Altered Expression of the Repair Genes in CD34+ Cells May be Responsible for Formation and Accumulation of Mutations in MDS Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4119-4119
Author(s):  
Jan Valka ◽  
Monika Belickova ◽  
Jitka Vesela ◽  
Eliska Stara ◽  
Barbora Pejsova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
High Risk ◽  
Expression Profile ◽  
Low Risk ◽  
Control Group ◽  
Dna Repair Genes ◽  
Altered Expression ◽  
Cd34 Cells ◽  
Repair Genes

Abstract Background and Aims Recent studies have demonstrated that in most cases of myelodysplastic syndrome (MDS) at least one mutation has been detected, suggesting that abnormal DNA repair may represent both cause and consequence of malignant transformation. In this study we investigated a possible role of different alterations in DNA repair genes in pathogenesis of MDS. Methods Gene expression of CD34+ cells was measured by RT² Profiler PCR Arrays (Qiagen). Polymorphisms and mutations were studied by targeting next generation sequencing (SeqCap EZ System, NimbleGen). Expression analysis of 84 DNA repair genes was performed in 18 MDS patients and subsequent analysis of selected genes was performed on a cohort of 80 patients. The enrichment resequencing of 84 genes was done in 16 patients. Results Five differentially expressed genes between CD34+ cells of patient and control samples were identified (p<0.05). The increased expression was detected in MPG and XPC genes and decreased expression in RAD51, RPA3 and XRCC2 genes. RAD51 gene showed significantly higher expression in the patients with low-risk MDS forms (RA, RARS, del5q) compared to control group (p=0.0005) and to contrast down-regulated expression was detected (p=0.0002) in high-risk MDS patients (RAEB-1 and -2, AML with myelodysplasia). The group of patients with RCMD showed the average expression at the level of the control group. The expression profile shown a gravity-related decreasing trend after dividing the patients according to IPSS-R, IPSS-R cytogenetic groups and blasts count. Expression profile of XRCC2 gene has similar characteristics as RAD51 (p<0.0001). The expression of RPA3 gene was generally decreased (p<0.0001) with a decreasing trend depending on the disease severity according to MDS forms, IPSS-R and cytogenetics. The RAD51, XRCC2 and RPA-3 genes are related to homologous recombination mechanism, with XRCC2 and RPA3 as a supporting factors for RAD51. The survival curves for all the three genes shown significant differences between groups with over-, intermediate- and down-expressed gene (p=0.0001 for RAD51; 0.0022 for XRCC-2 and 0.0054 for RPA3). MPG gene expression was up-regulated by all MDS types with no significant difference between each other (p=0.0015). Deeper testing of XPC gene expression demonstrated the up-regulation only in low-risk MDS group (p=0.0084). Gene-based analysis showed seven candidate SNPs significantly associated with the disease susceptibility using the HapMAP-CEU population from NCBI PubMed database as control group. With the presence of MDS, these polymorphisms are significantly connected: rs4135113 (p=0.03), rs12917 (p=0.003), rs2230641 (p=0.01), rs2228529 and rs2228526 (p=0.04, respectively p=0.03) and rs1799977 (p=0.04). Within the distribution of tested patient groups according to cytogenetics, we observed significant increase of dependency of these polymorphisms (OR 4.1-9.7, p<0.0001) and the presence of MDS in the group of patients with very-high risk cytogenetics. Conclusions DNA repair mechanisms are responsible for correcting DNA damage and preserving genomic integrity. Our study demonstrates, that altered expression of the repair genes in CD34+ cells may be responsible for the formation and accumulation of mutations in hematological malignancies. Furthermore, we have identified genetic variants that might contribute to the pathogenesis of MDS by modifying individual risk for the disease. Supported by grant (NT/13899, NT/14377, and NT/14539) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

DNA Repair Gene Expression in CD34+ Bone Marrow Cells in MDS Patients with Mutiple Chromosomal Abnormalities by Array-Based Comparative Genomic Hybridization (A-CGH).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3423-3423
Author(s):  
Lukasz P. Gondek ◽  
Christine L. O’Keefe ◽  
Matt Kalaycio ◽  
Anjali Advani ◽  
Mikkael M. Sekeres ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Dna Repair ◽  
Cumulative Exposure ◽  
Dna Repair Genes ◽  
Dna Repair Enzymes ◽  
Chromosomal Structure ◽  
Repair Enzymes ◽  
Cd34 Cells ◽  
Repair Genes

