Aleksandra Sałagacka

ABCB1/MDR1 gene polymorphisms as a prognostic factor in colorectal cancer

ObjectiveTo analyse the single-nucleotide polymorphisms (SNPs): ABCB11236C>T, ABCB12677G>T/A, ABCB13435C>T and haplotypes in the ABCB1/MDR1 gene, which could contribute to genetic risk of colorectal cancer (CRC). Disease association between the ABCB1/MDR1 genotype, allele, haplotype frequencies and histological features, such as TNM classification, localization of primary carcinoma, grade of malignancy, histological type of tumour, lymphoid infiltration and vessel invasion were estimated. In this study, the potential role of SNPs of the ABCB1/MDR1 gene as a prognostic marker for CRC was analysed.Materials and methodsTumour specimens of 95 patients with CRC were studied. Using automated sequencing or PCR-RFLP method, DNA for three common SNPs of ABCB1/MDR1 was extracted and analysed. The results of genotyping and haplotype analysis with histopathological features, grading and clinical staging of neoplasms were correlated.ResultsA statistically significant higher frequency of T1236 allele in T1/T2 (89.7%), M0 groups (81.6%) and I/II clinical staging (82.7%) in comparison with T3/T4 (68.2%), M1 groups (47.4%) and III/IV clinical staging (65.1%) was detected. Furthermore, multivariate analysis according to Coxs proportional hazard model indicated that the T1236 allele is a good, independent prognostic factor and the presence of this allele decreases the risk of death in comparison with a group without this allele (HR = 0.26; p = 0.0424). In addition, a statistically significant higher frequency of C3435 allele and significant differences in the C3435 allele distribution in N1/N2 group (91.7% and 62.5%, respectively) than N0 group (71.2% and 44.9%, respectively) was found. Each of the eight possible haplotypes was noted in M0 or I/II group and only seven in M1 or III/IV group. Haplotype T1236-G2677-C3435 only in less advanced CRC subjects (9.6% in I/II and 9.2% in M0 group) was detected. In addition, significant differences in haplotype distributions between M0 or I/II and M1 or III/IV group were found (p = 0.01 and p = 0.05, respectively).ConclusionsThese results suggest association between T1236 allele and T1236-G2677-C3435 haplotype and less advanced CRC, so these genetic markers may play a role as potentially good prognostic factors. Differences in haplotype distributions and degree of clinical staging may suggest that some other potential SNPs, especially in regulatory region of ABCB1/MDR1 gene, may influence P-glycoprotein function and CRC progression.

Polymorphisms and haplotypes in the multidrug resistance 1 gene (MDR1/ABCB1) and risk of multiple myeloma

MDR1(ABCB1) gene encodes for P-glycoprotein (P-gp, MDR1, ABCB1), an ATP-binding cassette superfamily member involved in the transport of xenobiotics. Here, we investigated whether common MDR1 single nucleotide polymorphisms (1236C>T, 2677G>A/T and 3435C>T) affect predisposition to multiple myeloma. Genotyping was performed in 111 myeloma patients and 96 controls by PCR-based assays. Haplotypes were inferred using PHASE algorithm. We found comparable allele and genotype frequencies among myeloma patients and controls. Moreover, patient and control groups did not differ regarding MDR1 haplotype distribution (p=0.18). In conclusion, our results do not support major influence of MDR1 variants on the risk of myeloma in Caucasians.

C3435T polymorphism of the ABCB1 gene: impact on genetic susceptibility to peptic ulcers

The functional single nucleotide polymorphism (SNP) C3435T in exon 26 of the ABCB1 gene encoding the xenobiotic transporter P-glycoprotein (P-gp) may influence susceptibility to several diseases, as well as the clinical outcome of treatment with P-gp substrates. Exposure to environmental chemicals is thought to be involved in peptic ulcer pathogenesis and then later in stomach cancer development. About 80% of ulcers are associated with Helicobacter pylori infection, one of the risk factors of stomach cancer. P-gp-transported drugs are used in treatment of H. pylori. Therefore, a lack of effectiveness in eradication therapy can lead to chronic stomach inflammation and promote cancerogenesis. In this study, 196 patients with peptic ulcers divided into two groups with and without H. pylori infection and combined with 96 healthy controls were genotyped for the ABCB1 C3435T SNP. A trend towards higher incidence of the 3435TT genotype among peptic ulcer patients than in controls (p = 0.0983) was observed. Likewise, the 3435T allele was more frequent in groups suffering from peptic ulcers. The association was near to statistical significance (p = 0.0538). Between analyzed genotypes and H. pylori infection, statistically significant dependence was found (p = 0.0372). In addition, the CT genotype was associated with 1.56 times and the TT with 2.45 times higher prevalence of infection compared to the CC genotype. Asimilar association was present in a subgroup of peptic ulcer men (p = 0.0090). The isolated C3435T ABCB1 SNP is not a major factor for genetic susceptibility to peptic ulcer, but in a group of men who suffered from peptic ulcer, this polymorphism seemed to be a risk factor for H. pylori infection development.

The Influence of C3435T Polymorphism of the ABCB1 Gene on Genetic Susceptibility to Depression and Treatment Response in Polish Population - Preliminary Report.

Background: Despite the high prevalence of depression, the mechanism of the origin of this disease as well as the causes of resistance to therapy in some patients are still not fully understood. Increasingly, the possible role of genetic factors is considered. One of them is polymorphisms in the ABCB1 (MDR1) gene which encodes P-glycoprotein, responsible for the transport of xenobiotics, including antidepressant drugs, through the blood-brain barrier. Methods: C3435T was evaluated in 90 patients with recurrent depressive disorders (rDD). Genotyping was performed using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Results: The obtained results indicate that the TT genotype occurred more frequently among patients with rDD than in healthy volunteers (p=0.0441). Also, at least one C allele was present significantly less frequent in the study group than in healthy individuals (p=0.0300). The severity of depressive symptoms was higher among patient with the CC genotype in comparison with the other genotypes (p=0.0106) but treatment response to antidepressants was better in this group than among patients with CT or TT genotypes (p=0.0301). Likewise, patients with the T allele have a significantly lower severity of symptoms (p=0.0026) and decreased therapy effectiveness (p=0.0142) than C allele carriers. Conclusions: This study suggests that C3435T polymorphisms in the ABCB1 gene are strongly associated with a predisposition to depression development, the severity of depressive symptoms and the effectiveness of therapy with using different groups of antidepressant agents.

Investigation of -308G>A and -1031T>C polymorphisms in the TNFA promoter region in Polish peptic ulcer patients.

Background/Aims Tumor necrosis factor α (TNF-α) encoded by TNFA is a key mediator in inflammation, a precursor condition for peptic ulceration. Promoter polymorphisms of TNFA that influence its transcriptional activity and TNF-α production are known. TNFA-308G>A (rs1800629) and TNFA-1031T>C (rs1799964), which are responsible for increased TNFA transcription, could influence the risk of peptic ulceration. This study aimed to investigate these polymorphisms and to evaluate their association with peptic ulcer disease and Helicobacter pylori infection in the Polish population. Methods Gastric mucosa specimens obtained from 177 Polish peptic ulcer patients were used to conduct rapid urease tests and to assess the investigated polymorphisms by polymerase chain reaction-restriction fragment length polymorphism. Genotyping data were compared with the results obtained from healthy individuals of Polish origin. Results There were no significant differences in genotype and allele frequency of the investigated polymorphisms between peptic ulcer patients and healthy individuals. No associations between the frequencies of particular genotypes and alleles for both single-nucleotide polymorphisms (SNPs) and the presence of H. pylori infection in peptic ulcer patients and in subgroups of men and women with peptic ulcer disease were found. Conclusions The investigated SNPs are not risk factors for either peptic ulcer or H. pylori infection development in the Polish population. The results require verification in a larger cohort.

