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Para-amino salicylic acid (PAS) was first reported by Lehmann in 1946 and used for tuberculosis treatment. However, due to its adverse effects, it is now used only as a second line anti-tuberculosis drug for treatment of multidrug resistant or extensively drug resistant M. tuberculosis. The structure of PAS is similar to para-amino benzoic acid (pABA), an intermediate metabolite in the folate synthesis pathway. The study has identified mutations in genes in folate pathway and their intergenic regions for their possibilities in responsible for PAS resistance. Genomic DNA from 120 PAS-resistant and 49 PAS-sensitive M. tuberculosis isolated from tuberculosis patients in Thailand were studied by whole genome sequencing. Twelve genes in the folate synthesis pathway were investigated for variants associated with PAS resistance. Fifty-one SNVs were found in nine genes and their intergenic regions (pabC, pabB, folC, ribD, thyX, dfrA, thyA, folK, folP). Functional correlation test confirmed mutations in RibD, ThyX, and ThyA are responsible for PAS resistance. Detection of mutation in thyA, folC, intergenic regions of thyX, ribD, and double deletion of thyA dfrA are proposed for determination of PAS resistant M. tuberculosis. [ABSTRACT FROM AUTHOR]

Neural tube defects (NTDs) are congenital abnormalities in the central nervous system. The exact etiology of NTDs is still not determined, but several genetic and epigenetic factors have been studied. Folate supplementation during gestation is recommended to reduce the risk of NTDs. In this review we examine single nucleotide polymorphisms (SNPs) of the genes in the folate pathway associated with NTD. We reviewed the literature for all papers discussing both NTDs and SNPs in the folate pathway. Data were represented through five different genetic models. Quality assessment was performed using the Newcastle–Ottawa Scale (NOS) and Cohen's Kappa inter-rater coefficient assessed author agreement. Fifty-nine papers were included. SNPs in MTHFR, MTRR, RFC genes were found to be highly associated with NTD risk. NOS showed that high quality papers were selected, and Kappa Q-test was 0.86. Our combined results support the notion that SNPs significantly influence NTDs across the population, particularly in Asian ethnicity. Additional high-quality research from diverse ethnicities is needed and meta-regression analysis based on a range of criteria may provide a more complete understanding of the role of folate metabolism in NTDs. [ABSTRACT FROM AUTHOR]

Background: Exist of folate pathway inhibitor genes among Acinetobacter spp. isolates are regarded as a significant mechanism of resistance for sulfa drugs in this pathogen. Objective: This study aimed to investigate genes associated with sulfa drugs resistance among Acinetobacter spp. using Polymerase Chain Reaction (PCR). Patients and Methods: This study included 928 specimens from patients who visited the main hospitals and private clinic laboratories of AlNajaf City-Iraq. All specimens were cultivated and bacterial diagnosis was done according to standard methods. Antibiotics susceptibility and molecular investigation for sul-1, sul-2, sul-3, dfr-A, dfr-B, dfr-G and dfr-K for all Acinetobacter spp. isolates were done. Results: The rate of Acinetobacter spp. isolates were 28 (5.4%). Results showed a high resistance towards antibiotics classes, 28 (100%) of isolates were resistance to piperacillin, cefotaxime, ceftriaxone, ceftazidime and cefepime, while the lowest resistance rate was against minocycline reached 42.85. PCR showed 28 (100%) of Acinetobacter spp isolates were harbored sul-1 and drf-G genes. 25 (89.29%) and 21 (75%) of isolates were positive for sul-2 and drf-A genes respectively, while sul-3, drf-B and drf-K genes were not detected. Conclusion: There is a great deal of concern about antimicrobial agent resistance and also about the number of drugassociated resistance genes contained in Acinetobacterspp. isolates especially sul-1, sul-2, dfr-A, and dfr-G, which have a significant role in sulfa drug resistance. [ABSTRACT FROM AUTHOR]

In an effort to comprehensively interrogate genetic variation in the folate pathway for risk of cleft lip with or without cleft palate (CLP), we evaluated 504 common and rare variants in 35 folate-related genes in a panel of 330 infants with CLP and 367 non-malformed controls. Odds ratios (OR) with 95% confidence intervals were computed for common genotypes. A Case-Control Difference metric was calculated for rare variants to highlight differentially occurring alleles. Interactions between variants and a maternal folate intake variable were also evaluated. In gene-only results, significant odds ratios were observed for multiple variants in the BHMT/BHMT2/DMGDH gene cluster, particularly in Hispanic infants. Also in this cluster, rare variant analysis highlighted a substantial case-control difference in BHMT rs60340837 (synonymous Y284Y). In Hispanics, the ALDH1L1 I812V variant (rs4646750) was the most significant risk allele: OR = 3.8 (95%CI = 1.6-9.2) when heterozygous. In non-Hispanic white infants, we observed significant risk for AHCYL2 rs1095423 (homozygous OR = 3.0, 95%CI 1.1-7.8) and the 68 bp CBS insertion (c.844ins68; heterozygous OR = 2.4, 95%CI = 1.1-5.3). Rare variant analysis in this group revealed case-control differences in MTRR and several other methionine cycle genes, a process implicated previously in clefting risk. In women with low folate intake specifically, increased risks were observed for CBS rs2851391 (OR = 3.6, 95%CI = 1.3-9.6) and the R259P nonsynonymous variant of TCN2 (rs1801198; OR = 2.8, 95%CI = 1.2-6.3). This comprehensive study provides further direction on candidate loci to help disentangle the folate-related developmental phenomena in human clefting risk. © 2016 Wiley Periodicals, Inc.

