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Background: The Alpe-DPD study (NCT02324452) demonstrated that prospective genotyping and dose-individualization using four alleles in DPYD (DPYD*2A/rs3918290, c.1236G > A/rs75017182, c.2846A > T/rs67376798 and c.1679 T > G/rs56038477) can mitigate the risk of severe fluoropyrimidine toxicity. However, this could not prevent all toxicities. The goal of this study was to identify additional genetic variants, both inside and outside DPYD, that may contribute to fluoropyrimidine toxicity. Methods: Biospecimens and data from the Alpe-DPD study were used. Exon sequencing was performed to identify risk variants inside DPYD. In silico and in vitro analyses were used to classify DPYD variants. A genome-wide association study (GWAS) with severe fluoropyrimidine-related toxicity was performed to identify variants outside DPYD. Association with severe toxicity was assessed using matched-pair analyses for the exon sequencing and logistic, Cox, and ordinal regression analyses for GWAS. Results: Twenty-four non-synonymous, frameshift, and splice site DPYD variants were detected in ten of 986 patients. Seven of these variants (c.1670C > T, c.1913 T > C, c.1925 T > C, c.506delC, c.731A > C, c.1740 + 1G > T, c.763 − 2A > G) were predicted to be deleterious. The carriers of either of these variants showed a trend towards a 2.14-fold (95% CI, 0.41–11.3, P = 0.388) increased risk of severe toxicity compared to matched controls (N = 30). After GWAS of 942 patients, no individual single nucleotide polymorphisms achieved genome-wide significance (P ≤ 5 × 10−8), however, five variants were suggestive of association (P < 5 × 10−6) with severe toxicity. Conclusions: Results from DPYD exon sequencing and GWAS analysis did not identify additional genetic variants associated with severe toxicity, which suggests that testing for single markers at a population level currently has limited clinical value. Identifying additional variants on an individual level is still promising to explain fluoropyrimidine-related severe toxicity. In addition, studies with larger samples sizes, in more diverse cohorts are needed to identify potential clinically relevant genetic variants related to severe fluoropyrimidine toxicity. [ABSTRACT FROM AUTHOR]

Background: Despite advances in the treatment of cancer, pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to the lack of effective therapies. Our previous study showed that Luteolin (Lut), a flavonoid, suppressed pancreatocarcinogenesis and reduced the expression of dihydropyrimidine dehydrogenase (DPYD), an enzyme that degrades pyrimidines such as 5‐fluorouracil (5‐FU), in PDACs. In this study, we investigated the role of DPYD and evaluated the therapeutic potential of combining 5‐FU with Lut in PDACs. Methods and Results: PDAC cells overexpressing DPYD showed increased proliferation, and invasiveness, adding to the resistance to 5‐FU. The xenograft tumors of DPYD‐overexpressing PDAC cells also exhibit enhanced growth and invasion compared to the control xenograft tumors. RNA‐seq analysis of the DPYD‐overexpressing PDAC xenograft tumors revealed an upregulation of genes associated with metallopeptidase activity—MMP9 and MEP1A. Furthermore, the overexpression of MEP1A in PDAC was associated with invasion. Next, we investigated the combined effects of Lut, a DPYD suppressor, and 5‐FU on DPYD‐overexpressing xenograft tumors and PDAC of Pdx1‐Cre; LSL‐KrasG12D/+; Trp53flox/flox(KPPC) mice. Neither single administration of 5‐FU nor Lut showed significant inhibitory effects; however, the combined administration of 5‐FU and Lut exhibited a significant tumor‐suppressive effect in both the xenograft tumors and KPPC models. Conclusion: We have elucidated that DPYD expression contributes to proliferation, invasiveness, and 5‐FU resistance, in PDACs. The combination therapy of Lut and 5‐FU holds the potential for enhanced efficacy against PDACs. [ABSTRACT FROM AUTHOR]

To reduce severe fluoropyrimidine-related toxicity, pharmacogenetic guidelines recommend a dose reduction for carriers of four high-risk variants in the DPYD gene (*2A, *13, c.2846A>T, HapB3). The polymorphism in the MIR27A gene has been shown to enhance the predictive value of these variants. Our study aimed to explore whether rs895819 in the MIR27A gene modifies the effect of five common DPYD variants: c.1129-5923C>G (rs75017182, HapB3), c.2194G>A (rs1801160, *6), c.1601G>A (rs1801158, *4), c.496A>G (rs2297595), and c.85T>C (rs1801265, *9A). The study included 370 Caucasian patients with gastrointestinal tumors who received fluoropyrimidine-containing chemotherapy. Genotyping was performed using high-resolution melting analysis. The DPYD*6 allele was associated with overall severe toxicity and neutropenia with an increased risk particularly pronounced in patients carrying the MIR27A variant. All carriers of DPYD*6 exhibited an association with asthenia regardless of their MIR27A status. The increased risk of neutropenia in patients with c.496G was only evident in those co-carrying the MIR27A variant. DPYD*4 was also significantly linked to neutropenia risk in co-carriers of the MIR27A variant. Thus, we have demonstrated the predictive value of the *6, *4, and c.496G alleles of the DPYD gene, considering the modifying effect of the MIR27A polymorphism. [ABSTRACT FROM AUTHOR]

Pharmacogenomics (PGx), examining the effect of genetic variation on interpatient variation in drug disposition and response, has been widely studied for several decades. However, as cost, as well as turnaround time associated with PGx testing, has significantly improved, the use of PGx in the clinical setting has been gaining momentum. Nevertheless, challenges have emerged in the broader clinical implementation of PGx. In this review, we will outline current models of PGx delivery and methodologies of evaluation, and discuss clinically relevant PGx tests and associated medications. Additionally, we will describe our approach for the broad implementation of pre‐emptive DPYD genotyping in patients taking fluoropyrimidines in Ontario, Canada, as an example of clinically actionable PGx testing with sufficient clinical evidence of patient benefit that can become a new standard of patient care. We will highlight challenges associated with PGx testing, including a lack of diversity in PGx studies as well as general limitations that impact the broad adoption of PGx testing. Lastly, we examine the future of PGx, discussing new clinical targets, methodologies and analysis approaches. [ABSTRACT FROM AUTHOR]

Abstract Background The Alpe-DPD study (NCT02324452) demonstrated that prospective genotyping and dose-individualization using four alleles in DPYD (DPYD*2A/rs3918290, c.1236G > A/rs75017182, c.2846A > T/rs67376798 and c.1679 T > G/rs56038477) can mitigate the risk of severe fluoropyrimidine toxicity. However, this could not prevent all toxicities. The goal of this study was to identify additional genetic variants, both inside and outside DPYD, that may contribute to fluoropyrimidine toxicity. Methods Biospecimens and data from the Alpe-DPD study were used. Exon sequencing was performed to identify risk variants inside DPYD. In silico and in vitro analyses were used to classify DPYD variants. A genome-wide association study (GWAS) with severe fluoropyrimidine-related toxicity was performed to identify variants outside DPYD. Association with severe toxicity was assessed using matched-pair analyses for the exon sequencing and logistic, Cox, and ordinal regression analyses for GWAS. Results Twenty-four non-synonymous, frameshift, and splice site DPYD variants were detected in ten of 986 patients. Seven of these variants (c.1670C > T, c.1913 T > C, c.1925 T > C, c.506delC, c.731A > C, c.1740 + 1G > T, c.763 − 2A > G) were predicted to be deleterious. The carriers of either of these variants showed a trend towards a 2.14-fold (95% CI, 0.41–11.3, P = 0.388) increased risk of severe toxicity compared to matched controls (N = 30). After GWAS of 942 patients, no individual single nucleotide polymorphisms achieved genome-wide significance (P ≤ 5 × 10−8), however, five variants were suggestive of association (P

Objective: Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme that degrades uracil, thymine, and 5-fluorouracil, which are important for treating gastric, colorectal, and breast cancers. In this study, we aimed to determine the association between chemotherapy-related toxicities and DPD gene variants; evaluate the consequences of these genetic differences; and integrate DPYD genetic screening into conventional cancer treatment regimens. Methods: Sixty-two patient files from 2015 to 2018 were retrospectively reviewed to investigate whether the DPYD gene causes toxicity before or during treatment. A total of 50 patients were enrolled after receiving informed consent and ethical clearance for the comprehensive examinations. The aim of this study was to reveal the genetic causes of adverse effects and better understand treatment responses. Results: Our analysis of 50 patients with cancer revealed that the severity of response to fluoropyrimidine compounds used in chemotherapy varied depending on DPYD gene polymorphisms. These mutations increased susceptibility to severe neutropenia -which can weaken immune systems- among other negative effects. It also found that IVS14 + 1G>A had a significant effect on treatment outcome, indicating that genetic screening should be included in planning therapy as it can prevent major side effects. Conclusion: Dihydropyrimidine connected to dehydrogenase gene polymorphism occurred in patients with gastrointestinal cancer who developed diarrhea, nausea, anemia, thrombostitopenia, and grade 3-4 neutropenia side effects while receiving 5-FU.

