April 2008
Volume 49, Issue 4
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Anatomy and Pathology/Oncology  |   April 2008
Epigenetic Regulation Identifies RASEF as a Tumor-Suppressor Gene in Uveal Melanoma
Author Affiliations
  • Willem Maat
    From the Departments of Ophthalmology and
  • Sigrid H. W. Beiboer
    Hogeschool Leiden, Leiden, The Netherlands.
  • Martine J. Jager
    From the Departments of Ophthalmology and
  • Gré P. M. Luyten
    From the Departments of Ophthalmology and
  • Nelleke A. Gruis
    Dermatology, Leiden University Medical Center (LUMC), Leiden, The Netherlands; and
  • Pieter A. van der Velden
    From the Departments of Ophthalmology and
    Dermatology, Leiden University Medical Center (LUMC), Leiden, The Netherlands; and
Investigative Ophthalmology & Visual Science April 2008, Vol.49, 1291-1298. doi:10.1167/iovs.07-1135
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      Willem Maat, Sigrid H. W. Beiboer, Martine J. Jager, Gré P. M. Luyten, Nelleke A. Gruis, Pieter A. van der Velden; Epigenetic Regulation Identifies RASEF as a Tumor-Suppressor Gene in Uveal Melanoma. Invest. Ophthalmol. Vis. Sci. 2008;49(4):1291-1298. doi: 10.1167/iovs.07-1135.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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; C→T) 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.

