Thiopurine intolerance-causing mutations in NUDT15 induce temperature-dependent destabilization of the catalytic site

Germline mutations in NUDT15 cause thiopurine intolerance during treatment of leukemia or autoimmune diseases. Previously, it has been shown that the mutations affect the enzymatic activity of the NUDT15 hydrolase due to decreased protein stability in vivo. Here we provide structural insights into protein destabilization in R139C and V18I mutants using thermolysin-based proteolysis and H/D exchange followed by mass spectrometry. Both mutants exhibited destabilization of the catalytic site, which was more pronounced at higher temperature. This structural perturbation is shared by the mutations despite their different positions within the protein structure. Reaction products of NUDT15 reverted these conformational abnormalities, demonstrating the importance of ligands for stabilization of a native state of the mutants. This study shows the action of pharmacogenetic variants in NUDT15 in a context of protein structure, which might open novel directions in personalized chemotherapy.     

Molecular mechanism of c‐Myc and PRPS1/2 against thiopurine resistance in Burkitt's lymphoma

Patients with relapsed/refractory Burkitt's lymphoma (BL) have a dismal prognosis. Current research efforts aim to increase cure rates by identifying high‐risk patients in need of more intensive or novel therapy. The 8q24 chromosomal translocation of the c‐Myc gene, a main molecular marker of BL, is related to the metabolism by regulating phosphoribosyl pyrophosphate synthetase 2 (PRPS2). In our study, BL showed significant resistance to thiopurines. PRPS2 homologous isoenzyme, PRPS1, was demonstrated to play the main role in thiopurine resistance. c‐Myc did not have direct effects on thiopurine resistance in BL for only driving PRPS2. PRPS1 wild type (WT) showed different resistance to 6‐mercaptopurine (6‐mp) in different metabolic cells because it could be inhibited by adenosine diphosphate or guanosine diphosphate negative feedback. PRPS1 A190T mutant could dramatically increase thiopurine resistance in BL. The interim analysis of the Treatment Regimen for Children or Adolescent with mature B cell non‐Hodgkin's lymphoma in China (CCCG‐B‐NHL‐2015 study) confirms the value of high‐dose methotrexate (MTX) and cytarabine (ARA‐C) in high‐risk paediatric patients with BL. However, there remains a subgroup of patients with lactate dehydrogenase higher than four times of the normal value (4N) for whom novel treatments are needed. Notably, we found that the combination of thiopurines and the phosphoribosylglycinamide formyltransferase (GART) inhibitor lometrexol could serve as a therapeutic strategy to overcome thiopurine resistance in BL.     

The frequency and distribution of thiopurine S-methyltransferase alleles in south Iranian population

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Variability in TPMT activity is mainly due to genetic polymorphism. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G) were determined in an Iranian population from south of Iran (n = 500), using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. Four hundred seventy four persons (94.8%) were homozygous for the wild type allele (TPMT*1/*1) and twenty five people were TPMT*1/*3C (5%). One patient was found to be heterozygous in terms TPMT*1 and *2 alleles with genotype of TPMT*1/*2 (0.2%). None of the participants had both defective alleles. The TPMT*3C and *2 were the only variant alleles observed in this population. The total frequency of variant alleles was 2.6% and the wild type allele frequency was 97.4%. The TPMT*3B and *3A alleles were not detected. Distributions of TPMT genotype and allele frequency in Iranian populations are different from the genetic profile found among Caucasian or Asian populations. Our findings also revealed inter-ethnic differences in TPMT frequencies between different parts of Iran. This view may help clinicians to choose an appropriate strategy for thiopurine drugs and reduce adverse drug reactions such as bone marrow suppression.     

How strong is the edge effect in the adsorption of anticancer drugs on a graphene cluster?

