RESUMO
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.
Assuntos
Epigênese Genética , Genes Supressores de Tumor/fisiologia , Melanoma/genética , Neoplasias Uveais/genética , Fatores ras de Troca de Nucleotídeo Guanina/genética , Linhagem Celular Tumoral , Metilação de DNA , Análise Mutacional de DNA , Regulação Neoplásica da Expressão Gênica/fisiologia , Inativação Gênica , Genótipo , Humanos , Melanoma/metabolismo , Mutação , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Neoplasias Uveais/mortalidadeAssuntos
Armazenamento de Sangue/métodos , Doadores de Sangue/classificação , União Europeia , Técnicas Genéticas , Bancos de Sangue/tendências , Antígenos de Grupos Sanguíneos/análise , Antígenos de Grupos Sanguíneos/genética , Ensaios Clínicos como Assunto , Técnicas Genéticas/tendências , Genótipo , HumanosRESUMO
BACKGROUND: In the Netherlands, 500,000 blood donors are active. Blood of all donors is currently typed serologically for ABO, the Rh phenotype, and K. Only a subset of donors is typed twice for a larger set of red cell (RBC) and/or platelet (PLT) antigens. To increase the direct availability of typed RBCs and PLTs, a high-throughput technique is being developed to genotype the whole donor cohort for all clinically relevant RBC and PLT antigens. STUDY DESIGN AND METHODS: A multiplex polymerase chain reaction was developed to both amplify and fluorescently label 19 gene fragments of RBC and PLT antigens in one reaction. To test the setup of the genotyping method by microarray, a pilot study with human PLT antigen (HPA)-typed donor samples was performed. On each slide, 12 arrays are present containing 20 probes per PLT antigen system (28 for HPA-3). The allele-specific oligohybridization method was used to discriminate between two different alleles. RESULTS: Two blinded panels encompassing 94 donors were genotyped for HPA-1 through -5 and -15; no discrepancies were found compared to their serologic typing (HPA-1, -2, -3, -4, and -5) and genotyping (HPA-15; TaqMan, Applied Biosystems). CONCLUSION: This study shows that the HPA microarray provides a reliable and fast genotyping procedure. With further development an automated throughput for complete typing of large donor cohorts can be obtained.
