Teodora Karan-Djurasevic

Expression of Bcl2L12 in chronic lymphocytic leukemia patients: association with clinical and molecular prognostic markers

Dysregulation of apoptosis is a distinctive feature of chronic lymphocytic leukemia (CLL), although a unique mechanism underlying apoptosis resistance of CLL B lymphocytes has not been identified yet. Aberrant expression as well as genetic and epigenetic alterations of numerous genes involved in different pathways of apoptosis regulation has been described in CLL. Here, we report the expression analysis of Bcl2L12 (Bcl2-like 12), a novel apoptotic gene belonging to Bcl2 family, in 58 Serbian CLL patients. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis revealed a significant overexpression of Bcl2L12 mRNA in CLL samples compared to non-leukemic samples, implying its role in the pathogenesis of the disease. Receiver operating characteristic (ROC) analysis showed that Bcl2L12 expression efficiently discriminates CLL cases from healthy controls. However, relatively homogenous Bcl2L12 mRNA expression among patients did not reflect their clinical characteristics (with the exception of lactate dehydrogenase status and time from diagnosis to treatment) and failed to show association with the most informative prognostic markers, namely the mutational status of rearranged immunoglobulin heavy chain variable region genes, CD38 and lipoprotein lipase gene (LPL) expression.

Novel transcriptional regulatory element in the phenylalanine hydroxylase gene intron 8

We present the first transcriptional regulatory element found in a PAH gene intron. The element is located in the PAH gene intron 8, acts as an enhancer specifically in the hepatoma cell line, and binds GATA-1 transcription factor. Herein the presented data could unlock a new area for the analysis of PAH gene expression and could contribute to refining genotype-phenotype correlation.

The influence of novel transcriptional regulatory element in intron 14 on the expression of Janus kinase 2 gene in myeloproliferative neoplasms

The expression of Janus kinase 2 (JAK2) gene is altered in myeloproliferative neoplasms (MPN) and the regulation of transcription could be a mechanism that modulates JAK2 gene expression. We analyzed the transcriptional potential of single-nucleotide polymorphism (SNP) rs12343867 T > C in JAK2 intron 14, tagging 46/1 haplotype, and its influence on JAK2 gene expression. Functional analysis of JAK2 intron 14 was performed using the pBLCAT5 reporter system in K562 cells. Identification of the proteins binding to the intron 14 regulatory element was accomplished by electrophoretic mobility shift assay (EMSA) and supershift assays. Quantification of the expression of JAK2 gene in a cohort of 51 MPN patients and 12 healthy controls was performed by real-time quantitative polymerase chain reaction (RQ-PCR). Functional analysis revealed that the intronic DNA element harboring SNP rs12343867 T > C acts as a transcriptional repressor in vitro. The repressor activity was significantly attenuated by the presence of nucleotide C. Supershift analysis showed the enrolment of transcriptional factor Meis1 in this process. RQ-PCR experiments showed increased JAK2 expression in patients with the JAK2V617F mutation, with a significant difference between essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) patients. SNP rs12343867 showed no statistically significant influence on the expression of JAK2 gene in MNP patients.

Thalassemia Syndromes in Serbia: An update

Thalassemia syndromes constitute a group of genetic disorders, widespread throughout the world. The present study contains data on thalassemia syndromes and their chromosomal environment obtained in Serbia over a period of 10 years. Ten different β-thalassemia (β-thal) mutations and two hemoglobin (Hb) variants were detected in 127 members of 68 families. Hb Lepore-Boston-Washington (Lepore-BW) (δ87Gln-β-IVS-II-8), a thalassemic Hb variant, was shown to be the most common cause of thalassemia in Serbia. Haplotype analyses of the β-globin gene clusters of healthy individuals as well as of individuals affected with β-thal showed that haplotype I was the most frequent haplotype in the Serbian population, followed by haplotypes II and IX. Two novel haplotypes were detected. Haplotype analyses showed the association between certain haplotypes and the most common thalassemic mutations. Results presented in this paper will update the Serbian national mutation database and contribute to a better understanding of genographic history of South European and Balkan populations.

