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PubMed2005

Lack of F8 mRNA: a novel mechanism leading to hemophilia A.

Hemophilia A (HA) is caused by partial or total deficiency of F8 protein activity. In a small group, about 1.8% of patients with HA, no mutation is found in the F8 gene. Among this group, we report here on one patient with severe HA in whom no mRNA of the F8 gene was detected. Using 2 common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother, and his sister was not detected by reverse transcription-polymerase chain reaction (RT-PCR) from total blood mRNA. Skewed X-chromosome inactivation in both the mother and the sister was excluded by studying the methylation profile of the androgen receptor gene (HUMARA locus). These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA, which points to a novel mechanism leading to HA.

Adolescent↗

PubMed2006

Involvement of ALF in human spermatogenesis and male infertility.

We conducted this study to explore functions of TF II Aalpha/beta-like factor (ALF) during human spermatogenesis, and the relationship of its expression levels with male infertility. The RT-PCR and Western blot analyses illustrated that ALF was highly expressed in adult testis. Immunohistochemistry and immunoflurescence showed that ALF is located in the spermatid nuclei and in the annulus of spermatozoa. Further, to reveal whether ALF is related to male infertility, we performed the same experiments in infertility patients. The changes in the expression levels of ALF in the male infertility samples lead us to believe that ALF may function in spermatogenesis, especially in spermiogenesis. We also detected the ALF DNA methylation level by real-time methylation-specific PCR (MSP) both in testes of adult, fetal and infertile patient. The differential expression level of ALF gene in different types of testes was regulated by DNA methylation. Our research identified ALF as a human spermatogenesis related gene, the abnormal expression of ALF might be the partial cause for human infertility.

Gene Expression Profiling↗

PubMed2026

A distinct subclade of AlkB family demethylases in ciliates safeguards the positional fidelity of eukaryotic N6-methyladenine (6mA).

DNA N6-methyladenine (6mA) is a newly recognized transcription-associated epigenetic mark in eukaryotes. While its methylation pathway has been well established, the identity of eukaryotic 6mA demethylase(s) responsible for its removal and dynamic regulation has remained elusive. Here, we identify and characterize DMT3 (TtALKBH5), an AlkB family dioxygenase in Tetrahymena thermophila, as a 6mA demethylase in ciliates and potentially several other unicellular eukaryotes with abundant 6mA and a defined AMT1 methyltransferase (MTase) complex, supported by genetic and molecular evidence. DMT3 acts on both fully and hemimethylated ApT dinucleotides, an activity partially facilitated by a naturally occurring cysteine-to-serine substitution. Genome profiling shows that DMT3 is enriched at transcription start sites (TSSs) of 6mA-enriched genes, complementary to the occupancy pattern of the AMT1 complex, where it selectively removes spurious 6mA deposited by AMT1. Genetic disruption of DMT3-mediated demethylation, either by knockout or catalytic inactivation, leads to aberrant 6mA accumulation at TSS regions, transcriptional dysregulation, altered chromatin accessibility, and impaired initiation of sexual reproduction. Notably, simultaneous removal of DMT3 and AMT1 eliminates these defects, indicating that spurious TSS 6mA underlies transcriptional and developmental impairment.

Adenine↗

PubMed2003

Nutritional links to plausible mechanisms underlying pancreatic cancer: a conference report.

