Abstract
BackgroundUveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantly originated from class II tumors or from tumors showing loss of an entire chromosome 3 (monosomy 3). We performed detailed EFS (embryonal Fyn-associated substrate) methylation analyses in UM, cultured uveal melanocytes and normal tissues, to explore the role of the differentially methylated EFS promoter region CpG island in tumor classification and metastatic progression.MethodsEFS methylation was determined by direct sequencing of PCR products from bisulfite-treated DNA or by sequence analysis of individual cloned PCR products. The results were associated with clinical features of tumors and tumor-related death of patients.ResultsAnalysis of 16 UM showed full methylation of the EFS CpG island in 8 (50%), no methylation in 5 (31%) and partial methylation in 3 (19%) tumors. Kaplan-Meier analysis revealed a higher risk of metastatic progression for tumors with EFS methylation (p = 0.02). This correlation was confirmed in an independent set of 24 randomly chosen tumors. Notably, only UM with EFS methylation gave rise to metastases. Real-time quantitative RT-PCR expression analysis revealed a significant inverse correlation of EFS mRNA expression with EFS methylation in UM. We further found that EFS methylation is tissue-specific with full methylation in peripheral blood cells, and no methylation in sperm, cultured primary fibroblasts and fetal muscle, kidney and brain. Adult brain samples, cultured melanocytes from the uveal tract, fetal liver and 3 of 4 buccal swab samples showed partial methylation. EFS methylation always affects both alleles in normal and tumor samples.ConclusionsBiallelic EFS methylation is likely to be the result of a site-directed methylation mechanism. Based on partial methylation as observed in cultured melanocytes we hypothesize that there might be methylated and unmethylated precursor cells located in the uveal tract. The EFS methylation of a UM may depend on which type of precursor cell the tumor originated from.
Highlights
Uveal melanoma (UM) is a rare eye tumor
We found the EFS CpG islands (CGIs) fully methylated in 8 tumors, unmethylated in 5 tumors and partially methylated in 3 tumors
EFS was fully methylated in blood DNA from healthy donors and partially methylated in DNA from cultured melanocytes isolated from the uveal tract
Summary
Uveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Hypermethylation of promoter-associated CGIs can result in transcriptional silencing of tumor suppressor genes (TSG) [10] In these instances - in line with the model of two hit inactivation - one mutational hit alters the methylation pattern of one allele and the second allele is either lost or inactivated by a structural mutation. An increasing number of nonimprinted, autosomal CGIs and CpG-rich regions have been identified that are already methylated in non-neoplastic cells [8,9,11] In some regions, this kind of CpG methylation establishes long-term gene inactivation and is part of the process of cell differentiation from pluripotent embryonic stem cells to terminally differentiated somatic cells [12,13,14]. A potential link between cell differentiation and cancer is suggested by the observation that genes that are preferentially hypermethylated in cancer are often marked for transcriptional repression through association with polycomb group proteins in embryonic stem cells [15,16]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