Dihydropyrimidine dehydrogenase (DPD) deficiency is the main cause of severe fluoropyrimidine-related toxicities. The best strategy for identifying DPD-deficient patients is still not defined. The EMA recommends targeted DPYD genotyping or uracilemia (U) testing. We analyzed the concordance between both approaches. This study included 19,376 consecutive French patients with pre-treatment plasma U, UH2 and targeted DPYD genotyping (*2A, *13, D949V, *7) analyzed at Eurofins Biomnis (2015–2022). Mean U was 9.9 ± 10.1 ng/mL (median 8.7, range 1.6–856). According to French recommendations, 7.3 % of patients were partially deficient (U 16–150 ng/mL) and 0.02 % completely deficient (U≥150 ng/mL). DPYD variant frequencies were *2A: 0.83 %, *13: 0.17 %, D949V: 1.16 %, *7: 0.05 % (2 homozygous patients with U at 22 and 856 ng/mL). Variant carriers exhibited higher U (median 13.8 vs. 8.6 ng/mL), and lower UH2/U (median 7.2 vs. 11.8) and UH2/U2 (median 0.54 vs. 1.37) relative to wild-type patients (p<0.00001). Sixty-six% of variant carriers exhibited uracilemia <16 ng/mL, challenging correct identification of DPD deficiency based on U. The sensitivity (% patients with a deficient phenotype among variant carriers) of U threshold at 16 ng/mL was 34 %. The best discriminant marker for identifying variant carriers was UH2/U2. UH2/U2<0.942 (29.7 % of patients) showed enhanced sensitivity (81 %) in identifying deleterious genotypes across different variants compared to 16 ng/mL U. These results reaffirm the poor concordance between DPD phenotyping and genotyping, suggesting that both approaches may be complementary and that targeted DPYD genotyping is not sufficiently reliable to identify all patients with complete deficiency. [ABSTRACT FROM AUTHOR]

Pre-treatment genotyping of four well-characterized toxicity risk-variants in the dihydropyrimidine dehydrogenase gene (DPYD) has been widely implemented in Europe to prevent serious adverse effects in cancer patients treated with fluoropyrimidines. Current genotyping practices are largely limited to selected commonly studied variants and are unable to determine phasing when more than one variant allele is detected. Recent evidence indicates that common DPYD variants modulate the functional impact of deleterious variants in a phase-dependent manner, where a cis- or a trans-configuration translates into different toxicity risks and dosing recommendations. DPYD is a large gene with 23 exons spanning nearly a mega-base of DNA, making it a challenging candidate for full-gene sequencing in the diagnostic setting. Herein, we present a time- and cost-efficient long-read sequencing approach for capturing the complete coding region of DPYD. We demonstrate that this method can reliably produce phased genotypes, overcoming a major limitation with current methods. This method was validated using 21 subjects, including two cancer patients, each of whom carried multiple DPYD variants. Genotype assignments showed complete concordance with conventional approaches. Furthermore, we demonstrate that the method is robust to technical challenges inherent in long-range sequencing of PCR products, including reference alignment bias and PCR chimerism. [ABSTRACT FROM AUTHOR]

Nucleobases are essential molecules for life, forming integral parts of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in all terrestrial life forms. Despite evidence of their abiotic synthesis in meteorites and laboratory simulations of interstellar medium (ISM) conditions, nucleobases have not been detected in the ISM. This study investigates the infrared spectra of uracil, cytosine, and thymine – pyrimidine nucleobases – embedded in an ice mixture simulating common volatiles found in protostellar discs. Our objective was to explore the feasibility of identifying unique infrared bands of pyrimidines in the ISM, despite significant overlapping absorption features from simpler, more abundant interstellar species such as H2O, CO, CH3OH, and NH3. Laboratory results revealed that although two common bands (1240 and 760 cm−1 in uracil; 1236 and 763 cm−1 in cytosine; and 1249 and 760 cm−1 in thymine) were identified, the detection of these bands in space is challenged by overlapping absorption features. Recent observations with the JWST have shown that interstellar organic species exhibit infrared signals within similar ranges, making it impossible to distinguish pyrimidine bands from these organics. Thus, detecting pyrimidines with current telescopes is infeasible, not due to sensitivity limitations or the need for more powerful instrumentation, but because of the intrinsic overlap in spectral features. This study complements previous research on purines by examining pyrimidines and including the impact of common ISM volatiles in the ice composition. The results highlight the significant challenges in detecting complex molecules in the ISM, underscoring the importance of understanding the spectral complexities and interactions to interpret astronomical observations accurately. [ABSTRACT FROM AUTHOR]

PURPOSE: Fluoropyrimidine-related toxicity and mortality risk increases significantly in patients carrying certain DPYD genetic variants with standard dosing. We implemented DPYD genotyping at a multisite cancer center and evaluated its impact on dosing, toxicity, and hospitalization. METHODS: In this prospective observational study, patients receiving (reactive) or planning to receive (pretreatment) fluoropyrimidine-based chemotherapy were genotyped for five DPYD variants as standard practice per provider discretion. The primary end point was the proportion of variant carriers receiving fluoropyrimidine modifications. Secondary end points included mean relative dose intensity, fluoropyrimidine-related grade 3+ toxicities, and hospitalizations. Fisher's exact test compared toxicity and hospitalization rates between pretreatment carriers, reactive carriers, and wild-type patients. Univariable and multivariable logistic regression identified factors associated with toxicity and hospitalization risk. Kaplan-Meier methods estimated time to event of first grade 3+ toxicity and hospitalization. RESULTS: Of the 757 patients who received DPYD genotyping (median age 63, 54% male, 74% White, 19% Black, 88% GI malignancy), 45 (5.9%) were heterozygous carriers. Fluoropyrimidine was modified in 93% of carriers who started treatment. In 442 patients with 3-month follow-up, 64%, 31%, and 30% of reactive carriers, pretreatment carriers, and wild-type patients had grade 3+ toxicity, respectively (P =.085); 64%, 25%, and 13% were hospitalized (P <.001). Reactive carriers had 10-fold higher odds of hospitalization compared with wild-type patients (P =.001), whereas no significant difference was noted between pretreatment carriers and wild-type patients. Time-to-event of toxicity and hospitalization were significantly different between genotype groups (P <.001), with reactive carriers having the earliest onset and highest incidence. CONCLUSION: DPYD genotyping prompted fluoropyrimidine modifications in most carriers. Pretreatment testing reduced toxicities and hospitalizations compared with reactive testing, thus normalizing the risk to that of wild-type patients, and should be considered standard practice. Real-world evidence showed DPYD genotyping to guide fluoropyrimidine dosing was feasible across a multisite cancer center. Pretreatment testing and genotype-guided dosing reduced severe toxicities and hospitalizations. #DPYD #Pharmacogenomics #PGx [ABSTRACT FROM AUTHOR]

The discovery of circadian clock genes greatly amplified the study of diurnal variations impacting cancer therapy, transforming it into a rapidly growing field of research. Especially, use of chronomodulated treatment with 5‐fluorouracil (5‐FU) has gained significance. Studies indicate high interindividual variability (IIV) in diurnal variations in dihydropyrimidine dehydrogenase (DPD) activity – a key enzyme for 5‐FU metabolism. However, the influence of individual DPD chronotypes on chronomodulated therapy remains unclear and warrants further investigation. To optimize precision dosing of chronomodulated 5‐FU, this study aims to: (i) build physiologically‐based pharmacokinetic (PBPK) models for 5‐FU, uracil, and their metabolites, (ii) assess the impact of diurnal variation on DPD activity, (iii) estimate individual DPD chronotypes, and (iv) personalize chronomodulated 5‐FU infusion rates based on a patient's DPD chronotype. Whole‐body PBPK models were developed with PK‐Sim(R) and MoBi(R). Sinusoidal functions were used to incorporate variations in enzyme activity and chronomodulated infusion rates as well as to estimate individual DPD chronotypes from DPYD mRNA expression or DPD enzymatic activity. Four whole‐body PBPK models for 5‐FU, uracil, and their metabolites were established utilizing data from 41 5‐FU and 10 publicly available uracil studies. IIV in DPD chronotypes was assessed and personalized chronomodulated administrations were developed to achieve (i) comparable 5‐FU peak plasma concentrations, (ii) comparable 5‐FU exposure, and (iii) constant 5‐FU plasma levels via "noise cancellation" chronomodulated infusion. The developed PBPK models capture the extent of diurnal variations in DPD activity and can help investigate individualized chronomodulated 5‐FU therapy through testing alternative personalized dosing strategies. [ABSTRACT FROM AUTHOR]