Adenocarcinoma of the pancreas is one of most catastrophic and least understood of cancers. Evidence from clinical studies indicates that the development of pancreas cancer progresses over many years before symptoms appear. Most people with pancreatic cancer die within six months of diagnosis. The lack of early disease markers, the paucity of direct subject/patient interview data and limited availability of high quality biological samples have slowed progress toward identifying environmental and genetic disease risk factors. Much remains to be learned about the development of pancreatic cancer and about potential interventions for disease prevention. Epidemiological and mechanistic studies examining risk factors for pancreatic cancer supply little consistent or strong evidence to provide a cohesive prevention strategy for this cancer, but offer clues for future research concerning the prevention and early detection of this devastating disease. This Executive Summary provides background discussion on pancreatic cancer and summaries of each of the topics discussed at the workshop, including 1) Molecular aspects, 2) Dietary and other risk factors for pancreatic cancer, 3) The metabolic hypothesis for pancreatic cancer, 4) Preclinical studies on pancreatic cancer, 5) Methylation, 6) Oxidative stress and 7) Biomarker Profiling. This document also contains a compilation of recommendations for future research, concluding remarks, a list of speakers and participants attending the workshop, and a selection of key references to aid future research into nutritional links to mechanisms underlying pancreas cancer. The recommendations section suggests gaps in current knowledge and articulates future directions for this area of investigation.

DNA Methylation↗

PubMed2005

Familial cancer associated with a polymorphism in ARLTS1.

BACKGROUND: The finding of hemizygous or homozygous deletions at band 14 on chromosome 13 in a variety of neoplasms suggests the presence of a tumor-suppressor locus telomeric to the RB1 gene. METHODS: We studied samples from 216 patients with various types of sporadic tumors or idiopathic pancytopenia, peripheral-blood samples from 109 patients with familial cancer or multiple cancers, and control blood samples from 475 healthy people or patients with diseases other than cancer. We performed functional studies of cell lines lacking ARLTS1 expression with the use of both the full-length ARLTS1 gene and a truncated variant. RESULTS: We found a gene at 13q14, ARLTS1, a member of the ADP-ribosylation factor family, with properties of a tumor-suppressor gene. We analyzed 800 DNA samples from tumors and blood cells from patients with sporadic or familial cancer and controls and found that the frequency of a nonsense polymorphism, G446A (Trp149Stop), was similar in controls and patients with sporadic tumors but was significantly more common among patients with familial cancer than among those in the other two groups (P=0.02; odds ratio, 5.7; 95 percent confidence interval, 1.3 to 24.8). ARLTS1 was down-regulated by promoter methylation in 25 percent of the primary tumors we analyzed. Transfection of wild-type ARLTS1 into A549 lung-cancer cells suppressed tumor formation in immunodeficient mice and induced apoptosis, whereas transfection of truncated ARLTS1 had a limited effect on apoptosis and tumor suppression. Microarray analysis revealed that the wild-type and Trp149Stop-transfected clones had different expression profiles. CONCLUSIONS: A genetic variant of ARLTS1 predisposes patients to familial cancer.

ADP-Ribosylation Factors↗

PubMed2005

Genomic profiling maps loss of heterozygosity and defines the timing and stage dependence of epigenetic and genetic events in Wilms' tumors.

To understand genetic and epigenetic pathways in Wilms' tumors, we carried out a genome scan for loss of heterozygosity (LOH) using Affymetrix 10K single nucleotide polymorphism (SNP) chips and supplemented the data with karyotype information. To score loss of imprinting (LOI) of the IGF2 gene, we assessed DNA methylation of the H19 5' differentially methylated region (DMR). Few chromosomal regions other than band 11p13 (WT1) were lost in Wilms' tumors from Denys-Drash and Wilms' tumor-aniridia syndromes, whereas sporadic Wilms' tumors showed LOH of several regions, most frequently 11p15 but also 1p, 4q, 7p, 11q, 14q, 16q, and 17p. LOI was common in the sporadic Wilms' tumors but absent in the syndromic cases. The SNP chips identified novel centers of LOH in the sporadic tumors, including a 2.4-Mb minimal region on chromosome 4q24-q25. Losses of chromosomes 1p, 14q, 16q, and 17p were more common in tumors presenting at an advanced stage; 11p15 LOH was seen at all stages, whereas LOI was associated with early-stage presentation. Wilms' tumors with LOI often completely lacked LOH in the genome-wide analysis, and in some tumors with concomitant 16q LOH and LOI, the loss of chromosome 16q was mosaic, whereas the H19 DMR methylation was complete. These findings confirm molecular differences between sporadic and syndromic Wilms' tumors, define regions of recurrent LOH, and indicate that gain of methylation at the H19 DMR is an early event in Wilms' tumorigenesis that is independent of chromosomal losses. The data further suggest a biological difference between sporadic Wilms' tumors with and without LOI.

