SEARCH · Search PubMed

Results for “Bisulfite sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources

PubMed2007

Correlation between hypermethylation of the RASSF2A promoter and K-ras/BRAF mutations in microsatellite-stable colorectal cancers.

Recently, RASSF2A was identified as a potential tumor suppressor epigenetically inactivated in human cancers. Here, we evaluated the methylation status of RASSF2A in colorectal cancer (CRC) and analyzed its correlation with K-ras/BRAF mutations, microsatellite instability status and other clinicopathological features. Using methylation-specific PCR and bisulfite sequencing, we analyzed the methylation status in primary CRC, adenomas and corresponding normal tissues and then compared it with the presence of K-ras and BRAF mutations. We also examined the expression and methylation status of RASSF2A in CRC cell lines. We found that aberrant methylation of RASSF2A promoter regions is associated with gene silencing in CRC cell lines. In primary CRC, the frequency of RASSF2A methylation was 72.6%, and it was found in 16 of 16 (100%) adenomas. In addition, there was a positive correlation between K-ras/BRAF mutations and RASSF2A methylation in primary CRC. Furthermore, a significant positive correlation between K-ras/BRAF mutations and RASSF2A methylation was also observed in microsatellite-stable (p = 0.033) and distal CRC (p = 0.025). These results show that RASSF2A methylation is a frequent event in colorectal tumorigenesis and positively correlates with K-ras/BRAF mutation in microsatellite-stable or distal CRC.

Adenoma↗

PubMed2007

EMP3 overexpression is associated with oligodendroglial tumors retaining chromosome arms 1p and 19q.

The epithelial membrane protein 3 (EMP3) gene located on chromosome 19q13 has been implicated as a candidate tumor suppressor gene (TSG) in neuroblastomas and gliomas. The aim of this study was to investigate whether EMP3 is involved in oligodendroglial tumors (OTs), which frequently carry combined chromosomes 1p and 19q deletion. We first investigated the transcript level of EMP3 in a cohort of 57 OTs by quantitative real-time RT-PCR. Our results showed that 10 (18%) tumors had reduced EMP3 expression level compared to normal brains. Six of these tumors carried chromosome 19q13 deletion but no statistical correlation was found between the 2 parameters. Intriguingly, a similar proportion (11 of 57, 19%) of tumors displayed EMP3 overexpression, with 8 of them having transcript level >10-fold higher than normal brain. All 11 OTs retained chromosomes 1p36 and 19q13, and a significant association was found between EMP3 overexpression and balanced chromosomes 1p36 and 19q13 (p = 0.004). The methylation status of EMP3 was evaluated by bisulfite sequencing in 29 OTs with diverse expression levels. All tumors except 3 showed aberrant methylation of EMP3 and no correlation was observed between transcript level and methylation status, suggesting that methylation alone does not mediate transcriptional down-regulation of EMP3 in OTs. In conclusion, our study demonstrates that EMP3 overexpression is involved in OTs retaining chromosomes 1p and 19q and does not support EMP3 as the target TSG on chromosome 19q13 in OTs.

Adolescent↗

PubMed2004

Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle.

BACKGROUND: Although the transient nature of transgene expression using first-generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain. METHODS: Rats were given intramuscular (i.m.) injections of a first-generation Ad containing the human fibroblast growth factor 4 (hFGF-4) gene driven by the cytomegalovirus (CMV) promoter and enhancer (CMV-PE). The copy number of hFGF-4 mRNA and viral DNA was measured in the same muscles by quantitative RT-PCR and quantitative PCR at times between 1 h and 84 days after virus injection. Quantitative Southern blot analysis for the intact hFGF-4 transcription unit DNA was also performed, and the methylation status of the CMV-PE DNA in the muscle was determined using bisulfite sequencing. RESULTS: The copy number of hFGF-4 mRNA peaked at 6 h then decreased 56-fold by 24 h, and a further 240-fold between days 3 and 28. Although the viral DNA copy number also decreased 23-fold between 6 h and 28 days, the ratio of copies of hFGF-4 mRNA per copy of viral DNA decreased 385-fold over this period. Methylation of the CMV-PE DNA in the muscle at both CpG and non-CpG sites was observed 24 h after virus administration and had increased at day 7. CONCLUSIONS: Decreased transcription associated with extensive methylation of the CMV-PE was the major mechanism responsible for the decrease in transgene mRNA levels. Strategies for preventing transcriptional silencing will be valuable for improving the duration of transgene expression from adenoviral vectors.

