RESUMO
BACKGROUND: MLH1 epimutation is characterised by constitutional monoallelic MLH1 promoter hypermethylation, which can cause colorectal cancer (CRC). Tumour molecular profiles of MLH1 epimutation CRCs were used to classify germline MLH1 promoter variants of uncertain significance and MLH1 methylated early-onset CRCs (EOCRCs). Genome-wide DNA methylation and somatic mutational profiles of tumours from two germline MLH1: c.-11C > T and one MLH1: c.-[28A > G; 7C > T] carriers and three MLH1 methylated EOCRCs (< 45 years) were compared with 38 reference CRCs. Methylation-sensitive droplet digital PCR (ddPCR) was used to detect mosaic MLH1 methylation in blood, normal mucosa and buccal DNA. RESULTS: Genome-wide methylation-based Consensus Clustering identified four clusters where the tumour methylation profiles of germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs clustered with the constitutional MLH1 epimutation CRCs but not with the sporadic MLH1 methylated CRCs. Furthermore, monoallelic MLH1 methylation and APC promoter hypermethylation in tumour were observed in both MLH1 epimutation and germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs. Mosaic constitutional MLH1 methylation in MLH1: c.-11C > T carriers and 1 of 3 MLH1 methylated EOCRCs was identified by methylation-sensitive ddPCR. CONCLUSIONS: Mosaic MLH1 epimutation underlies the CRC aetiology in MLH1: c.-11C > T germline carriers and a subset of MLH1 methylated EOCRCs. Tumour profiling and ultra-sensitive ddPCR methylation testing can be used to identify mosaic MLH1 epimutation carriers.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose , Neoplasias Colorretais , Humanos , Metilação de DNA , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/genética , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase , DNA , Neoplasias Colorretais/genética , Proteína 1 Homóloga a MutL/genéticaRESUMO
Pheochromocytomas (PC) and paragangliomas (PGL) are endocrine tumors for which the genetic and clinicopathological features of metastatic progression remain incompletely understood. As a result, the risk of metastasis from a primary tumor cannot be predicted. Early diagnosis of individuals at high risk of developing metastases is clinically important and the identification of new biomarkers that are predictive of metastatic potential is of high value. Activation of TERT has been associated with a number of malignant tumors, including PC/PGL. However, the mechanism of TERT activation in the majority of PC/PGL remains unclear. As TERT promoter mutations occur rarely in PC/PGL, we hypothesized that other mechanisms - such as structural variations - may underlie TERT activation in these tumors. From 35 PC and four PGL, we identified three primary PCs that developed metastases with elevated TERT expression, each of which lacked TERT promoter mutations and promoter DNA methylation. Using whole genome sequencing, we identified somatic structural alterations proximal to the TERT locus in two of these tumors. In both tumors, the genomic rearrangements led to the positioning of super-enhancers proximal to the TERT promoter, that are likely responsible for the activation of the normally tightly repressed TERT expression in chromaffin cells.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Biomarcadores Tumorais/genética , Mutação , Paraganglioma/genética , Feocromocitoma/genética , Regiões Promotoras Genéticas , Telomerase/genética , Neoplasias das Glândulas Suprarrenais/secundário , Metilação de DNA , Predisposição Genética para Doença , Humanos , Paraganglioma/patologia , Feocromocitoma/patologia , Prognóstico , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: DNA methylation is a complex epigenetic marker that can be analyzed using a wide variety of methods. Interpretation and visualization of DNA methylation data can mask complexity in terms of methylation status at each CpG site, cellular heterogeneity of samples and allelic DNA methylation patterns within a given DNA strand. Bisulfite sequencing is considered the gold standard, but visualization of massively parallel sequencing results remains a significant challenge. FINDINGS: We created a program called Methpat that facilitates visualization and interpretation of bisulfite sequencing data generated by massively parallel sequencing. To demonstrate this, we performed multiplex PCR that targeted 48 regions of interest across 86 human samples. The regions selected included known gene promoters associated with cancer, repetitive elements, known imprinted regions and mitochondrial genomic sequences. We interrogated a range of samples including human cell lines, primary tumours and primary tissue samples. Methpat generates two forms of output: a tab-delimited text file for each sample that summarizes DNA methylation patterns and their read counts for each amplicon, and a HTML file that summarizes this data visually. Methpat can be used with publicly available whole genome bisulfite sequencing and reduced representation bisulfite sequencing datasets with sufficient read depths. CONCLUSIONS: Using Methpat, complex DNA methylation data derived from massively parallel sequencing can be summarized and visualized for biological interpretation. By accounting for allelic DNA methylation states and their abundance in a sample, Methpat can unmask the complexity of DNA methylation and yield further biological insight in existing datasets.
