The association of folate pathway and DNA repair polymorphisms with susceptibility to childhood acute lymphoblastic leukemia.

Genetic factors may play an important role in susceptibility to childhood acute lymphoblastic leukemia (ALL). The aim of our study was to evaluate the associations of genetic polymorphisms in folate pathway and DNA repair genes with susceptibility to ALL. In total, 121 children with ALL and 184 unrelated healthy controls of Slovenian origin were genotyped for 14 polymorphisms in seven genes of folate pathway, base excision repair and homologous recombination repair (TYMS, MTHFR, OGG1, XRCC1, NBN, RAD51, and XRCC3). In addition, the exon 6 of NBN was screened for the presence of mutations using denaturing high performance liquid chromatography. Twelve polymorphisms were in Hardy-Weinberg equilibrium in controls and their genotype frequencies were in agreement with those reported in other Caucasian populations. Among the investigated polymorphisms and mutations, NBN Glu185Gln significantly decreased susceptibility to B-cell ALL (p=0.037), while TYMS 3R allele decreased susceptibility to T-cell ALL (p=0.011). Moreover, significantly decreased susceptibility to ALL was observed for MTHFR TA (p=0.030) and RAD51 GTT haplotypes (p=0.016). Susceptibility to ALL increased with the increasing number of risk alleles (ptrend=0.007). We also observed significant influence of hOGG-RAD51 and NBN-RAD51 interactions on susceptibility to ALL. Our results suggest that combination of several polymorphisms in DNA repair and folate pathways may significantly affect susceptibility to childhood ALL.

The influence of folate pathway polymorphisms on high-dose methotrexate-related toxicity and survival in children with non-Hodgkin malignant lymphoma.

BACKGROUND: We evaluated the influence of folate pathway polymorphisms on high-dose methotrexate (HD-MTX) related toxicity in paediatric patients with T-cell non-Hodgkin lymphoma (NHL). PATIENTS AND METHODS: In total, 30 NHL patients were genotyped for selected folate pathway polymorphisms. RESULTS: Carriers of at least one MTHFR 677T allele had significantly higher MTX area under the time-concentration curve levels at third MTX cycle (P = 0.003). These patients were also at higher odds of leucopoenia (P = 0.006) or thrombocytopenia (P = 0.041) and had higher number of different HD-MTX-related toxicity (P = 0.035) compared to patients with wild-type genotype. CONCLUSIONS: Our results suggest an important role of MTHFR 677C>T polymorphism in the development of HD-MTX-related toxicity in children with NHL.

Is survivin expression prognostic or predictive in malignant pleural mesothelioma?

Malignant pleural mesothelioma is an incurable cancer strongly associated with asbestos exposure and characterised by poor response to treatment. The inhibitor-of-apoptosis protein family member survivin is involved in apoptosis and proliferation and is expressed in cancer cells only. The aims of the present study were to elucidate whether survivin expression is associated with tumour cell apoptosis and proliferation and to assess the prognostic and predictive value of survivin expression in malignant pleural mesothelioma. Archival pleural mesothelioma tissue samples from 101 patients were immunohistochemically analysed for nuclear expression of survivin, for proliferation with the use of Ki-67 as marker and for apoptosis using active caspase-3 as a marker. Staining results and clinical data were included in a survival analysis. Survivin was highly expressed in tumour cell nuclei in all samples and this correlated positively with both apoptosis and proliferation, but did not have a significant prognostic value. We found significantly higher survivin expression in patients who responded to chemotherapy compared to patients with progressive disease. Survivin expression might contribute to treatment response prediction, but survivin expression in malignant pleural mesothelioma did not have prognostic significance.

DNA repair polymorphisms and treatment outcomes of patients with malignant mesothelioma treated with gemcitabine-platinum combination chemotherapy.

INTRODUCTION: Genetic polymorphisms that affect DNA repair capacity can modulate the efficacy and toxicity of cytotoxic agents. Therefore, the aim of our study was to evaluate the influence of genetic variability in DNA repair genes on treatment outcome in patients with malignant mesothelioma (MM) treated with gemcitabine-platinum combination chemotherapy. METHODS: In total, 109 patients with MM were genotyped for 10 polymorphisms in XRCC1, NBN, RAD51, and XRCC3 genes. The influence of selected polymorphisms on tumor response and occurrence of treatment-related toxicity was determined by logistic regression analysis, whereas their influence on survival was estimated by Cox proportional hazards model. RESULTS: There were no associations between the investigated polymorphisms and tumor response, but we observed a significant association between XRCC1 399Gln allele and reduced overall survival (hazards ratio = 1.70; 95% confidence interval [CI] 1.06-2.73; p = 0.028). Interaction between XRCC1 399Gln allele and C-reactive protein levels revealed that carriers of at least one XRCC1 399Gln allele with C-reactive protein levels above median had significantly shorter overall survival time compared with other patients (12.9 months versus 25.3 months, log-rank p < 0.001). We also observed an association between XRCC1 399Gln and lower frequency of leukopenia (odds ratio [OR] = 0.25; 95% CI 0.09-0.67; p = 0.006), neutropenia (OR = 0.24; 95% CI 0.09-0.68; p = 0.007), and thrombocytopenia (OR = 0.27; 95% CI 0.09-0.84; p = 0.024). In addition, NBN 3474A>C, XRCC3 -316A>G, and Thr241Met polymorphisms showed significant associations with treatment-related toxicity. CONCLUSIONS: Our results support the hypothesis that DNA repair gene polymorphisms, particularly XRCC1 Arg399Gln, may modify the response to gemcitabine-platinum combination chemotherapy and, for the first time, show this effect in patients with MM.

Genetic polymorphisms in homologous recombination repair genes in healthy Slovenian population and their influence on DNA damage.

BACKGROUND: Homologous recombination (HR) repair is an important mechanism involved in repairing double-strand breaks in DNA and for maintaining genomic stability. Polymorphisms in genes coding for enzymes involved in this pathway may influence the capacity for DNA repair. The aim of this study was to select tag single nucleotide polymorphisms (SNPs) in specific genes involved in HR repair, to determine their allele frequencies in a healthy Slovenian population and their influence on DNA damage detected with comet assay. MATERIALS AND METHODS: In total 373 individuals were genotyped for nine tag SNPs in three genes: XRCC3 722C>T, XRCC3 -316A>G, RAD51 -98G>C, RAD51 -61G>T, RAD51 1522T>G, NBS1 553G>C, NBS1 1197A>G, NBS1 37117C>T and NBS1 3474A>C using competitive allele-specific amplification (KASPar assay). Comet assay was performed in a subgroup of 26 individuals to determine the influence of selected SNPs on DNA damage. RESULTS: We observed that age significantly affected genotype frequencies distribution of XRCC3 -316A>G (P = 0.039) in healthy male blood donors. XRCC3 722C>T (P = 0.005), RAD51 -61G>T (P = 0.023) and NBS1 553G>C (P = 0.008) had a statistically significant influence on DNA damage. CONCLUSIONS: XRCC3 722C>T, RAD51 -61G>T and NBS1 553G>C polymorphisms significantly affect the repair of damaged DNA and may be of clinical importance as they are common in Slovenian population.

DNA repair polymorphisms influence the risk of second neoplasm after treatment of childhood acute lymphoblastic leukemia.

PURPOSE: Patients treated for childhood acute lymphoblastic leukemia (ALL) are considered to be at increased risk of developing second neoplasm. The aim of our study was to identify DNA repair polymorphisms contributing to the risk of second neoplasm in clinically well-characterized Slovenian patients treated for childhood ALL. METHODS: Pediatric patients diagnosed with ALL between 1971 and 2001 were included in the study. According to the identified clinical risk factors for second neoplasm, a matched set of cases with second neoplasm and controls was selected and genotyped for 11 DNA repair polymorphisms. RESULTS: Among 359 pediatric patients with ALL, 20 second neoplasms were observed. The dose of radiotherapy (P = 0.011), administration of epipodophyllotoxins (P = 0.006), and the dose of anthracyclines (P < 0.001) showed a significant association with the risk of second neoplasm. Among genetic factors, we observed a significant association of NBN 1197G allele with increased risk of second neoplasms (RR = 4.36; 95 % CI: 1.19-15.98; P = 0.026), while the risk was decreased in carriers of XRCC3-316G allele compared with patients with wild-type genotype (RR = 0.20; 95 % CI: 0.04-0.99; P = 0.049). CONCLUSIONS: Our results suggest an important role of NBN 1197A>G and XRCC3-316A>G polymorphisms in the development of second neoplasm in patients treated for childhood ALL.

