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At least 649 records · Page 36Linked to original sources
PubMed2003

Bisulfite conversion-specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation.

Methylation-specific PCR (MSP) is frequently used to distinguish methylated alleles in the genome. Sequences that have been incompletely converted during bisulfite treatment are frequently co-amplified during MSP. For accurate MSP, it is important to detect methylated sequences in a background of unconverted DNA with a high level of sensitivity. We report here sensitive techniques, bisulfite conversion-specific MSP (BS-MSP) to accurately evaluate CpG methylation. BS-MSP provides accurate results across a wide spectrum of bisulfite conversion levels. BS-MSP is also confirmed to be a useful technique for the routine analysis of clinical tumor specimens that were paraffin-embedded and microdissected. BS-MSP thus provides the powerful features of ease of use and compatibility with paraffin sections. We recommend that methylation analysis should include a step to eliminate unconverted DNA to avoid overestimation of the DNA methylation level in the samples.

Cadherins↗
PubMed2003

Strand-biased DNA methylation associated with centromeric regions in Arabidopsis.

The Arabidopsis genome project assembled 15 megabases of heterochromatic sequence, facilitating investigations of heterochromatin assembly, maintenance, and structure. In many species, large quantities of methylcytosine decorate heterochromatin; these modifications are typically maintained by methyltransferases that recognize newly replicated hemimethylated DNA. We assessed the extent and patterns of Arabidopsis heterochromatin methylation by amplifying and sequencing genomic DNA treated with bisulfite, which converts cytosine, but not methylcytosine, to uracil. This survey revealed unexpected asymmetries in methylation patterns, with one helix strand often exhibiting higher levels of methylation. We confirmed these observations both by immunoprecipitating methylated DNA strands and by restriction enzyme digestion of amplified, bisulfite-treated DNA. We also developed a primer-extension assay that can monitor the methylation status of an entire chromosome, demonstrating that strand-specific methylation occurs predominantly in the centromeric regions. Conventional models for methylation maintenance do not explain these unusual patterns; instead, new models that allow for strand specificity are required. The abundance of Arabidopsis strand-specific modifications points to their importance, perhaps as epigenetic signals that mark the heterochromatic regions that confer centromere activity.

Arabidopsis↗
PubMed1999

Progressive region-specific de novo methylation of the p16 CpG island in primary human mammary epithelial cell strains during escape from M(0) growth arrest.

CpG island methylation plays an important role in normal cellular processes, such as genomic imprinting and X-chromosome inactivation, as well as in abnormal processes, such as neoplasia. However, the dynamics of de novo CpG island methylation, during which a CpG island is converted from an unmethylated, active state to a densely methylated, inactive state, are largely unknown. It is unclear whether the development of de novo CpG island methylation is a progressive process, in which a subset of CpG sites are initially methylated with a subsequent increase in methylation density, or a single event, in which the initial methylation event encompasses the entire CpG island. The tumor suppressor gene p16/CDKN2a/INK4a (p16) is inactivated by CpG island methylation during neoplastic progression in a variety of human cancers. We investigated the development of methylation in the p16 CpG island in primary human mammary epithelial cell strains during escape from mortality stage 0 (M(0)) growth arrest. The methylation status of 47 CpG sites in the p16 CpG island on individual DNA molecules was determined by sequencing PCR clones of bisulfite-treated genomic DNA. The p16 CpG island was initially methylated at a subset of sites in three discrete regions in association with p16 transcriptional repression and escape from M(0) growth arrest. With continued passage, methylation gradually increased in density and methylation expanded to sites in adjacent regions. Thus, de novo methylation in the p16 CpG island is a progressive process that is neither site specific nor completely random but instead is region specific. Our results suggest that early detection of methylation in the CpG island of the p16 gene will require methylation analysis of the three regions and that the identification of region-specific methylation patterns in other genes may be essential for an accurate assessment of methylation-mediated transcriptional silencing.

Breast↗
PubMed2004

Hypermethylation of growth arrest DNA damage-inducible gene 45 beta promoter in human hepatocellular carcinoma.