The Mycobacterium abscessus drug development pipeline is poorly populated, with particularly few validated target-lead couples to initiate de novo drug discovery. Trimethoprim, an inhibitor of dihydrofolate reductase (DHFR) used for the treatment of a range of bacterial infections, is not active against M. abscessus . Thus, evidence that M. abscessus DHFR is vulnerable to pharmacological intervention with a small molecule inhibitor is lacking. Here, we show that the pyrrolo-quinazoline PQD-1, previously identified as a DHFR inhibitor active against Mycobacterium tuberculosis , exerts whole cell activity against M. abscessus . Enzyme inhibition studies showed that PQD-1, in contrast to trimethoprim, is a potent inhibitor of M. abscessus DHFR and over-expression of DHFR causes resistance to PQD-1, providing biochemical and genetic evidence that DHFR is a vulnerable target and mediates PQD-1's growth inhibitory activity in M. abscessus . As observed in M. tuberculosis , PQD-1 resistant mutations mapped to the folate pathway enzyme thymidylate synthase (TYMS) ThyA. Like trimethoprim in other bacteria, PQD-1 synergizes with the dihydropteroate synthase (DHPS) inhibitor sulfamethoxazole (SMX), offering an opportunity to exploit the successful dual inhibition of the folate pathway and develop similarly potent combinations against M. abscessus . PQD-1 is active against subspecies of M. abscessus and a panel of clinical isolates, providing epidemiological validation of the target-lead couple. Leveraging a series of PQD-1 analogs, we have demonstrated a dynamic structure-activity relationship (SAR). Collectively, the results identify M. abscessus DHFR as an attractive target and PQD-1 as a chemical starting point for the discovery of novel drugs and drug combinations that target the folate pathway in M. abscessus ., Competing Interests: C.J.B., A.E.M., E.B.N., D.B.O., and R.W. are employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.

Para-amino salicylic acid (PAS) was first reported by Lehmann in 1946 and used for tuberculosis treatment. However, due to its adverse effects, it is now used only as a second line anti-tuberculosis drug for treatment of multidrug resistant or extensively drug resistant M. tuberculosis. The structure of PAS is similar to para-amino benzoic acid (pABA), an intermediate metabolite in the folate synthesis pathway. The study has identified mutations in genes in folate pathway and their intergenic regions for their possibilities in responsible for PAS resistance. Genomic DNA from 120 PAS-resistant and 49 PAS-sensitive M. tuberculosis isolated from tuberculosis patients in Thailand were studied by whole genome sequencing. Twelve genes in the folate synthesis pathway were investigated for variants associated with PAS resistance. Fifty-one SNVs were found in nine genes and their intergenic regions (pabC, pabB, folC, ribD, thyX, dfrA, thyA, folK, folP). Functional correlation test confirmed mutations in RibD, ThyX, and ThyA are responsible for PAS resistance. Detection of mutation in thyA, folC, intergenic regions of thyX, ribD, and double deletion of thyA dfrA are proposed for determination of PAS resistant M. tuberculosis., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Plants rely on primary metabolism for flexible adaptation to environmental changes. Here, through a combination of chemical genetics and forward genetic studies in Arabidopsis plants, we identified that the essential folate metabolic pathway exerts a salicylic acid-independent negative control on plant immunity. Disruption of the folate pathway promotes enhanced resistance to Pseudomonas syringae DC3000 via activation of a primed immune state in plants, whereas its implementation results in enhanced susceptibility. Comparative proteomics analysis using immune-defective mutants identified a methionine synthase (METS1), in charge of the synthesis of Met through the folate-dependent 1C metabolism, acting as a nexus between the folate pathway and plant immunity. Overexpression of METS1 represses plant immunity and is accompanied by genome-wide global increase in DNA methylation, revealing that imposing a methylation pressure at the genomic level compromises plant immunity. Take together, these results indicate that the folate pathway represents a new layer of complexity in the regulation of plant defense responses. Folate metabolism interferes with plant immune responses to Pseudomonas syringae DC300 via a salicylic acid-independent negative control, which is exerted through the folate-dependent 1C methionine synthase (METS1). We show that the overexpression of METS1 promotes enhanced disease susceptibility toward P. syringae DC3000 that is accompanied by a genome-wide increase in DNA methylation level. [ABSTRACT FROM AUTHOR]