The adsorption of nucleobase-analog anticancer drugs (fluorouracil, thioguanine, and mercaptopurine) on a graphene flake (C₅₄H₁₈) was investigated by shifting the site at which adsorption occurs from one end of the sheet to the other end. The counterpoise-corrected M06-2X/cc-pVDZ binding energies revealed that the binding stability decreases in the sequence thioguanine?>?mercaptopurine?>?fluorouracil. We found that adsorption near the middle of the sheet is more favorable than adsorption near the edge due to the edge effect. This edge effect is stronger for the adsorption of thioguanine or mercaptopurine than for fluorouracil adsorption. However, the edge effect reduces the binding energy of the drug to the flake by only a small amount, <5 kcal/mol, depending on the adsorption site and the alignment of the drug at this site.     

Thiopurine Prodrugs for Plant Chemotherapy Purposes

Thiopurine prodrugs are antiviral chemicals used in medical therapy whose mechanisms of action are associated with inhibition of purine biosynthesis. In terms of plant chemotherapy, previous research of 6‐mercaptopurine (MP) administration in tobacco tissue culture infected by Tobacco mosaic virus (TMV) showed no inhibition of virus activity. Currently, not enough data exist to confirm thiopurine drug ineffectiveness against viruses in the plant kingdom. This paper presents a screening of MP, 6‐methylmercaptopurine riboside (MMPR), 6‐thioguanine (6‐TG) and 1‐amino‐6‐mercaptopurine (1A‐MP) against TMV and Cucumber mosaic virus (CMV) in in vitro tobacco explants and against Grapevine leafroll‐associated virus 3 (GLRaV 3) in in vitro grapevine explants. ELISA and RT‐PCR were used to evaluate antiviral activity. Higher toxicity levels of MP derivatives, compared to MP, were noted in tobacco and grapevine explants. 1A‐MP or 6‐TG treatment resulted CMV and GLRaV 3 virus‐eradicated explants as obtained with Inosine 5′‐monophosphate dehydrogenase inhibitors, whereas TMV was not eradicated by any of the studied drugs.     

Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

The thiopurine antimetabolites 6-thioguanine and 6-mercaptopurine are important chemotherapeutic drugs in the treatment of childhood acute lymphoblastic leukaemia. Measurement of metabolites of these thiopurines is important because correlations exist between levels of these metabolites and the prognosis in childhood acute lymphoblastic leukemia. The reversed-phase method for the determination of extracellular thiopurine nucleosides and bases was previously developed and has been modified such that methylthiopurine nucleosides, bases, thioxanthine and thiouric acid can be measured also. The anion-exchange method enables the determination of intracellular mono-, di- and triphosphate (methyl)thiopurine nucleotides in one run. Extraction on ice with perchloric acid and dipotassium hydrogenphosphate results in good recoveries for (methyl)thiopurine nucleotides in lymphoblasts and peripheral mononuclear cells and for methylthioinosine nucleotides in red blood cells. Measurement of the low concentrations of mono-, di- and triphosphate thioguanine nucleotides in red blood cells (detection limit 20 pmol/10⁹ cells) is possible after extraction with methanol and methylene chloride, followed by oxidation of thioguanine nucleotides with permanganate and fluorimetric detection.     

Synthesis of Novel Thiopurine Pyranonucleosides: Evaluation of Their Bioactivity

We report the synthesis of novel thiopurine pyranonucleosides. Direct coupling of silylated 6-mercaptopurine and 6-thioguanine with the appropriate pyranoses 1a–e via Vorbrüggen nucleosidation, gave the N-9 linked mercaptopurine 2a–e and thioguanine 4a–e nucleosides, while their N-7 substituted congeners 10a–e and 7a–e, were obtained through condensation of the same acetates with 6-chloro and 2-amino-6-chloropurines, followed by subsequent thionation. Nucleosides 3a–e, 5a–e, 8a–e, and 11a–e were evaluated for their cytostatic activity in three different tumor cell proliferative assays.     