This article provides a commentary on a study conducted by Samarasinghe et al. on the pharmacogenomic landscape of an Indigenous Australian population. The study focused on analyzing the allele frequency of important pharmacogenes in 473 Tiwi Indigenous people, specifically the DPYD and UGT1A1 genes. The findings revealed that the Tiwi population had a different distribution of DPYD variants compared to Caucasian populations, with only one clinically significant variant identified. The study also found that the UGT1A1 gene had variants that could impact the metabolism of irinotecan chemotherapy. The authors stress the importance of conducting genomic sequencing in diverse populations to enhance personalized medicine and prescribing guidelines. They highlight the significance of considering ethnic diversity in pharmacogenomic screening, particularly in relation to DPYD gene variants that are significant in European Caucasian populations. The authors advocate for further research to understand the clinical implications of DPYD variants in non-European ethnicities, including Indigenous communities in Australia. They propose that tailoring health interventions and genetic screening programs to reflect the genetic expression within local jurisdictions will enhance safe prescribing for all cancer patients. The authors also support collaborative research with Indigenous communities to deepen our understanding of genomic differences in diverse ethnic populations. [Extracted from the article]

We show that the nucleic acid bases adenine, cytosine, guanine, thymine, and uracil, as well as 2,6-diaminopurine, and the "core" nucleic acid bases purine and pyrimidine, are stable for more than one year in concentrated sulfuric acid at room temperature and at acid concentrations relevant for Venus clouds (81% w/w to 98% w/w acid, the rest water). This work builds on our initial stability studies and is the first ever to test the reactivity and structural integrity of organic molecules subjected to extended incubation in concentrated sulfuric acid. The one-year-long stability of nucleic acid bases supports the notion that the Venus cloud environment—composed of concentrated sulfuric acid—may be able to support complex organic chemicals for extended periods of time. [ABSTRACT FROM AUTHOR]

Abstract Background Despite advances in the treatment of cancer, pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to the lack of effective therapies. Our previous study showed that Luteolin (Lut), a flavonoid, suppressed pancreatocarcinogenesis and reduced the expression of dihydropyrimidine dehydrogenase (DPYD), an enzyme that degrades pyrimidines such as 5‐fluorouracil (5‐FU), in PDACs. In this study, we investigated the role of DPYD and evaluated the therapeutic potential of combining 5‐FU with Lut in PDACs. Methods and Results PDAC cells overexpressing DPYD showed increased proliferation, and invasiveness, adding to the resistance to 5‐FU. The xenograft tumors of DPYD‐overexpressing PDAC cells also exhibit enhanced growth and invasion compared to the control xenograft tumors. RNA‐seq analysis of the DPYD‐overexpressing PDAC xenograft tumors revealed an upregulation of genes associated with metallopeptidase activity—MMP9 and MEP1A. Furthermore, the overexpression of MEP1A in PDAC was associated with invasion. Next, we investigated the combined effects of Lut, a DPYD suppressor, and 5‐FU on DPYD‐overexpressing xenograft tumors and PDAC of Pdx1‐Cre; LSL‐KrasG12D/+; Trp53flox/flox(KPPC) mice. Neither single administration of 5‐FU nor Lut showed significant inhibitory effects; however, the combined administration of 5‐FU and Lut exhibited a significant tumor‐suppressive effect in both the xenograft tumors and KPPC models. Conclusion We have elucidated that DPYD expression contributes to proliferation, invasiveness, and 5‐FU resistance, in PDACs. The combination therapy of Lut and 5‐FU holds the potential for enhanced efficacy against PDACs.

This article is an editorial that discusses the state of the science of pharmacogenomics implementation in healthcare systems and communities. It highlights various programs and initiatives that have expanded the reach of pharmacogenomics testing across multiple countries and regions, including rural areas. The article presents case studies from Canada, the United States, Spain, the United Arab Emirates, and the Netherlands, showcasing the successful implementation of pharmacogenomics in different healthcare settings. The authors emphasize the importance of cross-border collaborations and responsible data usage to optimize the utility of pharmacogenomic applications for diverse populations. [Extracted from the article]

Fluoropyrimidines, crucial in cancer treatment, often cause toxicity concerns even at standard doses. Toxic accumulation of fluoropyrimidine metabolites, culminating in adverse effects, can stem from impaired dihydropyrimidine dehydrogenase (DPYD) enzymatic function. Emerging evidence underscores the role of single nucleotide polymorphisms (SNPs) in DPYD gene, capable of inducing DPYD activity deficiency. Consequently, DPYD genotyping’s importance is on the rise in clinical practice before initiating fluoropyrimidine treatment. Although polymerase chain reaction (PCR) followed by Sanger sequencing (SS; PCR-SS) is a prevalent method for DPYD genotyping, it may encounter limitations. In this context, there is reported a case in which a routine PCR-SS approach for genotyping DPYD SNP rs55886062 failed in a proband of African descent. The Clinical Pharmacogenetics Implementation Consortium (CPIC) categorizes the guanine (G) allele of this SNP as non-functional. The enforcement of whole genome sequencing (WGS) approach led to the identification of two adenine (A) insertions near the PCR primers annealing regions in the proband, responsible for a sequence frameshift and a genotyping error for rs55886062. These SNPs (rs145228578, 1-97981199-T-TA and rs141050810, 1-97981622-G-GA) were extremely rare in non-Finnish Europeans (0.05%) but prevalent in African populations (16%). Although limited evidence was available for these SNPs, they were catalogued as benign variants in public databases. Notably, these two SNPs exhibited a high linkage disequilibrium [LD; squared correlation coefficient (R2) = 0.98]. These findings highlighted the importance to consider the prevalence of genetic variants within diverse ethnic populations when designing primers and probes for SNP genotyping in pharmacogenetic testing. This preventive measure is essential to avoid sequence frameshifts or primer misalignments arising from SNP occurrences in the genome, which can compromise PCR-SS and lead to genotyping failures. Furthermore, this case highlights the significance of exploring alternative genotyping approaches, like WGS, when confronted with challenges associated with conventional techniques.

Abstract Background Dihydropyrimidine dehydrogenase (DPD), is the initial and rate-limiting enzyme in the catabolic pathway of pyrimidines. Deleterious variants in the DPYD gene cause DPD deficiency, a rare autosomal recessive disorder. The clinical spectrum of affected individuals is wide ranging from asymptomatic to severely affected patients presenting with intellectual disability, motor retardation, developmental delay and seizures. DPD is also important as the main enzyme in the catabolism of 5-fluorouracil (5-FU) which is extensively used as a chemotherapeutic agent. Even in the absence of clinical symptoms, individuals with either complete or partial DPD deficiency face a high risk of severe and even fatal fluoropyrimidine-associated toxicity. The identification of causative genetic variants in DPYD is therefore gaining increasing attention due to their potential use as predictive markers of fluoropyrimidine toxicity. Methods A male infant patient displaying biochemical features of DPD deficiency was investigated by clinical exome sequencing. Bioinformatics tools were used for data analysis and results were confirmed by MLPA and Sanger sequencing. Results A novel intragenic deletion of 71.2 kb in the DPYD gene was identified in homozygosity. The deletion, DPYD(NM_000110.4):c.850 + 23455_1128 + 8811del, eliminates exons 9 and 10 and may have resulted from a non-homologous end-joining event, as suggested by in silico analysis. Conclusions The study expands the spectrum of DPYD variants associated with DPD deficiency. Furthermore, it raises the concern that patients at risk for fluoropyrimidine toxicity due to DPYD deletions could be missed during pre-treatment genetic testing for the currently recommended single nucleotide polymorphisms.