Abstract Based on the high rate of chromosomal defects in MDS, inherent chromosomal instability (CIN) has been hypothesized as a key pathophysiologic factor of clonal evolution. Predisposition to DNA damage may be primarily due to acquired/inherited weakness in DNA repair machinery; such insufficiency may become manifest after a long latency following cumulative exposure to genotoxic agents. Subsequent changes in chromosomal structure and stepwise acquisition of neoplastic features could lead to leukemic progression. Previously, a number of allelic polymorphisms in DNA repair genes were observed. These variants may lead to altered expression of corresponding proteins. Conversely, if DNA damage is a primary defect, upregulation of specific DNA repair enzymes may be compensatory. Irrespective of the initial pathogenetic defect, we theorized that broad analysis of DNA repair machinery in MDS may point towards specific lesions that could be a subject of more targeted studies. Therefore, we examined levels of DNA repair enzymes using gene expression arrays. For proper comparisons, CD34 cells from 10 MDS patients (4 RA, 6 RAEB/RAEBt) and healthy controls were used. Expression array results were confirmed by Taqman PCR. Reference expression was established by pooling RNA from 12 controls. For more targeted analysis, A-CGH based genomic scan was used to better assess the extent of DNA damage in patients. The expression of 22 out of 113 DNA repair genes tested was detectable at levels &gt;1,5X background; 2-level normalization of gene expression was performed according to variation of mRNA input (housekeeping gene-ACTB) and inter-assay variation in the signal intensity (biotinylated artificial sequence -BAS2C). Our combined standard sample was validated against individual controls; signals &lt;1,5X pooled expression were obtained. Using expression levels of normal CD34 cells as a reference we found that 19 genes were upregulated in concordant fashion. The most dramatically increased genes included APEX, ATM, XRCC1, XRCC5 and MPG. This finding favors the theory that overexpression of the DNA repair machinery is a compensatory event to cope with a primarily increased level of DNA damage. When we subgrouped MDS patients according to FAB criteria, the expression of DNA repair genes (e.g., CIB1, ERCC1, SUMO1) increased with the malignant progression. For further analysis we have defined CIN phenotype by the presence of large or multiple small defects as determined by A-CGH. When patients with CIN vs. those with normal karyotype were compared, we found that chromosomal damage was not accompanied by a higher expression of DNA repair genes. MPG was most dramatically upregulated in all MDS patients. This gene involved in excision of methylated bases can induce single stranded breaks (SSB) and increase sensitivity to alkylating agents. Our finding suggest that either increased purine methylation induces a compensatory mechanism (MPG upregulation) or that overactivity of MPG itself results in increased base excision. Alternatively, overexpression of MPG may lead to SSB especially because downstream genes (e.g. XRCC3 or DNA ligase III) were not accordingly upregulated. In conclusion, our studies form a basis for further analysis of clinical phenotypes associated with upregulation of specific DNA repair genes and may indicate possible therapeutic targets in molecularly defined subtypes of MDS.

Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3553-3560 ◽  
Author(s):  
Wolf-K. Hofmann ◽  
Sven de Vos ◽  
Martina Komor ◽  
Dieter Hoelzer ◽  
William Wachsman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
High Risk ◽  
Bone Marrow Cells ◽  
Low Risk ◽  
Control Group ◽  
Expression Data ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Marrow Cells

Gene patterns of expression in purified CD34+ bone marrow cells from 7 patients with low-risk myelodysplastic syndrome (MDS) and 4 patients with high-risk MDS were compared with expression data from CD34+ bone marrow cells from 4 healthy control subjects. CD34+ cells were isolated by magnetic cell separation, and high-density oligonucleotide microarray analysis was performed. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction. Class membership prediction analysis selected 11 genes. Using the expression profile of these genes, we were able to discriminate patients with low-risk from patients with high-risk MDS and both patient groups from the control group by hierarchical clustering (Spearman confidence). The power of these 11 genes was verified by applying the algorithm to an unknown test set containing expression data from 8 additional patients with MDS (3 at low risk, 5 at high risk). Patients at low risk could be distinguished from those at high risk by clustering analysis. In low-risk MDS, we found that the retinoic-acid–induced gene (RAI3), the radiation-inducible, immediate-early response gene (IEX1), and the stress-induced phosphoprotein 1 (STIP1) were down-regulated. These data suggest that CD34+cells from patients with low-risk MDS lack defensive proteins, resulting in their susceptibility to cell damage. In summary, we propose that gene expression profiling may have clinical relevance for risk evaluation in MDS at the time of initial diagnosis. Furthermore, this study provides evidence that in MDS, hematopoietic stem cells accumulate defects that prevent normal hematopoiesis.

