64 MACROGLOSSIA IN CLONED PIGLETS IS ASSOCIATED WITH HYPOMETHYLATION AT THE KCNQ-OT1 CpG ISLAND

2011 ◽  
Vol 23 (1) ◽  
pp. 138
Author(s):  
C. Li ◽  
C. O'Gorman ◽  
R. S. Prather ◽  
J. A. Green ◽  
K. D. Wells
Keyword(s):  
Genomic Dna ◽  
Cpg Island ◽  
Methylation Status ◽  
Differentially Methylated Region ◽  
Sequencing Data ◽  
Chi Square ◽  
Cpg Sites ◽  
Loss Of Imprinting ◽  
Parent Of Origin ◽  
Cloned Pig

Beckwith-Wiedeman Syndrome (BWS) is a loss of imprinting (LOI) condition that is associated with macroglossia, midline abdominal defects, and neonatal gigantism among other symptoms. These symptoms have also been seen in animals produced by SCNT. A common feature of BWS is the loss of methylation at the KCNQ-OT1 differentially methylated region. We hypothesised that this locus would show a similar LOI in cloned piglets that display macroglossia. DNA sequence for the porcine KCNQ-OT1 region was assembled in silico from public genome sequencing data. A CpG island was noted as being similarly located in the swine sequence as one which has been described for the human differentially differentiated region. Primers were designed to amplify a portion of this region from bisulfite converted genomic DNA. The amplimer spanned 32 CpG sites. To confirm imprinting status of KCNQ-OT1 in swine, a non-cloned pig was evaluated as to the methylation status across this region using DNA isolated from muscle (M) and the proportion hypermethylated was evaluated by chi-square tests. As seen in humans, this region was hypermethylated in approximately half (12 of 24, P = 1) of the cloned, sequenced amplimers. This observation is consistent with a parent of origin imprint at this locus. Next, 2 cloned piglets that appeared normal were assessed for methylation at KCNQ-OT1. M DNA from each of these animals was consistent with normal methylation at this locus, (7 of 16 and 8 of 18 cloned, sequenced amplimers, P > 0.40). Next, M DNA was isolated from 2 cloned litter mates where 1 piglet presented with macroglossia and the other did not. The non-presenting piglet’s M DNA was methylated in approximately half of the cloned sequenced amplimers (9 of 17, P = 0.67) whereas the macroglossia piglet M DNA was devoid of the methylated allele (0 of 14, P < 0.001). An additional pair of macroglossia presenting and non-presenting cloned littermates was identified. In this pair, the non-presenting piglet showed a normal distribution of methylation at this allele (8 of 19, P = 0.77) and the macroglossia piglet deviated somewhat from normal (6 of 20, P < 0.05). These 2 case studies are consistent with the conclusion that the appearance of macroglossia in cloned pigs may be associated with hypomethylation at KCNQ-OT1 and may model BSW. However, additional abnormal pigs will need to be assessed to completely characterise the LOI in cloned piglets.

313. EPIGENETIC REGULATION OF THE CRH GENE PROMOTER INVOLVES SPECIFIC CpG DE-METHYLATION

2010 ◽  
Vol 22 (9) ◽  
pp. 113
Author(s):  
X. Pan ◽  
C. Abou-Seif ◽  
M. Allars ◽  
Y. Chen ◽  
R. C. Nicholson
Keyword(s):  
Epigenetic Regulation ◽  
Genomic Dna ◽  
Methylation Status ◽  
Gene Promoter ◽  
Bewo Cell ◽  
Gestational Length ◽  
Partial Methylation ◽  
Peripheral Tissues ◽  
Cpg Sites ◽  
Bewo Cells