Animals↗

PubMed2003

Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma.

Deletion of 11q23 is a common genetic aberration in nasopharyngeal carcinoma (NPC). Multiple candidate tumor suppressor genes (TSG) were mapped to this region but few of them were investigated in NPC. TSLC1 (tumor suppressor in lung cancer) is recently reported to be a putative TSG on 11q23. This gene was found to be inactivated by promoter hypermethylation in non-small cell lung carcinoma (NSCLC), liver cancer, and breast cancer. To study the role of TSLC1 gene in NPC tumorigenesis, we screened for mutations and aberrant methylation of TSLC1 gene in 5 NPC cell lines, 3 NPC xenografts, and 38 primary NPC cases. No somatic mutations of TSLC1 were detected in the NPC samples, but a 9-bp (CCACCACCA) deletion in exon 8 was found in a primary NPC and its corresponding blood sample. Bisulfite sequencing revealed aberrant methylation of TSLC1 promoter in four NPC cell lines. Loss of TSLC1 gene expression was found in two cell lines (HK-1 and CNE-2) with dense methylation. Expression of this gene was restored in these cell lines after treatment with demethylating agent 5-aza-2'-deoxycytidine. Our results showed that silencing of TSLC1 gene expression in NPC was associated with promoter hypermethylation. Promoter hypermethylation of TSLC1 gene was further illustrated in 34.2% (13/38) of primary NPCs. No aberrant promoter methylation was found in any of the four investigated normal nasopharyngeal epithelia. Frequent epigenetic inactivation of TSLC1 gene in NPC suggested that this gene is one of the target tumor suppressor genes of this endemic cancer.

Adult↗

PubMed2004

Epigenetic and gene expression changes related to transgenerational carcinogenesis.

Transgenerational carcinogenesis refers to transmission of cancer risk to the untreated progeny of parents exposed to carcinogens before mating. Accumulated evidence suggests that the mechanism of this process is epigenetic, and might involve hormonal and gene expression changes in offspring. To begin to test this hypothesis, we utilized a mouse model (NIH Swiss) in which exposure of fathers to Cr(III) chloride 2 wk before mating can alter incidence of neoplastic and nonneoplastic changes in offspring tissues. Utilizing a MS-RDA approach, we found that the sperm of these fathers had a significantly higher percentage of undermethylated copies of the 45S ribosomal RNA gene (rRNA); this finding was confirmed by bisulfite sequencing. Because gene methylation is a known mechanism of expression control in germ cells, and ribosomal RNA levels have been linked to cancer, these findings are consistent with the hypothesis. Secondly, we observed that offspring of Cr(III)-treated fathers were significantly heavier than controls, and had higher levels of serum T3. Possible effects of T3 levels on gene expression in the offspring were examined by microarray analysis of cDNAs from liver. A total of 58 genes, including 25 named genes, had expression ratios that correlated significantly with serum T3 ratios at P </= 0.001. Some of these genes have potential roles in growth and/or tumor suppression. These results also support the hypothesis of an epigenetic and/or gene expression-based mechanism for transgenerational carcinogenesis. Published 2004 Wiley-Liss, Inc.

Animals↗

PubMed2006

Aberrant DNA methylation of E-cadherin and p16 genes in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine.

The study aimed to assess the involvement of aberrant DNA methylation in lung carcinogenesis by measuring expressions of E-cadherin and p16 genes, and their DNA methylation status in the 5' upstream region in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week-old male Wistar rats were given 2000 ppm BHP in their drinking water for 12 wk, and maintained without further treatment until they were sacrificed at 25 wk. A total of nine lung adenocarcinomas were collected, and total RNA from each sample was extracted for assessment of gene expression by real-time reverse transcription (RT)-polymerase chain reaction (PCR). Expressions of E-cadherin and p16 genes were significantly reduced in lung adenocarcinomas (P < 0.05 and P < 0.005, respectively) compared with normal lung tissues. For methylation analysis, bisulfite sequencing was performed using two normal lung tissues and five tumors. The two normal lung tissues were all unmethylated in regions of E-cadherin and p16. In contrast, five adenocarcinomas were highly methylated, and these aberrant methylation patterns correlated with reduced expressions of E-cadherin and p16. These results suggest that aberrant DNA methylation of E-cadherin and p16 genes may play important roles in development of lung adenocarcinomas induced by BHP in rats.