Assuntos
Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA/métodos , Software , Linhagem Celular , Humanos , Neoplasias/genética , Especificidade de ÓrgãosRESUMO
BACKGROUND: DNA hypermethylation is reported as a frequent event and prognostic marker in head and neck squamous cell carcinomas (HNSCC). Methylation has been commonly assessed with non-quantitative methodologies, such as methylation-specific PCR (MSP). We investigated previously reported hypermethylated genes with quantitative methodology in oral tongue squamous cell carcinomas (OTSCC). RESULTS: The methylation status of 12 genes in 115 OTSCC samples was assessed by one or more of three quantitative analyses: methylation sensitive high resolution melting (MS-HRM), sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP), and bisulfite pyrosequencing. In contrast to much of the literature, either no or infrequent locus-specific methylation was identified by MS-HRM for DAPK1, RASSF1A, MGMT, MLH1, APC, CDH1, CDH13, BRCA1, ERCC1, and ATM. The most frequently methylated loci were RUNX3 (18/108 methylated) and ABO (22/107 methylated). Interrogation of the Cancer Genome Atlas (TCGA) HNSCC cohort confirmed the frequency of significant methylation for the loci investigated. Heterogeneous methylation of RUNX3 (18/108) and ABO (22/107) detected by MS-HRM, conferred significantly worse survival (P = 0.01, and P = 0.03). However, following quantification of methylation levels using pyrosequencing, only four tumors had significant quantities (>15%) of RUNX3 methylation which correlated with a worse patient outcome (P <0.001), while the prognostic significance of ABO hypermethylation was lost. RUNX3 methylation was not prognostic for the TCGA cohort (P = 0.76). CONCLUSIONS: We demonstrated the critical need for quantification of methylation levels and its impact on correlative analyses. In OTSCC, we found little evidence of significant or frequent hypermethylation of many loci reported to be commonly methylated. It is likely that previous reports have overestimated the frequency of significant methylation events as a consequence of the use of non-quantitative methodology.
RESUMO
Epigenetic silencing by promoter methylation of genes associated with cancer initiation and progression is a hallmark of tumour cells. As a consequence, testing for DNA methylation biomarkers in plasma or other body fluids shows great promise for detection of malignancies at early stages and/or for monitoring response to treatment. However, DNA from normal leukocytes may contribute to the DNA in plasma and will affect biomarker specificity if there is any methylation in the leukocytes. DNA from 48 samples of normal peripheral blood mononuclear cells was evaluated for the presence of methylation of a panel of DNA methylation biomarkers that have been implicated in cancer. SMART-MSP, a methylation specific PCR (MSP) methodology based on real time PCR amplification, high-resolution melting and strategic primer design, enabled quantitative detection of low levels of methylated DNA. Methylation was observed in all tested mononuclear cell DNA samples for the CDH1 and HIC1 promoters and in majority of DNA samples for the TWIST1 and DAPK1 promoters. APC and RARB promoter methylation, at a lower average level, was also detected in a substantial proportion of DNA samples. We found no BRCA1, CDKN2A, GSTP1 and RASSF1A promoter methylation in this sample set. Several individuals had higher levels of methylation at several loci suggestive of a methylator phenotype. In conclusion, methylation of many potential DNA methylation biomarkers can be detected in normal peripheral blood mononuclear cells, and is likely to affect their specificity for detecting low level disease. However, we found no evidence of promoter methylation for other genes indicating that panels of analytically sensitive and specific methylation biomarkers in body fluids can be obtained.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Leucócitos Mononucleares/metabolismo , Neoplasias/genética , Antígenos CD , Biomarcadores Tumorais , Caderinas/genética , Caderinas/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Leucócitos Mononucleares/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Metilação de DNA , DNA/química , Análise de Sequência de DNA/métodos , Proteínas Reguladoras de Apoptose/metabolismo , Sequência de Bases , Biotinilação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Associadas com Morte Celular , Técnicas Genéticas , Humanos , Transição de Fase , Reação em Cadeia da Polimerase , Sulfitos/química , Células Tumorais CultivadasRESUMO