Growth arrest DNA damage-inducible gene 45 beta (GADD45beta) has been known to regulate cell growth, apoptotic cell death, and cellular response to DNA damage. Down-regulation of GADD45beta has been verified to be specific in hepatocellular cancer (HCC) and consistent with the p53 mutant, and degree of malignancy of HCC. This observation was further confirmed by eight HCC cell lines and paired human normal and HCC tumor tissues by Northern blot and immunohistochemistry. To better understand the transcription regulation, we cloned and characterized the active promoter region of GADD45beta in luciferase-expressing vector. Using the luciferase assay, three nuclear factor-kappaB binding sites, one E2F-1 binding site, and one putative inhibition region were identified in the proximal promoter of GADD45beta from -865/+6. Of interest, no marked putative binding sites could be identified in the inhibition region between -520/-470, which corresponds to CpG-rich region. The demethylating agent 5-Aza-dC was used and demonstrated restoration of the GADD45beta expression in HepG2 in a dose-dependent manner. The methylation status in the promoter was further examined in one normal liver cell, eight HCC cell lines, eight HCC tissues, and five corresponding nonneoplastic liver tissues. Methylation-specific polymerase chain reaction and sequencing of the sodium bisulfite-treated DNA from HCC cell lines and HCC samples revealed a high percentage of hypermethylation of the CpG islands. Comparatively, the five nonneoplastic correspondent liver tissues demonstrated very low levels of methylation. To further understand the functional role of GADD45beta under-expression in HCC the GADD45beta cDNA constructed plasmid was transfected into HepG2 (p53 WT) and Hep3B (p53 null) cells. The transforming growth factor-beta was assayed by enzyme-linked immunosorbent assay, which revealed a decrease to 40% in transfectant of HepG2, but no significant change in Hep3B transfectant. Whereas, Hep3B co-transfected with p53 and GADD45beta demonstrated significantly reduced transforming growth factor-beta. The colony formation was further examined and revealed a decrease in HepG2-GADD45beta transfectant and Hep3B-p53/GADD45beta co-transfectant. These findings suggested that methylation might play a crucial role in the epigenetic regulation of GADD45beta in hepatocyte transformation that may be directed by p53 status. Thus, our results provided a deeper understanding of the molecular mechanism of GADD45beta down-regulation in HCC.

Apoptosis↗
PubMed1998

A 5'-CG-3'-rich region in the promoter of the transcriptionally frequently silenced RET protooncogene lacks methylated cytidine residues.

In a large proportion of familial and sporadic cases of Hirschsprung disease (HSCR) mutations in the RET (rearranged during transfection) protooncogene have been described. We have investigated the structure of the RET gene promoter and have analysed a region of approximately 1000 nucleotides in its promoter and 5'-upstream segments for the occurrence of 5-methyldeoxycytidine (5-mC) residues by using the bisulfite protocol of the genomic sequencing method. With an estimated sensitivity of about 93% of this technique, not a single 5-mC residue could be detected in the control region of a gene that seems to be silenced or exhibit low activity in many adult tissues. In these experiments, the DNAs of peripheral white blood cells (PWBC) from four healthy individuals, from seven patients with familial HSCR, as well as DNAs from different human tissues and from a human embryonic kidney (HEK) cell line have been included. In a DNA segment starting 790 nucleotides upstream of the transcriptional start site of the RET gene, a few 5-mC residues have been identified. This region possibly constitutes the transition site from an unmethylated promoter to a more extensively methylated region in the human genome. The data presented are remarkable in that a highly 5'-CG-3'-enriched segment of the human genome with 49 5'-CG-3' dinucleotide pairs in 400 bp within the putative promoter region is completely devoid of 5-mC residues, although this control region is not actively transcribed in most adult human tissues. By hybridization of a PCR-amplified RET protooncogene cDNA probe harboring exons 9-15 to a membrane (Clontech) containing poly-A selected RNAs from 50 different human tissues, weak RET protooncogene expression in many of the neural cell derived tissues has been detected. RNAs extracted from many other human tissues do not share sequence homologies to this 32P-labeled probe. Mechanisms other than DNA methylation obviously play the crucial role in the inactivation of the RET gene promoter in these tissues. We have also demonstrated by the in vitro premethylation of a RET promoter-chloramphenicol acetyltransferase (CAT) gene construct and transient transfection experiments into neuroblastoma cells that the transcriptional activity of the RET promoter is decreased by HpaII (5'-CCGG-3') methylation and abolished by SssI (5'-CG-3') methylation. Hence, the RET promoter region is sensitive to this regulatory signal. However in vivo, DNA methylation of the promoter region seems not to be the predominant regulatory mechanism for the RET protooncogene. Possibly, in adults the RET gene can be occasionally activated.

