Lázcoz Ripoll, Paula

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https://academica-e.unavarra.es/handle/2454/49387

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Lázcoz Ripoll

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Paula

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Ciencias de la Salud

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1172611

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7324

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Subject: RASSF1A ×

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Now showing 1 - 2 of 2
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    PublicationOpen Access
    Promoter methylation of RASSF1A associates to adult secondary glioblastomas and pediatric glioblastomas
    (Hindawi / Wiley, 2012) Muñoz, Jorge; Inda, María del Mar; Lázcoz Ripoll, Paula; Zazpe Cenoz, Idoya; Fan, Xing; Alfaro, Jorge; Tuñón Álvarez, Teresa; Rey, Juan A.; Castresana, Javier S.; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14ARF, and p16INK4A), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14ARF and p16INK4A did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14ARF and p16INK4A, in which other alterations (mutations, homozygous deletions) are prevalent.
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    PublicationOpen Access
    Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR
    (BioMed Central, 2008) Lorente, Aiala; Mueller, Wolf; Urdangarín, Edurne; Lázcoz Ripoll, Paula; Deimling, Andreas von; Sáez Castresana, Javier; Ciencias de la Salud; Osasun Zientziak
    Background: We present two melting curve analysis (MCA)-based semiquantitative real time PCR techniques to detect the promoter methylation status of genes. The first, MCA-MSP, follows the same principle as standard MSP but it is performed in a real time thermalcycler with results being visualized in a melting curve. The second, MCA-Meth, uses a single pair of primers designed with no CpGs in its sequence. These primers amplify both unmethylated and methylated sequences. In clinical applications the MSP technique has revolutionized methylation detection by simplifying the analysis to a PCR-based protocol. MCA-analysis based techniques may be able to further improve and simplify methylation analyses by reducing starting DNA amounts, by introducing an all-in-one tube reaction and by eliminating a final gel stage for visualization of the result. The current study aimed at investigating the feasibility of both MCA-MSP and MCA-Meth in the analysis of promoter methylation, and at defining potential advantages and shortcomings in comparison to currently implemented techniques, i. e. bisulfite sequencing and standard MSP. Methods: The promoters of the RASSF1A (3p21.3), BLU (3p21.3) and MGMT (10q26) genes were analyzed by MCA-MSP and MCA-Meth in 13 astrocytoma samples, 6 high grade glioma cell lines and 4 neuroblastoma cell lines. The data were compared with standard MSP and validated by bisulfite sequencing. Results: Both, MCA-MSP and MCA-Meth, successfully determined promoter methylation. MCA-MSP provided information similar to standard MSP analyses. However the analysis was possible in a single tube and avoided the gel stage. MCA-Meth proved to be useful in samples with intermediate methylation status, reflected by a melting curve position shift in dependence on methylation extent. Conclusion: We propose MCA-MSP and MCA-Meth as alternative or supplementary techniques to MSP or bisulfite sequencing.