Uveal melanoma is the most common primary intraocular neoplasm in adults, with an annual incidence of six to eight per million in Caucasian populations. 1 In contrast to cutaneous melanoma, clustering of uveal melanoma in families is extremely rare. 2 3 4 Occurrence of both uveal melanoma and cutaneous melanoma in a single family has been observed. 4 Recently, Jönsson et al. 5 revealed a genetic component in three such families, in which members are affected by either uveal or cutaneous melanoma. Linkage analysis in these families identified a potential uveal melanoma susceptibility locus on chromosome 9, area q21. 
This locus has a long history in melanoma that started with detection of isochromosome 9q with cytogenetic analysis. 6 7 Loss of heterozygosity (LOH) of markers at 9q22 was subsequently frequently reported and was shown to be associated with proliferation and tumor progression. 8 9 Recently, single nucleotide polymorphism (SNP) analysis has confirmed the LOH of this locus in melanoma, while genome-wide analysis in dizygotic twins for nevi numbers also showed linkage with this 9q region. 10 11 In addition, a gene slightly distal to RASEF, RMI1, has recently been shown to be a risk factor for cutaneous melanoma, whereas the locus for familial melanoma susceptibility is located on the short arm of chromosome 9. 12 13  
Cutaneous melanomas are often characterized by loss of the cell-cycle regulator p16 and/or activation of the RAS/RAF/ERK pathway. 14 15 These hallmarks of melanoma are also recognized in uveal melanoma, although the underlying mechanisms differ. 16 17 Whereas in cutaneous melanoma, p16 is commonly lost by chromosomal deletion of the CDKN2A gene, the preferential mechanism in uveal melanoma appears to be silencing of the p16-encoding CDKN2A promoter by methylation. 18 Mutations in BRAF, NRAS, or c-kit lead to constitutive ERK activation in most cutaneous melanomas. 19 20 However, mutations in BRAF have only rarely been reported in uveal melanoma, whereas activating NRAS and c-kit mutations have never been reported. 21 Still, ERK activation is also present in uveal melanoma, and this knowledge leads to the question of what causes ERK activation in the absence of activating mutations in BRAF, NRAS, or c-kit. 16 17 21  
The RASEF (RAS and EF hand domain containing) gene is located on chromosome 9, area q21, and encodes a protein with calcium-binding EF-hand and Ras GTPase (Rab family) motifs (http://www.genome.ucsc.edu/ provided in the public domain by the Genome Bioinformatics Group, University of Santa Cruz, CA); it is also known as RAB45 or FLJ31614. 22 Based on the functional domains in RASEF, the gene product may be engaged in the RAS pathway and in combination with evidence for linkage of the RASEF region with cutaneous and uveal melanoma, molecular analysis of this gene is warranted. 
In line with the analysis of cutaneous melanoma reported by Jönsson et al., 5 we therefore set out to analyze RASEF for mutations and for expression of the gene in uveal melanoma. 
Materials and Methods
Cell Lines and Primary Uveal Melanoma Specimens
In total, 11 cell lines derived from primary uveal melanomas (92.1; OCM-1, -3, and -8; and Mel-202, -270, -285, and -290) and uveal melanoma metastases (OMM-1, -2.3, and -2.5) were analyzed. All melanoma cell lines were cultured in RPMI 1640 medium (Invitrogen-Gibco, Paisley, Scotland, UK) supplemented with 3 mM l-glutamine (Invitrogen-Gibco), 2% penicillin-streptomycin, and 10% FBS (Hyclone, Logan, UT). All cell cultures were incubated at 37°C in a humidified 5% CO2 atmosphere. Archival frozen tumor specimens of primary uveal melanoma came from 35 patients who attended the Leiden University Medical Center between 1988 and 1996. All tumors were primary lesions with a tumor diameter greater than 12 mm, a prominence greater than 6 mm, and no treatment before enucleation. The validity of the diagnosis of uveal melanoma was confirmed histologically in all cases, and clinical and survival data were listed for use in the study (Table 1) . The research protocol followed the tenets of the current version of the Declaration of Helsinki (World Medical Association Declaration of Helsinki 1964; Ethical Principles for Medical Research Involving Human Subjects). 
DNA and RNA Extraction and Sodium-Bisulfite Modification
Using a column-based extraction kit (Genomic tip 100/G; Qiagen Benelux BV, Venlo, The Netherlands), DNA was extracted from the cell lines and frozen tumor material, according to the manufacturer’s guidelines. RNA was also extracted with a column-based extraction kit (RNeasy mini kit; Qiagen Benelux) from tumors in which enough frozen material was available (n =16). RNA was converted to cDNA (iScript cDNA synthesis kit; Bio-Rad Laboratories BV, Veenendaal, the Netherlands), according to the manufacturer’s guidelines. Genomic DNA was modified with sodium bisulfite (EZ Methylation Gold kit; Zymo Research Corp., Orange, CA). Enzymatically methylated human DNA (Chemicon Europe Ltd., Hampshire, UK) was used as the positive control in all experiments. DNA and RNA concentrations were determined by spectrophotometer (model ND-1000; NanoDrop Technologies Inc., Wilmington, DE). 
Mutation Screening and Genotyping
A 96-well light scanner (Idaho Technologies Inc., Salt Lake City, UT) for high-resolution melting-curve analysis was used to scan all amplicons of the RASEF gene. The primers are shown in Table 2 . DNA samples were amplified with a double-stranded DNA-binding dye (LC Green Plus; Idaho Technologies). Melting curves were analyzed in plots showing differences in fluorescence. The shift and curve shapes of melting profiles were used to distinguish between samples from control subjects and patients. PCR reaction with the green dye (LC Green; Idaho Technologies) contained PCR buffer (Invitrogen, Breda, The Netherlands), 1.5 mM MgCl2, 40 μM dNTPs, 1:10 diluted green dye (LC Green; Idaho Technologies), 0.4 μM of forward and reverse primers, and 1 unit Taq polymerase per 10-μL reaction (Fast Start; Roche Diagnostics BV, Almere, The Netherlands). PCR consisted of an initial denaturation at 94°C for 6 minutes followed by 40 cycles consisting of 15 seconds at 96°C, 30 seconds at 58°C, and 60 seconds at 72°C, and the PCR ended with a 1-minute denaturation at 94°C. After amplification, the amplified fragments (exon 5) were digested using 4 units of the restriction enzyme BstU1 (New England Biolabs, Beverly, MA) directly added to the PCR mixture. Analysis was performed by overnight digestion of the amplified fragments at 60°C. The BstU1 enzyme recognizes and cleaves the 5′-CG∧CG-3′ sequence. PCR products were separated on a 2% agarose gel in 1× TBE (0.09 M Tris-borate, 0.002 M EDTA; pH 8.2). 
RASEF Expression
The expression of RASEF in 16 tumors for which RNA was available was analyzed with real-time RT-PCR and specific primers, which are shown in Table 2 . PCR was performed as described earlier. 23  
Methylation Analysis
We applied bisulfite modification of tumor DNA in combination with PCR, as this introduces sequence differences between methylated and unmethylated DNA that can be analyzed with several methods. The sequence differences were initially determined with melting-temperature analysis, as this method provides both quantitative and qualitative measures of methylation (see 1 2 Fig. 3 ). The methylation status of the RASEF promoter region was determined by polymerase chain reaction with specific primers and by melting-temperature analysis and was further validated with a restriction digestion analysis. 
Primers were designed on computer (Beacon Designer Software ver. 5.0; Premier Biosoft International, Palo Alto, CA) using bisulfite-converted DNA sequences and amplified a CpG island in the RASEF gene promoter (Consensus CDS [Coding Sequence] accession number 6662.1; Gene ID 158158/ http://www.ncbi.nlm.nih.gov/ a genome database hosted by the National Center for Biotechnology Information, Bethesda, MD). The primers are shown in Table 2 . PCR was performed exactly as described earlier. 23  
Melting-Temperature Analysis
A melting-temperature analysis was performed as described earlier. 23  
Restriction Digestion Analysis and Sequence Analysis
After amplification with specific primers for bisulfite-converted DNA, the PCR-amplified fragments were digested with 4 units of restriction enzyme HinfI (Fermentas GmbH, St. Leon Rot, Germany) directly added to the PCR mixture (under conditions specified by the manufacturer). The HinfI enzyme recognizes and cleaves the 5′-G∧ANTC-3′ sequence. This sequence is not present in unmodified DNA and in modified unmethylated DNA. The RASEF amplicon of methylated DNA contains one HinfI recognition site and is dependent on both CT conversion and methylation of a CpG. The recognition site 5′-G∧ANTC-3′ appears only when the first C in the 5′-GANCC-3′ sequence is converted into thymine, whereas the second must be methylated and remains a cytosine. PCR products were separated on a 2% agarose gel in 1× TBE (0.09 M Tris-borate, 0.002 M EDTA; pH 8.2). DNA bands were excised from the gel, purified using a gel extraction kit (Nucleospin Extract II; Macherey-Nagel, GmbH, Düren, Germany) and sequenced (Prism 3700 DNA sequencing system; Applied Biosystems, Foster City, CA). 
Pyrophosphorolysis-Activated Polymerization
In the pyrophosphorolysis-activated polymerization (PAP) reaction, primers are used that contain a dideoxy-nucleotide (ddNTP) at their 3′ terminus and hence will not be extended. A polymerase with pyrophosphorolysis activity can remove the dideoxy-cytosine and thereby activate polymerization. Since this pyrophosphorolysis activity is dependent on double-stranded DNA, only primers that perfectly match the template will be activated. The specificity of pyrophosphorolysis allows us to amplify specifically the minute amounts of methylated RASEF DNA in the background of unmethylated DNA. The PAP products can be further validated with sequence analysis for internal CpGs. The primers are shown in Table 2 . The amplification was performed in a final volume of 25 μL containing 5 μL 5× PAP buffer (prepared as described by Liu and Sommer. 24 ), 0.3 μL (10 picomoles/μL) of each primer, 0.5 μL Taq polymerase (KLENtaq; DNA Polymerase Technology, Inc., St. Louis, MO), 17.9 μL H2O, and 1 μL DNA sample. 25 Amplification was initiated by hot start, followed by 40 cycles at 94°C for 15 seconds, 60°C for 40 seconds, 64°C for 40 seconds (T-anneal); 68°C for 40 seconds (pyrophosphorolysis activity); and 72°C for 40 seconds (elongation), and using a final melting curve from 70°C to 97°C with an increase in temperature of 0.2°C every 10 seconds. PCR products were separated on a 2% agarose gel in 1× TBE (0.09 M Tris-borate, 0.002 M EDTA; pH 8.2). 