Adenocarcinoma↗

PubMed2006

Aberrant methylation patterns of the Rassf1a gene in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine.

To clarify the involvement of the Rassf1a gene in lung carcinogenesis, we investigated the methylation status in the 5' upstream region of the RAS-association domain family 1, isoform A (Rassf1a) gene in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week-old male Wistar rats were given 2000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. A total of 15 lung adenocarcinomas were obtained and total RNAs were extracted from each for assessment of expression of the Rassf1a gene by reverse transcription (RT)-polymerase chain reaction (PCR) analysis. To measure the methylation status of the Rassf1a gene, five adenocarcinomas with a marked reduction of the Rassf1a expression and two normal lung tissues were used for a bisulfite sequencing analysis. While the normal lung tissue was unmethylated, all five adenocarcinomas were highly methylated in the 5' upstream region. Genomic DNAs were also extracted from 15 adenocarcinomas, and mutation analysis of the Rassf1a was performed with PCR-single-strand conformation polymorphism (SSCP) analysis. No mutations were detected throughout exons 1-6. The present results suggest that the aberrant methylation may be involved in the inactivation of the Rassf1a gene in the development of lung adenocarcinomas induced by BHP in rats.

Adenocarcinoma↗

PubMed2006

Reduced expression of the Connexin26 gene and its aberrant DNA methylation in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine.

Gap junctions are mediated by intercellular channels that connect adjacent cells and are composed of Connexin (Cx) proteins. A member of the Cx family, Cx26 is considered a potential tumor suppressor in several cancers. The expression of Cx26 gene and its methylation status in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) were investigated. Six-wk-old male Wistar rats were given 2,000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. A total of nine lung adenocarcinomas were obtained and total RNA was extracted to assess expression by real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). Five out of nine adenocarcinomas showed reduced expression compared with normal lung tissue. We next performed a bisulfite sequence analysis to measure the methylation status of the 5' upstream region of the Cx26 gene in two normal lung tissues and five lung adenocarcinomas that showed reduced expression of Cx26. All five adenocarcinomas were highly methylated in the 5' upstream region, while the two normal lung tissues were unmethylated. This suggests that aberrant methylation of the Cx26 gene may be involved in the development of lung adenocarcinomas induced by BHP in rats.

Adenocarcinoma↗

PubMedDate not supplied

5-Azacytidine modulates the response of sensitive and multidrug-resistant K562 leukemic cells to cytostatic drugs.

In an endeavor to improve responsiveness of tumor cells to drug combination treatments, we analyzed the effect of 5-azacytidine (5AC) as a model compound for a new class of drugs, DNA-demethylating agents. We used parental K562/WT chronic myelogenous leukemia cells and a multidrug-resistant subline thereof, K562/ADM. Multidrug-resistant cells were more resistant to daunorubicin, but more sensitive to cisplatin than parental K562 cells as measured by growth inhibition and apoptosis assays. Resistance to daunorubicin can be explained by amplification of the MDR1 drug transporter gene. Cisplatin induced more DNA damage in specific genes and in the entire genome of K562/ADM cells compared to K562/WT cells using PCR stop assays and atomic absorption spectroscopy. Pretreatment with 5AC modulated the response of K562/ADM cells toward MDR-type drugs (daunorubicin, vincristine, etoposide) and reduced function and expression of MDR1 as analyzed by flow cytometry and RT-PCR. Analysis of CpG island methylation in the promotor region of the MDR1 gene by bisulfite sequencing and a methylation-sensitive HpaII-digestion/PCR approach revealed that methylation of the MDR1 promotor of K562/ADM cells was greater than in K562/WT cells. 5AC treatment completely abolished MDR1 promotor methylation. The unexpected observation that DNA demethylation by 5AC rather decreases than increases MDR1 expression in K5612/ADM cells points to still unexplored sequences in the MDR1 promotor whose transcriptional activity may be affected by the methylation status. 5AC pretreatment also modulated K562/WT and K562/ADM cells to non-MDR-type drugs such as cisplatin and increased cisplatin-induced DNA damage.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Methylation dynamics of imprinted genes in mouse germ cells.