Blotting, Northern↗

PubMed2006

Biodegradability of imidazolium and pyridinium ionic liquids by an activated sludge microbial community.

Ionic liquids (ILs) are novel organic salts that have enormous potential for industrial use as green replacements for harmful volatile organic solvents. Varying the cationic components can alter the chemical and physical properties of ILs, including solubility, to suit a variety of industrial processes. However, to complement designer engineering, it is crucial to proactively characterize the biological impacts of new chemicals, in order to fully define them as environmentally friendly. Before introduction of ILs into the environment, we performed an analysis of the biodegradability of six ILs by activated sludge microorganisms collected from the South Bend, Indiana wastewater treatment plant. We examined biodegradability of 1-butyl, 1-hexyl and 1-octyl derivatives of 3-methyl-imidazolium and 3-methyl-pyridinium bromide compounds using the standard Organisation for Economic Cooperation and Development dissolved organic carbon Die-Away Test, changes in total dissolved nitrogen concentrations, and 1H-nuclear magnetic resonance analysis of initial and final chemical structures. Further, we examined microbial community profiles throughout the incubation period using denaturing gradient gel electrophoresis (DNA-PCR-DGGE). Our results suggest that hexyl and octyl substituted pyridinium-based ILs can be fully mineralized, but that imidazolium-based ILs are only partially mineralized. Butyl substituted ILs with either cation, were not biodegradable. Biodegradation rates also increase with longer alkyl chain length, which may be related to enhanced selection of a microbial community. Finally, DGGE analysis suggests that certain microorganisms are enriched by ILs used as a carbon source. Based on these results, we suggest that further IL design and synthesis include pyridinium cations and longer alkyl substitutions for rapid biodegradability.

Electrophoresis↗

PubMed2004

Age-associated alteration of gene expression patterns in mouse oocytes.

Decreasing oocyte competence with maternal aging is a major factor in human infertility. To investigate the age-dependent molecular changes in a mouse model, we compared the expression profiles of metaphase II oocytes collected from 5- to 6-week-old mice with those collected from 42- to 45-week-old mice using the NIA 22K 60-mer oligo microarray. Among approximately 11,000 genes whose transcripts were detected in oocytes, about 5% (530) showed statistically significant expression changes, excluding the possibility of global decline in transcript abundance. Consistent with the generally accepted view of aging, the differentially expressed genes included ones involved in mitochondrial function and oxidative stress. However, the expression of other genes involved in chromatin structure, DNA methylation, genome stability and RNA helicases was also altered, suggesting the existence of additional mechanisms for aging. Among the transcripts decreased with aging, we identified and characterized a group of new oocyte-specific genes, members of the human NACHT, leucine-rich repeat and PYD-containing (NALP) gene family. These results have implications for aging research as well as for clinical ooplasmic donation to rejuvenate aging oocytes.

Aging↗

PubMed2006

DNA adenine methylation regulates virulence gene expression in Salmonella enterica serovar Typhimurium.

Transcriptomic analyses during growth in Luria-Bertani medium were performed in strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic derivatives lacking Dam methylase. More genes were repressed than were activated by Dam methylation (139 versus 37). Key genes that were differentially regulated by Dam methylation were verified independently. The largest classes of Dam-repressed genes included genes belonging to the SOS regulon, as previously described in Escherichia coli, and genes of the SOS-inducible Salmonella prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were also identified. Invasion genes in pathogenicity island SPI-1 were activated by Dam methylation, while the fimbrial operon std was repressed by Dam methylation. Certain flagellar genes were repressed by Dam methylation, and Dam(-) mutants of S. enterica showed reduced motility. Altered expression patterns in the absence of Dam methylation were also found for the chemotaxis genes cheR (repressed by Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB (activated by Dam). The requirement for DNA adenine methylation in the regulation of specific virulence genes suggests that certain defects of Salmonella Dam(-) mutants in the mouse model may be caused by altered patterns of gene expression.