Introduction: One of the primary obstacles faced by individuals with advanced colorectal cancer (CRC) is the potential development of acquired chemoresistance as the disease advances. Studies have indicated a direct association between elevated levels of miR-92a-3p and the progression, metastasis, and chemoresistance observed in CRC. We proposed that miR-92a-3p impairs FOLFOX (fluorouracil/oxaliplatin) chemotherapy response by upregulating the expression of chemoresistance biomarker genes through the activation of ß-catenin and epithelial-mesenchymal transition (EMT). These FOLFOX biomarker genes include the pyrimidine biosynthesis pathway genes dihydropyrimidine dehydrogenase (DPYD), thymidylate synthase (TYMS), methylenetetrahydrofolate reductase (MTHFR), and the genes encoding the DNA repair complexes subunits ERCC1 and ERCC2, and XRCC1. Methods: To assess this, we transfected SW480 and SW620 colon cancer cell lines with miR-92a-3p mimics and then quantified the expression of DPYD, TYMS, MTHFR, ERCC1, ERCC2, and XRCC1, the expression of EMT markers and transcription factors, and activation of ß-catenin. Results and discussion: Our results reveal that miR-92a-3p does not affect the expression of DPYD, TYMS, MTHFR, and ERCC1. Furthermore, even though miR-92a-3p affects ERCC2, XRCC1, E-cadherin, and ß-catenin mRNA levels, it has no influence on their protein expression. Conclusion: We found that miR-92a-3p does not upregulate the expression of proteins of DNA-repair pathways and other genes involved in FOLFOX chemotherapy resistance. [ABSTRACT FROM AUTHOR]

A DPD deficiency should be considered in case of severe toxicity even in the absence of common risk variants in DPYD. [ABSTRACT FROM AUTHOR]

Background: Dihydropyrimidine dehydrogenase (DPD), is the initial and rate-limiting enzyme in the catabolic pathway of pyrimidines. Deleterious variants in the DPYD gene cause DPD deficiency, a rare autosomal recessive disorder. The clinical spectrum of affected individuals is wide ranging from asymptomatic to severely affected patients presenting with intellectual disability, motor retardation, developmental delay and seizures. DPD is also important as the main enzyme in the catabolism of 5-fluorouracil (5-FU) which is extensively used as a chemotherapeutic agent. Even in the absence of clinical symptoms, individuals with either complete or partial DPD deficiency face a high risk of severe and even fatal fluoropyrimidine-associated toxicity. The identification of causative genetic variants in DPYD is therefore gaining increasing attention due to their potential use as predictive markers of fluoropyrimidine toxicity. Methods: A male infant patient displaying biochemical features of DPD deficiency was investigated by clinical exome sequencing. Bioinformatics tools were used for data analysis and results were confirmed by MLPA and Sanger sequencing. Results: A novel intragenic deletion of 71.2 kb in the DPYD gene was identified in homozygosity. The deletion, DPYD(NM_000110.4):c.850 + 23455_1128 + 8811del, eliminates exons 9 and 10 and may have resulted from a non-homologous end-joining event, as suggested by in silico analysis. Conclusions: The study expands the spectrum of DPYD variants associated with DPD deficiency. Furthermore, it raises the concern that patients at risk for fluoropyrimidine toxicity due to DPYD deletions could be missed during pre-treatment genetic testing for the currently recommended single nucleotide polymorphisms. [ABSTRACT FROM AUTHOR]

Previous theoretical investigations of the reactions between an OH radical and a nucleobase have stated the most important pathways to be the C5-C6 addition for pyrimidines and the C8 addition for purines. Furthermore, the abstraction of a methyl hydrogen from thymine has also been proven an important pathway. The conclusions were based solely on gas-phase calculations and harmonic vibrational frequencies. In this paper, we supplement the calculations by applying solvent corrections through the polarizable continuum model (PCM) solvent model and applying anharmonicity in order to determine the importance of anharmonicity and solvent effects. Density functional theory (DFT) at the  ω B97-D/6-311++G(2df,2pd) level with the Eckart tunneling correction is used. The total reaction rate constants are found to be 1.48  × 10 − 13  cm3 molecules−1s−1 for adenine, 1.02  × 10 − 11  cm3 molecules−1s−1 for guanine, 5.52  × 10 − 13  cm3 molecules−1s−1 for thymine, 1.47  × 10 − 13  cm3 molecules−1s−1 for cytosine and 7.59  × 10 − 14  cm3 molecules−1s−1 for uracil. These rates are found to be approximately two orders of magnitude larger than experimental values. We find that the tendencies observed for preferred pathways for reactions calculated in a solvent are comparable to the preferred pathways for reactions calculated in gas phase. We conclude that applying a solvent has a larger impact on more parameters compared to the inclusion of anharmonicity. For some reactions the inclusion of anharmonicity has no effect, whereas for others it does impact the energetics. [ABSTRACT FROM AUTHOR]

Introduction: In April 2019, French authorities mandated dihydropyrimidine dehydrogenase (DPD) screening, specifically testing uracilemia, to mitigate the risk of toxicity associated with fluoropyrimidine‐based chemotherapy. However, this subject is still of debate as there is no consensus on a standardized DPD deficiency screening test. We conducted a real‐life retrospective study with the aim of assessing the impact of DPD screening on the occurrence of severe toxicity and exploring the potential benefits of complete genotyping using next‐generation sequencing. Methods: All adult patients consecutively treated with 5‐fluorouracil (5‐FU) or its oral prodrug at six cancer centers between March 2018 and February 2019 were considered for inclusion. Dihydropyrimidine dehydrogenase deficiency screening included gene encoding DPD (DPYD) genotyping using complete genome sequencing and DPD phenotyping (uracilemia or dihydrouracilemia/uracilemia ratio) or both tests. Associations between each DPD screening method and (i) severe (grade ≥3) early toxicity and (ii) fluoropyrimidine dose reduction in the second chemotherapy cycle were evaluated using multivariable logistic regression analysis. Furthermore, we assessed the concordance between DPD genotype and phenotype using Cohen's kappa. Results: A total of 551 patients were included. Most patients were tested for DPD deficiency (86%) including DPYD genotyping only (6%), DPD phenotyping only (8%), or both (72%). Complete DPD deficiency was not detected in the study population. Severe early toxicity events were observed in 73 patients (13%), with two patients (0.30%) presenting grade 5 toxicity. Despite the numerically higher toxicity rate in untested patients, the occurrence of severe toxicity was not significantly associated with the DPD screening method (p = 0.69). Concordance between the DPD genotype and phenotype was weak (Cohen's kappa of 0.14). Conclusion: Due to insufficient numbers, our study was not able to demonstrate any added value of DPYD genotyping using complete genome sequencing to prevent 5‐FU toxicity. The optimal strategy for DPD screening before fluoropyrimidine‐based chemotherapy requires further clinical evaluation. [ABSTRACT FROM AUTHOR]

Background/Aim: Renal dysfunction necessitates S-1 dose reduction. However, decreased dihydropyrimidine dehydrogenase (DPD) activity may lead to adverse events due to 5-FU. The guidelines provided by pharmaceutical companies state that total bilirubin (T-Bil) should be =upper limit of normal (ULN)×1.5 as a reference value for safely taking S-1. Nevertheless, the relationship between the degree of liver dysfunction and S-1 dose reduction has not been clearly established. Patients and Methods: This study focused on patients who received S-1 monotherapy for various types of cancer. The primary outcome was defined as the variation between blood sampling results on the test day and the subsequent test. The variation data were categorized based on the difference in T-Bil: Low T-Bil group (=2.25) and High T-Bil group (>2.25). Results: The number of patients that underwent S-1 monotherapy was 883 and the running number was 7,511; Low T-Bil group included 7,245 and High T-Bil group included 266. Examination of the effect of the T-Bil Group on clinical outcomes revealed a correlation with red blood cell (RBC) count, platelet (PLT) count, and T-Bil level. When the impact of the interaction between the T-Bil Group and any of the clinical outcomes, such as the RBC count, PLT count, and T-Bil level, was determined, each outcome showed a significant decrease in the High T-Bil group compared with the Low T-Bil group. Conclusion: S-1 administration in patients with liver dysfunction accompanied by elevated T-Bil levels may cause thrombocytopenia. [ABSTRACT FROM AUTHOR]

Background: Fluoropyrimidine drugs are widely used in chemotherapy to treat solid tumors. However, severe toxicity has been reported in 10% to 40% of patients. The DPYD gene encodes the rate-limiting enzyme dihydropyrimidine dehydrogenase responsible for fluoropyrimidine catabolism. The DPYD variants resulting in decreased or no enzyme activity are associated with increased risk of fluoropyrimidine toxicity. This study aims to develop a pharmacogenetic test for screening DPYD variants to guide fluoropyrimidine therapy. Methods: A multiplex allele-specific polymerase chain reaction (AS-PCR) assay, followed by capillary electrophoresis, was developed to detect 5 common DPYD variants (c.557A > G, c.1129–5923C > G, c.1679T > G, c.1905 + 1G > A, and c.2846A > T). Deidentified population samples were used for screening positive controls and optimizing assay conditions. Proficiency testing samples with known genotypes were analyzed for test validation. All variants detected were confirmed by Sanger sequencing. Results: From the deidentified population samples, 5 samples were heterozygous for c.557A > G, 2 samples were heterozygous for c.1129–5923C > G (HapB3), and 1 sample was heterozygous for c.2846A > T. The 20 proficiency samples matched with their assigned genotypes, including 13 wild-type samples, 3 samples heterozygous for c.1679T > G, 2 samples heterozygous for c.1905 + 1G > A, and 2 samples heterozygous for c.2846A > T. One of the 3 patient samples was heterozygous for c.1129–5923C > G (HapB3). All the variants detected by the multiplex AS-PCR assay were concordant with Sanger sequencing results. Conclusions: A robust multiplex AS-PCR assay was developed to rapidly detect 5 variants in the DPYD gene. It can be used for screening DPYD variants to identify patients with increased risk of toxicity when prescribed fluoropyrimidine therapy. [ABSTRACT FROM AUTHOR]