Somatic mutations of isocitrate dehydrogenases 1 and 2 are prognostic and follow-up markers in patients with acute myeloid leukaemia with normal karyotype.

Abstract Background Mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes are frequent molecular lesions in acute myeloid leukaemia with normal karyotype (AML-NK). The effects of IDH mutations on clinical features and treatment outcome in AML-NK have been widely investigated, but only a few studies monitored these mutations during follow-up. Patients and methods In our study samples from 110 adult de novo AML-NK were studied for the presence of IDH1 and IDH2 mutations, their associations with other prognostic markers and disease outcome. We also analyzed the stability of these mutations during the course of the disease in complete remission (CR) and relapse. Results IDH mutations were found in 25 (23%) patients. IDH+ patients tend to have lower CR rate compared to IDH-patients (44% vs 62.2%, p = 0.152), and had slightly lower disease free survival (12 months vs 17 months; p = 0.091). On the other hand, the presence of IDH mutations had significant impact on overall survival (2 vs 7 months; p = 0.039). The stability of IDH mutations were studied sequentially in 19 IDH+ patients. All of them lost the mutation in CR, and the same IDH mutations were detected in relapsed samples. Conclusions Our study shows that the presence of IDH mutations confer an adverse effect in AML-NK patients, which in combination with other molecular markers can lead to an improved risk stratification and better treatment. Also, IDH mutations are very stable during the course of the disease and can be potentially used as markers for minimal residual disease detection.

PARP-1 and YY1 are important novel regulators of CXCL12 gene transcription in rat pancreatic beta cells.

Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12) transcription. The roles of poly(ADP-ribose) polymerase-1 (PARP-1) and transcription factor Yin Yang 1 (YY1) in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ)-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the functional interplay of these proteins could finely balance Cxcl12 transcription.

Mutational Status and Gene Repertoire of IGHV-IGHD-IGHJ Rearrangements in Serbian Patients With Chronic Lymphocytic Leukemia

UNLABELLED The mutational status and configuration of immunoglobulin heavy variable (IGHV) gene rearrangements was analyzed in 85 Serbian patients with chronic lymphocytic leukemia (CLL). We found that 55.3% of cases belonged to mutated and 44.7% to unmutated CLL, progressive disease predominating in the unmutated subset. IGHV gene use resembled that obtained for Mediterranean countries, except for underrepresentation of the IGHV4 subgroup in our cohort. BACKGROUND Chronic lymphocytic leukemia (CLL) results from the clonal expansion of mature B lymphocytes and is characterized by extreme clinical heterogeneity. One of the most reliable prognostic markers in chronic lymphocytic leukemia (CLL) is the mutational status of immunoglobulin heavy variable (IGHV) genes, which defines 2 subsets, mutated CLL (M-CLL) and unmutated CLL (U-CLL), with different clinical courses. Biased IGHV gene use between M-CLL and U-CLL clones, as well as population differences in the IGHV gene repertoire have been reported. PATIENTS AND METHODS In this study, mutational status and configuration of IGHV-IGHD-IGHJ rearrangements in 85 Serbian patients were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing methodology. RESULTS We found that 55.3% of cases belonged to M-CLL and 44.7% belonged to U-CLL, with progressive disease predominating in the unmutated subset. Most frequently expressed was the IGHV3 subgroup (55.7%), followed by IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2 (1.1%), and IGHV6 (1.1%). The distribution of IGHD subgroups was as follows: IGHD3, 39.1%; IGHD2, 21.8%; IGHD6, 12.6%; IGHD1, 10.3%; IGHD4, 8%; IGHD5, 6.9%; and IGHD7, 1.1%. The most frequent IGHJ gene was IGHJ4 (48.9%), followed by IGHJ6 (28.4%), IGHJ3 (11.4%), and IGHJ5 (11.4%). In 15.3% of cases, heavy complementarity-determining region 3 (VH CDR3) amino acid sequences could be assigned to previously defined stereotyped clusters. CONCLUSIONS Our study showed a strong correlation between IGHV gene mutational status and clinical course of CLL. IGHV gene use was comparable to that obtained for Mediterranean countries, with the exception of the IGHV4 subgroup, which was underrepresented in our cohort.