Statistical Analysis
Survival analysis for RASEF promoter methylation was performed using a Kaplan Meier analysis and log rank test (SPSS ver. 14.0 for Windows; SPSS Inc., Chicago, IL). For a comparison between the presence or absence of RASEF methylation and metastatic disease and tumor characteristics, the χ2 test and analysis of variance were performed. 
Results
Mutation Screening in Uveal Melanoma
To analyze RASEF as a tumor-suppressor gene (TSG) candidate, we first investigated the gene for mutations. Mutation screening was performed in two steps: first, the 17 exons were prescreened by high-resolution melting-curve analysis. Though melting-curve analysis showed few variations, we nevertheless generated sequences for 2 tumor samples of each exon both sequenced with the forward and the backward PCR primer. We detected a sequence variation, which was a known polymorphism in exon 5 of RASEF encoding a R262C (C→T; Arginine→Cysteine) substitution (Fig. 1) . This SNP occurs frequently in the population, with reported frequencies between 50% and 58% of the Caucasian population. 22 In 10 of the 11 cell lines, a homozygous genotype of the T allele was observed. The primary uveal melanomas (n = 35) displayed a normal frequency of the SNP (54%), with 16 uveal melanomas presenting a heterozygous genotype (Table 1) . However, both melting-curve analysis and restriction enzyme analysis revealed imbalance of the alleles in two samples. Whereas in gel analysis of BstUI digested RASEF, exon 5 PCR indicated the presence of the C-allele, melting-curve analysis indicated that the relative concentration of the C-allele was at least 10-fold lower than the T-allele in UM13 and -21 (Fig. 1)
Expression analysis
The allelic imbalance observed in the primary uveal melanoma was followed up by RASEF RT-PCR expression analysis. In the cell lines, two groups were distinguishable, based on expression levels. In 6 of 11 uveal melanoma cell lines (92.1; Mel 202 and -270; and OMM-1, -2.3, and -2.5) an approximately 30- to 100-fold reduced expression of RASEF was displayed compared with the other cell lines (OCM1, -3, and -8; Mel285 and -290; Fig. 2 ). Cell lines Mel270 and OMM-2.3 and -2.5 are derived from the same patient and fell into the same group. Among the uveal melanomas cell lines with low RASEF expression, a homozygous (TT) genotype prevailed. When primary tumors were analyzed, variable levels of RASEF expression were also observed, but clustering into two groups was not as marked, and the absolute expression levels differed even more. One sample failed in the expression analysis (UM15). Correlating the expression levels with the genotypes of the tumors revealed that the homozygous tumors tended to present a lower RASEF expression (P = 0.015), as was the case in one tested uveal melanoma presenting allelic imbalance (UM13). 
Methylation Analysis
Because we did not detect mutations that could explain the low RASEF expression in the primary uveal melanomas and the cell lines, we considered epigenetic regulation as the possible mechanism of downregulation. All five RASEF-expressing cell lines contained an unmethylated promoter while hypermethylation of all CpGs within the amplicon was present in all six cell lines that lacked RASEF expression. The analysis of methylation with melting temperature was confirmed by sequence analysis (Fig. 3) . In primary uveal melanomas, methylation was much more heterogeneous and never reached the level of methylation observed in the cell lines. Uveal melanoma samples 15 (failed in expression analysis) and 21 displayed the highest methylation but also contained an equimolar level of unmethylated RASEF. In the other uveal melanoma samples with methylated RASEF, a minor fraction of the CpGs was methylated. Still, there was a correlation between methylation and expression of RASEF in the primary tumor samples, although not as obvious as in the cell lines. 
To validate methylation in primary tumors, we used restriction-enzyme analysis. By HinfI digestion, we were able to confirm RASEF methylation in primary uveal melanoma (data not shown). Next, we set out to isolate the methylated fraction and applied PAP. By applying PAP, we were able to show completely methylated alleles and thereby validate melting-temperature analysis in five tumors that had shown a methylated fraction in the background of unmethylated DNA. In one sample, a methylated allele was detected in a tumor that had shown a normal curve with melting-temperature analysis, suggesting a very low level of the methylated allele (Fig. 3)
Survival
The mean follow-up of the 35 patients was 78 months (2–210 months), and 20 patients had died of tumor-related metastasis at the time of analysis. Two patients had died of a metastasis from another primary tumor (UM7 and UM25), one patient was lost to follow-up (UM22) after 2 months, and two patients had died of unknown causes. The presence of methylation within the RASEF promoter region correlated with death due to metastatic disease (P = 0.024; log rank test). The genotype of the 35 tumors did not correlate to cell type, methylation status, or the development of metastatic disease (P = 0.441; Pearson χ2). 
Although the genotype itself was not associated with metastatic death, patients with a homozygous genotype and methylation of the RASEF gene (n = 7) had a significantly higher risk of development of metastasis than did patients with a heterozygous genotype and no methylation (survival 51 ± 15.5 vs. 161 ± 19.0 months; P = 0.019). 
Discussion
Linkage analysis in uveal and cutaneous melanoma families identified the 9q21 region as a locus for a potential TSG involved in the development of melanoma. In addition, LOH analysis in two uveal melanomas from members of the families in which linkage was identified indicated 9q21 to be the possible region for a TSG. 5 The 9q21 region harbors the RASEF gene, which is potentially involved in the RAS pathway prominent in the development of melanoma. 26 27 As patients with melanoma from the family just mentioned had been analyzed for RASEF mutations, we set out to analyze sporadic uveal melanoma and uveal melanoma cell lines. 
In line with the findings of Jönsson et al., 5 we did not detect any mutations in the RASEF gene other than a known SNP. 5 22 Using this SNP, we detected allelic imbalance in some of the tumors that were heterozygous for this marker (UM13 and -21). Because the imbalances were not complete, we suspect tumor heterogeneity in the primary tumors in contrast to the cell lines, all of which, with one exception, displayed a homozygous genotype. Gene expression analysis revealed that 5 of 11 uveal melanoma cell lines had high RASEF expression, whereas the others hardly showed expression. As almost all the low-expression cell lines displayed the homozygous T-allele, there appears to be an association between expression and genotype. This apparent association, however, could also be based on the small number of cell lines that were tested and the fact that three cell lines were derived from the same patient (Mel 270, OMM 2.3, and OMM 2.5). In the primary tumors, expression varied widely and often exceeded the expression seen in the cell lines. Among the uveal melanomas with low RASEF expression a homozygous genotype prevailed, but this fact does not favor a specific allele. This finding may indicate that there is no risk factor linked to either allele and that the low expression is more likely due to a somatic alteration. As we had not observed any mutations in the cell lines, we subsequently considered epigenetic modifications as the cause of low RASEF expression. Indeed, all cell lines that did not express RASEF contained a methylated promoter, whereas all cell lines with expression lacked this methylation, confirming our hypothesis. Hereafter, we performed demethylation experiments with 5-azacytidine, which revealed a highly induced expression in a cell line with methylated RASEF. Demethylation of an unmethylated cell line resulted in the opposite effect. The demethylating agent is highly toxic and may explain downregulation of RASEF expression in the unmethylated cell line. Toxicity of 5-azacytidine and demethylation of all the other genes during treatment are the reasons that we reserve functional analysis using genetically modified cell lines for follow-up research. 
The primary uveal melanomas displayed heterogeneity for RASEF methylation but never reached levels above ∼50% methylation, and most commonly only a part of the CpGs present in the promotor region was methylated. Furthermore, methylation not only coincided with low expression but also with a homozygous genotype, which suggests a combination of methylation and LOH being the mechanism of loss of expression. The additional effect of LOH seems to be associated with the aggressiveness of the tumor, because homozygous tumors with a methylated RASEF promoter region tended to have a decreased survival compared with heterozygous tumors without methylation (P = 0.019; Fig. 4 ). To confirm the suggested mechanism, we compared the RASEF homozygous genotype of four tumors with the genotype of their peripheral blood leukocytes, also obtained at time of enucleation. One person, in whom the tumor showed a nearly complete homozygous T-allele genotype, revealed a heterozygous genotype in leukocyte DNA, confirming the mechanism of LOH in tumor tissue (Fig. 5) . Of interest, in the tumor tissue of this patient, the RASEF gene was methylated, furthermore confirming our conclusion. 
We conclude that homozygosity in combination with methylation is the mechanism that targets RASEF in uveal melanoma, appointing RASEF as a bona fide tumor suppressor that is epigenetically silenced in uveal melanoma. Allelic imbalance at this locus supports a tumor-suppressor role for RASEF; however, analysis of RASEF in proliferation, survival, and migration of uveal melanoma is needed to confirm this. 
 
