated and in four tumors DIRAS3 was unmethylated. As we could not detect any aberrant methylation pattern of the imprinted SNRPN promoter/exon 1 region on chromosome 15, it is unlikely that loss of imprinting is due to a general impairment of the imprinting/methylation process in these tumors. Based on the observation of hypomethylated as well as hypermethylated DIRAS3 alleles in primary tumors we conclude that LOI of DIRAS3 is a random rather than specific alteration in uveal melanomas. Furthermore, a specific role of DIRAS3 in metastatic progression of disease is unlikely as tumors with disomy 3 and monosomy 3 are similarly affected by LOI and down regulation of DIRAS3 expression. We also determined the DIRAS3 methylation status in neuroblastomas and retinoblastomas, which are early childhood tumors, and could not find altered methylation in any of these tumors. Therefore, we suggest that LOI of DIRAS3 might be related to the age of the patients.