Corticotropin Releasing Hormone (CRH), is expressed in many regions of the central nervous system and in some peripheral tissues, and plays an important role in determining gestational length. In placenta, a cAMP regulatory site (CRE) is crucial for CRH gene regulation. The promoter of CRH gene has 9 CpG sites, which should be the targets of epigenetic regulation by DNA methylation. The BeWo cell line, derived from human gestational choriocarcinoma, has been widely used as an in vitro model for the placenta. BeWo cells only produce CRH after exposure to cAMP. The DNA methyl transferase (DNMT) inhibitor 5-aza-cytidine stimulates CRH expression 5-fold in camp treated BeWo cells, indicating the CRH promoter as a target of DNMTs. To evaluate methylation differences of the 9 CpG sites in CRH gene promoter in BeWo cells after treatment with cAMP. Genomic DNA was extracted from BeWo cells treated or not with cAMP. Sodium bisulfite conversion was used to modify the genomic DNA. PCR was used to amplify the CRH promoter region with primers that did not contain CpG sites. The PCR products were cloned and sequenced. The CpG methylation status of each sample was obtained by comparing the sequencing results with the original sequence. In non-stimulated cells (control) CpG -4 was methylated in 50% of the clones and CpG -6 was methylated in 75% of the clones, but the other 7 sites were methylated in every clone. In the cAMP treated cells however there was 100% methylation at CpG sites 6 through 9, but only partial methylation at CpG-1 and 3 (60%), CpG-4 and 5 (40%). Most interestingly, there was no methylation found at CpG-2 in any of the clones from cAMP treated cells, indicating that specific CpG de-methylation around the CRE is required for CRH gene expression.

scholarly journals Quantitative High-Resolution CpG Island Mapping with Pyrosequencing™ Reveals Disease-Specific Methylation Patterns of the CDKN2B Gene in Myelodysplastic Syndrome and Myeloid Leukemia

2007 ◽  
Vol 53 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Kai Brakensiek ◽  
Luzie U Wingen ◽  
Florian Länger ◽  
Hans Kreipe ◽  
Ulrich Lehmann
Keyword(s):  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Cpg Island ◽  
Methylation Status ◽  
Low Grade ◽  
Myeloid Malignancies ◽  
Cpg Sites ◽  
Methylation Patterns ◽  
Cdkn2b Gene ◽  
Disease Specific

Abstract Background: Gene silencing through aberrant CpG island methylation is the most extensively analyzed epigenetic event in human tumorigenesis and has huge diagnostic and prognostic potential. Methylation patterns are often very heterogeneous, however, presenting a serious challenge for the development of methylation assays for diagnostic purposes. Methods: We used Pyrosequencing™ technology to determine the methylation status of 68 CpG sites in the CpG island of the CDKN2B gene [cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)], frequently hypermethylated in myeloid malignancies, in a series of bone marrow samples from patients with myelodysplasia and myeloid leukemia (n = 82) and from 32 controls. A total of 7762 individual methylation sites were quantitatively evaluated. Precision and reproducibility of the quantification was evaluated with several overlapping primers. Results: The use of optimized sequencing primers and the new Pyro Q-CpG™ software enabled precise and reproducible quantification with a single sequencing primer of up to 15 CpG sites distributed over ∼100 bp. Extensive statistical analyses of the whole CpG island revealed for the first time disease-specific methylation patterns of the CDKN2B gene in myeloid malignancies and small regions of differential methylation with high discriminatory power that enabled differentiation of even low-grade myelodysplastic syndrome samples from the controls, a result that was confirmed in an independent group of 9 control and 36 patient samples. Conclusion: The precise quantitative methylation mapping of whole CpG islands is now possible with Pyrosequencing software in combination with optimized sequencing primers. This method reveals disease-specific methylation patterns and enables the development of specific diagnostic assays.

Identification of Frequent Differentially Methylated Region in Sporadic Bladder Cancers

2014 ◽  
Vol 94 (4) ◽  
pp. 479-484 ◽  
Author(s):  
Ryo Hasegawa ◽  
Kyoko Fujiwara ◽  
Daisuke Obinata ◽  
Hiroyuki Kawashima ◽  
Yui Shinojima ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Bladder Cancer ◽  
Genomic Dna ◽  
Differentially Methylated Region ◽  
Low Grade ◽  
Aberrant Methylation ◽  
High Grade ◽  
Cpg Sites ◽  
Intron 1 ◽  
Exon 1