This article discusses the impact of hemolysis on uracilemia in the context of dihydropyrimidine dehydrogenase (DPD) deficiency testing. DPD deficiency is a known cause of severe toxicities in patients receiving fluoropyrimidine anticancer drugs. The French Health Authorities recommend screening for DPD deficiency by measuring plasma uracil concentration before administering these drugs. The study found that sample hemolysis can significantly affect uracilemia levels, potentially leading to misinterpretation of DPD activity. The authors recommend careful evaluation of sample hemolysis and requesting a new blood sample if uracilemia is above the threshold value in hemolyzed samples. [Extracted from the article]

The DPYD gene encodes dihydropyrimidine dehydrogenase, the rate‐limiting enzyme for the metabolism of fluoropyrimidines 5‐fluorouracil and capecitabine. Genetic variants in DPYD have been associated with altered enzyme activity, therefore accurate detection and interpretation is critical to predict metabolizer status for individualized fluoropyrimidine therapy. The most commonly observed deleterious variation is the causal variant linked to the previously described HapB3 haplotype, c.1129‐5923C>G (rs75017182) in intron 10, which introduces a cryptic splice site. A benign synonymous variant in exon 11, c.1236G>A (rs56038477) is also linked to HapB3 and is commonly used for testing. Previously, these single‐nucleotide polymorphisms (SNPs) have been reported to be in perfect linkage disequilibrium (LD); therefore, c.1236G>A is often utilized as a proxy for the function‐altering intronic variant. Clinical genotyping of DPYD identified a patient who had c.1236G>A, but not c.1129‐5923C>G, suggesting that these two SNPs may not be in perfect LD, as previously assumed. Additional individuals with c.1236G>A, but not c.1129‐5923C>G, were identified in the Children's Mercy Data Warehouse and the All of Us Research Program version 7 cohort substantiating incomplete SNP linkage. Consequently, testing only c.1236G>A can generate false‐positive results in some cases and lead to suboptimal dosing that may negatively impact patient therapy and prospect of survival. Our data show that DPYD genotyping should include the functional variant c.1129‐5923C>G, and not the c.1236G>A proxy, to accurately predict DPD activity. [ABSTRACT FROM AUTHOR]

Simple Summary: Feedstuff deficiency has forced researchers to find new natural alternatives. Distillers grain (DG) is a high-quality feed alternative due to its rich nutrients. However, the use of DG is limited in livestock diets due to the presence of some antinutritional compounds. The probiotic fermentation of DG can improve its nutritional composition, eliminating the antinutrient composition of DG. In this study, feeding with 15% probiotic-fermented distillers grains (PFDG) improved the antioxidant capacity, immune status, and rumen enzyme activity, without affecting the ADG, of finishing cattle, regulating rumen microbiota abundance and showing a positive effect on rumen phenylalanine metabolism, tryptophan metabolism, and pyrimidine metabolism. The current results indicate that PFDG could be incorporated, at a 15% inclusion rate, into the diet in finishing cattle. A total of 30 Simmental crossbred cattle (6.50 months old, 265.0 ± 22.48 kg) were randomly divided into three groups, with 10 heads per group, and fed for 45 days. The diet treatments consisted of the Control group without PFDG supplementation, the PFDG-15% group with 15% PFDG substituting for 15% concentrate, and PFDG-30% group with 30% PFDG substituting for 30% concentrate. The results showed that compared with the Control group, the average daily gain (ADG) of the cattle in the PFDG-30% group decreased significantly (0.890 vs. 0.768 kg/d, p = 0.005). The serum malondialdehyde content of cattle in the PFDG-15% and PFDG-30% groups decreased significantly (p = 0.047) compared to that of the Control group. However, the serum superoxide dismutase activity of cattle in the PFDG-30% group was significantly higher than that of the Control group (p = 0.047). Meanwhile, both the PFDG-15% and PFDG-30% groups (1758.47 vs. 2061.30 μg/mL) showed higher serum levels of immunoglobulin G, while the interleukin-10 concentration was lower in the PFDG-30% group (p = 0.027). In addition, the PFDG-15% and PFDG-30% groups shifted the rumen microbiota by improving the abundances of F082 (related to propionic acid production) and fiber-degrading bacteria (Lachnospiraceae_UGG-009 and Prevotellaceae_UCG-001) and reducing the abundance of the disease-associated bacteria Selenomonas. A Kyoto encyclopedia of genes and genomes (KEGG) analysis illustrated that three key metabolic pathways, including phenylalanine metabolism, pyrimidine metabolism, and tryptophan metabolism, were enriched in the PFDG-15% group, but eight key metabolic pathways, including arachidonic acid metabolism, were enriched in the PFDG-30% group. Importantly, both the PFDG-15% and PFDG-30% groups increased (p < 0.01) the activities of cellulase, lipase, and protease in the rumen. Finally, the different bacterial abundance in the rumen was associated with changes in the ADG, serum antioxidant capacity, immune status, rumen enzyme activity, and metabolites. These results suggest that PFDG alters rumen microbiome abundance, metabolome, and enzyme activity for enhancing serum antioxidant capacity and the immune status, but when the supplemental level reaches 30%, it has a negative effect on ADG and the anti-inflammatory factors in finishing cattle. [ABSTRACT FROM AUTHOR]

The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human β-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer–dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer–dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human β-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer–dimer interfaces are highly conserved between human and fruit fly enzymes. [ABSTRACT FROM AUTHOR]

Hepatitis E is a disease associated with acute inflammation of the liver. It is related to several dysregulated metabolic pathways and alterations in the concentration of several metabolites. However, longitudinal analysis of the alterations in metabolites and lipids is generally lacking. This study investigated the changes in levels of metabolites and lipids over time in sera from men with acute hepatitis E compared to healthy controls similar in age and gender. Untargeted measurement of levels of various metabolites and lipids was done using mass spectrometry on 65 sera sequentially sampled from 14 patients with acute hepatitis E and 25 serum samples from five controls. Temporal changes in intensities of metabolites and lipids were determined over different times at 3‐day periods for the hepatitis E virus (HEV) group. In carbohydrate metabolism, glucose levels, fructose 1‐6‐bisphosphate and ribulose‐5‐phosphate were increased in the HEV‐infected persons compared to the healthy controls. HEV infection is significantly associated with decreased levels of inosine, guanosine, adenosine and urate in purine metabolism and thymine, uracil and β‐aminoisobutyrate in pyrimidine metabolism. Glutamate, alanine and valine levels were significantly lower in the HEV group than in healthy individuals. Homogentisate of tyrosine metabolism and cystathionine of serine metabolism were increased, whereas kynurenate of tryptophan metabolism decreased in the HEV group. Metabolites of the bile acid biosynthesis, urea cycle (arginine and citrulline) and ammonia recycling (urocanate) were significantly altered. Co‐enzymes, pantothenate and pyridoxal, and co‐factors, lipoamide and FAD, were elevated in the HEV group. The acylcarnitines, sphingomyelins, phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysoPC and lysoPE tended to be lower in the HEV group. In conclusion, acute hepatitis E is associated with altered metabolite and lipid profiles, significantly increased catabolism of carbohydrates, purines/pyrimidines and amino acids, and decreased levels of several glycerophospholipids. [ABSTRACT FROM AUTHOR]

Thiazolidin‐4‐one is a versatile nucleus that has been reported to exhibit a diverse array of biological activities, including anticancer and antimicrobial activities. In the current research study, a series of C2‐(p‐substituted phenyl)‐thiazolidin‐4‐one compounds conjugated with anilines were designed and tested for drug‐likeness behaviour. Subsequently, the designed compounds were synthesized, characterized, and tested for anticancer activity against three cancer cell lines, namely liver (HEP−G2 cells), breast (MDA‐MB‐231), and glioblastoma (U87MG cells), with gemcitabine as the reference positive control drug. Initially, the compounds were tested at 50 micromolar, while the MIC was determined for the selected active compounds. Among the three cell lines, MDA‐MB‐231 showed relatively higher sensitivity towards the tested compounds. Four compounds of the series exhibited comparable or superior cytotoxicity to the reference drug gemcitabine. Particularly, N‐(p‐tolyl)‐2‐(2‐(4‐methoxyphenyl)‐4‐oxothiazolidin‐3‐yl)acetamide (6 d) displayed highest cytotoxicity among the series. The anticancer activity of the selected compounds was also tested against 3D spheroid models, in which compound N‐(4‐chlorophenyl)‐2‐(2‐(4‐methoxyphenyl)‐4‐oxothiazolidin‐3‐yl)acetamide (6 a) induced apoptosis in brain tumour spheroids (MTS). Docking studies of compound 6 d were performed against different targets, namely dihydrofolate reductase, phosphoinositide 3‐kinase, epidermal growth factor receptor, dihydroorotate dehydrogenase, and murine double minute 2, which illustrated inhibition of dihydroorotate dehydrogenase as a possible mechanism of toxicity. The compounds were also evaluated in vitro for antibacterial activity against two bacterial strains (E. coli and P. aeruginosa) and a Candida albicans fungal strain. [ABSTRACT FROM AUTHOR]