Adult↗
PubMed2000

Methylation mosaicism of 5'-(CGG)(n)-3' repeats in fragile X, premutation and normal individuals.

Fragile X syndrome (FRAXA) is characterized at the molecular level by an expansion of a naturally occurring 5'-(CGG)(n)-3' repeat in the promoter and 5'-untranslated region (5'-UTR) of the fragile X mental retardation (FMR1) gene on human chromosome Xq27.3. When expanded, this region is usually hypermethylated. Inactivation of the FMR1 promoter and absence of the FMR1 protein are the likely cause of the syndrome. By using the bisulfite protocol of the genomic sequencing method, we have determined the methylation patterns in this region on single chromosomes of healthy individuals and of selected premutation carriers and FRAXA patients. In control experiments with unmethylated or M- Sss I-premethylated DNAs, this protocol has been ascertained to reliably detect all cytidines or 5-methylcytidines as unmethylated or methylated nucleotides, respectively. Analyses of the DNA from FRAXA patients reveal considerable variability in the lengths of the 5'-(CGG)(n)-3' repeats and in the levels of methylation in the repeat and the 5'-UTR. In one patient (OEl) with high repeat length hetero-geneity ( n = 15 to >200), shorter repeats (n = 20-80) were methylated or unmethylated, longer repeats ( n = 100-150) were often completely methylated, but one repeat with n = 160 proved to be completely unmethylated. This type of methylation mosaicism was observed in several FRAXA patients. In healthy females, methylated 5'-CG-3' sequences were found in some repeats and 5'-UTRs, as expected for the sequences from one of the X chromosomes. The natural FMR1 promoter is methylation sensitive, as demonstrated by the loss of activity in transfection experiments using the unmethylated or M- Sss I-premethylated FMR1 promoter fused to the luciferase gene as an activity indicator.

5' Untranslated Regions↗
PubMed1997

A 5' 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development.

The imprinted mouse H19 gene is hypermethylated on the inactive paternal allele in somatic tissues and sperm. Previous observations from a limited analysis have suggested that methylation of a few CpG dinucleotides in the region upstream from the start of transcription may be the mark that confers parental identity to the H19 alleles. Here we exploit bisulfite mutagenesis coupled with genomic sequencing to derive the methylation status of 68 CpGs that reside in a 4-kb region 5' to the start of transcription. This method reveals a 2-kb region positioned between 2 and 4 kb upstream from the start of transcription that is strikingly differentially methylated in midgestation embryos. At least 12 of the cytosine residues in this region are exclusively methylated on the paternal allele in blastocysts. In contrast, a 350-bp promoter-proximal region is less differentially methylated in midgestation embryos and, like most of the genome, is largely devoid of methylation on both alleles in blastocysts. We also demonstrate exclusive expression of the maternal H19 allele in the embryos that exhibit paternal methylation of the upstream 2-kb region. These data suggest that the 2-kb differentially methylated region acts as a key regulatory domain for imprinted H19 expression.

Alleles↗
PubMed1987

Transition mutations within the Xenopus borealis somatic 5S RNA gene can have independent effects on transcription and TFIIIA binding.

Base-pair changes were introduced into the Xenopus borealis somatic 5S RNA gene by treatment with sodium bisulfite. Mutants were screened by sequence determination. The collection of mutants permitted a detailed investigation of the fine structure of the intragenic control region that binds the transcription factor TFIIIA. Selected mutants were recloned in tandem with a somatic 5S RNA maxigene to permit sensitive measurement of their relative transcription activities. The transcription efficiencies of a number of mutations at the borders of the control region were correlated with TFIIIA binding by using DNase I protection (footprinting) assays. Mutations affecting transcription and TFIIIA binding extended from gene residues 46 to 91. The results reinforce a model in which the distal half of the protected region constitutes a tight binding domain for TFIIIA. A number of transitions in the 5' domain led to significant increases or decreases in transcription efficiency, but resulted in barely detectable changes in TFIIIA binding. Two mutants with C----T transitions at gene residues 52 and 53 were transcribed with increased efficiencies (up phenotype). These results suggest that TFIIIA must make appropriate contacts within the 5' domain of the control region to permit subsequent binding of other factors in stable transcription complexes.

Animals↗
PubMed2005

Both hypomethylation and hypermethylation in a 0.2-kb region of a DNA repeat in cancer.