Highlights • Exogenous phenylalanine application confers chilling tolerance in tomato fruits. • Conferring chilling tolerance is accompanying by maintaining membrane integrity. • Employing higher MTHFD enzyme activity is crucial for ensuring sufficient supply of NADPH. • Higher ROS scavenging system activity may arise from ensuring sufficient supply of NADPH. • Ensuring sufficient supply of NADPH giving rise to higher phenols and flavonoids accumulation and DPPH scavenging capacity. Abstract In this experiment, the mechanisms displayed by exogenous phenylalanine application to confer chilling tolerance in tomato fruits (Lycopersicon esculentum cv. Izmir) during storage at 4 °C for 28 days were studied. Phenylalanine application at 5 mM significantly confers chilling injury tolerance in tomato fruits associated with membrane integrity maintenance reflected by a lower degree of electrolytes leakage and malondialdehyde (MDA) accumulation. Membrane integrity maintenance in tomato fruits in response to exogenous phenylalanine application at 5 mM may arise from lower H 2 O 2 accumulation due to higher activity of reactive oxygen species (ROS) scavenging enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), concurrent with higher endogenous lycopene and proline accumulation. In addition, higher phenols and flavonoids accumulation arising from higher phenylalanine ammonia-lyase (PAL) enzyme activity is concurrent with higher ascorbic acid (AA) accumulation giving rise to higher DPPH scavenging capacity, which may be vital for the maintenance of membrane integrity in tomato fruits treated with exogenous phenylalanine. Ensuring supply of reducing power NADPH by triggering of folates pathway demonstrated by higher methylenetetrahydrofolate dehydrogenase (MTHFD) enzyme activity may be responsible for activation of ROS scavenging systems in tomato fruits in response to exogenous phenylalanine application, contributing to membrane integrity maintenance. Accordingly, exogenous phenylalanine application may serve as a safe promising procedure to confer chilling tolerance in tomato fruits during cold storage. [ABSTRACT FROM AUTHOR]

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.

Genetic variation in drug detoxification pathways may influence outcomes in pediatric acute lymphoblastic leukemia (ALL). We evaluated relapse risk and 24 variants in 17 genes in 714 patients in CCG-1961. Three TPMT and 1 MTR variant were associated with increased risks of relapse (rs4712327, OR 3.3, 95%CI 1.2–8.6; rs2842947, OR 2.7, 95%CI 1.1–6.8; rs2842935, OR 2.5, 95%CI 1.1–5.0; rs10925235, OR 4.9, 95%CI 1.1–25.1). One variant in SLC19A1 showed a protective effect (rs4819128, OR 0.5, 95%CI 0.3–0.9). Our study provides data that relapse risk in pediatric ALL is associated with germline variations in TPMT, MTR and SLC19A1.

The expression levels of one-carbon metabolic enzymes were investigated and observed to be correlated with clinicopathological parameters in patients with pancreatic cancer. Mitochondrial one-carbon metabolism comprises a network of biological reactions that integrate nutrient status with nucleotide synthesis, amino acid metabolism, antioxidant reduced nicotinamide adenine dinucleotide phosphate production and epigenetic methylation processes. Previous studies have reported that the hyper-activation of mitochondrial one-carbon metabolism serves a significant role in malignant cancer phenotypes. A total of 103 patients underwent surgical resection of pancreatic ductal adenocarcinomas (PDAC) at Osaka University Hospital between April 2007 and December 2013 and were enrolled in this study. Subsequently, the expression of the one-carbon metabolic enzymes methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), aldehyde dehydrogenase 1 family member L2 (ALDH1L2), and serine hydroxymethyltransferase (SHMT2) was examined using immunohistochemical analysis. The immunohistochemical analyses demonstrated that patients with high expression levels of MTHFD2, ALDH1L2 or SHMT2 had significantly poor overall survival (OS) and disease-free survival (DFS) rates, as compared with patients with low expression levels. Furthermore, multivariate Cox proportional hazards analysis indicated that MTHFD2 and ALDH1L2 were independent prognostic factors for OS and DFS, whereas SHMT2 was not predictive of DFS. However, high and low expression levels of all three folate metabolic enzymes were significantly associated with improved OS and DFS, compared with the high expression of one or two folate metabolic enzymes. The expression levels of mitochondrial one-carbon metabolic enzymes are independent prognostic factors and potential therapeutic targets for future pancreatic cancer treatments. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is one of the major health issues worldwide. 5-Fluorouracil (5-FU) is a cornerstone of chemotherapy for CRC and the major targets of 5-FU are folate-metabolizing enzymes.Methods: A total of 103 CRC patients with complete clinical data were included in this prospective cohort study. Genotyping was performed using polymerase chain reaction (PCR) followed by sequencing. Using Kaplan-Meier curves, log-rank tests, and Cox proportional hazard models, we evaluated associations between functional polymorphisms in four genes MTHFR (1298A>C and 677C>T), DPYD (496A>G and 85T>C), DHFR 19 bp del, and MTR (2756 A>G) with disease-free survival (DFS).Results: The minor allele frequencies of MTHFR 1298A>C, MTHFR 677C>T, DPYD 496A>G, DPYD 85T>C, DHFR 19 bp del, and MTR 2756 A>G were 0.364, 0.214, 0.116, 0.209, 0.383, and 0.097, respectively. CRC patients carrying the homozygous GG genotype in DPYD 496A>G had 4.36 times shorter DFS than wild-type AA carriers, (DFSGG vs AA: 8.0 ± 4 vs 69.0 ± 10 months; HR 4.36, 95% CI 1.04-18; p = 0.04). Moreover, female carriers of homozygous CC genotype of DPYD 85T>C had shorter DFS compared to either heterozygous or wild-type genotypes, and were 12.7 times shorter than wild-type TT carriers (DFSCC vs TT: 5.0 ± 1.5 vs 42.0 ± 7.6 months; HR 12.7, 95% CI 2.2-71.4; p = 0.004). However, there were no significant associations with the other studied polymorphisms.Conclusion: Genetic polymorphism in DPYD seems to be associated with DFS in CRC patients receiving an adjuvant regimen of 5-FU/capecitabine-based chemotherapy. Further studies are needed to verify these findings. [ABSTRACT FROM AUTHOR]