Table 1.
 
Tumor Characteristics and Survival Data of 35 Uveal Melanoma Patients Sorted by Methylation Status and RASEF Genotype
Table 1.
 
Tumor Characteristics and Survival Data of 35 Uveal Melanoma Patients Sorted by Methylation Status and RASEF Genotype
Tumor ID Cell Type Survival (mo) Present Status Hypermethylated RASEF Genotype
UM1 Spindle 210 Alive Not present Hom C
UM16 Epithelioid 115 Alive Not present Hom C
UM29 Spindle 24 Died, due to metastases Not present Hom C
UM3 Mixed 95 Died, due to metastases Not present Hom C
UM33 Mixed 24 Died, due to metastases Not present Hom C
UM12 Epithelioid 137 Died, due to metastases Not present Hom T
UM19 Spindle 15 Died, due to metastases Not present Hom T
UM26 Spindle 122 Alive Not present Hom T
UM31 Mixed 34 Died, due to metastases Not present Hom T
UM32 Epithelioid 63 Died, due to metastases Not present Hom T
UM4 Spindle 31 Died, due to metastases Not present Hom T
UM6 Epithelioid 57 Died, due to metastases Not present Hom T
UM27 Mixed 23 Died, due to metastases Present Hom C
UM28 Epithelioid 33 Died, due to metastases Present Hom C
UM30 Spindle 113 Alive Present Hom C
UM11 Mixed 13 Died, due to metastases Present Hom T
UM18 Spindle 12 Died, due to metastases Present Hom T
UM5 Spindle 50 Died, due to metastases Present Hom T
UM35 Mixed 94 Alive Present Het/homT*
UM21 Epithelioid 167 Died, due to metastases Present Het/homT*
UM13 Epithelioid 30 Died, due to metastases Present Het/homT*
UM15 Mixed 23 Died, due to metastases Present Het
UM17 Epithelioid 33 Died, due to metastases Present Het
UM2 Mixed 29 Died, due to metastases Not present Het
UM14 Mixed 42 Died, due to metastases Not present Het
UM25 Mixed 29 Died, other cause Not present Het
UM7 Mixed 63 Died, other cause Not present Het
UM23 Spindle 152 Died, unknown cause Not present Het
UM22 Spindle 2 Lost to follow up Not present Het
UM8 Spindle 191 Alive Not present Het
UM9 Mixed 131 Alive Not present Het
UM10 Mixed 136 Alive Not present Het
UM20 Mixed 187 Alive Not present Het
UM24 Mixed 143 Alive Not present Het
UM34 Mixed 106 Alive Not present Het
Table 2.
 
RASEF Primers Used for Mutation Detection, Expression Analysis, and Methylation Analysis
Table 2.
 