Haplotype Analysis of TNFA Gene in Peptic Ulcer Patients

Abstract The TNFA gene product TNF-? is a cytokine promoting an immune response after bacterial infections. An excessive production of the cytokine is thought to be connected with Helicobacter pylori-induced disorders like gastritis, peptic ulcer disease (PUD), intestinal metaplasia, or even gastric cancer. The synthesis of TNF-á is controlled at transcriptional level and dependent on single nucleotide polymorphisms (SNPs) of TNFA promoter region. SNPs assembled in haplotype have been implicated as potential risk factors for various autoimmune and infectious diseases. The aim of this study was to determine the frequencies of TNFA haplotypes composed of the four common single-nucleotide polymorphisms of the gene (-1031C>T: rs1799964, -863C>A: rs1800630, - 857C>T: rs1799724, -308G>A: rs1800629) in peptic ulcer patients and to assess the significance of the haplotypes as the risk factors of H. pylori infection development. 203 peptic ulcer patients were genotyped using polymerase chain reaction-restriction fragment length polymorphism method. Haplotypes and degree of linkage disequilibrium (LD) were inferred with PHASE 2.1 and EMLD software. There was no statistically significant difference in haplotype frequencies between the H. pylori-infected and-uninfected peptic ulcer cases (p=0.62). Analogous association was also absent in the subgroups: peptic ulcer woman (p=0.69), peptic ulcer men (p=0.17). The locus pairs -308_-857, -863_-1031, and -857_-1031 was found to be in very strong L¯D , -308_-1031 and -857 and -863 in strong LD, and -308_-863 - in modest LD. TNFA haplotype structure is not connected with individual differences in susceptibility to development of H. pylori infection in peptic ulcer patients.

Is the ABCB1 gene associated with the increased risk of gastric cancer development?--preliminary research.

One of the most common malignant diseases, both worldwide and in Poland, is gastric cancer. The pathogenesis of gastric cancer development is not entirely clear. Next to the environmental risk factors, such as Helicobacter pylori infection or dietary habits, the host genetic factors as predispositions to gastric cancer development are discussed. A transmembrane protein that could be associated with predisposition to cancer development is P-glycoprotein (P-gp). Physiologically, P-gp is present in normal tissue of the gastrointestinal tract, where it plays a protective role by transporting xenobiotics from a cell into extracellular environment. P-gp is encoded by the highly polymorphic ABCB1 gene. The most frequent polymorphisms at positions 1236, 2677, and 3435 may affect both the function and amount of protein, thereby leading to a loss of its physiological function, which could increase the predisposition to development of many diseases, including cancer. In this study, the potential significance of the ABCB1 gene in the development and progression of gastric cancer was evaluated. In 19 tissue samples collected from patients with gastric cancer, the ABCB1 gene polymorphisms were identified at positions 1236 and 2677 by automated sequencing and SNP 3435 by the RFLP method. The relative level of ABCB1 expression was measured in 10 samples of gastric cancer and morphologically normal tissues by real-time PCR. For SNPs at positions 1236, 2677, and 3435, no statistically significant differences in genotype frequencies between gastric cancer patients and healthy individuals were found. However, genotype TT for all studied polymorphisms occurred more frequently in the group of gastric cancer patients (31.6, 26.3, 42.1%, respectively) than in the group of healthy individuals (14.6, 13.5, 21.9%, respectively). The lowest relative expression levels of ABCB1 mRNA were observed for genotypes CC of SNP 1236, CC of SNP 3435, and GG of SNP 2677 (median: 0.215, 0.160, 0.160, respectively). There was a tendency that mutant homozygote TT for SNPs at positions 1236, 2677, and 3435 occurred more frequently in the subgroup of patients with Tis or stage I of TNM classification (SNP 1236 p = 0.0760; SNP 2677 p = 0.0813; SNP 3435 p = 0.0760) than in the subgroup of patients with stage II or III. Also the expression levels were lowest (median 0.740) in the group of patients with the less advanced clinical stage of cancer (Tis or I). Preliminary research showed that the ABCB1 gene polymorphisms at positions 1236, 2677, and 3435 were not related to an increased susceptibility of gastric cancer development. However, they may be associated with the inhibition of gastric cancer progression.