Introduction: Aberrant methylation levels in the cytosine-phosphate-guanine island (CpGi) region from exon 1 to intron 1 of the zygote arrest 1 (ZAR1) gene have been reported in several types of human cancers, including melanoma, brain tumor, and hepatocellular carcinoma. In the present study, methylation levels at the CpGi of ZAR1 exon 1/intron 1 in bladder cancer specimens were analyzed using mass spectrometry. Materials and Methods: Genomic DNA was extracted from 20 sporadic bladder cancers, and the methylation levels at ZAR1 CpGi were quantitatively examined by the MassARRAY EpiTYPER method. Result: The methylation levels at specific CpG sites of the ZAR1 CpGi were significantly lower in high-grade bladder cancers than in low-grade tumors. Conclusions: The results of the present study indicated a decreased methylation level at CpG sites of ZAR1 exon 1/intron 1. CpGi could serve as a biomarker for invasive bladder cancer.

Epigenetic Regulation of ZAP70 in Chronic Lymphocytic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2246-2246
Author(s):  
Anton Parker ◽  
Shilu Amin ◽  
Tricia Zwiefelhofer ◽  
Ben Gregory ◽  
Helen White ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cpg Island ◽  
Methylation Status ◽  
Differentially Methylated Region ◽  
Leukocyte Populations ◽  
Expression Status

Abstract ZAP70 expression has been shown to be involved in enhanced signalling and more aggressive disease in a subset of CLL. Mechanisms regulating ZAP70 expression are unknown. We have shown previously that despite the absence of a 5’ CpG island, the methylation status of a small region of CpG dinucleotides (CpGs) correlates with the transcriptional state of the gene in both normal lymphocytes and B cell leukemias. Quantitative methylation analysis of 605 CpGs across the 28kb genomic region spanning ZAP70 was performed by MassARRAY on a panel of 17 CLL tumor cell samples, 4 lymphoid cell lines and B cell, T cell and myeloid cell samples pooled from 3 normal individuals. All samples showed hypermethylation of the gene body and of the gene’s two 3’ CpG islands. However, there was variability between samples in the methylation of 12 consecutive CpGs within a 1kb predicted promoter region (PPR), spanning the transcription start site (TSS) and in the methylation of 24 consecutive CpGs in an adjacent 1kb differentially methylated region (DMR), downstream of the TSS. The methylation of the PPR and DMR, together with the expression status of the samples, suggested four different states for the gene (Table 1). Table 1 - ZAP70 gene states defined by ZAP70 expression status and methylation of the PPR and DMR. MEAN CpG METHYLATION (%) SAMPLE ZAP70 EXPRESSION STATUS PPR DMR GENE STATE NAMALWA − 65 82 I B CELLS − 48 82 I MYELOID − 53 80 I CLL6 − 4 86 II CLL7 − 5 75 II CLL8 − 12 78 II CLL10 − 12 62 II CLL11 − 4 62 II CLL12 − 21 77 II HBL2 − 18 60 II CLL13 + 4 40 III CLL14 + 5 46 III CLL15 + 7 45 III CLL16 + 9 43 III CLL17 + 5 56 III NALM6 + 8 52 III CLL1 + 3 4 IV CLL2 + 3 4 IV CLL9 + 4 6 IV CLL4 + 3 8 IV CLL5 + 4 16 IV CLL3 + 4 17 IV JURKAT + 3 4 IV T CELLS + 9 13 IV Bisulphite cloning and sequencing of a PCR amplicon spanning an exon1 C/A SNP (rs2276645) and the PPR/DMR junction was performed together with cDNA pyrosequencing of rs2276645 on the five CLL tumor samples identified with gene state III. All samples showed allele specific methylation (ASM) of the A allele within the DMR and almost complete restriction of ZAP70 expression to the hypomethylated C allele. Bisulphite pyrosequencing of two DMR CpGs in purified leukocyte populations from these cases showed that ASM appears restricted to CLL cells, with hypermethylation and hypomethylation of the myeloid and T cells respectively (Table2). This suggests that while methylation of the DMR is sufficient for allele restriction, ASM does not result from imprinting and may be restricted to CLL tumor cells. Table 2 – Mean methylation of 2 DMR CpGs in leukocyte populations from CLL patients with known ASM of the DMR in their tumor cells. MYELOID CELLS CLL CELLS T CELLS PATIENT CD15 (%) METHYLATION(%) CD19 (%) METHYLATION (%) CD2 (%) METHYLATION (%) CLL13 83 90 98 59 71 21 CLL14 98 99 98 50 88 10 CLL15 88 84 93 45 85 24 CLL16 99 96 99 52 82 18 CLL17 92 89 98 49 90 22 Native chromatin immunoprecipitation (N-ChIP) using anti-AcH3, H3K14Ac and H3K14Me2 antibodies was performed on the 4 cell lines and tumor cells from CLLs 1, 2, 6, 7, 13 and 14 from the MassARRAY series. PCR for amplicons across the PPR and DMR showed the presence of all 3 histone modifications in ZAP70 expressing JURKAT and NALM6 cells but these modifications were absent in the ZAP70 negative NAMALWA and HBL2 cells. In contrast, all 6 CLL samples showed enrichment for all 3 modifications, regardless of gene state, suggesting an open, active/permissive chromatin structure, despite clear differences in methylation of the DMR. Further bisulphite pyrosequencing and N-ChIP of NAMALWA and HBL2 cells cultured for 6 days in the presence of 0.5μM Decitibine showed concomitant DMR demethyaltion, increased AcH3 within the DMR and up regulation of ZAP70 expression, all of which were reversed when the drug stimulus was removed. Taken together this data suggests that ZAP70 is regulated by epigenetic mechanisms, with the methylation status of a small DMR playing a key role, sufficient to differentiate the transcriptional activity of two alleles within a single cell. It is apparent that the gene is primed for expression in all CLL cells and that methylation of the DMR is part of the key switching process between active transcription and silencing. The differences in DMR methylation between an individual’s expressing T cells and CLL cells, suggests that differences may exist in the mechanism of regulation between T and B cells and raises the possibility that such differences could be exploited as targets for therapy.