Prognostic Impact of NPM1 Mutations in Serbian Adult Patients with Acute Myeloid Leukemia

Based on current findings, the presence of NPM1 mutations in acute myeloid leukemia (AML) patients is associated with an increased probability of complete remission (CR) and better overall survival (OS). We determined the incidence and prognostic relevance of NPM1 mutations, their association with FLT3 and IDH mutations, and other clinical characteristics in Serbian adult AML patients. Samples from 111 adult de novo AML patients, including 73 AML cases with a normal karyotype (NK-AML), were studied. NPM1, FLT3, and IDH mutations were detected by PCR and direct sequencing. NPM1 mutations were detected in 22.5% of patients. The presence of NPM1 mutations predicted a low CR rate and shorter OS. NPM1 mutations showed an association with both FLT3 and IDH mutations. Survival analysis based on NPM1/FLT3 mutational status revealed a lower OS for NPM1+/FLT3– compared to the NPM1–/FLT3– group in NK-AML patients. The lack of impact or unfavorable prognostic effect of NPM1 mutations found in this study can be assigned to a small cohort of analyzed AML patients, as can the presence of FLT3 and IDH mutations or other genetic lesions that cooperate with NPM1 mutations influencing prognosis.

Gene Mutation Profiles in Primary Diffuse Large B Cell Lymphoma of Central Nervous System: Next Generation Sequencing Analyses.

The existence of a potential primary central nervous system lymphoma-specific genomic signature that differs from the systemic form of diffuse large B cell lymphoma (DLBCL) has been suggested, but is still controversial. We investigated 19 patients with primary DLBCL of central nervous system (DLBCL CNS) using the TruSeq Amplicon Cancer Panel (TSACP) for 48 cancer-related genes. Next generation sequencing (NGS) analyses have revealed that over 80% of potentially protein-changing mutations were located in eight genes (CTNNB1, PIK3CA, PTEN, ATM, KRAS, PTPN11, TP53 and JAK3), pointing to the potential role of these genes in lymphomagenesis. TP53 was the only gene harboring mutations in all 19 patients. In addition, the presence of mutated TP53 and ATM genes correlated with a higher total number of mutations in other analyzed genes. Furthermore, the presence of mutated ATM correlated with poorer event-free survival (EFS) (p = 0.036). The presence of the mutated SMO gene correlated with earlier disease relapse (p = 0.023), inferior event-free survival (p = 0.011) and overall survival (OS) (p = 0.017), while mutations in the PTEN gene were associated with inferior OS (p = 0.048). Our findings suggest that the TP53 and ATM genes could be involved in the molecular pathophysiology of primary DLBCL CNS, whereas mutations in the PTEN and SMO genes could affect survival regardless of the initial treatment approach.

Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia

Summary Background: In chronic lymphocytic leukemia (CLL), in vivo apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the Bcl2 family members play a central role. Aim: The aim of this study was to investigate the association of pro-apoptotic Bax gene expression and Bcl2/Bax ratio with the clinical features of CLL patients as well as with molecular prognostic markers, namely the mutational status of rearranged immunoglobulin heavy variable (IGHV) genes and lipoprotein lipase (LPL) gene expression. Methods: We analyzed the expression of Bax mRNA and Bcl2/Bax mRNA ratio in the peripheral blood mononuclear cells of 58 unselected CLL patients and 10 healthy controls by the quantitative reverse-transcriptase polymerase chain reaction. Results: We detected significant Bax gene overexpression in CLL samples compared to non-leukemic samples (p=0.003), as well as an elevated Bcl2/Bax ratio (p=<0.001). Regarding the association with prognostic markers, the Bcl2/Bax ratio showed a negative correlation to lymphocyte doubling time (r=−0.307; p=0.0451), while high-level Bax expression was associated with LPL-positive status (p=0.035). Both the expression of Bax and Bcl2/Bax ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance. Conclusions: Our results suggest that dysregulated expression of Bcl2 and Bax, which leads to a high Bcl2/Bax ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL.