Mateusz Kurzawski

Effect of CYP2C19*17 gene variant on Helicobacter pylori eradication in peptic ulcer patients

ObjectiveEradication of Helicobacter pylori is an important treatment strategy in peptic ulcer patients. Current regimens of eradication consist of proton pump inhibitor (PPI) and two antibiotics. The principal enzyme involved in PPIs metabolism is CYP2C19, which exhibits an interindividual variability of activity, mainly due to genetic polymorphism. Two alleles (CYP2C19*2 and CYP2C19*3), responsible for slow PPIs metabolism, were previously associated with higher efficacy of eradication. Recently, a novel CYP2C19 gene variant (CYP2C19*17), associated with faster metabolism of CYP2C19 substrates, was described. In the present study, a potential association between CYP2C19*17 allele and lower H. pylori eradication efficacy was tested in a group of peptic ulcer patients.MethodsA total of 125 peptic ulcer patients, positive for H. pylori infection, were treated with triple therapy (pantoprazole+amoxicillin+metronidazole). Subsequently, the patients were divided into two groups (group 1 – success, and group 2 – failure of eradication after therapy) and genotyped for the presence of CYP2C19 variant alleles (*2, *3, and *17).ResultsFrequency of CYP2C19 alleles in two groups of patients were: 56.4 versus 65 (p=0.060) for *1, 15.4 versus 5.3 (p=0.015) for *2, and 28.2 versus 25.5 (p=0.663) for *17 allele, respectively. CYP2C19*3 was not detected in the evaluated population. No significant differences in frequency nor distribution of *17 allele were found between two groups of patients. CYP2C19*2 allele was associated with successful H. pylori eradication (p<0.02), *2 allele carriers were found to be over 4-times more prone to the treatment compared to *1/*1 homozygotes (OR=4.2, p=0.015).ConclusionOur results suggest that, contrary to CYP2C19*2, CYP2C19*17 allele has no impact on efficacy of H. pylori eradication in peptic ulcer patients treated with pantoprazole.

Reduced folate carrier-1 80G>A polymorphism affects methotrexate treatment outcome in rheumatoid arthritis.

The folate antagonist methotrexate (MTX) is a drug currently used in the treatment of rheumatoid arthritis (RA). MTX enters the cells through the reduced folate carrier (RFC-1) and is activated to polyglutamates. Previous studies have shown that RFC-1 expression may influence the efficacy of therapy with MTX. The studies suggest that G80A polymorphism in RFC-1 is associated with altered folate/antifolate levels and the subjects carrying homozygous mutant 80AA genotype tend to have higher plasma folate and MTX concentrations and higher erythrocyte polyglutamate levels compared with those with the wild type or heterozygous genotype. It is possible that this polymorphism might influence MTX treatment outcome in patients with RA. In the present study, we examined the association between RFC-1 G80A polymorphism and treatment outcome in patients with RA administered MTX. The study was carried out on 174 patients diagnosed with RA treated with MTX (7.5–15.0 mg weekly) plus low doses of methylprednisone. The RFC-1 80G>A polymorphism (resulting in a histidine-to-arginine substitution at codon 27 of RFC-1) was detected using a polymerase chain reaction-restriction fragment length polymorphism method. The probability of remission of RA symptoms was 3.32-fold higher in carriers of 80AA genotype as compared with patients with 80GG genotype (P=0.021, OR=3.32, 95% CI: 1.26–8.79). The frequency of A allele among MTX responders was 62.1, compared to 47.8% in a group of poor MTX responders (P=0.013, OR=1.78, 95% CI: 1.13–2.81). Moreover, the increase of aminotransferase activity was noted more frequently in carriers of 80AA genotype. The present data suggest that evaluation of RFC-1 gene 80G>A polymorphism may be a useful tool to optimize MTX therapy in patients with RA.