DNA methylation differences between maternal and paternal alleles of many imprinted genes are inherited from the male and female gametes and subsequently maintained during development. However, the stages of gametogenesis during which methylation imprints are established have not been well defined. In this study, we used bisulfite sequencing to determine the methylation dynamics of the imprinted genes small nuclear ribonucleoprotein N (Snrpn), insulin-like growth factor 2 receptor (Igf2r), mesoderm-specific transcript (Mest; formerly Peg1), paternally expressed gene 3 (Peg3), and H19 fetal liver mRNA (H19). We identified regions in the maternally imprinted genes (Snrpn, Mest, and Peg3) that were unmethylated in sperm but 100% methylated in mature oocytes. Igf2r, which is expressed from the maternal allele, was completely methylated within intronic differentially methylated region 2 in oocytes and unmethylated in sperm. The 5' region of H19, a paternally imprinted gene, was completely methylated in sperm and unmethylated in oocytes. We examined the methylation status of Snrpn during oocyte growth and maturation. Whereas the DNA of non-growing oocytes was mostly unmethylated, mid-size growing oocytes had a mosaic pattern of allelic methylation, and full acquisition of the methylation imprint was complete by metaphase II. We have identified regions within imprinted genes that show gamete-specific methylation patterns in mature germ cells and demonstrated that maternal methylation imprints on at least one imprinted gene, Snrpn, are established during the postnatal growth phase of oogenesis. Thus, whereas paternal imprints seem to be established early (in diploid gonocytes well before the onset of meiosis), maternal imprints are established late (in growing oocytes that are arrested in the diplotene stage of meiosis). These findings raise the possibility that assisted reproductive technologies that involve in vitro maturation of oocytes may result in developmental abnormalities due to incomplete methylation imprints in immature oocytes.

Animals↗

PubMed2002

Paradoxical methylation of the tyrosine hydroxylase gene in mouse preimplantation embryos.

The mouse tyrosine hydroxylase (Th) gene is located in an evolutionarily conserved imprinted gene cluster on distal chromosome 7. It is associated with a CpG island that spans the promoter of the gene. Using a bisulfite sequencing method we show that the Th promoter is fully methylated in both male and female mouse germ cells and in human spermatozoa, suggesting that it belongs to the newly identified category of CpG islands, the similarly methylated regions (SMRs). Contrary to other tissue-specific gene sequences, the mouse Th promoter escapes the initial wave of genome demethylation during the first few cell cycles, but becomes demethylated between the morula and the blastocyst stages. This unusual methylation ontogeny may be a characteristic of the SMRs and/or related to the localization of the Th gene in an imprinted gene cluster.

Animals↗

PubMed2006

Gene expression and methylation patterns in cloned embryos.

A considerable proportion of the offspring, in particular in ruminants and mouse, born from nuclear transfer (NT)-derived and in vitro-produced (IVP) embryos are affected by multiple abnormalities, of which a high birthweight and an extended gestation length are the predominant features; a phenomenon that has been termed "Large Offspring syndrome" (LOS). According to a current hypothesis, LOS is caused by persistent aberrations of expression patterns of developmentally important genes starting as early as at the preimplantation stages. The underlying mechanisms are widely unknown at present, but epigenetic modifications of embryonic and fetal gene expression patterns, primarily caused by alterations in DNA methylation are thought to be involved in this syndrome. Appropriate DNA methylation is essential for regular transcription during mammalian development and differentiation. Sensitive reverse transcription polymerase chain reaction assays allow the study of messenger RNA (mRNA) expression levels of specific genes in single embryos. The methylation status of a specific gene can be assessed by bisulfite sequencing. Studies to unravel mRNA expression patterns from IVP- and NT-derived embryos have revealed numerous aberrations ranging from suppression of expression to de novo overexpression or more frequently to a significant upregulation or downregulation of a specific gene. mRNA expression patterns from in vivo-derived embryos are essential as the "physiological standard" against which the findings for IVP and NT-derived embryos are to be compared. Unraveling the underlying molecular mechanisms will contribute to the production of viable embryos and aid to improve biotechnologies applied to early mammalian embryos.

Animals↗

PubMed2003

Differential methylation pattern of the X-linked lymphoproliferative (XLP) disease gene SH2D1A correlates with the cell lineage-specific transcription.