Pyrimidine biosynthesis and ribonucleoside metabolism in species of Pseudomonas was the focus of this review, in relation to their current taxonomic assignments in different homology groups. It was of interest to learn whether pyrimidine biosynthesis in taxonomically related species of Pseudomonas was regulated in a similar fashion by pyrimidine base supplementation or by pyrimidine limitation of pyrimidine auxotrophic strains. It was concluded that the regulation of pyrimidine biosynthesis in Pseudomonas species could not be correlated with their taxonomic assignment into a specific homology group. Pyrimidine ribonucleoside metabolism in Pseudomonas species primarily involved the pyrimidine ribonucleoside salvage enzymes nucleoside hydrolase and cytosine deaminase, independently of the Pseudomonas homology group to which the species was assigned. Similarly, pyrimidine base catabolism was shown to be active in different taxonomic homology groups of Pseudomonas. Although the number of studies exploring the catabolism of the pyrimidine bases uracil and thymine was limited in scope, it did appear that the presence of the pyrimidine base reductive pathway of pyrimidine catabolism was a commonality observed for the species of Pseudomonas investigated. There also appeared to be a connection between pyrimidine ribonucleoside degradation and the catabolism of pyrimidine bases in providing a cellular source of carbon or nitrogen independently of which homology group the species of Pseudomonas were assigned to. [ABSTRACT FROM AUTHOR]

Clinical identification and fundamental study of viruses rely on the detection of viral proteins or viral nucleic acids. Yet, amplification-based and antigen-based methods are not able to provide precise compositional information of individual virions due to small particle size and low-abundance chemical contents (e.g., ~ 5000 proteins in a vesicular stomatitis virus). Here, we report a widefield interferometric defocus-enhanced mid-infrared photothermal (WIDE-MIP) microscope for high-throughput fingerprinting of single viruses. With the identification of feature absorption peaks, WIDE-MIP reveals the contents of viral proteins and nucleic acids in single DNA vaccinia viruses and RNA vesicular stomatitis viruses. Different nucleic acid signatures of thymine and uracil residue vibrations are obtained to differentiate DNA and RNA viruses. WIDE-MIP imaging further reveals an enriched β sheet components in DNA varicella-zoster virus proteins. Together, these advances open a new avenue for compositional analysis of viral vectors and elucidating protein function in an assembled virion. The study of viruses relies on the detection of viral proteins or viral nucleic acids. Here, the authors present a widefield interferometric defocus-enhanced mid-infrared photothermal (WIDE-MIP) microscope for high-throughput fingerprinting of single viruses. [ABSTRACT FROM AUTHOR]

Phenylpyrazolo[3,4-d]pyrimidine is considered a milestone scaffold known to possess various biological activities such as antiparasitic, antifungal, antimicrobial, and antiproliferative activities. In addition, the urgent need for selective and potent novel anticancer agents represents a major route in the drug discovery process. Herein, new aryl analogs were synthesized and evaluated for their anticancer effects on a panel of cancer cell lines: MCF-7, HCT116, and HePG-2. Some of these compounds showed potent cytotoxicity, with variable degrees of potency and cell line selectivity in antiproliferative assays with low resistance. As the analogs carry the pyrazolopyrimidine scaffold, which looks structurally very similar to tyrosine and receptor kinase inhibitors, the potent compounds were evaluated for their inhibitory effects on three essential cancer targets: EGFRWT, EGFRT790M, VGFR2, and Top-II. The data obtained revealed that most of these compounds were potent, with variable degrees of target selectivity and dual EGFR/VGFR2 inhibitors at the IC50 value range, i.e., 0.3–24 µM. Among these, compound 5i was the most potent non-selective dual EGFR/VGFR2 inhibitor, with inhibitory concentrations of 0.3 and 7.60 µM, respectively. When 5i was tested in an MCF-7 model, it effectively inhibited tumor growth, strongly induced cancer cell apoptosis, inhibited cell migration, and suppressed cell cycle progression leading to DNA fragmentation. Molecular docking studies were performed to explore the binding mode and mechanism of such compounds on protein targets and mapped with reference ligands. The results of our studies indicate that the newly discovered phenylpyrazolo[3,4-d]pyrimidine-based multitarget inhibitors have significant potential for anticancer treatment. [ABSTRACT FROM AUTHOR]

This issue of I Clinical Pharmacology and Therapeutics i features an impressive article by Hertz I et al i .[1] who vehemently call for a change of the current policy of the US Federal Food and Drug Administration (FDA) not to recommend genetic testing of the dihydropyrimidine dehydrogenase ( I DPYD i ) gene prior to initiation of systemic fluoropyrimidine therapy. Patients with certain homozygous or certain compound heterozygous mutations in the DPYD gene locus that cause complete or near complete absence of DPD activity, are at the highest risk for severe, life-threatening, or fatal adverse reactions caused by fluorouracil. Effectiveness and safety of reduced-dose fluoropyrimidine therapy in patients carrying the DPYD*2A variant: a matched pair analysis. Why is There Still Debate About Recommending DPYD-Testing Before Fluoropyrimidine Treatment?. [Extracted from the article]

Introduction: Dihydropyrimidine dehydrogenase (DPD), encoded by DPYD gene, is the rate-limiting enzyme responsible for fluoropyrimidine (FP) catabolism. DPYD gene variants seriously affect DPD activity and are well validated predictors of FPassociated toxicity. DPYD variants rs3918290, rs55886062, rs67376798, and rs75017182 are currently included in FP genetic-based dosing guidelines and are recommended for genotyping by the European Medicines Agency (EMA) before treatment initiation. In Greece, however, no data exist on DPYD genotyping. The aim of the present study was to analyze prevalence of DPYD rs3918290, rs55886062, rs67376798, rs75017182, and, additionally, rs1801160 variants, and assess their association with FP-induced toxicity in Greek cancer patients. Methods: Study group consisted of 313 FP-treated cancer patients. DPYD genotyping was conducted on QuantStudio ™ 12K Flex Real-Time PCR System (ThermoFisher Scientific) using the TaqMan® assays C__30633851_20 (rs3918290), __11985548_10 (rs55886062), C_;_27530948_10 (rs67376798), C_104846637_10 (rs75017182) and C__11372171_10 (rs1801160). Results: Any grade toxicity (1-4) was recorded in 208 patients (66.5%). Out of them, 25 patients (12%) experienced grade 3-4 toxicity. DPYD EMA recommended variants were detected in 9 patients (2.9%), all experiencing toxicity (p = 0.031, 100% specificity). This frequency was found increased in grade 3-4 toxicity cases (12%, p = 0.004, 97.9% specificity). DPYD deficiency increased the odds of grade 3-4 toxicity (OR: 6.493, p = 0.014) and of grade 1-4 gastrointestinal (OR: 13.990, p = 0.014), neurological (OR: 4.134, p = 0.040) and nutrition/metabolism (OR: 4.821, p = 0.035) toxicities. FP dose intensity was significantly reduced in DPYD deficient patients (β = -0.060, p <0.001). DPYD rs1801160 variant was not associated with FP-induced toxicity or dose intensity. Triple interaction of DPYD*TYMS*MTHFR was associated with grade 3-4 toxicity (OR: 3.725, p = 0.007). Conclusion: Our findings confirm the clinical validity of DPYD reduced function alleles as risk factors for development of FP-associated toxicity in the Greek population. Pre-treatment DPYD genotyping should be implemented in clinical practice and guide FP dosing. DPYD*gene interactions merit further investigation as to their potential to increase the prognostic value of DPYD genotyping and improve safety of FP-based chemotherapy. [ABSTRACT FROM AUTHOR]