NBL2 is a tandem 1.4-kb DNA repeat, whose hypomethylation in hepatocellular carcinomas was shown previously to be an independent predictor of disease progression. Here, we examined methylation of all cytosine residues in a 0.2-kb subregion of NBL2 in ovarian carcinomas, Wilms' tumors, and diverse control tissues by hairpin-bisulfite PCR. This new genomic sequencing method detects 5-methylcytosine on covalently linked complementary strands of a DNA fragment. All DNA clones from normal somatic tissues displayed symmetrical methylation at seven CpG positions and no methylation or only hemimethylation at two others. Unexpectedly, 56% of cancer DNA clones had decreased methylation at some normally methylated CpG sites as well as increased methylation at one or both of the normally unmethylated sites. All 146 DNA clones from 10 cancers could be distinguished from all 91 somatic control clones by assessing methylation changes at three of these CpG sites. The special involvement of DNA methyltransferase 3B in NBL2 methylation was indicated by analysis of cells from immunodeficiency, centromeric region instability, and facial anomalies syndrome patients who have mutations in the gene encoding DNA methyltransferase 3B. Blot hybridization of 33 cancer DNAs digested with CpG methylation-sensitive enzymes confirmed that NBL2 arrays are unusually susceptible to cancer-linked hypermethylation and hypomethylation, consistent with our novel genomic sequencing findings. The combined Southern blot and genomic sequencing data indicate that some of the cancer-linked alterations in CpG methylation are occurring with considerable sequence specificity. NBL2 is an attractive candidate for an epigenetic cancer marker and for elucidating the nature of epigenetic changes in cancer.

5-Methylcytosine↗
PubMed2005

DNA methylation in promoter regions of red cell membrane protein genes in healthy individuals and patients with hereditary membrane disorders.

The methylation state of 5'-CG-3' sites is known to be linked to the regulation of promoter function by modulating DNA-protein interactions and to the structure of chromatin. As part of a project to determine methylation patterns in the human genome, we examined the methylation profiles of several genes for human erythroid membrane proteins: ELB42 (protein 4.2), EPB3 (band 3), SPTB gene (beta-spectrin), and ANK1 (ankyrin). The bisulfite protocol of the genomic sequencing method was applied. The number of 5'-CG-3' dinucleotides was the most abundant in SPTB and ANK1, much less in EPB3, and the least in ELB42. In the DNA of peripheral blood mononuclear cells from healthy individuals, the promoter regions of EPB3 and ELB42 were extensively methylated, but the SPTB and ANK1 promoters were totally unmethylated. We also investigated methylation profiles in peripheral blood mononuclear cells from patients with red cell membrane diseases, such as complete protein 4.2 deficiency due to ELB42 mutations, hereditary spherocytosis with EPB3 mutations, and hereditary elliptocytosis with SPTB mutations. The DNA methylation states in these genes of erythroid cells, which we obtained at the second phase of the 2-phase liquid culture of erythroid precursor cells in the peripheral blood, were essentially identical or very similar to those of peripheral blood mononuclear cells. In disease states, the DNA methylation profiles of these red cell membrane protein genes were essentially not different from those in healthy individuals (statistically not significant).

Anemia↗
PubMed2001

CpG methylation as a basis for breast tumor-specific loss of NES1/kallikrein 10 expression.

The normal epithelial cell-specific-1 (NES1)/kallikrein 10 gene is expressed in normal mammary epithelial cells, but its expression is dramatically decreased in breast cancer cell lines. Now, we have cloned and characterized the active promoter region of NES1. Using a luciferase reporter system, we demonstrate that most tumor cell lines are able to support full or partial transcription from the NES1 promoter, suggesting a role for promoter-independent cis-acting mechanisms of loss of NES1 expression. We show that hypermethylation of the NES1 gene represents one such mechanism. Using methylation-specific PCR and sequence analysis of sodium bisulfite-treated genomic DNA, we demonstrate a strong correlation between exon 3 hypermethylation and loss of NES1 mRNA expression in a panel of breast cancer cell lines and in primary tumors. Treatment of NES1-nonexpressing cells with a demethylating agent led to reexpression of NES1, suggesting an important role of hypermethylation in the loss of NES1 expression. We suggest that hypermethylation is responsible for tumor-specific loss of NES1 gene expression. Our results also suggest that hypermethylation of the NES1 gene may serve as a potential marker for breast cancer.

Antimetabolites, Antineoplastic↗
PubMed2002

A hereditary nonpolyposis colorectal carcinoma case associated with hypermethylation of the MLH1 gene in normal tissue and loss of heterozygosity of the unmethylated allele in the resulting microsatellite instability-high tumor.