Abstract: Folate metabolism genes are pivotal to critical biological processes and are related to several conditions, including developmental, cognitive, and cardiovascular anomalies. A systematic catalog of genetic polymorphisms in protein coding regions, regulatory transcription factor binding sites, and miRNA binding sites associated with folate pathway genes may contribute to personalized medicine. We performed a comprehensive computational survey of single nucleotide polymorphisms (SNPs) of folate pathway genes to highlight functional polymorphisms in the coding region, transcription factor binding sites, and miRNAs binding sites. Folate pathway genes were searched through PubMed and Kyoto Encyclopedia of Genes and Genomes pathway databases. SNPs were identified and characterized using the University of California, Santa Cruz genome browser and SNPnexus tool. Functional characterization of nonsynonymous SNPs (nsSNPS) was performed using bioinformatics tools, and common deleterious nsSNPs were identified. We identified 48 genes of folate pathway containing 287 SNPs in the coding regions. Out of these SNPs, rs5742905, rs45511401, and rs1801133 were predicted to be deleterious through four different bioinformatics tools. Three‐dimensional structures of two proteins with and without deleterious nsSNPs were predicted by SWISSPDB viewer and SuperPose. Besides, a total of 237 SNPs was identified in transcription factor binding sites using the Genomatix software suite and six miRNA target site SNPs using miRNASNP. This systematic and extensive in silico analysis of functional SNPs of folate pathway may provide a foundation for future targeted mechanistic, structure‐function, and genetic epidemiological studies. [ABSTRACT FROM AUTHOR]

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuestTM tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection. [ABSTRACT FROM AUTHOR]

Background Multi-target approaches are necessary to properly analyze or modify the function of a biochemical pathway or a protein family. An example of such a problem is the repurposing of the known human anti-cancer drugs, antifolates, as selective anti-parasitic agents. This requires considering a set of experimentally validated protein targets in the folate pathway of major pathogenic trypanosomatid parasites and humans: ( i ) the primary parasite on-targets: pteridine reductase 1 (PTR1) (absent in humans) and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR–TS), ( ii ) the primary off-targets: human DHFR and TS, and ( iii ) the secondary on-target: human folate receptor β , a folate/antifolate transporter. Methods We computationally compared the structural, dynamic and physico-chemical properties of the targets. We based our analysis on available inhibitory activity and crystallographic data, including a crystal structure of the bifunctional T. cruzi DHFR–TS with tetrahydrofolate bound determined in this work. Due to the low sequence and structural similarity of the targets analyzed, we employed a mapping of binding pockets based on the known common ligands, folate and methotrexate. Results Our analysis provides a set of practical strategies for the design of selective trypanosomatid folate pathway inhibitors, which are supported by enzyme inhibition measurements and crystallographic structures. Conclusions The ligand-based comparative computational mapping of protein binding pockets provides a basis for repurposing of anti-folates and the design of new anti-trypanosmatid agents. General significance Apart from the target-based discovery of selective compounds, our approach may be also applied for protein engineering or analyzing evolutionary relationships in protein families. [ABSTRACT FROM AUTHOR]

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.