Background: Fluoropyrimidine toxicity is often due to variations in the gene (DPYD) encoding dihydropyrimidine dehydrogenase (DPD). DPYD genotyping can be used to adjust doses to reduce the likelihood of fluoropyrimidine toxicity while maintaining therapeutically effective drug levels. Methods: A multiplex QPCR assay was locally developed to allow genotyping for six DPYD variants. The test was offered prospectively for all patients starting on fluoropyrimidines at the BC Cancer Centre in Vancouver and then across B.C., Canada as well as retrospectively for patients suspected to have had an adverse reaction to therapy. Dose adjustments were made for variant carriers. The incidence of toxicity in the first three cycles was compared between DPYD variant allele carriers and non-variant carriers. Subsequent to an initial implementation phase, this test was made available province-wide. Results: In 9 months, 186 patients were tested and 14 were found to be heterozygous variant carriers. Fluoropyrimidine-related toxicity was higher in DPYD variant carriers. Of 127 non-variant carriers who have completed chemotherapy, 18 (14%) experienced severe (grade =3, Common Terminology Criteria for Adverse Events version 5.0). Of note, 22% (3 patients) of the variant carriers experienced severe toxicity even after DPYD-guided dose reductions. For one of these carriers who experienced severe thrombocytopenia within the first week, DPYD testing likely prevented lethal toxicity. In DPYD variant carriers who tolerate reduced doses, a later 25% increase led to chemotherapy discontinuation. As a result, a recommendation was made to clinicians based on available literature and expert opinion specifying that variant carriers who tolerated two cycles without toxicity can have a dose escalation of only 10%. Conclusion: DPYD-guided dose reductions were a feasible and acceptable method of preventing severe toxicity in DPYD variant carriers. Even with dose reductions, there were variant carriers who still experienced severe fluoropyrimidine toxicity, highlighting the importance of adhering to guideline-recommended dose reductions. Following the completion of the pilot phase of this study, DPYD genotyping was made available province-wide in British Columbia. [ABSTRACT FROM AUTHOR]

Investigation of the interaction of radiations with biomolecules has several applications in the fields of radiation biophysics, radiation protection, medical biology, and medical physics. In this study, a comprehensive radiological parameterization of proton and alpha particles interactions at the energy range of (1 keV–10 GeV) and (1 keV–1 GeV), respectively for nucleotide bases (adenine, guanine, cytosine, uracil, thymine), carbohydrates (glucose, sucrose, raffinose), lipids (cholesterol, retinol, progesterone, cortisone, and phylloquinone), and fatty acids (lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic, arachidonic, and arachidic) biomolecules have been carried out using PAGEX computer software. The radiological parameters calculated are the mass stopping power (S(E)/ρ), mass stopping cross-section (Sc), effective atomic number (Zeff), and effective electron density (Neff). The highest values of S(E)/ρ and Sc of proton and alpha particle interactions with the biomolecules occurred at the kinetic energy of about 0.1 and 0.5 MeV, respectively while that of the Zeff and Neff occurred at 0.1 MeV and 1 MeV, respectively. The S(E)/ρ and Sc similarly vary with the kinetic energy due to their relations. As expected, the dependence of the Zeff and Neff of the biomolecules on the incidence energy was found to be similar. The comparisons of the Zeff and Neff calculated in this study, using PAGEX software with some studies in the literature that used the interpolation method showed variations that account for differences between the two methods. [ABSTRACT FROM AUTHOR]

Dihydropyrimidine dehydrogenase (DPYD) is the rate-limiting enzyme involved in the metabolism of fluoropyrimidine-based chemotherapy. However, single-nucleotide variants (SNVs) in DPYD only partially explain fluoropyrimidine-induced toxicity. The expression of DPYD has previously been shown to be regulated by microRNA-27a (miR-27a) and a common miR-27a SNV (rs895819) has been associated with an increased risk of toxicity in patients harboring a DPYD variant who received standard fluoropyrimidine dosing. We investigated if the miR-27a rs895819 SNV was associated with toxicity in DPYD wildtype patients and carriers of DPYD variants who received a reduced dose. The regulation of DPYD using miR-27a was investigated in HepG2 cells utilizing a miR-27a mimic. miR-27a overexpression decreased DPYD mRNA expression compared to control cells (p < 0.0001). In a cohort of patients that received pre-emptive DPYD genotyping, 45 patients had a DPYD variant and 180 were wildtype. Patients heterozygous for rs895819 had an increased risk of toxicity, which was seen in both patients who were wildtype for DPYD variants (OR (95%CI) = 1.99 (1.00–3.99)) and DPYD variant carriers (OR (95%CI) = 8.10 (1.16–86.21)). Therefore, miR-27a rs895819 may be a clinically relevant predictor of fluoropyrimidine-associated toxicities. Furthermore, toxicity was more profound in DPYD variant carriers, even after DPYD genotype-guided dose reduction. This suggests that patients may benefit from miR-27a genotyping to guide fluoropyrimidine dosing. [ABSTRACT FROM AUTHOR]

This study used computational methods to investigate the multitarget mechanisms of 5‐hydroxy ferulic acid in preventing cancer. The antiradical activity of 5‐hydroxy ferulic acid was systematically studied by using the M06‐2X/6‐311++G(d,p) method in both pentyl ethanoate (lipid‐like) and water media. The results show that 5‐hydroxy ferulic acid is more effective at scavenging the HOO• radical (koverall=5.2×108 M−1s−1) than Trolox (koverall=1.3×105 M−1s−1) in aqueous solution. The complexation ability of 5‐hydroxy ferulic acid with Cu(II) ions was investigated at the M06/6‐311++G(d,p) level of theory under physiological pH conditions. The most thermodynamically stable complexes can slow down about 107 times the Haber‐Weiss cycle's first reaction (from 4.2×109 M−1s−1 to 3.8×102 M−1s−1) and mitigate the potential damage caused by ⋅OH radical production. Furthermore, by performing molecular docking simulations on protein‐ligand adducts and comparing their activity to that of 5‐fluorouracil (a commercial anticancer drug), the existing forms of 5‐hydroxy ferulic acid at physiological pH were extremely effective at inhibiting the thymidylate synthase enzyme. Based on these findings, 5‐hydroxy ferulic acid may be an effective cancer treatment. [ABSTRACT FROM AUTHOR]

Aim: Dihydropyrimidine dehydrogenase (DPD) deficiency can be detected by phenotyping (measurement of plasma uracil [U], with U ≥ 16 μg/L defining a partial deficiency) and/or by genotyping (screening for the four most frequent DPYD variants). We aimed to determine the proportion of discrepancies between phenotypic and genotypic approaches and to identify possible explanatory factors. Methods: Data from patients who underwent both phenotyping and genotyping were retrospectively collected. Complementary genetic analyses (genotyping of the variant c.557A>G and DPYD sequencing) were performed for patients with U ≥ 16 μg/L without any common variants. The characteristics of patients classified according to the congruence of the phenotyping and genotyping approaches were compared (Kruskal–Wallis test), and determinants of U levels were studied in the whole cohort (linear model). Results: Among the 712 included patients, phenotyping and genotyping were discordant for 12.5%, with 63 (8.8%) having U ≥ 16 μg/L in the absence of a common variant. Complementary genetic investigations marginally reduced the percentage of discrepancies to 12.1%: Among the nine additional identified variants, only the c.557A>G variant, carried by three patients, had been previously reported to be associated with DPD deficiency. Liver dysfunction could explain certain discordances, as ASAT, ALP, GGT and bilirubin levels were significantly elevated, with more frequent liver metastases in patients with U ≥ 16 μg/L and the absence of a DPYD variant. The impact of cytolysis was confirmed, as ASAT levels were independently associated with increased U (p < 0.001). Conclusion: The frequent discordances between DPD phenotyping and genotyping approaches highlight the need to perform these two approaches to screen for all DPD deficiencies. [ABSTRACT FROM AUTHOR]

The nucleobases, namely the purines adenine and guanine and the pyrimidines cytosine, thymine, and uracil, are the key monomers of the information polymers DNA and RNA. All the nucleobases were recently detected in the analysis of certain meteorites known as carbonaceous chondrites. The concentration of the nucleobases found in meteorites is smaller than the concentration of amino acids. However, the nucleobases were successfully synthesized abiotically by ultraviolet irradiation of interstellar ice analogues at 10 K. Consequently, it is possible that the purines and pyrimidines may be present in the interstellar medium or in other space environments and may be detectable by the new orbiting telescopes that indeed are able to cover not only the mid-infrared spectral range but also the far-infrared. This work supplies the reference spectra of the five nucleobases used by the terrestrial biochemistry in the mid-infrared and in the far-infrared with the purpose to facilitate the search and identification of these astrobiological and astrochemical relevant molecules in space. The mid-infrared spectra of all nucleobases were recorded at three different temperatures from −180 °C to nearly ambient temperature and at +250 °C. Furthermore, the molar extinction coefficients (ε) of all mid- and far-infrared bands were determined, as well as the integrated molar absorptivities (ψ). With the reported infrared spectra together with the ε and ψ values, it will be possible to identify qualitatively the nucleobases and estimate their relative abundance in space. [ABSTRACT FROM AUTHOR]

