Regulatory network analysis of microRNAs and genes in imatinib-resistant chronic myeloid leukemia.

Targeted therapy in the form of selective breakpoint cluster region-abelson (BCR/ABL) tyrosine kinase inhibitor (imatinib mesylate) has successfully been introduced in the treatment of the chronic myeloid leukemia (CML). However, acquired resistance against imatinib mesylate (IM) has been reported in nearly half of patients and has been recognized as major issue in clinical practice. Multiple resistance genes and microRNAs (miRNAs) are thought to be involved in the IM resistance process. These resistance genes and miRNAs tend to interact with each other through a regulatory network. Therefore, it is crucial to study the impact of these interactions in the IM resistance process. The present study focused on miRNA and gene network analysis in order to elucidate the role of interacting elements and to understand their functional contribution in therapeutic failure. Unlike previous studies which were centered only on genes or miRNAs, the prime focus of the present study was on relationships. To this end, three regulatory networks including differentially expressed, related, and global networks were constructed and analyzed in search of similarities and differences. Regulatory associations between miRNAs and their target genes, transcription factors and miRNAs, as well as miRNAs and their host genes were also macroscopically investigated. Certain key pathways in the three networks, especially in the differentially expressed network, were featured. The differentially expressed network emerged as a fault map of IM-resistant CML. Theoretically, the IM resistance process could be prevented by correcting the included errors. The present network-based approach to study resistance miRNAs and genes might help in understanding the molecular mechanisms of IM resistance in CML as well as in the improvement of CML therapy.

Downregulation of miR-451 in Tunisian chronic myeloid leukemia patients: potential implication in imatinib resistance.

OBJECTIVES: Resistance to imatinib has been recognized as a major challenge for the treatment of chronic myeloid leukemia (CML). Aberrant expression of miR-451 has been reported to participate in anticancer drug resistance. However, the role of miR-451 in imatinib resistance has not been investigated. The present study was undertaken to determine the expression of miR-451 in order to find a possible association between the expression of this miRNA and imatinib resistance in Tunisian CML patients. METHODS: First, real-time RT-PCR was performed to identify the expression of miR-451 in peripheral leukocytes of 59 CML patients treated with imatinib. Then, bioinformatics analysis was carried out to understand the regulatory roles of miR-451 in imatinib-resistant process. RESULTS: Downregulated miR-451 was observed in imatinib-resistant CML cases. In silico analysis identified MYC as a potential target of miR-451. We further revealed the existence of an MYC-binding site in MiR-451 promoter region. On the other hand, increased level of MYC was detected in imatinib-resistant CML cases which may explain the causative role of MYC in CML cases and the downregulation of miR-451. CONCLUSION AND DISCUSSION: Taken together, our findings suggest that miR-451 and MYC form together a regulatory loop which may act as a potential therapeutic target, and disruption of suggested regulatory loop could help to improve CML therapy.

Fetal Hemoglobin in Tunisian Sickle Cell Disease Patient: Relationship with Polymorphic Sequences Cis to the ß-Globin Gene.

Fetal hemoglobin (HbF) plays a dominant role in ameliorating morbidity and mortality of hemoglobinopathies. We evaluated the effects of polymorphic markers within the ß-globin gene cluster to identify the genetic mechanics that influence HbF on Tunisian sickling patients (n = 242). Haplotype analysis was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the framework polymorphism was established by PCR-sequencing, four independent regions of interest were identified: the 5' region of ß-LCR-HS2 site, the intervening sequence II (IVSII) region of two fetal (Gγ and Aγ) genes and the 5' region of ß-globin gene. The correlation of these various Haplotypes and SNPs with HbF expression and clinical data was studied. Our data showed that among the various polymorphic markers analyzed, only the sequence (AT)xN12(AT)y in LCR HS2 region was significantly associated (p < 0.05) with increased HbF levels, suggesting that the ß-globin gene cluster exerts a significant effect on HbF in sickle cell patients. This study can improve understanding of the physiopathology of the disease and aid to increase our ability to predict clinical severity.

Two new ß+ -thalassemia mutation [ß -56 (G → C); HBBc. -106 G → C] and [ß -83 (G → A); HBBc. -133 G → A] described among the Tunisian population.

OBJECTIVES: Different thalassemia mutations have been reported in various ethnic groups and geographical regions in Tunisia. In the present study, we have investigated two rare ß(+) -thalassemia mutations, that have not previously been reported in the Tunisian population [ß -56 (G > C); HBBc. -106 G > C] and [ß -83 (G > A); HBBc. -133 G > A]. METHODS: The whole ß-globin gene was directly sequenced, and haplotype analysis was conducted through a PCR/RFLP method. RESULTS: Two new mutations were identified for the first time in Tunisia. They are located within the promoter region of ß-globin gene at position -56 (G > C) and -83 (G > A). Linkage analysis using ß-globin gene cluster haplotypes showed that these two mutations were associated with Mediterranean ß-haplotype IX [- + - + + + +] and framework 2 (FW2) [CCTCT]. CONCLUSIONS: The two newly described mutations lead to the ß(+) -thalassemia among Tunisian patients. The haplotype analysis and framework assignment have helped to identify the chromosomal background associated with these mutations, and determine their origin and spread.

Polymorphisms in XPC, XPD and XPG DNA repair genes and leukemia risk in a Tunisian population.

Human DNA repair mechanisms protect the genome from DNA damage caused by endogenous and environmental agents. Polymorphisms in DNA repair genes and differences in repair capacity between individuals have been widely reported in different cancers. In this study we aimed to evaluate the associations between XPC Lys939Gln (rs2228001), XPD Lys751Gln (rs13181) and XPG Asp1104His (rs17655) polymorphisms and leukemia risk in a Tunisian population. Genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) in 206 patients with leukemia and 206 healthy controls. We found increased risk of leukemia among subjects carrying the XPC 939Gln/Gln genotype (odds ratio [OR] = 2.48, 95% confidence interval [CI] = 1.353-4.560, p = 0.0042). Moreover, in subgroup analysis according to clinical types, patients with chronic myeloid leukemia (CML) showed a higher risk than patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) (OR = 3.87, 95% CI = 1.820-8.237, p = 0.0003). However, the XPD 751Gln allele may be protective against CML and AML development, and no significant differences in genotype frequencies were observed for the XPG gene between patients and controls. Further studies with larger samples and risk factor information are needed.

Haplotype map of sickle cell anemia in Tunisia.

ß-Globin haplotypes are important to establish the ethnic origin and predict the clinical development of sickle cell disease patients (SCD). To determine the chromosomal background of ß (S) Tunisian sickle cell patients, in this first study in Tunisia, we have explored four polymorphic regions of ß-globin cluster on chromosome 11. It is the 5' region of ß-LCR-HS2 site, the intervening sequence II (IVSII) region of two fetal ((G)γ and (A)γ) genes and the 5' region of ß-globin gene. The results reveal a high molecular diversity of a microsatellite configuration describing the sequences haplotypes. The linkage disequilibrium analysis showed various haplotype combinations giving 22 "extended haplotypes". These results confirm the utility of the ß-globin haplotypes for population studies and contribute to knowledge of the Tunisian gene pool, as well as establishing the role of genetic markers in physiopathology of SCD.

Microsatellite and single nucleotide polymorphisms in the ß-globin locus control region-hypersensitive Site 2: SPECIFICITY of Tunisian ßs chromosomes.