Influence of folate pathway polymorphisms on high-dose methotrexate-related toxicity and survival in childhood acute lymphoblastic leukemia.

The prediction of high-dose methotrexate (HD-MTX) toxicity is a key issue in the individualization of treatment in childhood acute lymphoblastic leukemia (ALL). Our aim was to evaluate the influence of MTX pathway polymorphisms on HD-MTX treatment outcome in children with ALL. In total, 167 children with ALL were genotyped for methylenetetrahydrofolate dehydrogenase (MTHFD1) 1958G > A, methylenetetrahydrofolate reductase (MTHFR) 677C > T and 1298A > C and thymidylate synthase (TYMS) 2R > 3R polymorphisms. The MTHFD1 1958A allele significantly reduced the odds of hepatotoxicity (adjusted p = 0.009), while the TYMS 3R allele significantly reduced the odds of leukocytopenia and thrombocytopenia (adjusted p = 0.005 and adjusted p = 0.002, respectively). MTHFR polymorphisms did not influence HD-MTX-related toxicity, but a significant effect of MTHFR 677C > T-TYMS 2R > 3R and MTHFD1 1958G > A-MTHFR 677C > T interactions on HD-MTX-related toxicity was observed. None of the investigated polymorphisms influenced survival. Our study suggests an important role of polymorphisms and gene-gene interactions within the folate pathway in HD-MTX-related toxicity in childhood ALL.

The influence of gemcitabine pathway polymorphisms on treatment outcome in patients with malignant mesothelioma.

OBJECTIVE: Identification of biomarkers that could predict gemcitabine efficacy and toxicity is a key issue in the development of individualized therapy. The aim of our study was to evaluate the influence of gemcitabine pathway polymorphisms on treatment outcome in patients with malignant mesothelioma (MM). METHODS: In total, 107 patients with MM, treated with gemcitabine-platinum chemotherapy, were genotyped for 11 polymorphisms in deoxycytidine kinase, ribonucleotide reductase M1 (RRM1), and cytidine deaminase genes using KASPar assays. Binary logistic regression was used to evaluate the influence of selected polymorphisms on tumor response and occurrence of treatment-related toxicity, while their influence on survival was estimated by Cox proportional hazards model. A haplotype analysis was carried out to assess the combined effect of RRM1 polymorphisms. RESULTS: Deoxycytidine kinase and cytidine deaminase polymorphisms did not influence treatment outcome in patients with MM. In multivariable analysis, RRM1 2927A>C polymorphism significantly decreased overall survival probability [hazard ratio (HR)=2.02; 95% confidence interval (CI)=1.11-3.65; P=0.021]. Two promoter polymorphisms, RRM1 -524T>C and -37C>A, decreased the odds of nausea/vomiting grade≥2 occurrence [odds ratio (OR)=0.25; 95% CI=0.10-0.60; P=0.002 and OR=0.26; 95% CI=0.11-0.63; P=0.003, respectively]. RRM1 TTCCA haplotype was associated with worse tumor response (OR=16.67; 95% CI=2.38-100.00; P=0.004) and worse overall survival (HR=2.97; 95% CI=1.46-6.06; P=0.003) compared with the most frequent TTCAA haplotype, while TCACA haplotype influenced nausea/vomiting grade≥2 occurrence (OR=0.27; 95% CI=0.12-0.60; P=0.001). CONCLUSION: RRM1 polymorphisms as well as haplotypes showed an association with gemcitabine treatment efficacy and toxicity; therefore, they should be validated as potential markers for the prediction of clinical outcome in patients with MM.

BIRC5 promoter SNPs do not affect nuclear survivin expression and survival of malignant pleural mesothelioma patients.