RASEF Primers Used for Mutation Detection, Expression Analysis, and Methylation Analysis
Primer Sequence
Exon 1 Forward 3′-GGCAAGCAGCGGTGGACTC-5′
Reverse 5′-GTAGGTGAAGGAAGACAAGCAACTC-3′
Exon 2 Forward 3′-TCTTCCCTTCCTTCCGTTCATTCTG-5′
Reverse 5′-GTCCACCTATATCATAGTGTGACAATGC-3′
Exon 3 Forward 3′-TTCTCTTCATCTGTAATATATAGGGCTTAACG-5′
Reverse 5′-CCCTCTCCGTAGAAACCACCTC-3′
Exon 4 Forward 3′-TCACCTTCCCTGTGTAGGAGAAC-5′
Reverse 5′-CTGAGATGCTGAGGCTGTTCC-3′
Exon 5 Forward 3′-CAAAGCAATTCAAAGTGAGTTTGTAAGC-5′
Reverse 5′-TGAGGATGTGGTCTAACAGGAAGTG-3′
Exon 6 Forward 3′-GTGTGGGAGGGTGACAGGAC-5′
Reverse 5′-AAATCATTAGAAAGTAAAGAAGATATTAGCAAAG-3′
Exon 7 Forward 3′-AAAGGGTCTGGGAGGGTAGG-5′
Reverse 5′-AAACAAGTGAAATGTAAATGTAATGAGC-3′
Exon 8 Forward 3′-CCCAATGATACTTTCCTTGTCTCTCTTTC-5′
Reverse 5′-ACTTACTTGAGGCTCTCCTTTAAGAAATTAC-3′
Exon 9 Forward 3′-TAGTTACATTAGAAGTTTGAGTAGTGTGC-5′
Reverse 5′-TTAACATACCTGTCATAGCCTAGAGG-3′
Exon 10 Forward 3′-AGCCCTCAGGTAAATTGGTCTTCC-5′
Reverse 5′-TGACAGATAGAAGGCAAATAAGGTGAC-3′
Exon 11–12 Forward 3′-TGACATAAGGGATGAAGAGACATTTGG-5′
Reverse 5′-TTATCAACCGAAATACGAGCCATACC-3′
Exon 13 Forward 3′-CAATGGAATTATTTACATCGTGCTCTC-5′
Reverse 5′-TTTGAGTATGAAGAACATCAAGTGG-3′
Exon 14 Forward 3′-GGCAACACAAACTGACTGATGATG-5′
Reverse 5′-TTTCTGTTTCTCCATTATGATTTCTTACCTC-3′
Exon 15 Forward 3′-TGTTGCTGTTGTTCTGTGGTCATC-5′
Reverse 5′-ACCGACTTCAAAGCCATTAAACCC-3′
Exon 16 Forward 3′-AAGGGCTTCATTTAATTGTGTGTATTTC-5′
Reverse 5′-CCACCATGACTGACAGATAAGAGAG-3′
Exon 17 Forward 3′-TATGAAGATTAAGTCAAGACCTATAAAGC-5′
Reverse 5′-GACTTTGTGGGTAACCTAATTCAGC-3′
RASEF QPCR Forward 3′-ATCAGACTTCAAAGCACAGAAATGG-5′
RASEF QPCR Reverse 5′-TTCCTCTTCCAACTCACTCAACTG-3′
RASEF Bisulfite Forward 3′-GGGATGGAGGCGGATGGG-5′
RASEF Bisulfite Reverse 5′-CCGCAACTCCGTACACAATACC-3′
RASEF PAP Forward 5′-GGACGGAGAGGAGTTGGTTCGGTTG-ddC-3′
RASEF PAP Reverse 5′-CCGCAACTCCGTACACAATACCCGAAA-ddC-3′
Figure 1.
 
(A) Melting-curve analysis of the RASEF R262C polymorphism in control and primary uveal melanoma and cell lines. Red: heterozygous control and tumor samples; blue: heterozygous primary uveal melanoma samples with a lowered difference plot that is not seen in control samples. (B) A calibration curve was created with dilutions of the T allele in a constant background of the C allele. Based on this curve it was estimated that the relative abundance of the alleles in the primary uveal melanoma samples 13 and 21 was decreased at least 10-fold. (CE) RASEF exon 5 sequence analysis of primary uveal melanomas shows the R262C polymorphism at position 37. The cytosine from the consensus sequence is substituted for a thymine.
Figure 1.
 
(A) Melting-curve analysis of the RASEF R262C polymorphism in control and primary uveal melanoma and cell lines. Red: heterozygous control and tumor samples; blue: heterozygous primary uveal melanoma samples with a lowered difference plot that is not seen in control samples. (B) A calibration curve was created with dilutions of the T allele in a constant background of the C allele. Based on this curve it was estimated that the relative abundance of the alleles in the primary uveal melanoma samples 13 and 21 was decreased at least 10-fold. (CE) RASEF exon 5 sequence analysis of primary uveal melanomas shows the R262C polymorphism at position 37. The cytosine from the consensus sequence is substituted for a thymine.
Figure 2.
 
Expression analysis for RASEF in cell lines and primary uveal melanoma (UM), measured with real-time RT-PCR. Expression was normalized with the control gene RPS11. The change (x-fold) of expression is calculated compared with the median expression level. The RASEF genotypes of the samples are indicated in the graph. UM13 displays loss of the C allele indicated by a lowercase c. UM15 failed in the expression analysis.
Figure 2.
 
Expression analysis for RASEF in cell lines and primary uveal melanoma (UM), measured with real-time RT-PCR. Expression was normalized with the control gene RPS11. The change (x-fold) of expression is calculated compared with the median expression level. The RASEF genotypes of the samples are indicated in the graph. UM13 displays loss of the C allele indicated by a lowercase c. UM15 failed in the expression analysis.
Figure 3.
 
(A) Melting-temperature analysis of amplified RASEF product reveals the methylation of primary uveal melanoma samples and cell lines. Blue: methylated samples; green and purple: samples with a mixed methylation pattern; red: unmethylated samples. (B) Methylation in the promotor region of the RASEF gene in primary uveal melanoma sample 25, as shown by sequence analysis after PAP. After bisulfite treatment and PCR, unmethylated cytosines converted into thymidine. Methylated cytosines remained unchanged.
Figure 3.
 
(A) Melting-temperature analysis of amplified RASEF product reveals the methylation of primary uveal melanoma samples and cell lines. Blue: methylated samples; green and purple: samples with a mixed methylation pattern; red: unmethylated samples. (B) Methylation in the promotor region of the RASEF gene in primary uveal melanoma sample 25, as shown by sequence analysis after PAP. After bisulfite treatment and PCR, unmethylated cytosines converted into thymidine. Methylated cytosines remained unchanged.
Figure 4.
 