Fourteen suspected hereditary nonpolyposis colorectal carcinoma cases with microsatellite unstable(microsatellite instability-high; MSI-H) tumors but no germ-line MSH2, MSH6, or MLH1 mutations were examined for hypermethylation of CpG sites in the critical promoter region of MLH1. The methylation patterns were determined using methylation-specific PCR and by sequence analysis of sodium bisulfite-treated genomic DNA. In one case, DNA hypermethylation of one allele was detected in DNA isolated from blood. In the MSI-H tumor from this case, the unmethylated MLH1 allele was eliminated by loss of heterozygosity, and the methylated allele was retained. This biallelic inactivation resulted in loss of expression of MLH1 in the tumor as confirmed by immunohistochemistry. These results suggest a novel mode of germ-line inactivation of a cancer susceptibility gene.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR.

We report a novel method for rapid quantification of the degree of DNA methylation of a specific gene. Our method combined bisulfite-mediated PCR and quantification of deoxyribonucleoside monophosphate (dNMP) contents in the PCR product through capillary electrophoresis. A specific bisulfite-PCR product was enzymatically hydrolyzed to dNMP monomers which were quantitatively analyzed through subsequent capillary electrophoresis. PCR following bisulfite treatment converts unmethylated cytosines to thymines while leaving methyl-cytosines unchanged. Then the ratio of cytosine to thymine determined by capillary electrophoresis represents the ratio of methyl-cytosine to cytosine in genomic locus of interest. Pure oligonucleotides with known sequences were processed in parallel as standards for normalization of dNMP peaks in capillary electrophoresis. Sources of quantification uncertainty such as carryovers of dNTPs or primers and incomplete hydrolysis were examined and ruled out. When the method was applied to samples with known methylation levels (by bisulfite-mediated sequencing) as a validation, deviations were within +/-5%. After bisulfite-PCR, the analytical procedure can be completed within 1.5 h.

Cytosine↗
PubMed2001

Methylation and mutational analysis of p27(kip1) in prostate carcinoma.

BACKGROUND: We have previously identified 12p12-13 as a region of frequent genetic loss in prostate carcinoma. A candidate tumor suppressor gene at this locus is the cyclin dependent kinase inhibitor p27(kip1), which has been implicated as a marker of aggressive prostate carcinoma. Herein, we examine metastatic prostate tumors, xenografts, and cell lines for gene inactivation via mutational inactivation or promoter hypermethylation. METHODS: Mutation analysis was performed on metastatic prostate tumors of 18 patients, eight prostate carcinoma cell lines, and 18 xenografts by PCR amplification of the entire open reading frame of p27(kip1). PCR products were sequenced directly using internal primers. Methylation analysis was performed on four cell lines and nine xenografts using direct sequencing of cloned PCR products of bisulfite treated DNA. Presence of a CpG was consistent with methylation of that cytosine in the original sample. RESULTS: With the exception of the previously reported homozygous deletion, no additional mutations were identified. Methylated CpG residues were identified in three xenografts (LuCAP23, LuCAP35, and PC82) and the methylated residues clustered at six sites; the cytosines 69, 149, 191, 286, 349, and 487 base pairs 5' of the ATG start codon. However, no sample demonstrated promotor methylation in all sequenced clones and the number of methylated base pairs ranged from seven to three, not the level usually associated with gene silencing. CONCLUSIONS: Mutational inactivation of p27(kip1) is a rare event in metastatic prostate carcinoma. While CpG methylation does occur, it is an infrequent event and does not appear to be the mechanism of p27(kip1) down regulation in prostate carcinoma.

Cell Cycle Proteins↗
PubMed2002

Reduced Brca1 protein expression in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary carcinomas.

Reduced expression of BRCA1 protein, caused by the hypermethylation of its gene promoter and by other mechanisms, is observed in most sporadic human breast cancers, whereas its somatic mutations are rare. In the present study, we demonstrate that immunoreactivity of Brca1 was reduced in almost all rat mammary carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), whereas hypermethylation of its promoter, mutations in its entire coding region, and decreased mRNA expression were absent. Using two kinds of polyclonal antibodies against human BRCA1 that recognized the N and C termini, respectively, immunoreactivity of Brca1 was found to be reduced in all 17 carcinomas examined and, especially, to be almost completely lost in 8 or 10 carcinomas. The reduction was confirmed by immunoblot analysis of five mammary carcinomas and normal mammary epithelial cells. Sequencing of the Brca1 promoter region after bisulfite modification in 10 PhIP-induced mammary carcinomas showed that the region was completely unmethylated in all of them. No mutations were detected in the entire coding region by direct sequencing of the Brca1 cDNA. Decrease in mRNA levels was not detected by quantitative real-time reverse transcription-polymerase chain reaction. These data suggested that PhIP-induced mammary carcinomas could model human breast cancers that show reduced BRCA1 immunoreactivity without promoter hypermethylation and with normal mRNA expression. As underlying mechanisms, alterations in posttranscriptional regulation or stability of Brca1 protein were suggested.