DNA methylation based biomarkers have considerable potential for molecular diagnostics, both as tumor specific biomarkers for the early detection or post-therapeutic monitoring of cancer as well as prognostic and predictive biomarkers for therapeutic stratification. Particularly in the former, the accurate estimation of DNA methylation is of compelling importance. However, quantification of DNA methylation has many traps for the unwary, especially when heterogeneous methylation comprising multiple alleles with varied DNA methylation patterns (epialleles) is present. The frequent occurrence of heterogeneous methylation as distinct from a simple mixture of fully methylated and unmethylated alleles is generally not taken into account when DNA methylation is considered as a cancer biomarker. When heterogeneous DNA methylation is present, the proportion of methylated molecules is difficult to quantify without a method that allows the measurement of individual epialleles. In this article, we critically assess the methodologies frequently used to investigate DNA methylation, with an emphasis on the detection and measurement of heterogeneous DNA methylation. The adoption of digital approaches will enable the effective use of heterogeneous DNA methylation as a cancer biomarker.
Assuntos
Alelos , Biomarcadores Tumorais/metabolismo , Metilação de DNA/fisiologia , Heterogeneidade Genética , Neoplasias/diagnóstico , Ilhas de CpG/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Metilação de DNA/genética , Humanos , Espectrometria de Massas/métodos , Neoplasias/metabolismo , Reação em Cadeia da Polimerase/métodos , Mapeamento por Restrição/métodos , Análise de Sequência de DNA/métodos , Sulfitos , Temperatura de TransiçãoRESUMO
Methylation of the CpG island in the MGMT promoter region is a frequent event in several cancer types including colorectal cancer, lung cancer, lymphoma, and glioblastoma. A correlation between methylation and the T allele of the rs16906252 single nucleotide polymorphism (SNP) in colorectal carcinomas has previously been reported. As aberrant MGMT methylation can be an early event in tumor development, we tested the hypothesis that normal individuals possessing the T allele may be predisposed to somatic methylation at the MGMT promoter. Peripheral blood monononuclear cell DNA from 89 normal, healthy individuals was genotyped at rs1690625 and assessed for the methylation status of the MGMT promoter region using independent quantitative methodologies capable of detecting low-level methylation: MethyLight and Sensitive Melting Analysis after Real-time Methylation-Specific PCR (SMART-MSP). There was a strong association between presence of the T allele and detectable methylation (P = 0.00005) in the peripheral blood DNA. Furthermore, when a MSP assay flanking the SNP was used to amplify methylated sequences in heterozygotes, only the T allele was methylated. Thus, detectable somatic methylation of the MGMT promoter in normal individuals is strongly associated with the T allele of the rs16906252 MGMT promoter SNP.
Assuntos
Alelos , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Proteínas Supressoras de Tumor/genética , Sequência de Bases , Predisposição Genética para Doença , Genótipo , Humanos , Neoplasias/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the CDKN2B (p15) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies of the myeloid lineage. Its promoter region usually shows heterogeneous methylation and is only rarely fully methylated. The methylation status of CDKN2B can be used as a biomarker of response to treatment. Therefore the accurate characterisation of its methylation is desirable. RESULTS: MS-HRM was used to assess CDKN2B methylation in acute myeloid leukaemia (AML) samples. All the AML samples that were methylated at the CDKN2B promoter (40/93) showed varying degrees of heterogeneous methylation. Six representative samples were selected for further study. dMS-HRM was used to simultaneously count the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM. CONCLUSION: dMS-HRM is a powerful technique for the analysis of methylation in CDKN2B and other heterogeneously methylated genes. It eliminates both PCR and cloning bias towards either methylated or unmethylated DNA. Potentially complex information is simplified into a digital output, allowing counting of methylated and unmethylated alleles and providing an overall picture of methylation at the given locus. Downstream sequencing is minimised as dMS-HRM acts as a screen to select only methylated clones for further analysis.