Kaplan-Meier analysis and log rank test showed the difference in survival according to genotype and presence or absence of methylation of the promotor region of the RASEF gene (P = 0.019).
Figure 4.
 
Kaplan-Meier analysis and log rank test showed the difference in survival according to genotype and presence or absence of methylation of the promotor region of the RASEF gene (P = 0.019).
Figure 5.
 
Digestion analysis of DNA from a patient in whom the tumor (UM35, right) showed a nearly complete homozygous T-allele genotype revealed a heterozygous genotype in leukocyte DNA (left), confirming the mechanism of LOH in tumor tissue.
Figure 5.
 
Digestion analysis of DNA from a patient in whom the tumor (UM35, right) showed a nearly complete homozygous T-allele genotype revealed a heterozygous genotype in leukocyte DNA (left), confirming the mechanism of LOH in tumor tissue.
The authors thank Bruce R. Ksander (Schepens Eye Institute, Harvard Medical School, Boston, MA) for cell lines Mel-202, -270, -285, and -290, and OMM-2.3 and -2.5, June Kan-Mitchell (Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI) for cell lines OCM-1, -3, and -8, and Rolf H. A. M. Vossen (Department of Human and Clinical Genetics, LUMC, Leiden, The Netherlands) for technical assistance. 
EganKM, SeddonJM, GlynnRJ, GragoudasES, AlbertDM. Epidemiologic aspects of uveal melanoma. Surv Ophthalmol. 1988;32:239–251. [CrossRef] [PubMed]
KodjikianL, NguyenK, LumbrosoL, et al. Familial uveal melanoma: a report on two families and a review of the literature. Acta Ophthalmol Scand. 2003;81:389–395. [CrossRef] [PubMed]
SmithJH, Padnick-SilverL, NewlinA, RhodesK, RubinsteinWS. Genetic study of familial uveal melanoma: association of uveal and cutaneous melanoma with cutaneous and ocular nevi. Ophthalmology. 2007;114:774–779. [CrossRef] [PubMed]
van HeesCL, JagerMJ, BleekerJC, KemmeH, BergmanW. Occurrence of cutaneous and uveal melanoma in patients with uveal melanoma and their first degree relatives. Melanoma Res. 1998;8:175–180. [CrossRef] [PubMed]
JönssonG, BendahlPO, SandbergT, et al. Mapping of a novel ocular and cutaneous malignant melanoma susceptibility locus to chromosome 9q21.32. J Natl Cancer Inst. 2005;97:1377–1382. [CrossRef] [PubMed]
KopfI, StiernerU, IslamQ, DelleU, KindblomLG, MartinssonT. Characterization of four melanoma cell lines with electron microscopy, immunocytochemistry, cytogenetics, flow cytometry, and southern analysis. Cancer Genet Cytogenet. 1992;62:111–123. [CrossRef] [PubMed]
AlbinoAP, SozziG, NanusDM, JhanwarSC, HoughtonAN. Malignant transformation of human melanocytes: induction of a complete melanoma phenotype and genotype. Oncogene. 1992;7:2315–2321. [PubMed]
KumarR, SmedsJ, LundhRB, HemminkiK. Loss of heterozygosity at chromosome 9p21 (INK4–p14ARF locus): homozygous deletions and mutations in the p16 and p14ARF genes in sporadic primary melanomas. Melanoma Res. 1999;9:138–147. [CrossRef] [PubMed]
BoniR, VortmeyerAO, BurgG, HofbauerG, ZhuangZ. The PTEN tumour suppressor gene and malignant melanoma. Melanoma Res. 1998;8:300–302. [CrossRef] [PubMed]
ZhuG, MontgomeryGW, JamesMR, et al. A genome-wide scan for naevus count: linkage to CDKN2A and to other chromosome regions. Eur J Hum Genet. 2007;15:94–102. [CrossRef] [PubMed]
StarkM, HaywardN. Genome-wide loss of heterozygosity and copy number analysis in melanoma using high-density single-nucleotide polymorphism arrays. Cancer Res. 2007;67:2632–2642. [CrossRef] [PubMed]
BrobergK, HoglundM, GustafssonC, et al. Genetic variant of the human homologous recombination-associated gene RMI1 (S455N) impacts the risk of AML/MDS and malignant melanoma. Cancer Lett. 2007;258:38–44. [CrossRef] [PubMed]
Cannon-AlbrightLA, GoldgarDE, MeyerLJ, et al. Assignment of a locus for familial melanoma, MLM, to chromosome 9p13–p22. Science. 1992;258:1148–1152. [CrossRef] [PubMed]
CohenC, Zavala-PompaA, SequeiraJH, et al. Mitogen-activated protein kinase activation is an early event in melanoma progression. Clin Cancer Res. 2002;8:3728–3733. [PubMed]
SatyamoorthyK, LiG, GerreroMR, et al. Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation. Cancer Res. 2003;63:756–759. [PubMed]
CalipelA, MouriauxF, GlotinAL, MalecazeF, FaussatAM, MascarelliF. Extracellular signal-regulated kinase-dependent proliferation is mediated through the protein kinase A/B-Raf pathway in human uveal melanoma cells. J Biol Chem. 2006;281:9238–9250. [CrossRef] [PubMed]
WeberA, HenggeUR, UrbanikD, et al. Absence of mutations of the BRAF gene and constitutive activation of extracellular-regulated kinase in malignant melanomas of the uvea. Lab Invest. 2003;83:1771–1776. [CrossRef] [PubMed]
van der VeldenPA, Metzelaar-BlokJA, BergmanW, et al. Promoter hypermethylation: a common cause of reduced p16(INK4a) expression in uveal melanoma. Cancer Res. 2001;61:5303–5306. [PubMed]
DaviesH, BignellGR, CoxC, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949–954. [CrossRef] [PubMed]
van ElsasA, ZerpSF, van der FlierS, et al. Relevance of ultraviolet-induced N-ras oncogene point mutations in development of primary human cutaneous melanoma. Am J Pathol. 1996;149:883–893. [PubMed]
ZuidervaartW, van NieuwpoortF, StarkM, et al. Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS. Br J Cancer. 2005;92:2032–2038. [CrossRef] [PubMed]
SweetserDA, PeniketAJ, HaalandC, et al. Delineation of the minimal commonly deleted segment and identification of candidate tumor-suppressor genes in del(9q) acute myeloid leukemia. Genes Chromosomes Cancer. 2005;44:279–291. [CrossRef] [PubMed]
MaatW, van der VeldenPA, Out-LuitingC, et al. Epigenetic inactivation of RASSF1a in uveal melanoma. Invest Ophthalmol Vis Sci. 2007;48:486–490. [CrossRef] [PubMed]
LiuQ, SommerSS. Pyrophosphorolysis by Type II DNA polymerases: implications for pyrophosphorolysis-activated polymerization. Anal Biochem. 2004;324:22–28. [CrossRef] [PubMed]
LiuQ, SommerSS. PAP: detection of ultra rare mutations depends on P* oligonucleotides: “sleeping beauties” awakened by the kiss of pyrophosphorolysis. Hum Mutat. 2004;23:426–436. [CrossRef] [PubMed]
PaduaRA, BarrassN, CurrieGA. A novel transforming gene in a human malignant melanoma cell line. Nature. 1984;311:671–673. [CrossRef] [PubMed]
RimoldiD, SalviS, LienardD, et al. Lack of BRAF mutations in uveal melanoma. Cancer Res. 2003;63:5712–5715. [PubMed]
Figure 1.
 