scholarly journals Expression of DNA repair and metabolic genes in response to a flavonoid-rich diet

2007 ◽  
Vol 98 (3) ◽  
pp. 525-533 ◽  
Author(s):  
Simonetta Guarrera ◽  
Carlotta Sacerdote ◽  
Laura Fiorini ◽  
Rosa Marsala ◽  
Silvia Polidoro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
Significant Negative Correlation ◽  
Dna Repair Genes ◽  
Metabolic Genes ◽  
Damage Repair ◽  
Flavonoid Intake ◽  
Repair Mechanisms ◽  
Repair Genes

A diet rich in fruit and vegetables can be effective in the reduction of oxidative stress, through the antioxidant effects of phytochemicals and other mechanisms. Protection against the carcinogenic effects of chemicals may also be exerted by an enhancement of detoxification and DNA damage repair mechanisms. To investigate a putative effect of flavonoids, a class of polyphenols, on the regulation of the gene expression of DNA repair and metabolic genes, a 1-month flavonoid-rich diet was administered to thirty healthy male smokers, nine of whom underwent gene expression analysis. We postulated that tobacco smoke is a powerful source of reactive oxygen species. The expression level of twelve genes (APEX, ERCC1, ERCC2, ERCC4, MGMT, OGG1, XPA, XPC, XRCC1, XRCC3, AHR, CYP1A1) was investigated. We found a significant increase (P < 0·001) in flavonoid intake. Urinary phenolic content and anti-mutagenicity did not significantly change after diet, nor was a correlation found between flavonoid intake and urinary phenolic levels or anti-mutagenicity. Phenolic levels showed a significant positive correlation with urinary anti-mutagenicity. AHR levels were significantly reduced after the diet (P = 0·038), whereas the other genes showed a generalized up regulation, significant for XRCC3 gene (P = 0·038). Also in the context of a generalized up regulation of DNA repair genes, we found a non-significant negative correlation between flavonoid intake and the expression of all the DNA repair genes. Larger studies are needed to clarify the possible effects of flavonoids in vivo; our preliminary results could help to better plan new studies on gene expression and diet.

scholarly journals DNA Repair Gene Expression Adjusted by the PCNA Metagene Predicts Survival in Multiple Cancers

2019 ◽  
Author(s):  
Leif Peterson ◽  
Tatiana Kovyrshina
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
Gene Selection ◽  
P Value ◽  
Survival Prediction ◽  
Dna Repair Genes ◽  
Repair Gene ◽  
A Genome ◽  
Dna Repair Gene ◽  
Repair Genes

Removal of the proliferation component of gene expression by PCNA adjustment has been addressed in numerous survival prediction studies for breast cancer and all cancers in the TCGA. These studies indicate that widespread co-regulation of proliferation upwardly biases survival prediction when gene selection is performed on a genome-wide basis. In addition, removal of the correlative effects of proliferation does not reduce the random bias associated with survival prediction using random gene selection. Since most cancers become addicted to DNA repair as a result of forced cellular replication, increased oxidation, and repair deficiencies from oncogenic loss or genetic polymorphisms, we pursued an investigation to remove the proliferation component of expression in DNA repair genes to determine survival prediction. This translational hypothesis-driven focus on DNA repair genes is directly amenable to finding new sets of DNA repair genes that could potentially be studied for inhibition therapy. Overall survival (OS) prediction was evaluated in 18 cancers by using normalized RNA-Seq data for 126 DNA repair genes with expression available in TCGA. Transformations for normality and adjustments for age at diagnosis, stage, and PCNA metagene expression were performed for all DNA repair genes. We also analyzed genomic event rates (GER) for somatic mutations, deletions, and amplification in driver genes and DNA repair genes. After performing empirical p-value testing with use of randomly selected gene sets, it was observed that OS could be predicted significantly by sets of DNA repair genes for 61% (11/18) of the cancers. Interestingly, PARP1 was not a significant predictor of survival for any of the 11 cancers. Results from cluster analysis of GERs indicates that the most opportunistic cancers for inhibition therapy may be AML, colorectal, and renal papillary, because of potentially less confounding due to lower GERs for mutations, deletions, and amplifications in DNA repair genes. However, the most opportunistic cancer for inhibition therapy is likely to be AML, since it showed the lowest GERs for mutations, deletions, and amplifications in DNA repair genes. In conclusion, our hypothesis-driven focus to target DNA repair gene expression adjusted for the PCNA metagene as a means of predicting OS in various cancers resulted in statistically significant sets of genes.