Preliminary study on polymorphism analysis of SpRunt-1 gene by PCR–SSCP in <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> and its association with growth traits

In current study, the SpRunt-1 gene was screened for the polymorphisms using PCR-single strand conformation polymorphism (PCR–SSCP) and DNA sequencing methods in Strongylocentrotus intermedius (S. intermedius). Three polymorphisms were found with two site mutations, G833A and C1505T mutation, and an insertion mutation (GT) between 931 nt and 932 nt. These two site mutations are synonymous mutations and the insertion mutation is frame shift mutation. One way ANOVA analysis of correlation between the single nucleotide polymorphisms (SNPs) and growth traits showed that the gonad weight of AA genotype (G at 833 nt) was significantly higher than that of BB genotype (A at 833 nt) (P = 0.029). The body weight of the CC genotype (C at 1505 nt) was significantly higher than that of DD genotype (T at 1505 nt) (P < 0.01) and the shell height of CD genotype was significantly higher than that of DD genotype (P = 0.032). These results provided the evidence that S. intermedius SpRunt-1 gene could be selected as a candidate gene for the growth traits.     

Genetic analysis of C5a receptors in neutrophils from patients with familial Mediterranean fever

Familial Mediterranean fever (FMF) is an autoinflammatory disease, characterized by MEFV gene mutations and self-limited recurrent episodes of fever and localized serositis. Complement system is a key regulator of the inflammatory process. The aim of this study was to investigate the genetic alterations and mRNA expression pattern of C5aR and C5L2 genes in neutrophils from attack-free FMF patients. No mutations were observed in the two receptors’ genes, while the genetic alteration observed in the C5aR1 gene was identified as N279 K polymorphic variant. Furthermore, lower mRNA expression of C5L2 gene was observed in neutrophils from FMF patients compared to control subjects. The binding capacity of rhC5a and the ability to produce reactive oxygen species was similar in neutrophils from healthy subjects and FMF patients and independent of the presence of N279 K polymorphism or mRNA expression of C5L2.     

Effect of genetic variants of chemokine receptors on the development of myocardial infarction in Turkish population

Inflammation is a crucial component of coronary atherosclerosis and myocardial infarction (MI). Chemokine receptors are important modulators of inflammation. Polymorphisms in genes coding for chemokine receptors, CCR2 and CCR5, have been studied as genetic markers of coronary artery disease. In the present study, we investigated whether genetic variants of CCR2-V64I and CCR5-delta32 chemokine receptors have any effect on the development of myocardial infarction. A total of 146 MI patients and 202 control subjects were genotyped for CCR2 and CCR5. CCR2-V64I genotypes were not significantly different between patients with MI and controls (P > 0.05). CCR5-delta32 genotype distribution in cases was significantly different from that of controls (P = 0.042). The CCR5-delta32 wt/deletion genotype frequencies for controls and cases were 0.10 and 0.19, respectively and individuals with CCR5-delta32 wt/deletion genotype had a 2.13-fold increased risk of myocardial infarction (P = 0.0013). Individuals carrying the CCR5-delta32 heterozygote or homozygous variant genotype (deletion/deletion + wt/deletion) had a 1.96-fold increased risk of myocardial infarction compared with the wild-type genotype (wt/wt) (p: 0.016). In conclusion, our data have suggested that genetic variant of CCR5 might be associated with the development of MI. Further larger sample size studies are required to confirm our findings.     

Polymorphism in exon 6 of the human NT5E gene is associated with aortic valve calcification

ABSTRACT

NT5E encodes ecto-5′-nucleotidase (e5NT, CD73) which hydrolyses extracellular AMP to adenosine. Adenosine has been shown to play a protective role against aortic valve calcification (AVC). We identified two nonsynonymous missense single nucleotide polymorphisms (c.1126A > G, p.T376A and c.1136T > C, p.M379T) in exon 6 of the human NT5E gene. Since both substitutions might affect e5NT activity and consequently alter extracellular adenosine levels, we evaluated the association between NT5E alleles and calcific aortic valve disease in 119 patients (95 patients with AVC and 24 controls). In AVC patients, the frequency of the G allele at c.1126 and the frequency of the GG genotype as well as the frequency of the C allele at c.1136, and the frequencies of CC and TC genotypes tended to be higher as compared to controls. The allele and genotype frequencies in AVC patients and controls were also compared to those calculated from the 1000 Genomes Project data for control individuals of European ancestry (n = 503). We found that the frequency of the C allele at c.1136 is significantly higher in patients with AVC than in the European controls (0.111 vs. 0.054, P = 0.0052). Moreover, e5NT activity in aortic valves showed a trend toward lower levels in AVC patients with CC and TC genotypes than in those with the TT genotype. Our findings indicate that the genetic polymorphism of NT5E may contribute to the pathogenesis of calcific aortic valve disease and that the C allele of SNP c.1136 is associated with an increased risk of AVC.     