(A) Melting-curve analysis of the RASEF R262C polymorphism in control and primary uveal melanoma and cell lines. Red: heterozygous control and tumor samples; blue: heterozygous primary uveal melanoma samples with a lowered difference plot that is not seen in control samples. (B) A calibration curve was created with dilutions of the T allele in a constant background of the C allele. Based on this curve it was estimated that the relative abundance of the alleles in the primary uveal melanoma samples 13 and 21 was decreased at least 10-fold. (CE) RASEF exon 5 sequence analysis of primary uveal melanomas shows the R262C polymorphism at position 37. The cytosine from the consensus sequence is substituted for a thymine.
Figure 1.
 
(A) Melting-curve analysis of the RASEF R262C polymorphism in control and primary uveal melanoma and cell lines. Red: heterozygous control and tumor samples; blue: heterozygous primary uveal melanoma samples with a lowered difference plot that is not seen in control samples. (B) A calibration curve was created with dilutions of the T allele in a constant background of the C allele. Based on this curve it was estimated that the relative abundance of the alleles in the primary uveal melanoma samples 13 and 21 was decreased at least 10-fold. (CE) RASEF exon 5 sequence analysis of primary uveal melanomas shows the R262C polymorphism at position 37. The cytosine from the consensus sequence is substituted for a thymine.
Figure 2.
 
Expression analysis for RASEF in cell lines and primary uveal melanoma (UM), measured with real-time RT-PCR. Expression was normalized with the control gene RPS11. The change (x-fold) of expression is calculated compared with the median expression level. The RASEF genotypes of the samples are indicated in the graph. UM13 displays loss of the C allele indicated by a lowercase c. UM15 failed in the expression analysis.
Figure 2.
 
Expression analysis for RASEF in cell lines and primary uveal melanoma (UM), measured with real-time RT-PCR. Expression was normalized with the control gene RPS11. The change (x-fold) of expression is calculated compared with the median expression level. The RASEF genotypes of the samples are indicated in the graph. UM13 displays loss of the C allele indicated by a lowercase c. UM15 failed in the expression analysis.
Figure 3.
 
(A) Melting-temperature analysis of amplified RASEF product reveals the methylation of primary uveal melanoma samples and cell lines. Blue: methylated samples; green and purple: samples with a mixed methylation pattern; red: unmethylated samples. (B) Methylation in the promotor region of the RASEF gene in primary uveal melanoma sample 25, as shown by sequence analysis after PAP. After bisulfite treatment and PCR, unmethylated cytosines converted into thymidine. Methylated cytosines remained unchanged.
Figure 3.
 
(A) Melting-temperature analysis of amplified RASEF product reveals the methylation of primary uveal melanoma samples and cell lines. Blue: methylated samples; green and purple: samples with a mixed methylation pattern; red: unmethylated samples. (B) Methylation in the promotor region of the RASEF gene in primary uveal melanoma sample 25, as shown by sequence analysis after PAP. After bisulfite treatment and PCR, unmethylated cytosines converted into thymidine. Methylated cytosines remained unchanged.
Figure 4.
 
Kaplan-Meier analysis and log rank test showed the difference in survival according to genotype and presence or absence of methylation of the promotor region of the RASEF gene (P = 0.019).
Figure 4.
 
Kaplan-Meier analysis and log rank test showed the difference in survival according to genotype and presence or absence of methylation of the promotor region of the RASEF gene (P = 0.019).
Figure 5.
 
Digestion analysis of DNA from a patient in whom the tumor (UM35, right) showed a nearly complete homozygous T-allele genotype revealed a heterozygous genotype in leukocyte DNA (left), confirming the mechanism of LOH in tumor tissue.
Figure 5.
 
Digestion analysis of DNA from a patient in whom the tumor (UM35, right) showed a nearly complete homozygous T-allele genotype revealed a heterozygous genotype in leukocyte DNA (left), confirming the mechanism of LOH in tumor tissue.
Table 1.
 
Tumor Characteristics and Survival Data of 35 Uveal Melanoma Patients Sorted by Methylation Status and RASEF Genotype
Table 1.
 