The diversity of sickle cell disease severity is attributed to several cis acting factors, among them the single nucleotide polymorphisms (SNPs) and (AT) rich region in the ß-locus control region (ß-LCR). This contains five DNase I hypersensitive sites (HS) located 6 to 22 kb upstream to the ϵ gene. The most important of these is the HS2 (5' ß-LCR-HS2), characterized by the presence of three different SNPs and a microsatellite region known to be in association with ß(S) chromosomes in various populations. The aim of this study was to present the molecular investigation of the 5' ß-LCR-HS2 site in normal and sickle cell disease individuals in order to determine if there is any correlation or specificity between these molecular markers, the ß(S) Tunisian chromosomes and phenotypical expression of sickle cell disease. One hundred and twenty-four chromosomes from Tunisian individuals (49 ß(S) carriers and 13 normal individuals) were screened by polymerase chain reaction (PCR) and sequencing for the polymorphic short tandem microsatellite repeats (AT)(X)N(12)(AT)(Y) and the three SNPs (rs7119428, rs9736333 and rs60240093) of the 5' ß-LCR-HS2. Twelve configurations of the microsatellite motif were found with an ancestral configuration elaborated by ClustalW software. Normal and mutated alleles were observed at the homozygous and heterozygous states for the three SNPs. Correlation between microsatellites and SNPs suggests that mutant SNP alleles were mainly associated, in the homozygous sickle cell disease phenotype, with the (AT)(8)N(12)GT(AT)(7) configuration, whereas, normal SNP alleles were associated with the (AT)(X)N(12)(AT)(11) configurations in normal ß(A) chromosomes. The correlation of these various configurations with Hb F expression was also investigated. The principal component analysis (PCA) showed the correlation between the homozygous sickle cell disease phenotype, mutated SNP alleles and the Benin microsatellite configuration (AT)(8)N(12)GT(AT)(7), which confirmed the specificity of this configuration to the ß(S) chromosomes. In addition, the observed high level of Hb F (14.6%) could play a protective role against Hb S to justify the modulation of sickle cell disease severity within the Benin haplotype compared to the other haplotypes. This study highlights the fact that the ß-LCR-HS2 could be a genetic marker to identify the ethnic Tunisian ß(S) chromosomes and facilitate the molecular diagnosis of sickle cell disease.

KIT mutation detection in Tunisian patients with newly diagnosed myelogenous leukemia: prevalence and prognostic significance.

The KIT gene encodes a class III tyrosine kinase receptor in which specific somatic mutations have been found to be associated with many diseases. In this work, we investigated the prevalence of KIT mutations in patients with chronic and acute myelogenous leukemia (CML and AML) and their prognostic significance. A total of 157 subjects were included in the present study (84 patients with CML, 33 with AML, and 40 healthy controls). Patients were analyzed at the first diagnosis, and the KIT mutations were screened by polymerase chain reaction (PCR) and direct sequencing technologies. The results demonstrated the presence of a G/A transition at codon 796, which is associated with the R796K protein variation. This mutation was detected at 21.42% in the CML subgroup and was absent in both AML patients and healthy controls; however, no correlation was found between this mutation and clinical parameters such as the molecular response to Gleevec. In conclusion, we retain that the KIT gene is highly mutated in the CML subgroup, but its role as a prognostic factor needs to be further elucidated.

Influence of genetic polymorphisms of xenobiotic metabolizing enzymes on the risk of developing leukemia in a Tunisian population.

Leukemia is a type of cancer of the blood or bone marrow that is characterized by an abnormal increase of white blood cells. Leukemia is clinically and pathologically subdivided into a variety of large groups. The risk of developing leukemia may be influenced by polymorphisms of xenobiotic metabolizing enzymes. In this work, we conduct a case-control study to assess the impact of polymorphisms in GSTM1, GSTT1 and NAT2 genes on the risk of developing leukemia. Our data have shown that GSTM1*0 and GSTT1*0 were respectively associated with 2.05 and 4.36 increased risk for acute lymphoblastic leukemia (ALL). We have also shown that GSTM1*0 and GSTT1*0 act additively to increase the risk for ALL. Indeed, patients harbouring the "GSTM1*0/GSTT1*0" genotype were at 11.81-fold increased risk for developing ALL (P = 2 10(-5)). The risk for developing acute myeloid leukemia (AML) increases on patients with "rapid or intermediate NAT2 genotypes". Finally, the comparison of leukemia subgroups according to GSTM1, GSTT1 and NAT2 genotypes, suggests that leukemogenesis of different leukemia subgroups is very distinct. In conclusion, our findings suggest that leukemogenesis is associated with carcinogen metabolism and consequently related to environmental exposures.