Abstract 4199: Genetic variation in the in vitro genotoxic response to glycidamide and gene expression of DNA repair genes

2011 ◽  
Author(s):  
Marta Pingarilho ◽  
Nuno G. Oliveira ◽  
Célia Martins ◽  
Bruno C. Gomes ◽  
Ana S. Fernandes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
Genetic Variation ◽  
Dna Repair Genes ◽  
Repair Genes

Azoospermic infertility is associated with altered expression of DNA repair genes

DNA Repair ◽  
2019 ◽  
Vol 75 ◽  
pp. 39-47 ◽  
Author(s):  
Vertika Singh ◽  
Deepika Jaiswal ◽  
Kanhaiya Singh ◽  
Sameer Trivedi ◽  
Neeraj K Agrawal ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Repair Genes ◽  
Altered Expression ◽  
Repair Genes

Let-7a, Mir-17 and Mir-20a Expression Levels in CD34+ Bone Marrow Cells of Patients with Myelodysplastic Syndromes (MDS) Are Associated with Established Prognostic Factors, Supporting Their Implication in the Pathogenesis of the Disease,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3792-3792 ◽  
Author(s):  
Christos K Kontos ◽  
Vassiliki Pappa ◽  
Diamantina Vasilatou ◽  
Maria-Angeliki S Pavlou ◽  
Frida Kontsioti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Prognostic Factors ◽  
High Risk ◽  
Mirna Expression ◽  
Who Classification ◽  
Low Risk ◽  
Expression Levels ◽  
Cd34 Cells ◽  
Bone Marrow Blasts