Association of Nrf2-encoding NFE2L2 haplotypes with Parkinson's disease

BackgroundOxidative stress is heavily implicated in the pathogenic process of Parkinsons disease. Varying capacity to detoxify radical oxygen species through induction of phase II antioxidant enzymes in substantia nigra may influence disease risk. Here, we hypothesize that variation in NFE2L2 and KEAP1, the genes encoding the two major regulators of the phase II response, may affect the risk of Parkinsons disease.MethodsThe study included a Swedish discovery case-control material (165 cases and 190 controls) and a Polish replication case-control material (192 cases and 192 controls). Eight tag single nucleotide polymorphisms representing the variation in NFE2L2 and three representing the variation in KEAP1 were chosen using HapMap data and were genotyped using TaqMan Allelic Discrimination.ResultsWe identified a protective NFE2L2 haplotype in both of our European case-control materials. Each haplotype allele was associated with five years later age at onset of the disease (p = 0.001) in the Swedish material, and decreased risk of PD (p = 2 × 10-6), with an odds ratio of 0.4 (95% CI 0.3-0.6) for heterozygous and 0.2 (95% CI 0.1-0.4) for homozygous carriers, in the Polish material. The identified haplotype includes a functional promoter haplotype previously associated with high transcriptional activity. Genetic variation in KEAP1 did not show any associations.ConclusionThese data suggest that variation in NFE2L2 modifies the Parkinsons disease process and provide another link between oxidative stress and neurodegeneration.

677C>T and 1298A>C MTHFR polymorphisms affect methotrexate treatment outcome in rheumatoid arthritis

INTRODUCTION Methotrexate (MTX), widely used in the treatment of rheumatoid arthritis (RA), inhibits dihydrofolate reductase and folate-dependent enzymes. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and has been shown to be polymorphic, affecting the enzyme activity. METHODS To examine the association between 677C>T and 1298A>C MTHFR polymorphisms and MTX efficacy in the treatment of RA, a total of 174 RA patients, treated with MTX plus methylprednisone 4 mg and folic acid 5 mg were analyzed. RESULTS In univariate regression analysis model, the MTHFR 677T allele was associated with significantly higher frequency of remission, whereas in the case of the 1298C allele, a tendency for higher remission rate was observed. In multivariate regression analysis, the presence of both 677T and 1298C alleles was associated with an increased frequency of remission. CONCLUSION The results of our study suggest that the MTHFR 677T and 1298C alleles may be associated with an increased rate of RA remission in patients treated with MTX receiving high doses of folic acid supplementation.

The association of functional catechol-O-methyltransferase haplotypes with risk of Parkinson's disease, levodopa treatment response, and complications.

Introduction Differences in catechol-O-methyltransferase (COMT) activity and genotype may determine individual variations in the therapeutic response to levodopa or Parkinsons disease (PD) susceptibility. The role of functional COMT haplotypes in PD susceptibility and treatment response has not been examined. Objectives In this case–control study, we investigated the association of the most common COMT gene haplotypes (formed by single nucleotide polymorphisms (SNPs): rs6269:A>G; rs4633C>T; rs4818:C>G; and rs4680:A>G) with PD risk and the association of the COMT haplotypes with the dose and complications of levodopa therapy in PD patients. Methods A total of 679 study participants (322 PD and 357 controls) were included. Each participant was genotyped for four SNPs in the COMT gene, located in a common haploblock, that has been shown to influence COMT enzymatic activity. The influence of COMT haplotypes on the dose of levodopa administered during fifth year of treatment, and occurrence of motor complications were examined in PD patients. The EH program (Jurg Ott, Rockefeller University, New York, USA) was used to estimate haplotype frequencies. Results The estimated frequencies of low (A_C_C_G) and medium (A_T_C_A) activity haplotypes tended to be slightly lower among PD patients when compared with controls (P=0.09, G_C_G_G-high activity haplotype as reference). The frequency of G_C_G_G (high activity) haplotype carriers was higher in late onset PD patients (P=0.04) compared with controls. The mean levodopa dose increased with the activity of the functional haplotypes (low<medium<high). Doses prescribed for G_C_G_G (high activity) haplotype carriers (mean 604.2±261.9 mg) were significantly higher than those for the noncarriers (mean 512.2±133.5 mg, P<0.05). The COMT haplotype seemed to have little influence on the development of levodopa-induced dyskinesias. Conclusion Our study showed a possible association of functional COMT haplotypes with the risk of PD. Both nonsynonymous and synonymous SNPs within functional COMT haplotype blocks may be more relevant than individual SNPs in conferring PD susceptibility. The doses of levodopa treatment can be influenced by specific COMT haplotypes and this may be useful in instituting individualized therapy for PD patients.