Animals↗
PubMed1995

Sequencing of the rat light neurofilament promoter reveals differences in methylation between expressing and non-expressing cell lines, but not tissues.

The DNA methylation pattern of the promoter (pNF-L) region of the rat light-neurofilament-encoding gene (NF-L), a neuron-specific gene, was assessed in NF-L expressing and non-expressing cell lines and tissues by genomic sequencing using PCR amplification of bisulfite-modified DNA. We analysed twenty-five potential CpG methylation sites between nucleotide (nt) positions -311 and +103 of pNF-L, containing Sp1- and AP-2-binding sites, a CGCCCCCGC box and a cAMP-responsive element. Six out of 25 possible CpG methylation sites are within these elements. The pNF-L promoter was unmethylated in NF-L-expressing rat brain, as well as in liver not expressing NF-L. In NF-L-expressing PC12 cells, the promoter was unmethylated, whereas in non-expressing glioma C6 cells intensive methylation occurred. A cluster of methylated CpG dinucleotides spanned the region from nt -176 to -67 bp. Thus, methylation of this promoter region could play a role in silencing NF-L in the glioma cell line in vitro, but not in liver tissue in vivo. In a non-CpG sequence, in the CpApG trinucleotide at nt position -114, cytosine was found to be partially methylated. It is thus possible to describe the methylation state of each cytosine present in the area of genomic DNA of interest.

Animals↗
PubMed2006

Role of the BRAF mutations in the microsatellite instability genetic pathway in sporadic colorectal cancer.

BACKGROUND AND AIMS: Between 10 and 15% of all cases of colorectal cancer are the result of microsatellite instability (MSI) in the genetic pathway due to an alteration in the DNA repair genes. Tumors with high MSI are characterized by a better prognosis. The BRAF oncogene has been linked to the MSI pathway in tumorogenesis. The objective of this study was to determine whether alterations in BRAF are related to MSI and whether they can result in differences in survival rates. METHODS: The study cohort comprised 351 patients diagnosed with sporadic colorectal cancer. MSI was determined in accordance with the National Cancer Institute's (NCI) recommendations by means of PCR and sequence analyses. Mutational analysis of the BRAF gene was performed by real-time PCR and subsequent sequencing of the altered samples. The methylation pattern of the hMLH1 gene was determined using methylation-specific PCR analyses of bisulfite-treated DNA and the results confirmed by sequencing. RESULTS: Of the patients tested, 6.9% showed high MSI and 3.7% showed a BRAF gene mutation. hMLH1 methylation was observed in 67.2% of the patients with MSI and/or the BRAF alteration. The BRAF mutation was related to the MSI genetic pathway (P < 0.0001) and with hMLH1 methylation. In the analysis of overall survival only MSI had an independent prognostic value for the risk of death. Patients with the BRAF mutation showed a higher risk of death, although this association was found not to be statistically significant. CONCLUSIONS: There is a subgroup of carcinomas which develop via the MSI pathway that carry an alteration of the BRAF gene. This alteration confers a poorer outcome on these patients within the total group of patients with MSI who have a better prognosis. This hypothesis should be further investigated in a larger study population due to the low incidence of BRAF mutations in colorectal cancer.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Methylation sequencing from limiting DNA: embryonic, fixed, and microdissected cells.

It is frequently useful to determine the methylation state of samples containing limited amounts of DNA such as from embryos, or from fixed tissue samples in which DNA is degraded or difficult to isolate. By modification of the standard protocols for DNA preparation and bisulfite treatment, it is possible to obtain DNA methylation sequence data for such samples. We present methods for bisulfite treatment of embryos, fixed sections, and samples obtained by laser capture microdissection, and discuss the additional experimental considerations required when working with small numbers of cells or degraded DNA samples.

3T3 Cells↗