Hyper-Methylation DAZAP2 May Suppress Its Expression in Specific Subtypes of Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4421-4421
Author(s):  
Wei-Xin Hu ◽  
Qiang Qu ◽  
Jiang Li ◽  
Wei Ren
Keyword(s):  
Transcriptional Activity ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Myeloma Cell ◽  
Positive Control ◽  
Newly Diagnosed ◽  
Cpg Sites

Abstract Abstract 4421 Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. We previously reported that DAZAP2 was down-regulated in newly diagnosed myeloma (MM) and this may influence MM cell growth and survival. This study was undertaken to evaluate the mechanism of down-regulation DAZAP2 and determine the methylation status and loci of its promoter by using bisulphite genomic-sequencing (BGS) method in KM3 myeloma cell line. Two islands with rich GC box in the promoter of DAZAP2 gene were identified and amplified by using bisulfite-sequencing PCR (BSP). Island 1 spans -472 to -247 including 9 CpG sites (CpGs) and island 2 covers -226 to -13 including 23 CpG sites. The ratio of methylated CpGs in two CpG islands was 46.25%. The above sequences were demethylated and inserted into a pGL2-basic vector. COS-7 cells were transfected with recombinant plasmids and the activity of luciferase was evaluated. The results showed that the CpG island 1 had a weakly transcriptional activity, whereas the CpG island 2 had a strong transcriptional activity (2.38 folds compared with the positive control). The other sequence which covered CpG island 1 and 2 showed a remarkable activity (15.1 folds compared with the positive control). These data indicated that CpG island 2 of DAZAP2 gene may be hypermethylated and suppressed its expression in MM cells. We further correlated DAZAP2 expression with normal plasma cells and malignant myeloma cells, as well as the molecular subtypes which the dataset includes 8 genetic subtypes (MY, PR, LB, MS, HP, CD-1, CD-2, and MF) from 351 newly diagnosed myeloma cases (Zhan, 2006). Interestingly, we extended and confirmed our previous discovery that DAZAP2 was significantly down-regulated in MM cells by using a large uniform dataset (P = 0.004). The low expression of DAZAP2 was especially significant in the subgroups of MY, PR, LB, HP, and CD-1. This study warrants further investigation of DAZAP2 and its potential role in myeloma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hypermethylation of the IGF2 differentially methylated region 2 is a specific event in insulinomas leading to loss-of-imprinting and overexpression