The need for new antimicrobials is great in face of a growing pool of resistant pathogenic organisms. This review will address the potential for antimicrobial therapy based on polypharmacological activities within the currently utilized bacterial biosynthetic folate pathway. The folate metabolic pathway leads to synthesis of required precursors for cellular function and contains a critical node, dihydrofolate reductase (DHFR), which is shared between prokaryotes and eukaryotes. The DHFR enzyme is currently targeted by methotrexate in anti-cancer therapies, by trimethoprim for antibacterial uses, and by pyrimethamine for anti-protozoal applications. An additional anti-folate target is dihyropteroate synthase (DHPS), which is unique to prokaryotes as they cannot acquire folate through dietary means. It has been demonstrated as a primary target for the longest standing antibiotic class, the sulfonamides, which act synergistically with DHFR inhibitors. Investigations have revealed most DHPS enzymes possess the ability to utilize sulfa drugs metabolically, producing alternate products that presumably inhibit downstream enzymes requiring the produced dihydropteroate. Recent work has established an off-target effect of sulfonamide antibiotics on a eukaryotic enzyme, sepiapterin reductase, causing alterations in neurotransmitter synthesis. Given that inhibitors of both DHFR and DHPS are designed to mimic their cognate substrate, which contain shared substructures, it is reasonable to expect such “off-target” effects. These inhibitors are also likely to interact with the enzymatic neighbors in the folate pathway that bind products of the DHFR or DHPS enzymes and/or substrates of similar substructure. Computational studies designed to assess polypharmacology reiterate these conclusions. This leads to hypotheses exploring the vast utility of multiple members of the folate pathway for modulating cellular metabolism, and includes an appealing capacity for prokaryotic-specific polypharmacology for antimicrobial applications.

Gene polymorphisms involved in homocysteine-methionine pathway result in hyperhomocysteinemia, a predisposing condition to several diseases. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate and homocysteine metabolism. The two known functional polymorphisms of MTHFR gene, 677C>T and 1298A>C have been implicated in a variety of multifactorial diseases: cardio-cerebrovascular and neurodegenerative disorders, autoimmune diseases, birth defects, diabetes, neuropsychiatric disorders, cancer and renal disease. C667T, and to a lesser extent A1298C polymorphisms, have been also reported to have a pharmacogenetic role in predicting drug toxicity in cancer and rheumatoid arthritis treatment. We review here the principal effects of the MTHFR gene variations in different clinical conditions. [ABSTRACT FROM AUTHOR]

Gastric cancer is ranked as the most common cancer in Koreans. A recent molecular biological study about the folate pathway gene revealed the correlation with a couple of cancer types. In the folate pathway, several genes are involved, including methylenetetrahydrofolate reductase (MTHFR), methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), and methyltetrahydrofolate-homocysteine methyltransferase (MTR). The MTHFR gene has been reported several times for the correlation with gastric cancer risk. However, the association of the MTRR or MTR gene has not been reported to date. In this study, we investigated the association between the single nucleotide polymorphisms (SNPs) of the MTHFR, MTRR, and MTR genes and the risk of gastric cancer in Koreans. To identify the genetic association with gastric cancer, we selected 17 SNPs sites in folate pathway-associated genes of MTHFR, MTR, and MTRR and tested in 1,261 gastric cancer patients and 375 healthy controls. By genotype analysis, estimating odds ratios and 95% confidence intervals (CI), rs1801394 in the MTRR gene showed increased risk for gastric cacner, with statistical significance both in the codominant model (odds ratio [OR], 1.39; 95% CI, 1.04 to 1.85) and dominant model (OR, 1.34; 95% CI, 1.02 to 1.75). Especially, in the obese group (body mass index ≥ 25 kg/m2), the codominant (OR, 9.08; 95% CI, 1.01 to 94.59) and recessive model (OR, 3.72; 95% CI, 0.92 to 16.59) showed dramatically increased risk (p < 0.05). In conclusion, rs1801394 in the MTRR gene is associated with gastric cancer risk, and its functional significance need to be validated.

Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNA fMet to fMet-tRNA fMet is crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene tetrahydrofolate (5,10-CH 2 -THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using in vivo and in vitro approaches, we show that 10-CHO-DHF may also be utilized by Fmt as an alternative substrate (formyl group donor) to formylate Met-tRNA fMet . Dihydrofolate (DHF) formed as a by-product in the in vitro assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expressing strains were more sensitive to trimethoprim (TMP) than the ∆ fmt strain, suggesting that the domino effect of TMP leads to inhibition of protein synthesis and strain growth. Antifolate treatment to Escherichia coli showed a decrease in the reduced folate species (THF, 5,10-CH 2 -THF, 5-CH 3 -THF, 5,10-CH + -THF and 5-CHO-THF) and increase in the oxidized folate species (folic acid and DHF). In cells, 10-CHO-DHF and 10-CHO-folic acid were enriched in the stationary phase. This suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway for generating other folate intermediates and fMet-tRNA fMet .