Bacterial Proteins↗

PubMed1998

Bisulfite genomic sequencing-derived methylation profile of the xist gene throughout early mouse development.

Differential epigenetic modification by methylation of CpG dinucleotides is a candidate mechanism that may identify the alleles of imprinted genes and result in monoallelic expression of either the maternal or the paternal allele. Determination of the allelic methylation status of imprinted genes in the gametes and during early development is constrained by the limiting quantities of genomic DNA available from these early developmental stages. To circumvent this problem we have used bisulfite genomic sequencing to determine the allelic methylation status of the minimal promoter and a 1-kb region within the Xist gene during preimplantation development. We find that the parental Xist alleles are not differentially methylated in these regions. Our findings are discussed in the context of previous conflicting data obtained using methylation-sensitive restriction enzyme digestion followed by PCR amplification to assay for methylation.

Animals↗

PubMed2026

Epigenetic activation of NK-cell effector programs and caspase-8-dependent apoptosis mediates the antitumor activity of LGP in NSCLC.

BACKGROUND: Effective activation of natural killer (NK) cell cytotoxicity and caspase-8-dependent extrinsic apoptosis remains a major challenge in non-small cell lung cancer (NSCLC). Epigenetic mechanisms regulating NK cell function within the tumor microenvironment are poorly understood and rarely targeted therapeutically. METHODS: The antitumor activity of Li-Ginseng Powder (LGP), a specifically processed Panax ginseng formulation enriched in rare ginsenosides (Rh4, Rg3, Rg5, Rk1, and Rk3), was evaluated in human lung cancer A549 cells and A549 xenograft mouse models. NK cell infiltration and activation were assessed by flow cytometry, immunoblotting, and immunohistochemistry. Whole-genome bisulfite sequencing (WGBS) was performed to analyze DNA methylation changes. The effects of LGP ginsenosides (LGG) on tumor cell apoptosis and death receptor signaling were examined in vitro. RESULTS: LGP significantly suppressed tumor growth and enhanced systemic and intratumoral NK cell activation. Promoter demethylation of NK cell effector genes, including Ncr1, Gzmb, Nktr, and Itgal, was associated with increased NK cell infiltration and activation, elevated granule-mediated cytotoxicity, and enhanced IFN-γ signaling. In parallel, LGP treatment induced caspase-8-dependent apoptosis associated with increased expression of membrane death receptors, their ligands, FADD, and procaspase-8 in tumor tissues. In vitro, LGG upregulated these apoptosis-initiating proteins and triggered caspase-8 activation in A549 cells independent of promoter methylation changes. Collectively, these immune-associated and tumor-intrinsic responses contributed to robust tumor suppression with a favorable systemic safety profile. CONCLUSIONS: LGP exerts dual antitumor effects characterized by enhanced NK-cell activation and increased sensitivity of tumor cells to caspase-8-dependent extrinsic apoptosis. These coordinated immune-associated and apoptosis-sensitizing effects underscore the therapeutic potential of LGP for the treatment of NSCLC.

DNA methylation↗

PubMed2006

Between genotype and phenotype.

Genome-wide profiling of cytosine methylation for three human chromosomes in a variety of normal tissues provides a reference resource to begin to decode the epigenetic regulation of gene expression in normal development and to understand gene silencing in cancer.

Chromosomes, Human↗

PubMed2026

Cell-free DNA methylation biomarkers for the early detection and tumor burden monitoring of gastric cancer.