2009 ◽  
Vol 16 (3) ◽  
pp. 939-952 ◽  
Author(s):  
Emelyne Dejeux ◽  
Robert Olaso ◽  
Bertrand Dousset ◽  
Anne Audebourg ◽  
Ivo G Gut ◽  
...  
Keyword(s):  
Methylation Status ◽  
Differentially Methylated Region ◽  
Pancreatic Tumors ◽  
Early Event ◽  
Endocrine Tumors ◽  
Loss Of Imprinting ◽  
Pancreatic Endocrine Tumors ◽  
Degree Of Malignancy ◽  
Tumor Types ◽  
Methylation Patterns

Prediction of the evolution of endocrine pancreatic tumors remains difficult based on histological criteria alone. We have previously demonstrated that epigenetic changes are an early event in a mouse model developing insulinomas. Particularly, overexpression of the imprinted IGF2 was caused by the hypermethylation of CpGs in the differentially methylated region 2 (DMR2). Here, we investigated whether IGF2 hypermethylation is also observed in human insulinomas and whether this alteration is common to other human endocrine tumors of the pancreas and the digestive tract. We analyzed the methylation status of 40 CpGs located in the DMR0 and DMR2 of the IGF2 as well as in the H19 DMR by pyrosequencing in a cohort of 62 patients with pancreatic or small intestine endocrine tumors. Altered methylation patterns were observed in all tumor types for the different regions of IGF2, but not for H19. However, hypermethylation of the IGF2 DMR2 was specific for insulinomas and did not occur in any of the other types of tumors which were characterized by a loss of methylation in this region. Gain of methylation in the IGF2 DMR2 in insulinomas correlated with loss-of-imprinting and promoter 4 mediated overexpression of IGF2 at the RNA and protein level. Furthermore, a decreasing degree of methylation in the different regions of IGF2 correlated well with increasing degree of malignancy according to the WHO classification of pancreatic endocrine tumors (PETs), suggesting that methylation of IGF2 might be a useful biomarker for classification and staging of PETs.

DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss

2020 ◽  
Author(s):  
Kushaan Khambata ◽  
Sanketa Raut ◽  
Sharvari Deshpande ◽  
Sweta Mohan ◽  
Shobha Sonawane ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Pregnancy Loss ◽  
Recurrent Pregnancy Loss ◽  
Methylation Status ◽  
Differentially Methylated Region ◽  
Imprinted Genes ◽  
Epigenetic Factors ◽  
Cpg Sites ◽  
Sperm Dna ◽  
Sperm Dna Methylation