Abstract Abstract 3792 Introduction: MicroRNAs are single, small non-coding RNA molecules of approximately 21–26 nucleotides, which regulate the expression of numerous genes. miRNAs may act either at the post-transcriptional or the post-translational level to repress gene expression; still, upregulation of gene expression has been noticed in some cases as a direct effect of miRNA function. The importance of miRNAs in carcinogenesis is emphasized by the association of cancers with alterations in miRNA expression. Many miRNAs, including let-7a and those of the miR-17-92 cluster (miR-17, miR-20a, etc.), have been shown or are predicted to affect the activities of targeted mRNAs encoding proteins that have oncogenic or anti-oncogenic functions. let-7a downregulates KRAS, while miR-17 and miR-20a downregulate E2F1. Both these proteins are overexpressed in myelodysplastic syndromes (MDS) and have been shown to be involved in the pathobiology of the disease. Purpose: In the current study, we examined the prognostic value of let-7a, miR-17 and miR-20a levels in MDS and their potential as novel molecular biomarkers. Furthermore, we investigated the protein expression levels of validated targets of these three miRNAs in bone marrow CD34+ cells of MDS patients. Material and Methods: We evaluated 43 patients with MDS (34 men, 9 women) with a median age of 73 years (range 45–87). According to WHO classification, 12 patients (27.9%) were diagnosed with RA, 6 (13.9%) RCMD, 8 (18.6%) with RAEB-I, 7 (16.3%) with RAEB-II, 8 (18.6%) with AML, and 2 (4.7%) with CMML. According to IPSS, 13 patients (32.5%) had low risk, 14 (35.0%) intermediate I risk, 6 (15.0%) intermediate II, and 7 (17.5%) high risk disease. WPSS classification was: 8 (23.5%) very low risk, 5 (14.7%) low risk, 8 (23.5%) intermediate, 9 (26.5%) high risk, and 4 (11.8%) very high risk. We isolated CD34+ cells from bone marrow mononuclear cells from MDS patients, as well as from peripheral blood of donors of CD34+ cells for stem cell transplantation, using magnetic beads. Extraction of small RNA-containing total RNA from CD34+ cells was performed and cDNA of let-7a, miR-17 and miR-20a was synthesized using specific primers. miRNA expression levels were determined using quantitative real-time PCR, the TaqMan® chemistry and the relative quantification (2−ΔΔCT) method. The snoRNA RNU48 was used as reference gene. Furthermore, total protein was extracted from CD34+ cells using a lysis buffer and subsequently quantified using the Bradford assay. Western blot analysis was carried out for MYC, E2F1, Cyclin D1 (CCND1), BCL2 and KRAS, while Actin was used as reference protein. Results: In MDS patients, let-7a expression levels were 0.053–506.1 copies/RNU48 copies, while miR-17 and miR-20a expression levels were 0.005–2694.5 and 0.003–3116.7 copies/103RNU48 copies, respectively. No significant differences were found between patients and controls regarding let-7a, miR-17 and miR-20a expression. let-7a underexpression was associated with high (>10%) bone marrow blasts percentage (P =0.036), presence of WHO classification subtypes with poor prognosis (RAEB-I, RAEB-II and AML) (P =0.020), and high IPSS (P =0.037). Furthermore, miR-17 underexpression was related to high (>10%) bone marrow blasts percentage (P =0.008), intermediate and/or high risk karyotype (P =0.018) and high IPSS (P =0.016). Moreover, miR-20a underexpression was associated with high IPSS (P =0.037) and WPSS (P =0.013). Interestingly, protein expression levels of all targets analyzed in the current study were shown to be lower in samples overexpressing let-7a, miR-17 and/or miR-20a, in comparison with the corresponding protein levels noticed in specimens showing lower expression of these three miRNAs. Conclusion: To the best of our knowledge, this is the first study showing that expression levels of let-7a, miR-17 and miR-20a are associated with established prognostic factors in MDS, including IPSS and WPSS. Furthermore, these three miRNAs seem to be implicated in the pathogenesis of the disease, most probably by finely tuning the expression of target proteins that are involved in highly important molecular pathways, therefore affecting key cellular functions, such as cell cycle control, apoptosis, cell proliferation, and regulation of gene expression. Undoubtedly, further studies are needed to confirm the present findings and clarify their association with the pathogenesis of different MDS subgroups. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Molecular genetic markers of sensitivity to industrial environment factors at a miners

2020 ◽  
Author(s):  
Anna A. Timofeeva ◽  
Varvara I. Minina ◽  
Evgeniya Anatol'evna Astaf'eva ◽  
Tatyana A. Golovina ◽  
Vladislav Igorevich Fedoseev ◽  
...  
Keyword(s):  
Dna Repair ◽  
Chromosomal Aberrations ◽  
Evaluation Method ◽  
Ribosomal Gene ◽  
Ribosomal Genes ◽  
Control Group ◽  
Average Dose ◽  
Dna Repair Genes ◽  
Chromosomal Disorders ◽  
Repair Genes

SUMMARY: Background. Active ribosomal genes and DNA repair genes play an important role in restoring genome integrity. Therefore we were studied relationship of active ribosomal genes dose and DNA repair genes polymorphisms with high level of chromosomal disorders in miners. Materials and methods. The DNA damage level was assessed using chromosomal aberrations (CA) at 288 coal miners and 676 men in the control group. The dose of active ribosomal gene (AcRG) has been analyzed using Ag-NORS staining regions of chromosomes and cytogenetic semi-quantitative evaluation method. Real-time PCR and allele-specific PCR techniques were used to analyze polymorphic variants of the XPG (rs17655), XPD (rs13181), XRCC2 (rs3218536) and XRCC3 (rs861539) genes. Results. A statistically significant (p=0.0001) increase of the СА level at miners was found in comparison with the control group. The association XPD 2251TG locus with increasing CA level is revealed of in recessive inheritance model (Padj=0,0001). The association XPG 3310GC locus with increasing СА level is revealed at the smoking workers (Padj=0,017). An average dose of AcRG was registered a statistically significant increase in the frequency of single fragments (p=0.016) at the miners. Conclusion. The obtained data on associations of chromosomal aberrations with different variants of DNA repair genes and the dose of active ribosomal genes are useful for the formation of high-risk groups.

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