In the current study, an artificial neural network (ANN)-based breast cancer prediction model was developed from the data of folate and xenobiotic pathway genetic polymorphisms along with the nutritional and demographic variables to investigate how micronutrients modulate susceptibility to breast cancer. The developed ANN model explained 94.2% variability in breast cancer prediction. Fixed effect models of folate (400 μg/day) and B 12 (6 μg/day) showed 33.3% and 11.3% risk reduction, respectively. Multifactor dimensionality reduction analysis showed the following interactions in responders to folate: RFC1 G80A × MTHFR C677T (primary), COMT H108L × CYP1A1 m2 (secondary), MTR A2756G (tertiary). The interactions among responders to B 12 were RFC1G80A × cSHMT C1420T and CYP1A1 m2 × CYP1A1 m4. ANN simulations revealed that increased folate might restore ER and PR expression and reduce the promoter CpG island methylation of extra cellular superoxide dismutase and BRCA1. Dietary intake of folate appears to confer protection against breast cancer through its modulating effects on ER and PR expression and methylation of EC-SOD and BRCA1. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a multi-factorial disorder with high-penetrant mutations accounting for small percentage of PD. Our previous studies demonstrated individual association of genetic variants in folate, xenobiotic, and dopamine metabolic pathways with PD risk. The rational of the study was to develop a risk prediction model for PD using these genetic polymorphisms along with synuclein (SNCA) polymorphism. We have generated additive, multifactor dimensionality reduction (MDR), recursive partitioning (RP), and artificial neural network (ANN) models using 21 SNPs as inputs and disease outcome as output. The clinical utility of all these models was assessed by plotting receiver operating characteristics curves where in area under the curve (AUC) was used as an index of diagnostic utility of the model. The additive model was the simplest and exhibited an AUC of 0.72. The MDR model showed significant gene-gene interactions between SNCA, DRD4VNTR, and DRD2A polymorphisms. The RP model showed SHMT C1420T as important determinant of PD risk. This variant allele was found to be protective and this protection was nullified by MTRR A66G. Inheritance of SHMT wild allele and SNCA intronic polymorphism was shown to increase the risk of PD. The ANN model showed higher diagnostic utility (AUC = 0.86) compared to all the models and was able to explain 56.6 % cases of sporadic PD. To conclude, the ANN model developed using SNPs in folate, xenobiotic, and dopamine pathways along with SNCA has higher clinical utility in predicting PD risk compared to other models. [ABSTRACT FROM AUTHOR]

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 ( p trend = 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. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common primary bone malignancy that occurs mostly in adolescents. Treatment protocols usually include multiagent preoperative and postoperative chemotherapy based on methotrexate, cisplatin, doxorubicin and ifosfamide. Despite a favourable prognosis, there are considerable interindividual differences in treatment outcome. Genetic variability of enzymes involved in the metabolism and transport of methotrexate could contribute towards observed differences in response to chemotherapy. Our aim was to evaluate how polymorphisms in the folate pathway and transporter genes influence treatment outcome in osteosarcoma patients.In total, 44 osteosarcoma patients treated with methotrexate were genotyped for eleven polymorphisms in four folate pathway and five folate transporter genes. Cox regression was used in survival analysis. Logistic regression was used to assess the influence of polymorphisms on treatment efficacy and toxicity and nonparametric tests were used to determine the influence on serum methotrexate levels.Polymorphic SLCO1B1 rs4149056 and rs11045879 alleles were associated with significantly higher serum methotrexate area under the curve (P=0.001 and 0.011, respectively). Carriers of at least one polymorphic SLCO1B1 rs4149056 and rs11045879 allele tended to have longer event-free survival compared with patients with two wild-type alleles [P=0.040, hazard ratio (HR)=0.26, 95% confidence interval (CI)=0.07-0.94; and P=0.034, HR=0.20, 95% CI=0.05-0.89, respectively]. Compared with the most common haplotype, carriers of both polymorphic alleles had significantly longer event-free survival (P=0.009, HR=0.27, 95% CI=0.10-0.72).We have shown that SLCO1B1 polymorphisms influence methotrexate disposition and survival in methotrexate-treated osteosarcoma patients and therefore might serve as pharmacogenetic markers of treatment outcome. [ABSTRACT FROM AUTHOR]

Introduction. A combination of pemetrexed and cisplatin has been shown to improve the outcome in patients with malignant pleural mesothelioma (MPM), however, there is a great heterogeneity in treatment response among patients. The aim of our study was to evaluate the influence of polymorphisms in folate pathway and transporter genes on pemetrexed treatment outcome in Slovenian patients with MPM. Methods. MPM patients treated with pemetrexed in the course of a prospective randomized clinical trial were genotyped for nineteen polymorphisms in five genes of folate pathway and six transporter genes. Logistic regression was used to assess the influence of polymorphisms on treatment efficacy and toxicity, while Cox regression was used to determine their influence on progression-free and overall survival. Results. Patients with at least one polymorphic MTHFD1 rs2236225 allele had a significantly lower response rate (p = 0.005; odds ratio [OR] = 0.12; 95% confidence interval [CI] = 0.03−0.54) and shorter progression-free survival (p = 0.032; hazard ratio [HR] = 3.10; 95% CI = 1.10−8.74) than non-carriers. Polymorphisms in transporter genes did not influence survival; however, several were associated with toxicity. Liver toxicity was significantly lower in carriers of polymorphic ABCC2 rs2273697 (p = 0.028; OR = 0.23; 95% CI = 0.06−0.85), SLCO1B1 rs4149056 (p = 0.028; OR = 0.23; 95% CI = 0.06−0.85) and rs11045879 (p = 0.014; OR = 0.18; 95% CI = 0.05−0.71) alleles compared to non-carriers, as well as in patients with SLCO1B1 GCAC haplotype (p = 0.048; OR = 0.17; 95% CI = 0.03−0.98). Gastrointestinal toxicity was much more common in patients with polymorphic ABCC2 rs717620 allele (p = 0.004; OR = 10.67; 95% CI = 2.15−52.85) and ABCC2 CAG haplotype (p = 0.006; OR = 5.67; 95% CI = 1.64−19.66). Conclusions. MTHFD1 polymorphism affected treatment response and survival, while polymorphisms in ABCC2 and SLCO1B1 transporter genes influenced the risk for toxicity. These polymorphisms could serve as potential markers of pemetrexed treatment outcome in patients with MPM. [ABSTRACT FROM AUTHOR]