SH2D1A, the X-linked lymphoproliferative disease (XLP) gene, encodes a cytoplasmic protein that plays an essential role in controlling Epstein-Barr virus infection. It is expressed in T and NK cells, but not in B cells or in granulocytes. The promoter, the regulatory regions, as well as the mechanisms controlling its tissue-specific expression, are still unknown. We tested the hypothesis that DNA methylation might contribute to tissue-specific SH2D1A gene expression and analyzed the methylation status of 2,300 bp upstream of the ATG starting codon, the coding region and part of intron 1. By bisulfite sequencing and methylation-sensitive restriction enzyme digestion, we show that a differential methylation pattern of CpG-rich regions in the 5' region and the adjacent exon 1 of the SH2D1A gene indeed correlates with the tissue-specific gene transcription.

Animals↗

PubMed2005

Increased plasticity of genomic imprinting of Dlk1 in brain is due to genetic and epigenetic factors.

The expression of six imprinted genes (Dlk1, Gt12, Igf2r, Kcnq1, Nnat, and Peg1) was examined in brains of 21 mice derived from N2 x N2 intercrosses between C57BL/6 and MOLF/Ei strains. Imprinting of Igf2r, Kcnq1, Gt12, and Dlk1 varied among individuals. As three of these genes are implicated in cell-cell signaling or cell-environment interactions, variation in their imprinting may influence a wide range of biological processes from cell differentiation to behavior. To elucidate the mechanisms underlying the interindividual imprinting variation in the brain, we focused our effort on the paternally expressed gene Dlk1. We investigated expression of Dlk1 in the brains of animals from N9 and N10 backcrosses and found that reactivation of the normally silent maternal Dlk1 allele in the N9 and N10 mice occurred less often than in N2 x N2 animals. Our data suggest that trans-acting genetic factors of MOLF/Ei origin facilitate the reactivation of the normally silent maternal allele of Dlk1. We mapped one of these factors to the proximal part of Chr 7. The results of bisulfite sequencing methylation analysis show that reactivation of the maternal allele was also associated with hypermethylation of the intragenic differentially methylated region (IG DMR), which is the imprinting control region for the Dlk1-Gt12 domain. Thus, the imprinting status of Dlk1 in the brain depends upon trans-acting genetic influences and correlates with the methylation status of a specific subregion of the IG DMR.

Animals↗

PubMed2006

Relationship between DNA methylation and histone acetylation levels, cell redox and cell differentiation states in sugarbeet lines.

In order to evaluate the permanent chromatin remodeling in plant allowing their high developmental plasticity, three sugarbeet cell lines (Beta vulgaris L. altissima) originating from the same mother plant and exhibiting graduate states of differentiation were analyzed. Cell differentiation has been estimated by the cell redox state characterized by 36 biochemical parameters as reactive oxygen species steady-state levels, peroxidation product contents and enzymatic or non-enzymatic protective systems. Chromatin remodeling has been estimated by the measurement of levels of DNA methylation, histone acetylation and corresponding enzyme activities that were shown to differ between cell lines. Furthermore, distinct loci related to proteins involved in cell cycle, gene expression regulation and cell redox state were shown by restriction landmark genome scanning or bisulfite sequencing to display differential methylation states in relation to the morphogenic capacity of the lines. DNA methylating, demethylating and/or histone acetylating treatments allowed to generate a collection of sugarbeet cell lines differing by their phenotypes (from organogenic to dedifferentiated), methylcytosine percentages (from 15.0 to 43.5%) and acetylated histone ratios (from 0.37 to 0.52). Correlations between methylcytosine or acetylated histone contents and levels of various parameters (23 or 7, respectively, out of 36) of the cell redox state could be established. These data lead to the identification of biomarkers of sugarbeet morphogenesis in vitro under epigenetic regulation and provide evidence for a connection between plant morphogenesis in vitro, cell redox state and epigenetic mechanisms.

Acetylation↗

PubMed2026

Scion-based drought stress memory affects potato response to water deficit.