Abstract STUDY QUESTION What is the sperm DNA methylation status of imprinted genes in male partners from couples experiencing recurrent pregnancy loss (RPL)? SUMMARY ANSWER Aberrations in sperm DNA methylation status of several imprinted genes, such as insulin like growth factor-2-H19 differentially methylated region (IGF2-H19 DMR), intergenic differentially methylated region (IG-DMR), mesoderm specific transcript (MEST), zinc finger protein which regulates apoptosis and cell cycle arrest (ZAC), DMR in intron 10 of KCNQ1 gene (KvDMR), paternally expressed gene 3 (PEG3) and paternally expressed gene 10 (PEG10), as well as decreased sperm global 5-methylcytosine (5mC) levels, are associated with RPL. WHAT IS KNOWN ALREADY RPL is defined as loss of two or more pregnancies, affecting 1–2% of couples of reproductive age. Although there are several maternal and paternal aetiological factors contributing to RPL, nearly 50% of the cases remain idiopathic. Thus, there is a need to identify putative paternal factors that could be contributing towards pregnancy loss in cases of idiopathic RPL. STUDY DESIGN, SIZE, DURATION In this case–control study, 112 couples undergoing RPL with no identifiable cause were recruited from September 2015 to May 2018. The control group comprised of 106 healthy proven fertile couples with no history of infertility or miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS In this study, we investigated the paternal genetic and epigenetic factors that could be associated with RPL. We studied DNA methylation, by pyrosequencing, of selected imprinted genes implicated in embryo development, such as IGF2-H19 DMR, IG-DMR, MEST, ZAC, KvDMR, PEG3, PEG10 and small nuclear ribonucleoprotein polypeptide N (SNRPN) in sperm of men whose partners present RPL. Global DNA methylation in sperm was evaluated by studying 5mC content and long interspersed nuclear element 1 (LINE1) promoter methylation. We also studied polymorphisms by pyrosequencing in the IGF2-H19 DMR as well in the IGF2 promoter in both groups. MAIN RESULTS AND THE ROLE OF CHANCE In the RPL group, we found a significant decrease in the global sperm 5mC levels and significant decrease in DNA methylation at three CpG sites in LINE1 promoter. For IGF2-H19 DMR and IG-DMR, a significant decrease in sperm DNA methylation at specific CpG sites was observed in RPL group. For maternally imprinted genes like MEST, ZAC, KvDMR, PEG3 and PEG10 hypermethylation was noted. Polymorphism studies for IGF2-H19 DMR and IGF2 revealed significant differences in the genotypic frequencies in males. LIMITATIONS, REASONS FOR CAUTION In this study, we analysed the methylation levels of selected candidate imprinted genes implicated in embryo development. Detection of methylation changes occurring at the genome-wide level may reveal further candidate genes having a better distinction between the control and study groups. WIDER IMPLICATIONS OF THE FINDINGS Our study demonstrates that certain polymorphisms and aberrant sperm methylation status in imprinted genes are associated with RPL and could contribute to the aetiology of RPL. This study suggests that investigation of paternal genetic and epigenetic factors could be useful in identification of possible causes of idiopathic RPL. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Department of Science and Technology-Science and Engineering Research Board (EMR/2014/000145) and National Institute for Research in Reproductive Health intramural funds (RA/872/01-2020). All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

scholarly journals Contribution of Dopamine Transporter Gene Methylation Status to Cannabis Dependency

2020 ◽  
Vol 10 (6) ◽  
pp. 400
Author(s):  
Anna Grzywacz ◽  
Wojciech Barczak ◽  
Jolanta Chmielowiec ◽  
Krzysztof Chmielowiec ◽  
Aleksandra Suchanecka ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
Dna Sequences ◽  
Blood Cells ◽  
Cpg Island ◽  
Methylation Status ◽  
Gene Promoter ◽  
Transporter Gene ◽  
Dopamine Transporter Gene ◽  
Cpg Sites ◽  
Dat1 Gene

The susceptibility to cannabis dependency results from the influence of numerous factors such as social, genetic, as well as epigenetic factors. Many studies have attempted to discover a molecular basis for this disease. However, our study aimed at evaluating the connection between altered methylation of the dopamine transporter gene (DAT1) promoter CpG sites and cannabis dependency. In the cases of some DNA sequences, including the DAT1 gene region, their methylation status in blood cells may reflect a systemic modulation in the whole organism. Consequently, we isolated the DNA from the peripheral blood cells from a group of 201 cannabis-dependent patients and 285 controls who were healthy volunteers and who were matched for age and sex. The DNA was subjected to bisulfite conversion and sequencing. Our analysis revealed no statistical differences in the general methylation status of the DAT1 gene promoter CpG island between the patients and controls. Yet, the analysis of individual CpG sites where methylation occurred indicated significant differences. These sites are known to be bound by transcription factors (e.g., SP1, p53, PAX5, or GR), which, apart from other functions, were shown to play a role in the development of the nervous system. Therefore, DAT1 gene promoter methylation studies may provide important insight into the mechanism of cannabis dependency.