Interleukin-10 (IL10) and tumor necrosis factor α (TNF) gene polymorphisms in Parkinson's disease patients

Recent studies revealed that inflammatory processes might play an important role in the pathogenesis of Parkinsons disease (PD). We hypothesized that genetically determined differences in the immune response, especially in anti- and pro-inflammatory cytokines production might influence the risk for the development and/or onset of sporadic PD. In the present study, we investigated the genetic polymorphisms of the IL10 (-1082 and -519) and TNF (-308) genes in relation to the risk of PD, and their associations with age of PD onset in a group of 316 patients, divided into two subgroups: Group 1: patients with early onset PD (EOPD), i.e. before 50 years of age (102 patients), and group 2: patients with onset of PD after 50 years of age comprising 214 subjects. Control samples were obtained from 300 randomly selected healthy individuals from the same geographical region with no signs of Parkinsonism as evaluated by a neurologist. PCR-RFLP methods were used for genotyping. No statistically significant differences between PD patients and controls were found in the frequency of a single locus of IL10 promoter. We found TNF -308A allele significantly more frequent in EOPD patients compared to the controls (p=0.007). The overrepresentation of the A allele was reflected by a significant increase in AA homozygous individuals in EOPD patients compared to the controls (p=0.0021). The results from our study revealed that the TNF -308AA genotype might increase the risk of early onset of PD.

Frequency distribution of thiopurine S-methyltransferase alleles in a Polish population

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability mainly a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. It has previously been reported that 3 variant alleles:TPMT*2, *3A, and *3C are responsible for over 95% cases of lower enzyme activity. The purpose of this study was to determine the frequency of TPMT variant alleles in a Polish population. DNA samples were obtained from 358 unrelated healthy Polish subjects of white origin, and TPMT genetic polymorphism was determined using PCR-RFLP and allele-specific PCR methods. The results showed that allelic frequencies were 0.4% for TPMT*2, 2.7% for TPMT*3A, and 0.1% for TPMT*3C, respectively. A TPMT*3B allele was not found in the studied population. The general pattern of TPMT allele disposition in the Polish population is similar to those determined for other white populations, but the frequency of total variant alleles is lower than in other European populations studied to date.

Association of COMT, MTHFR, and SLC19A1(RFC-1) polymorphisms with homocysteine blood levels and cognitive impairment in Parkinson's disease.

Introduction Elevated plasma homocysteine (Hcy) concentration is an independent risk factor for cardiovascular disease, and its involvement in endothelial cell dysfunction is well established. However, the role of Hcy and folate in the pathogenesis of Parkinson’s disease (PD) remains controversial. Objectives The study was aimed at evaluating the relationships between Hcy, vitamin B12, and folic acid levels in the blood and cognitive status in PD patients with the genetic polymorphisms of MTHFR (rs1801133: C>T-677C>T, rs1801131: A>C-1298A>C), COMT (rs4680: A>G-Val158Met, rs6269: A>G, rs4633: C>T, rs4818: C>G), or SLC19A1 (rs1051266: G>A-80G>A). Methods A total of 502 participants (248 with PD and 254 age-matched and sex-matched controls) were included in the study. The Unified Parkinson’s Disease Rating Scale score, Hoehn–Yahr staging, and the Schwab–England scale were used to assess motor abilities and activity during daily life. Complex psychological examination with a battery of tests was used to classify patients into groups with (PDD) and without (nPDD) dementia. Blood samples were examined for Hcy, vitamin B12, and folic acid levels, as well as polymorphisms in genes related to Hcy metabolism, such as COMT, MTHFR, and SLC19A1(RFC-1). Results The frequency of homozygous COMT rs4680G and rs4633C allele carriers was significantly decreased in PD patients in comparison with the controls (P=0.015; odds ratio=0.60; 95% confidence interval 0.41–0.90 and P=0.020; odds ratio=0.619; 95% confidence interval 0.42–0.92, respectively). No significant differences in the distribution of MTHFR 677C>T, 1298A>C, and SLC19A1 80G>A alleles and genotypes between PD patients and the controls were found. Hcy levels were significantly increased in PD patients (18±7.8 &mgr;mol/l) as compared with the controls (14.0±9.6 &mgr;mol/l, P=10–8) and were significantly associated with the MTHFR 677C>T polymorphism both in PD patients and controls, in which T allele carriers were characterized by markedly elevated Hcy plasma concentrations. No association was observed between Hcy plasma level and COMT and SLC19A polymorphisms. The results of multivariate logistic regression analysis revealed age (P=0.0003) and Hcy plasma levels (P=0.07) as independent risk factors predisposing individuals to PD dementia. The studied polymorphisms were not associated with cognitive status in PD patients. Conclusion The genetic factors studied were not associated with cognitive status in PD patients. Only age and Hcy plasma levels were found to be independent risk factors predisposing individuals to PD dementia. However, COMT: rs4680: A>G and rs4633: C>T polymorphisms were found to significantly affect PD risk, and the MTHFR 677C>T polymorphism helped determine plasma Hcy concentrations.