A scion-based stress memory signal, which was derived from drought-primed potato plants, was transmitted to new potato plants generated through vegetative reproduction. This affected potato tuber yield. Drought is one of the most significant threats to agricultural productivity worldwide. The cultivated potato (Solanum tuberosum L.) is a crop species that is sensitive to drought stress. This study investigated the impact of scion-based drought stress memory on tuber yield, physiological parameters, gene expression, and DNA methylation in the vegetative progeny of grafted plants. The tuber progeny plants remembered the drought stress signal transmitted from the drought-primed scion. Significant changes were observed in the expression of genes, primarily those related to photosynthetic metabolic pathways, as well as those associated with chromatin remodeling, DNA repair, and the plant's response to abiotic stresses. The gene expression landscape corresponded with variability in chlorophyll fluorescence parameters. In the first and the second generation of vegetatively propagated plants, scion-based memory had a positive effect on tuber yield. This was achieved by buffering the decline in yield caused by drought, as compared to plants grown under control conditions. Whole-genome bisulfite sequencing analysis revealed no correlation between changes in DNA methylation and gene expression. Drought-induced alterations in DNA methylation were erased in the second progeny generation. We propose that there is a direct causal relationship between scion-based memory of drought stress and photosynthetic efficiency, as well as potato tuber productivity.

Solanum tuberosum↗

PubMed2025

Methylated ARHGAP40 in renal cell carcinoma associated with tumor necrosis and grade: a potential biomarker for non-invasive early detection.

This study investigated the expression, methylation patterns, and clinicopathological implications of ARHGAP40 in renal cell carcinoma (RCC), the most common urinary malignancy. A total of 60 clear cell renal cell carcinomas (ccRCC), 30 papillary renal cell carcinomas (pRCC), 30 chromophobe renal cell carcinomas (chRCC), and 13 other RCC subtypes were enrolled. ARHGAP40 expression was analyzed in both RCC tissues and matched paracancerous normal tissues using immunohistochemistry (IHC). The methylation status of the ARHGAP40 promoter region was assessed in both normal and tumor samples by bisulfite sequencing PCR (BSP). Circulating tumor DNA (ctDNA) extracted from peripheral blood samples of RCC patients (20), patients with benign renal tumors (1), and healthy controls (14) was quantitatively analyzed for methylation using quantitative methylation-specific PCR (qMSP). ARHGAP40 expression was significantly downregulated in RCC compared to matched normal tissues (P < 0.001). This reduced expression correlated with tumor necrosis (P = 0.009) but showed no significant association with age, gender, tumor location, tumor diameter, TNM stage, or vascular invasion. In the ccRCC subtype, ARHGAP40 expression exhibited a progressive decrease with larger tumor diameter (P = 0.045), advancing histological grade (P = 0.032), and tumor necrosis (P = 0.011). The methylation status of ARHGAP40 was consistent with its expression level in both tumor and adjacent normal tissues. Methylated ARHGAP40 DNA was detectable only in RCC patient ctDNA samples. ARHGAP40 is epigenetically silenced in RCC through methylation-mediated downregulation, which correlates with tumor necrosis and grade. The detection of methylated ARHGAP40 in ctDNA holds promise as a potential biomarker for early RCC diagnosis.

Humans↗

PubMed2006

Epigenetic silencing of the putative tumor suppressor gene testisin in testicular germ cell tumors.

PURPOSE: Testisin, a serine protease abundantly expressed only in normal testes, is thought to be a tumor suppressor gene silenced by aberrant methylation in testicular germ cell tumors (TGCT). This study aimed at identifying the CpG sites relevant for testisin gene silencing when methylated and evaluating the potential of aberrant methylation as a biomarker in TGCT. METHODS: Bisulfite sequencing and subsequent real-time RT-PCR were carried out in germ cell tumor cell lines and a cervical carcinoma cell line to reveal CpG sites implicated in testisin gene silencing. The normalized index of methylation (NIM) and the relative gene expression (RGE) were calculated in 33 TGCT and 19 normal testicular tissue samples by quantitative methylation-specific PCR (QMSP) and real-time RT-PCR. RESULTS: CpG sites in the 5'untranslated region proved to be relevant for testisin gene silencing when methylated. Targeting these CpG sites by QMSP, we demonstrated that the median NIM was 8.6 times higher in the TGCT group than in normal testicular samples. This was associated with a median 23.6 times lower RGE in the TGCT probes. Non-seminomas had a significantly higher NIM than seminomas, but RGE was downregulated to comparable levels in both subgroups. QMSP was highly sensitive and distinguished TGCT from normal samples with excellent specificity. CONCLUSIONS: Our results demonstrate for the first time that aberrant methylation of specific CpG sites near the transcription initiation site is an important factor in testisin gene silencing in TGCT.

5' Untranslated Regions↗