Tumor Characteristics and Survival Data of 35 Uveal Melanoma Patients Sorted by Methylation Status and RASEF Genotype
Tumor ID Cell Type Survival (mo) Present Status Hypermethylated RASEF Genotype
UM1 Spindle 210 Alive Not present Hom C
UM16 Epithelioid 115 Alive Not present Hom C
UM29 Spindle 24 Died, due to metastases Not present Hom C
UM3 Mixed 95 Died, due to metastases Not present Hom C
UM33 Mixed 24 Died, due to metastases Not present Hom C
UM12 Epithelioid 137 Died, due to metastases Not present Hom T
UM19 Spindle 15 Died, due to metastases Not present Hom T
UM26 Spindle 122 Alive Not present Hom T
UM31 Mixed 34 Died, due to metastases Not present Hom T
UM32 Epithelioid 63 Died, due to metastases Not present Hom T
UM4 Spindle 31 Died, due to metastases Not present Hom T
UM6 Epithelioid 57 Died, due to metastases Not present Hom T
UM27 Mixed 23 Died, due to metastases Present Hom C
UM28 Epithelioid 33 Died, due to metastases Present Hom C
UM30 Spindle 113 Alive Present Hom C
UM11 Mixed 13 Died, due to metastases Present Hom T
UM18 Spindle 12 Died, due to metastases Present Hom T
UM5 Spindle 50 Died, due to metastases Present Hom T
UM35 Mixed 94 Alive Present Het/homT*
UM21 Epithelioid 167 Died, due to metastases Present Het/homT*
UM13 Epithelioid 30 Died, due to metastases Present Het/homT*
UM15 Mixed 23 Died, due to metastases Present Het
UM17 Epithelioid 33 Died, due to metastases Present Het
UM2 Mixed 29 Died, due to metastases Not present Het
UM14 Mixed 42 Died, due to metastases Not present Het
UM25 Mixed 29 Died, other cause Not present Het
UM7 Mixed 63 Died, other cause Not present Het
UM23 Spindle 152 Died, unknown cause Not present Het
UM22 Spindle 2 Lost to follow up Not present Het
UM8 Spindle 191 Alive Not present Het
UM9 Mixed 131 Alive Not present Het
UM10 Mixed 136 Alive Not present Het
UM20 Mixed 187 Alive Not present Het
UM24 Mixed 143 Alive Not present Het
UM34 Mixed 106 Alive Not present Het
Table 2.
 
RASEF Primers Used for Mutation Detection, Expression Analysis, and Methylation Analysis
Table 2.
 
RASEF Primers Used for Mutation Detection, Expression Analysis, and Methylation Analysis
Primer Sequence
Exon 1 Forward 3′-GGCAAGCAGCGGTGGACTC-5′
Reverse 5′-GTAGGTGAAGGAAGACAAGCAACTC-3′
Exon 2 Forward 3′-TCTTCCCTTCCTTCCGTTCATTCTG-5′
Reverse 5′-GTCCACCTATATCATAGTGTGACAATGC-3′
Exon 3 Forward 3′-TTCTCTTCATCTGTAATATATAGGGCTTAACG-5′
Reverse 5′-CCCTCTCCGTAGAAACCACCTC-3′
Exon 4 Forward 3′-TCACCTTCCCTGTGTAGGAGAAC-5′
Reverse 5′-CTGAGATGCTGAGGCTGTTCC-3′
Exon 5 Forward 3′-CAAAGCAATTCAAAGTGAGTTTGTAAGC-5′
Reverse 5′-TGAGGATGTGGTCTAACAGGAAGTG-3′
Exon 6 Forward 3′-GTGTGGGAGGGTGACAGGAC-5′
Reverse 5′-AAATCATTAGAAAGTAAAGAAGATATTAGCAAAG-3′
Exon 7 Forward 3′-AAAGGGTCTGGGAGGGTAGG-5′
Reverse 5′-AAACAAGTGAAATGTAAATGTAATGAGC-3′
Exon 8 Forward 3′-CCCAATGATACTTTCCTTGTCTCTCTTTC-5′
Reverse 5′-ACTTACTTGAGGCTCTCCTTTAAGAAATTAC-3′
Exon 9 Forward 3′-TAGTTACATTAGAAGTTTGAGTAGTGTGC-5′
Reverse 5′-TTAACATACCTGTCATAGCCTAGAGG-3′
Exon 10 Forward 3′-AGCCCTCAGGTAAATTGGTCTTCC-5′
Reverse 5′-TGACAGATAGAAGGCAAATAAGGTGAC-3′
Exon 11–12 Forward 3′-TGACATAAGGGATGAAGAGACATTTGG-5′
Reverse 5′-TTATCAACCGAAATACGAGCCATACC-3′
Exon 13 Forward 3′-CAATGGAATTATTTACATCGTGCTCTC-5′
Reverse 5′-TTTGAGTATGAAGAACATCAAGTGG-3′
Exon 14 Forward 3′-GGCAACACAAACTGACTGATGATG-5′
Reverse 5′-TTTCTGTTTCTCCATTATGATTTCTTACCTC-3′
Exon 15 Forward 3′-TGTTGCTGTTGTTCTGTGGTCATC-5′
Reverse 5′-ACCGACTTCAAAGCCATTAAACCC-3′
Exon 16 Forward 3′-AAGGGCTTCATTTAATTGTGTGTATTTC-5′
Reverse 5′-CCACCATGACTGACAGATAAGAGAG-3′
Exon 17 Forward 3′-TATGAAGATTAAGTCAAGACCTATAAAGC-5′
Reverse 5′-GACTTTGTGGGTAACCTAATTCAGC-3′
RASEF QPCR Forward 3′-ATCAGACTTCAAAGCACAGAAATGG-5′
RASEF QPCR Reverse 5′-TTCCTCTTCCAACTCACTCAACTG-3′
RASEF Bisulfite Forward 3′-GGGATGGAGGCGGATGGG-5′
RASEF Bisulfite Reverse 5′-CCGCAACTCCGTACACAATACC-3′
RASEF PAP Forward 5′-GGACGGAGAGGAGTTGGTTCGGTTG-ddC-3′
RASEF PAP Reverse 5′-CCGCAACTCCGTACACAATACCCGAAA-ddC-3′
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