Tissue Factor Pathway Inhibitor 2 (TFPI2) Is Commonly Methylated in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4617-4617
Author(s):  
Eleftheria Hatzimichael ◽  
Aggeliki Dasoula ◽  
Valentinos Kounnis ◽  
Andreas Katsenos ◽  
Tim Crook ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Genomic Dna ◽  
Tissue Factor Pathway Inhibitor ◽  
Cpg Island ◽  
Methylation Status ◽  
Tissue Factor Pathway ◽  
Beta 2 ◽  
Beta 2 Microglobulin

Abstract Abstract 4617 Background: Tissue Factor Pathway Inhibitor 2 (TFPI2) is a member of the Kunitz-type serine proteinase inhibitor family that regulates extracellular tissue homeostasis by inhibiting matrix metalloproteinases. Transcriptional silencing of TFPI2 due to DNA methylation of the CpG island of its promoter has been reported in several tumors and has been suggested to facilitate tumor cell invasion and angiogenesis. We investigated the DNA methylation status of TFPI2 in multiple myeloma (MM) patients as part of a program of epigenetic profiling of multiple myeloma. Methods: Genomic DNA extracted from two human MM cell lines (U-266 and RPMI-8226) and bone marrow aspirate samples from a well chracterized series of MM patients was modified by sodium bisulphite using the EZ DNA methylation kit, ZymoResearch. Bisulfite modified DNA was used as a template for Methylation Specific PCR with primers specific for unmethylated and methylated alleles. Control methylated (CpG Genome™ Universal Methylated, Chemicon International) and unmethylated genomic DNA was included in each experiment. Chi-square test, logistic regression analysis and unpaired t-test were used to investigate associations between gene methylation and ISS stage, presence of extramedullary disease, bone disease, anemia (hemoglobin ≤10 mg/dL), renal failure, serum albumin and beta (2)-microglobulin level. Kaplan-Meier curves were used to estimate the probabilities of survival and the Log-rank test to assess the statistical significance of differences in event rates. Results: Forty six patients with MM (26 male, 20 female; mean age 64) and two human MM cell lines were studied. According to the International Staging System (ISS) 21 patients were classified as stage I, 10 stage II and 15 as stage III disease. Methylation in the CpG island of the promoter of TFPI2 was detected in 30 patients (65%, 95%CI= 52,6–78,7%) and in both cell lines (100%). Patients with methylated TFPI2 had significantly elevated beta 2 microglobulin levels (5553 vs 3787 mg/L mean values, p=0.0002) but we did not detect a statistically significant difference in overall survival among patients with methylated versus non methylated TFPI2. No other relevant correlations were detected. Conclusion: TFPI2 is commonly methylated in MM patients. Transcriptional silencing of TFPI2 may play a role in the mechanism of myelomatogenesis and its methylation status warrants further evaluation as a diagnostic co-marker for this disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DNA methylation in the OPG/RANK/RANKL pathway is associated with steroid-induced osteonecrosis of the femoral head

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Menghu Sun ◽  
Yuju Cao ◽  
Xiaolong Yang ◽  
Feimeng An ◽  
Huiqiang Wu ◽  
...  
Keyword(s):  
Femoral Head ◽  
Case Control Study ◽  
Methylation Status ◽  
Cpg Islands ◽  
Signalling Pathway ◽  
Chi Square ◽  
Cpg Sites ◽  
Chi Square Test ◽  
The Relationship ◽  
Control Study

Abstract Background Dysregulation of the OPG/RANK/RANKL signalling pathway is a key step in the occurrence of steroid-induced osteonecrosis of the femoral head (ONFH). This study aims to understand the degree of methylation of the OPG, RANK, and RANKL genes in steroid-related ONFH. Methods A case-control study was designed, including 50 patients (25 males and 25 females) and 50 matched controls. The European Molecular Biology Open Software Suite (EMBOSS) was used to predict the existence and location of CpG islands in the OPG, RANK, and RANKL genes. The Agena MassARRAY platform was used to detect the methylation status of the above genes in the blood of subjects. The relationship between the methylation level of CpG sites in each gene and steroid-related ONFH was analysed by the chi-square test, logistic regression analysis, and other statistical methods. Results In the CpG islands of the OPG, RANK, and RANKL genes in patients with steroid-related ONFH, several CpG sites with high methylation rates and high methylation levels were found. Some hypermethylated CpG sites increase the risk of steroid-related ONFH. In addition, a few hypermethylated CpG sites have predictive value for the early diagnosis of steroid-related ONFH. Conclusion Methylation of certain sites in the OPG/RANK/RANKL signalling pathway increases the risk of steroid-related ONFH. Some hypermethylated CpG sites may be used as early prediction and diagnostic targets for steroid-related ONFH.

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