Development of sensitive biomarkers is required to achieve early detection and tumor burden monitoring in gastric cancer (GC). We performed genome-wide methylation sequencing on 78 tissue and 241 plasma samples from 171 GC patients and 114 healthy controls from two independent clinical centers. Differentially methylated regions (DMRs) were screened using paired GC and normal tissues, and refined through cfDNA profiles with LASSO regression to construct a cfDNA-based biomarker, the GCML-score. The GCML-score, consisting of 13 DMRs, demonstrated excellent diagnostic performance (AUC: 0.95/0.99/0.95 overall and 0.96/0.99/0.82 in early GC for training/internal validation/external validation cohorts). In 12 patients receiving neoadjuvant chemotherapy, dynamic changes in GCML-score were consistent with radiological tumor burden, highlighting its monitoring potential. The GCML-score, derived from genome-wide cfDNA methylation profiling, provides a robust tool for early GC detection and real-time tumor burden monitoring, facilitating improved prognosis and personalized therapeutic strategies.

Journal Article↗

PubMed2004

Timing of establishment of paternal methylation imprints in the mouse.

Imprinted genes are characterized by predominant expression from one parental allele and differential DNA methylation. Few imprinted genes have been found to acquire a methylation mark in the male germ line, however, and only one of these, H19, has been studied in detail. We examined methylation of the Rasgrf1 and Gtl2 differentially methylated regions (DMR) to determine whether methylation is erased in male germ cells at e12.5 and when the paternal allele acquires methylation. We also compared their methylation dynamics with those of H19 and the maternally methylated gene Snrpn. Our results show that methylation is erased on Rasgrf1, H19, and Snrpn at e12.5, but that Gtl2 retains substantial methylation at this stage. Erasure of methylation marks on Gtl2 appears to occur later in female germ cells to give the unmethylated profile seen in mature MII oocytes. In the male germ line, de novo methylation of Rasgrf1, Gtl2, and H19 occurs in parallel between e12.5 and e17.5, but the DMR are not completely methylated until the mature sperm stage, suggesting a methylation dynamic different from that of IAP, L1, and minor satellite sequences, which have been shown to become fully methylated by e17.5 in male germ cells. This study also indicates important differences between different imprinted DMR in timing and extent of methylation in the germ cells.

Alleles↗

PubMed2006

Prognostic factors for anaplastic astrocytomas.

Anaplastic astrocytomas (WHO grade III) constitute about 10% of all gliomas. Definitive data on predictive and prognostic factors are lacking for these neoplasms that are considered the most enigmatic entity among the whole spectrum of astrocytic tumors because of their unclear biologic behavior and variable clinical outcome. Currently, only few factors have been identified as useful for prognosis of anaplastic astrocytoma: age and Karnofsky Performance Status. Attempts have been made to identify biological prognostic factors for response to therapy and clinical outcome, as well as potential targets for new therapies. Potential prognostic biomarkers concern tumor suppressor genes on chromosome 9q that are involved in the RB1 pathway; PTEN, the PI3k/Akt/p70s6k cascade, survivin gene, Formylpeptide receptor, minichromosome maintenance protein 3 and genes on chromosome 7. Furthermore, some angiogenic factors (e.g. hypoxia-inducible factor-1alpha, vascular endothelial growth factor and scatter factor/hepatocyte growth factor) and the methylation status of O6-methylguanine-DNA methyltransferase gene (one of the main effectors of DNA repair system) are emerging novel putative determinants of prognosis. Moreover, recent studies on magnetic resonance imaging characteristics give prognostic significance to the presence of necrosis and enhancement. The state of the art pictured here underlie the recent interest on gene expression profile to identify aberrations useful to understand the biologic behavior of astrocytic tumors. Our knowledge in this field is still limited, and remains an issue of great concern.

Adult↗

PubMed1996

Reclassification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Beijerinckia sp. strain B1, as Sphingomonas yanoikuyae by fatty acid analysis, protein pattern analysis, DNA-DNA hybridization, and 16S ribosomal DNA sequencing.