PURPOSE: Malignant pleural mesothelioma is an incurable, asbestos-associated cancer. Its incidence is rapidly increasing and survival remains short. Apoptosis deregulation is an important feature of cancer and survivin, a member of the inhibitor-of-apoptosis-protein family encoded by the BIRC5 gene, has been suggested to have a role in the development and progression of several cancers. Genetic variability, in particular single nucleotide polymorphisms in the BIRC5 promoter, may affect the protein's expression levels. The aim of our study was to elucidate the effects of BIRC5 promoter single nucleotide polymorphisms on survivin expression, patient survival and age at diagnosis in malignant pleural mesothelioma. METHODS: Archival mesothelioma samples from 101 Slovenian patients were immunohistochemically analysed for survivin expression. DNA was extracted from tumour samples and genotyped for three BIRC5 promoter single nucleotide polymorphisms (-31G > C, -241C > T and -625G > C). Genotypes were associated with nuclear survivin expression. Nuclear survivin expression, genotypes, haplotypes, histological type, gender and asbestos exposure were included in univariate Cox survival analyses. RESULTS: Survivin expression was detected in both tumour cell nuclei and cytoplasms in all analysed samples. No association between BIRC5 promoter polymorphism genotypes or haplotypes and nuclear survivin expression was found. Polymorphism -241C > T affected patients' age at diagnosis. Survival analysis confirmed that younger age at diagnosis and epitheloid histological type improved survival, but no significant effects of nuclear survivin expression or genotype/haplotype on overall survival were observed. CONCLUSION: Our findings indicate no relationship between BIRC5 genotypes and survivin expression or overall survival in mesothelioma patients. We observed that BIRC5 -241C > T polymorphism had a significant effect on patient age at diagnosis.

Genetic polymorphisms in base excision repair in healthy slovenian population and their influence on DNA damage.

Genetic polymorphisms in DNA repair genes may result in altered DNA damage. The aim of our study was to determine the frequencies of common functional single nucleotide polymorphisms (SNPs) in specific base-excision DNA repair (BER) genes in healthy Slovenian population and evaluate their influence on DNA damage established by comet assay. In total 141 unrelated healthy subjects were genotyped for hOGG1 Ser326Cys, XRCC1 Arg194Trp and Arg399Gln polymorphisms by real-time TaqMan assay. The frequencies of the hOGG1 326Ser/Ser, 326Ser/Cys and 326Cys/Cys genotypes were 63.8, 29.8, and 6.4%, respectively. The frequency distribution of XRCC1 polymorphism was 88.7% for 194Arg/Arg, 9.2% for 194Arg/Trp, 2.1% for 194Trp/Trp and 46.8% for 399Arg/Arg 40.4% for 399Arg/Gln, 12.8% for 399Gln/Gln. The influence of selected BER polymorphisms on the percentage of comet tail DNA (% TD) was determined in a subgroup of 26 subjects. We found that% TD was significantly increased among individuals with hOGG1 326Ser/Cys heterozygous variant genotype as compared to 326Ser/Ser wild-type genotype (% TD = 8.9 ± 4.2 vs. % TD = 6.9 ± 1.4, P = 0.017). No significant associations between XRCC1 Arg194Trp and Arg399Gln polymorphisms and% TD were found. Our results confirmed that DNA damage is modulated by hOGG1 Ser326Cys polymorphism.

A versatile bacterial expression vector based on the synthetic biology plasmid pSB1.

We have developed an Escherichia coli expression vector that is particularly useful for construction and production of fusion proteins. Based on the synthetic biology pSB1C3 platform, the resulting vector offers a combination of useful features: the strong T7 promoter combined with lac operator, OmpA signal sequence, a selection of cloning sites located at convenient positions and a 3'-terminal His-10 tag. Each of these regions is flanked by a restriction site that allows for easy vector modification, including removal of the signal sequence without perturbation of the reading frame. All the elements were assembled by stepwise addition of three cassettes for which the design was made de novo. To prove the efficiency of the new vector, named pMD204, we successfully produced a cysteine proteinase inhibitor variant in the periplasm and in the cytoplasm of E. coli, in both cases as a soluble and active protein.