Allele Frequency of Inosine Triphosphate Pyrophosphatase Gene Polymorphisms in a Japanese Population

The enzyme inosine triphosphate pyrophosphatase (ITPase) catalyses the pyrophosphohydrolysis of ITP to IMP. ITPase deficiency is a clinically benign autosomal recessive condition characterised by the abnormal accumulation of ITP in erythrocytes. A deficiency of ITPase may predict adverse reactions to therapy with the thiopurine drug 6‐mercaptopurine and its prodrug azathioprine. In this study, we examine the frequencies of ITPA polymorphisms in 100 healthy Japanese individuals. The allele frequency of the 94C > A variant in the Japanese sample was 0.135 (Caucasian allele frequency 0.06). The IV2 + 21A > C polymorphism was not found in Japanese (Caucasian allele frequency 0.130). Allele frequencies of the 138G > A, 561G > A and 708G > A polymorphisms were 0.57, 0.18 and 0.06 respectively in the Japanese population, and with the exception of the 138G > A polymorphism, similar to allele frequencies in Caucasians.     

A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides

MutT-related proteins, including the Escherichia coli MutT and human MutT homologue 1 (MTH1) proteins, degrade 8-oxo- 7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to a monophosphate, thereby preventing mutations caused by the misincorporation of 8-oxoguanine into DNA. Here, we report that human cells have another mechanism for cleaning up the nucleotide pool to ensure accurate DNA replication. The human Nudix type 5 (NUDT5) protein hydrolyses 8-oxo-dGDP to monophosphate with a K_m of 0.77 µM, a value considerably lower than that for ADP sugars, which were originally identified as being substrates of NUDT5. NUDT5 hydrolyses 8-oxo-dGTP only at very low levels, but is able to substitute for MutT when it is defective. When NUDT5 is expressed in E. coli mutT⁻ cells, the increased frequency of spontaneous mutations is decreased to normal levels. Considering the enzymatic parameters of MTH1 and NUDT5 for oxidized guanine nucleotides, NUDT5 might have a much greater role than MTH1 in preventing the occurrence of mutations that are caused by the misincorporation of 8-oxoguanine in human cells.     

Thiopurine methyltransferase predicts the extent of cytotoxicty and DNA damage in astroglial cells after thioguanine exposure

Thiopurine methyltransferase (Tpmt) is the primary enzyme responsible for deactivating thiopurine drugs. Thiopurine drugs (i.e., thioguanine [TG], mercaptopurine, azathioprine) are commonly used for the treatment of cancer, organ transplant, and autoimmune disorders. Chronic thiopurine therapy has been linked to the development of brain cancer (most commonly astrocytomas), and Tpmt status has been associated with this risk. Therefore, we investigated whether the level of Tpmt protein activity could predict TG-associated cytotoxicity and DNA damage in astrocytic cells. We found that TG induced cytotoxicity in a dose-dependent manner in Tpmt(+/+), Tpmt(+/-) and Tpmt(-/-) primary mouse astrocytes and that a low Tpmt phenotype predicted significantly higher sensitivity to TG than did a high Tpmt phenotype. We also found that TG exposure induced significantly more DNA damage in the form of single strand breaks (SSBs) and double strand breaks (DSBs) in primary astrocytes with low Tpmt versus high Tpmt. More interestingly, we found that Tpmt(+/-) astrocytes had the highest degree of cytotoxicity and genotoxicity (i.e., IC(50), SSBs and DSBs) after TG exposure. We then used human glioma cell lines as model astroglial cells to represent high (T98) and low (A172) Tpmt expressers and found that A172 had the highest degree of cytoxicity and SSBs after TG exposure. When we over-expressed Tpmt in the A172 cell line, we found that TG IC(50) was significantly higher and SSB's were significantly lower as compared to mock transfected cells. This study shows that low Tpmt can lead to greater sensitivity to thiopurine therapy in astroglial cells. When Tpmt deactivation at the germ-line is considered, this study also suggests that heterozygosity may be subject to the greatest genotoxic effects of thiopurine therapy.     