Background Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. Methods Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ΔfolE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. Results Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. Conclusions Functional assays for P. falciparum GCH1 based on enzymatic activity and genetic complementation were successfully developed. The assays in combination with a homology model characterized the enzymatic activity of P. falciparum GCH1 and the importance of its key amino acid residues. The potential to use the assay for inhibitor screening was validated by 8-oxo-GTP, a known GTP analogue inhibitor. [ABSTRACT FROM AUTHOR]

Introduction: Trypanosomatidic parasitic infections in humans and animals caused by Trypanosoma brucei, Trypanosoma cruzi , and Leishmania species pose a significant health and economic burden in developing countries. There are few effective and accessible treatments for these diseases, and the existing therapies suffer from problems, such as parasite resistance and side effects. Structure-based drug design (SBDD) is one of the strategies that has been applied to discover new compounds targeting trypanosomatid-borne diseases., Areas Covered: We review the current literature (mostly over the last 5 years, searched in the PubMed database on 11 November 2021) on the application of structure-based drug design approaches to identify new anti-trypanosomatidic compounds that interfere with a validated target biochemical pathway, the trypanosomatid folate pathway., Expert Opinion: The application of structure-based drug design approaches to perturb the trypanosomatid folate pathway has successfully provided many new inhibitors with good selectivity profiles, most of which are natural products or their derivatives or have scaffolds of known drugs. However, the inhibitory effect against the target protein(s) often does not translate to anti-parasitic activity. Further progress is hampered by our incomplete understanding of parasite biology and biochemistry, which is necessary to complement SBDD in a multiparameter optimization approach to discovering selective anti-parasitic drugs.

Are the maternal gene variants MTHFR: c.665C>T, MTHFR: c.1286A>C, MTR: c.2756A>G, MTRR: c.66A>G, RFC1: c.80C>T and TCN2: c.776G>C and blood markers of the folate pathway important factors in assessing the risk of fetal trisomy 21 (fetal-T21)? Twenty pregnant women with a high risk and twenty with a low risk of fetal-T21 underwent prenatal examination. Selected gene variants and folate pathway markers and pregnancy-associated plasma protein A (PAPP-A) and free β-subunit of human chorionic gonadotropin β (free-β-hCG) multiple of the medians (MoMs) were determined. The distributions of the alternative alleles and genotypes of the gene variants did not differ between the studied groups. There was no relationship between PAPP-A and β-hCG MoM values and the presence of allele alternative genotype variants. The occurrence of alternative variants of the selected genes and concentrations of most of the studied folate pathway markers may not play a crucial role in the risk of fetal-T21 in pregnant women. However, the relationships between erythrocyte folate concentrations and the occurrence of alternative variants: c.665C>T MTHFR and c.776G>C TCN2, as well as the methylmalonic acid concentration and the occurrence of alternative variant c.776G>C TCN2 in pregnant women with fetal-T21, encourage further research. So far, of the biochemical markers, maternal PAPP-A and β-hCG MoM values remain independent risk factors for fetal-T21. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: This study aims to determine the possible association between folate pathway gene polymorphisms and idiopathic pulmonary fibrosis. This represents the first study carried out on folate pathway gene polymorphisms as possible risk factors in this kind of pathology. The premise is that several polymorphisms mapping on genes responsible for folate uptake are associated with the risk of numerous diseases occurring between pregnancy and old age, and that too little is currently known about idiopathic pulmonary fibrosis. Design and methods: We genotyped 9 single nucleotide polymorphisms and 1 polymorphic insertion in 7 essential genes belonging to the folate pathway in 32 Italian idiopathic pulmonary fibrosis patients and 81 control subjects. This was done by PCR and restriction analysis. Results: Allelic and genotypic association tests indicated that for all the analysed polymorphisms there were no significant differences between patients and controls. Nevertheless, the haplotype association analysis revealed a significant association between idiopathic pulmonary fibrosis and transcobalamin II gene polymorphisms: specifically the haplotype 776G (rs1801198)–c.1026-394G (rs7286680)–444C (rs10418) (OR=2.84; 95% C.I. 1.36–5.93, P value=0.004). Conclusions: This small-scale preliminary study would suggest the importance of further research focusing on the role of folate in the onset of idiopathic pulmonary fibrosis. [Copyright &y& Elsevier]