A bacterium isolated from a polluted stream, capable of metabolizing biphenyl, naphthalene, phenanthrene, and higher-molecular-weight polycyclic aromatic hydrocarbons (D. Gibson, V. Mahadevan, D. Jerina, H. Yagi, and H. Yeh, Science 189:295-297, 1975), was previously identified as Beijerinckia sp. strain B1. In this investigation, 16S rRNA gene sequencing, biochemical tests, fatty acid methyl ester analysis, polyacrylamide gel electrophoresis of protein, and DNA-DNA hybridization were used to determine the taxonomic relationship of Beijerinckia sp. strain B1. The sequence of the 16S rRNA gene of B1 was identical to that of Sphingomonas yanoikuyae ATCC 51230T. The biochemical tests, fatty acid analysis, and sodium dodecyl sulfate-polacrylamide gel electrophoresis profile of soluble proteins of strain B1 showed results similar to those of S. yanoikuyae. DNA-DNA hybridization indicated that B1 and S. yanoikuyae ATCC 51230T are 75% homologous at the DNA level. We propose that Beijerinckia sp. strain B1 be reclassified as S. yanoikuyae.

Bacterial Proteins↗

PubMed2006

Promoter methylation profile in gallbladder cancer.

BACKGROUND: Methylation in the promoter region of genes is an important mechanism of inactivation of tumor suppressor genes. Our objective was to analyze the methylation pattern of some of the genes involved in carcinogenesis of the gallbladder, examining the immunohistochemical expression of proteins, clinical features, and patient survival time. METHODS: Twenty cases of gallbladder cancer were selected from the frozen tumor bank. The DNA extracted was analyzed by means of a methylation-specific polymerase chain reaction test for the CDKN2A (p16), MLH1, APC, FHIT, and CDH1 (E-cadherin) genes. Morphological and clinical data and follow-up information were obtained. RESULTS: All cases were in an advanced stage: histologically moderate or poorly differentiated tumors (95%). Methylation of the promoter area of genes was observed in 5%, 20%, 30%, 40%, and 65% of cases, and an altered immunohistochemical pattern (AIP) in 5%, 35%, 21%, 25%, and 66% for the MLH1, CDKN2A, FHIT, APC, and CDH1 genes, respectively. The Kappa concordance index between methylation of the promoter area and AIP for the MLH1 and CDH1 genes was very high (K > 0.75) and substantial for APC (K > 0.45). No correlation was found between survival time and the methylation of the genes studied. CONCLUSIONS: The high frequency of gene methylation (with the exception of MLH1) and the high agreement between AIP and methylation of the gene promoter area for the MLH1, APC, and CDH1 genes suggest that the inactivation of tumor suppressor genes and of the genes related to the control of cellular proliferation through this mechanism is involved in gallbladder carcinogenesis.

Acid Anhydride Hydrolases↗

PubMedDate not supplied

The mouse Necdin gene is expressed from the paternal allele only and lies in the 7C region of the mouse chromosome 7, a region of conserved synteny to the human Prader-Willi syndrome region.

Prader-Willi syndrome (PWS) is a neurogenetic disorder resulting from the loss of paternal expression of gene(s) localized in the 15q11-q12 region. A new human gene encoding a putative protein with high homology to the mouse NECDIN protein has recently been characterized and mapped to chromosome 15q11-q12. It is expressed from the paternal allele only, suggesting its potential involvement in PWS. We now report the localization of the mouse Necdin gene in a region of conserved synteny to the human PWS region. We demonstrate the paternal specific expression of Necdin in the mouse central nervous system, and show that parental alleles display a differential methylation profile in the coding region. Finally, fluorescence in situ hybridization analysis reveals an asynchronous pattern of replication at the Necdin locus. These results clearly demonstrate imprinting of the mouse Necdin gene. Mouse models will be powerful tools in the study of human PWS phenotype and imprinting mechanisms.

Alleles↗