Quantitative analysis of FJ 194940.1 gene expression in colon cancer and its association with clinicopathological parameters

Aim of the study The FJ 194940.1 gene is located on chromosome 1 and consists of 6 exons and 5 introns. The gene undergoes alternative splicing and its isoforms appear during cancer development. Evidence suggests that expression of FJ 194940.1 splice variants relate to colorectal cancer progression. This paper discusses the quantitative analysis of the exon V expression level of FJ 194940.1 in colon cancer. The aim of the study is to carry out quantitative analysis by real-time PCR in a series of 102 colon cancer samples that had previously shown presence of exon V expression. To compare the exon V expression level with certain histological parameters and clinical staging of the neoplasm in order to assess its potential role as a prognostic factor in colon cancer. Material and methods Tissue specimens of colorectal cancer were obtained from the Oncological Centre of Lodz, Poland.Total RNA isolation was performed in accordance with the protocol enclosed in the Total RNA Prep Plus Minicolumn Kit (A&A Biotechnology, Poland). Reverse Transcriptase-PCR reaction was carried out using Enhanced Avian HSRT-PCR Kit, Sigma, according to the manufacturers protocol.. Presence of cDNA in each sample was checked by PCR amplification of β-actin. Only samples showing the PCR product of this housekeeping gene were included in further tests. The amount of FJ 194940.1 transcript containing exon V was analysed by means of real-time PCR. Results Exon V expression level is not significantly related to any clinicopathological features in colon cancer. However, there was a tendency towards a lower exon V expression level in a group of cases where vessel invasion was present (p = 0.0697). Additionally, the risk of death in patients with a low exon V expression level was more than two times higher when compared to patients with a high exon V expression level. Conclusions FJ 194940.1 gene expression correlates with cancer progression independently of analysed clinicopathological parameters.

Changes in the apoptotic gene expression profile in CLL patients treated with rituximab combined with cladribine and cyclophosphamide-preliminary results

The study was aimed to investigate modifications of apoptotic gene expression profile by microarray technique in 10 patients with chronic lymphocytic leukemia by treatment with rituximab, cladribine and cyclophosphamide (RCC) according to IGHV mutational status. The TaqMan Low Density Array for 96 gene transcripts was used. Those modifications followed two distinctive patterns largely overlapping the IGHV mutational status. In the IGHV-mutated group, the expression of many proapoptotic genes increased after treatment as compared to initial value. Our results suggest that RCC drugs may act through influence on the expression of some apoptosis-involved genes dependently on the IGVH mutational status.

Characterisation of selected molecular mechanisms influencing pharmacokinetics and pharmacodynamics of antidepressants

Over 350 millions of people suffer from depression worldwide, and an increase in the incidence of the disease is expected in the coming years. Therefore, there is a strong need of knowing the mechanisms of development this disease and its effective treatment. Pharmacological therapy is an essential element of antidepressant therapy and its failure is a serious problem in clinical psychiatry. In spite of large number of available medicines, only 50% of patients achieved remission after single-drug treatment. Genetic factors which predispose to depression development and predict an outcome of its pharmacotherapy, probably play a substantial role in these phenomena. In spite of this, they are still not characterized enough and applied in practice. Therefore, the aim of this study is to present the current knowledge of the impact of selected genes on pharmacokinetics and pharmacodynamics of antidepressants by changing function and/or structure of encoded proteins. In the review the best known polymorphisms of selected genes encoding isoenzymes of cytochrome P-450, responsible for metabolism of popular antidepressant drugs, namely CYP2C19, CYP2D6, CYP1A2, and CYP3A4/5 are described. Further, 4 polymorphisms of ABCB1 gene (rs 1045642, rs 2032582, rs 1128503 and rs 2032583) encoding glycoprotein P, which play a key role in transportation of large number of drugs used in treatment of depression. For benefit of treatment of patients with depression, it is worthy to estimate and to take on the board all so far known mechanisms of planning therapy.