Abstract: Background: The development of non-syndromic congenital heart defects (CHDs) involves a complex interplay of genetics, metabolism, and lifestyle. Previous studies have implicated maternal single nucleotide polymorphisms (SNPs) and altered metabolism in folate-related pathways as CHD risk factors. Objective: We sought to discover associations between maternal SNPs and metabolites involved in the homocysteine, folate, and transsulfuration pathways, and determine if these associations differ between CHD cases and controls. Design: Genetic, metabolic, demographic, and lifestyle information was available for 335 mothers with CHD-affected pregnancies and 263 mothers with unaffected pregnancies. Analysis was conducted on 1160 SNPs, 13 plasma metabolites, and 2 metabolite ratios. A two-stage multiple linear regression was fitted to each combination of SNP and metabolite/ratio. Results: We identified 4 SNPs in the methionine adenosyltransferase II alpha (MAT2A) gene that were associated with methionine levels. Three SNPs in tRNA aspartic acid methyltransferase 1 (TRDMT1) gene were associated with total plasma folate levels. Glutamylcysteine (GluCys) levels were associated with multiple SNPs within the glutathione peroxidase 6 (GPX6) and O-6-methylguanine-DNA methyltransferase (MGMT) genes. The regression model revealed interactions between genotype and case–control status in the association of total plasma folate, total glutathione (GSH), and free GSH, to SNPs within the MGMT, 5,10-methenyltetrahydrofolate synthetase (MTHFS), and catalase (CAT) genes, respectively. Conclusions: Our study provides further evidence that genetic variation within folate-related pathways accounts for inter-individual variability in key metabolites. We identified specific SNP–metabolite relationships that differed in mothers with CHD-affected pregnancies, compared to controls. Our results underscore the importance of multifactorial studies to define maternal CHD risk. [Copyright &y& Elsevier]

No drug therapy is completely risk free, and the costs associated with non-response and adverse effects can exceed the cost of the therapy. The ultimate goal of pharmacogenetic research is to find robust genetic predictors of drug response that enable the development of prospective genetic tests to reliably identify patients at risk of non-response or of developing an adverse effect prior to the drug being prescribed. Currently, thiopurine S-methyltransferase (TPMT) deficiency is the only pharmacogenetic factor that is prospectively assessed before azathioprine or 6-mercaptopurine immunomodulation is commenced in patients with Crohn's disease (CD). As yet no other inherited determinant of drug response has made the transition from bench to bedside for the management of this disease. In this review we summarize what is known about TPMT deficiency and explore whether there is evidence to support a role of other genetic polymorphisms in predicting the response of CD patients to thiopurine drugs, methotrexate, and anti-tumor necrosis factor α (TNFα) therapy. [ABSTRACT FROM AUTHOR]

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 treat- ment 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. [ABSTRACT FROM AUTHOR]

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg2+-dependent transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP), forming 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, which is a critical step in the de novo folic acid-biosynthesis pathway. Diffraction-quality crystals of HPPK from the medically relevant species Staphylococcus aureus were grown in the presence of ammonium sulfate or sodium malonate and diffracted to better than 1.65 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 36.8, b = 76.6, c = 51.5 Å, α = γ = 90.0, β = 100.2°. The crystals contained two molecules per asymmetric unit, with a volume per protein weight ( VM) of 2.04 Å3 Da−1 and an estimated solvent content of 39.6%. [ABSTRACT FROM AUTHOR]

Abstract: Folate, a vitamin of the B group involved in one-carbon group metabolism, plays an important role in DNA synthesis and methylation. Several polymorphisms in the genes involved in folate uptake and biotransformations have been shown to be associated to the risk of cancer and to anticancer drug response. We studied common polymorphisms in MTHFR (N5,10-methylene-tetrahydrofolate reductase), MTHFD1 (N5,10-methylene-tetrahydrofolate dehydrogenase), MTR (methionine synthetase) and SLC19A1 (reduced folate carrier) in the panel of 60 human tumour cell lines established by the NCI for anticancer drug screening and we tentatively associated these polymorphisms with gene expression and drug cytotoxicity as extracted from the public database of the Developmental Therapeutic Programme. We observed a consistent and highly significant association between the presence of the variant C allele of the A>C1298 polymorphism of MTHFR and the sensitivity to many anticancer drugs belonging to the classes of antifolates, antimetabolites, alkylating agents and, to a lesser extent, topoisomerase inhibitors. In contrast, the T variant allele of the C>T677 variation of MTHFR was rather associated to lower sensitivity of the cell lines towards anticancer drugs (alkylating agents, antifolates and antimetabolites) but with much lower effects than the A>C1298 variation. The polymorphisms of the other genes studied were not associated with differences in anticancer drug sensitivity, but the expression of the SLC19A1 gene was significantly correlated with the sensitivity to several drugs (antifolates, thiopurines, nitrosoureas, and DACH-platinum drugs). We concluded that the NCI-60 panel may constitute a good starting point for implementing clinical studies aimed at discovering and validating predictive genetic markers of drug efficacy and/or toxicity. [Copyright &y& Elsevier]