Occurrence of a New Orange Variant of Curtobacterium flaccumfaciens pv. flaccumfaciens,Causing Common Bean Wilt in Iran

A new orange variant of Curtobacterium flaccumfaciens pv. flaccumfaciens was isolated from seeds of common bean cv. Daneshkadeh and Dehghan stored in the seed banks in Khomein Bean Research Station, and field plants (cv. Local Khomein) in Arak, Iran. The pathogenicity of the isolates was confirmed on 5‐ to 7‐day‐old seedlings of cv. Daneshkadeh. Marginal necrosis and interveinal chlorosis on first trifoliate leaves were observed 10–15 days after inoculation. Amplification of 306 bp fragment of orange‐pigmented strains using CffFOR2‐ and CffREV4‐specific prime pair characterized them as C. flaccumfaciens pv. flaccumfaciens. Although the yellow‐pigmented variant of the causal agent was previously reported on cowpea, this is the first report of orange variant of C. flaccumfaciens pv. flaccumfaciens causing bacterial wilt on common bean in Iran.     

<Emphasis Type="Italic">IGFBP3</Emphasis> polymorphisms and risk of cancer: a meta-analysis

Until now, there were several studies evaluating the association between the polymorphisms in the IGFBP3 gene and cancer risk in diverse populations and in multiple types of cancer, but their outcomes have been contradictory and need to be investigated further. Here, we performed a meta-analysis from all eligible case–control studies to address the association of IGFBP3 A-202C and Gly32Ala polymorphisms to cancer. 20 articles including 41 studies for A-202C variant including 28,322 cancer patients and 36,772 healthy controls and six articles for Gly32Ala variant including 4,477 cases and 5,443 controls were selected in our analysis. Overall, A-202C polymorphism was appeared to be a risk factor of cancer (OR = 0.98, P = 0.05). A allele of IGFBP3 A-202C SNP was significantly less common in the cancer patients than in controls and AA genotype significantly decreased the cancer risk in additive genetic model when comparing to CC genotype (OR = 0.93, P = 0.004). Another SNP, Gly32Ala, seemed to be in linkage equilibrium with A-202C SNP. However, no significance was found when we analyzed the relation of cancer risk and Gly32Ala polymorphism (OR = 0.93, P = 0.36). Further, we compared the distributions of A-202C SNP in different types of cancer, significant association was found in additive genetic model in breast cancer (OR = 0.93, P = 0.01) and prostate cancer (OR = 0.88, P = 0.05). In the analysis of the variants in different population, A-202C variant was significantly associated with cancer risk in Africans (OR = 0.90, P = 0.05), but not in Caucasians (OR = 0.98, P = 0.12) or in Asians (OR = 1.03, P = 0.61). These results indicated that polymorphisms of IGFBP3 might have different effect in different types of cancer and different population. Further large study combining both IGFBP3 A-202C and Gly32Ala SNPs on different types of cancer in different populations were needed to validate former results.     

Novel SNPs of the bovine <Emphasis Type="Italic">CACNA2D1</Emphasis> gene and their association with carcass and meat quality traits