The impact of thiopurine S-methyltransferase polymorphism on azathioprine-induced myelotoxicity in renal transplant recipients

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability, mainly as a result of genetic polymorphism. Patients with intermediate or deficient TMPT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. It has previously been reported that 3 variant alleles: TPMT*2, *3A, and *3C are responsible for over 95% cases of low enzyme activity. The purpose of this study was to explore the association between these polymorphisms and the occurrence of azathioprine adverse effects in 112 renal transplant recipients undergoing triple immunosuppressive therapy including azathioprine, cyclosporine, and prednisone. TPMT genetic polymorphism was determined using PCR-RFLP and allele-specific PCR methods. Azathioprine dose, leukocyte, erythrocyte, and platelet counts, graft rejection episodes, as well as cyclosporine levels were analyzed throughout the first year after organ transplantation. We found the frequency of leukopenia episodes (WBC < 4.0 × 109/L) significantly higher in heterozygous patients (53.8%) compared with those with TPMT wild-type genotype (23.5%). One patient, who was a compound homozygote (3A/*3C), experienced severe azathioprine-related myelotoxicity each time after receiving the standard drug dose. Our results suggest that polymorphisms in TPMT gene may be responsible for approximately 12.5% of all leukopenia episodes in renal transplant recipients treated with azathioprine. Genotyping for the major TPMT variant alleles may be a valuable tool in preventing AZA toxicity and optimization of immunosuppressive therapy.

TPMT but not ITPA gene polymorphism influences the risk of azathioprine intolerance in renal transplant recipients

PurposeThiopurine drugs have to be withdrawn in 10–30% of cases due to side effects, and it has been presented that genetic factors may be responsible for some of reported toxicity cases. Among polymorphic enzymes of thiopurines’ metabolic pathway, thiopurine S-methyltransferase (TPMT) has been studied most extensively, and some recent studies point to inosine triphosphate pyrophosphohydrolase (ITPA) polymorphism as an additional toxicity risk factor.MethodsThe aim of the current study was to evaluate an association between TPMT and ITPA gene polymorphisms and drug intolerance in a cohort of 157 renal transplant recipients treated with azathioprine (AZA). Each subject was genotyped for the presence of variant TPMT (*2, *3A, *3B, and *3C) and ITPA (94C>A and IVS2+21A>C) alleles.ResultsMean AZA dose, mean white-blood-cell count, and platelet count in the course of treatment were lower in carriers of variant TPMT alleles compared to patients with TPMT wild-type genotype. Leukocyte numbers fell below 4.0 × 109/L in 41.2% of TPMT heterozygous renal transplant recipients, compared to only 18.0% of wild-type patients (P < 0.01). In contrast, ITPA genotype did not influence AZA dose, hematological parameters, or leucopenia risk.ConclusionsOur results suggest that routine genotyping of renal transplant recipients for TPMT variants may be useful in reducing the risk of AZA-related myelotoxicity, but there is not enough evidence to introduce ITPA testing into clinical practice.