Calcium channel, voltage-dependent, alpha-2/delta subunit 1 (CACNA2D1) gene encodes a member of the alpha-2/delta subunit family, proteins are accessory molecules associated with voltage-gated calcium channels, and increase the density at the plasma membrane of calcium channels activated by high voltage. The main objective of the present study was to identify polymorphisms of CACNA2D1 gene, and to analyze associations between these polymorphisms and carcass and meat quality traits in cattle. In this study, through PCR-SSCP and DNA sequencing methods, two new allelic variant corresponding to the C → G and G → T mutations at positions 526740 and 537917 in the exon25 and exon35 of bovine CACNA2D1 gene, respectively, could be detected. SNP C526740G is a nonsynonymous mutation, resulting in a Cysteine (Cys) to Tryptophan (Trp) amino acid replacement and SNP G537917T resulting in an Aspartic (Asp) to Tyrosine (Tyr) amino acid replacement. The gene-specific SNP markers association analysis was investigated. The C526740G was significantly associated with Meat color (MC) (P = 0.0297) and Backfat thickness (BF) (P < 0.001). The G537917A indicated significant association with Dressing percentage (DP) (P = 0.0485). No significant association, however, was detected between any of the marker genotype and other traits measured in this study. Results from this study initially suggested that CACNA2D1 gene is one of the potential candidate genes influencing carcass and meat quality traits and gene-specific SNPs may be a useful marker for MAS programs in cattle breeding.     

Association of p73 G4C14‐to‐A4T14 polymorphism at exon 2 with the response of human lung adenocarcinoma cell lines to chemotherapy

In order to assess the effect of p73 gene polymorphism G4C14‐A4T14 on cisplatin‐based chemosensitivity of human lung adenocarcinoma cell lines, we examined the differences in biological character and drug sensitivity affected by cisplatin between human lung adenocarcinoma cell lines A549 and P15. The allelic expression ofp73 in A549 and P15 was studied by Sty I polymorphism analysis. MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay was used to analyse the response of these two cell lines to cisplatin. The changes in the biological behaviour of the cells were observed by colony formation assay. The drug‐induced apoptosis of cells was measured by Hoechst and TUNEL techniques. Homozygous allelic expression was demonstrated in the two cell lines. AT/AT genotype appeared in A549, GC/GC genotype was detected in P15. Although the colony formation number decreased with an increasing cisplatin dose (P<0.05), there was no significant difference in colony‐formation rate in these two cell lines (P>0.05). MTT assay also determined that the 50% inhibitory concentration (IC₅₀) for A549 and P15 was 8.9 and 11.6 μmol/l, respectively; the IC₅₀ value did not differ significantly between A549 and P15 (P>0.05). The cell apoptosis induced by cisplatin was demonstrated in both A549 and P15. P73 G4C14‐A4T14 polymorphisms at exon 2 existed in human NSCLC (non‐small‐cell lung cancer) cell lines. Our data in vitro suggest that p73 G4C14‐A4T14 polymorphism has no significant relationship to the cisplatin‐based chemosensitivity in human lung adenocarcinoma.     

Mechanism of cytotoxic action of azaguanine and thioguanine in wild-type V79 cell lines and their relative efficiency in selection of structural gene mutants

The cytotoxic effects of azaguanine and thioguanine have been compared in two wild-type V79 cells. To achieve equitoxic effects in both cell lines a 10–20-fold higher concentration of azaguanine than thioguanine was required. Affinity of HGPRT for azaguanine was 10-fold lower than for hypoxanthine in both cell lines and was similar to that for thioguanine in V79S cells. Affinity for thioguanine differed by a factor of 3 in the two cell lines. The rate of cell kill by azaguanine was markedly slower than by thioguanine in both cell lines. Reduction of whole cell uptake of [¹⁴C]hypoxanthine incorporation by unlabelled azaguanine was only demonstrable after prolonged incubation periods as was incorporation of [¹⁴C]azaguanine into acid-insoluble material. Experiments with cell-free extracts indicated that hypoxanthine acts as a non-competitive inhibitor of the enzyme. The slow rate of dissociation of the HGPRT—azaguanine complex is reflected in the slow rate of killing of wild-type cells. Clones resistant to the cytotoxic effects of these analogues have been selected from both cell lines and have been shown to possess HGPRT with altered kinetic properties. Our data suggest that azaguanine and thioguanine may select for mutations at different sites on the HGPRT molecule in V79 cells and provide possible explanations for the differences in effectiveness of these two agents reported in other cell lines.