Background: Capecitabine is an oral chemotherapy prodrug of 5-fluorouracil (5-FU) with unpredictable toxicity, especially in older adults. The aim of this study was to evaluate the pharmacokinetics (PK) of capecitabine and its metabolites in younger adults (< 70 years) and older adults (≥ 70 years) receiving capecitabine for solid cancer. Methods: Eligible participants receiving capecitabine had 2 venous samples collected on day 14 of cycle 1 and cycle 2 of their treatment. Capecitabine and metabolite concentrations were determined using liquid chromatography with tandem mass spectrometry. A Bayesian estimation approach was used to generate individual estimates of PK parameters for 5-FU. A linear mixed-effect analysis of variance (ANOVA) model was used to compare dose-normalised log-transformed PK parameters between age groups. Correlations were determined by linear regression and logistic regression analyses. Results: Of the total 26 participants, 58% were male with a median age of 67 years (range, 37–85) with 54% aged < 70 years and 46% aged ≥ 70 years. Participants aged ≥ 70 years, compared to those aged < 70 years, had a greater 5-FU exposure based on area under the concentration–time curve (AUC) of 17% (90% CI 103–134%; 0.893 vs. 0.762 mg h/L) and 14% increase in maximal concentration, Cmax (90% CI 82.1–159%; 0.343 vs. 0.300 mg/L). The 5-FU Cmax was positively associated with time up and go (TUG) (Pearson's correlation 0.77, p = 0.01), but not other geriatric assessment domains or severe toxicity. Conclusion: 5-FU exposure was significantly increased in older adults compared to younger adults receiving equivalent doses of capecitabine, and is a possible cause for increased toxicity in older adults. [ABSTRACT FROM AUTHOR]

Abstract Background Dihydropyrimidine dehydrogenase (DPD) is a key enzyme in the metabolism of fluoropyrimidines. Variations in the encoding DPYD gene are associated with severe fluoropyrimidine toxicity and up-front dose reductions are recommended. We conducted a retrospective study to evaluate the impact of implementing DPYD variant testing for patients with gastrointestinal cancers in routine clinical practice in a high volume cancer centre in London, United Kingdom. Methods Patients receiving fluoropyrimidine chemotherapy for gastrointestinal cancer prior to, and following the implementation of DPYD testing were identified retrospectively. After November 2018, patients were tested for DPYD variants c.1905+1G>A (DPYD*2A), c.2846A>T (DPYD rs67376798), c.1679T>G (DPYD*13), c.1236G>A (DPYD rs56038477), c.1601G>A (DPYD*4) prior to commencing fluoropyrimidines alone or in combination with other cytotoxics and/or radiotherapy. Patients with a DPYD heterozygous variant received an initial dose reduction of 25–50%. Toxicity by CTCAE v4.03 criteria was compared between DPYD heterozygous variant and wild type carriers. Results Between 1st December 2018 and 31st July 2019, 370 patients who were fluoropyrimidine naïve underwent a DPYD genotyping test prior to receiving a capecitabine (n = 236, 63.8%) or 5FU (n = 134, 36.2%) containing chemotherapy regimen. 33 patients (8.8%) were heterozygous DPYD variant carriers and 337 (91.2%) were wild type. The most prevalent variants were c.1601G > A (n = 16) and c.1236G > A (n = 9). Mean relative dose intensity for the first dose was 54.2% (range 37.5–75%) for DPYD heterozygous carriers and 93.2% (42.9–100%) for DPYD wild type carriers. Overall grade 3 or worse toxicity was similar in DPYD variant carriers (4/33, 12.1%) as compared to wild-type carriers (89/337, 25.7%; P = 0.0924). Conclusions Our study demonstrates successful routine DPYD mutation testing prior to the initiation of fluoropyrimidine chemotherapy with high uptake. In patients with DPYD heterozygous variants with pre-emptive dose reductions, high incidence of severe toxicity was not observed. Our data supports routine DPYD genotype testing prior to commencement of fluoropyrimidine chemotherapy.

RNA structure can be essential for its cellular function. Therefore, methods to investigate the structure of RNA in vivo are of great importance for understanding the role of cellular RNAs. RNA structure probing is an indirect method to asess the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. Dimethyl sulfate (DMS) is a well-established compound that reports on base pairing context of adenine (A) and cytidine (C) in-vitro and in-vivo, but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in plant, bacteria, and human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyze the effectiveness of guanine modification by the glyoxal family in Saccharomyces cerevisiae and Candida albicans. We show that within glyoxal family of compounds, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. Further, we show that PGO treatment does not affect the processing of different RNA species in the cell and is not toxic for the cells under the conditions we have established for RNA structural probing. We also explore the effectiveness of uracil modification by Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluenesulfonate (CMCT) in vivo and demonstrate that uracils can be modified by CMCT in S. cerevisiae in vivo. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil nucleotides in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems. [ABSTRACT FROM AUTHOR]

Esophageal cancer is a cancer with poor prognosis and the standard 1st line treatment for metastatic or recurrent EC is systemic chemotherapy with doublet chemotherapy based on platinum and 5-fluorouracil (5-FU). However, 5-FU could be a source of severe treatment-related toxicities due to deficiency of dihydropyrimidine dehydrogenase (DPD). In this case report, a 74-year-old man with metastatic esophageal cancer was found to have partial DPD deficiency based on uracilemia measurements (about 90 ng/mL). Despite this, 5-FU was safely administered thanks to therapeutic drug monitoring (TDM). The case report highlights the importance of TDM in administering 5-FU to patients with partial DPD deficiency, as it allows individualized dosing and prevents severe toxicity. [ABSTRACT FROM AUTHOR]

Plasma uracil is a new biomarker to assess the activity of dihydropyrimidine dehydrogenase before cancer treatment with fluoropyrimidine drugs. Knowledge on the biological variation of plasma uracil is important to assess the applicability of plasma uracil as a biomarker of drug tolerance and efficacy. A total of 33 apparently healthy individuals were submitted to sequential blood draws for three days. On the second day, blood draws were performed every third hour for 12 h. Plasma uracil was quantified by LC-MS/MS. The within-subject (CVI) and between-subject (CVG) biological variation estimates were calculated using linear mixed-effects models. The overall median value of plasma uracil was 10.6 ng/mL (range 5.6–23.1 ng/mL). The CVI and CVG were 13.5 and 22.1%, respectively. Plasma uracil remained stable during the day, and there was no day-to-day variation observed. No differences in biological variation components were found between sex and no correlation to age was found. Four samples were calculated to be required to estimate the homeostatic set-point ±15% with 95% confidence. Plasma uracil is subject to tight homeostatic regulation without semidiurnal and day-to-day variation, however between-subject variation exists. This emphasizes plasma uracil as a well-suited biomarker for evaluation of dihydropyrimidine dehydrogenase activity, but four samples are required to establish the homeostatic set-point in a patient. [ABSTRACT FROM AUTHOR]

Platin-based chemotherapy is the standard treatment for patients with non-small cell lung cancer (NSCLC). However, resistance to this therapy is a major obstacle in successful treatment. In this study, we aimed to investigate the impact of several pharmacogenetic variants in patients with unresectable NSCLC treated with platin-based chemotherapy. Our results showed that DPYD variant carriers had significantly shorter progression-free survival and overall survival compared to DPYD wild-type patients, whereas DPD deficiency was not associated with a higher incidence of high-grade toxicity. For the first time, our study provides evidence that DPYD gene variants are associated with resistance to platin-based chemotherapy in NSCLC patients. Although further studies are needed to confirm these findings and explore the underlying mechanisms of this association, our results suggest that genetic testing of DPYD variants may be useful for identifying patients at a higher risk of platin-based chemotherapy resistance and might be helpful in guiding future personalized treatment strategies in NSCLC patients. [ABSTRACT FROM AUTHOR]

Chrysin is a naturally occurring bioflavonoid found in honey and propolis, with anti-cancer and anti-inflammatory properties. This study aimed to investigate the therapeutic effect of chrysin combined with 5-fluorouracil (5-FU) in the treatment of mice with colorectal cancer (CRC). Moreover, the effects of these two compounds on the expression of β-catenin and inducible nitric oxide synthase (iNOS) were investigated. The CRC was induced in the mice by azoxymethane (AOM). The co-administration of 5-FU and chrysin in the treatment of mice reduced the number of aberrant crypt foci (ACF) and the pathologic lesion percentage compared to other treatment groups (P <0.05). The co-administration of 5-FU and chrysin resulted in a reduction in β-catenin and iNOS (P <0.05). We showed that a combination of 5-FU and chrysin is superior to 5-FU or chrysin alone in the treatment of mice with CRC. Our approach opens an avenue to introduce a useful therapeutic option for colorectal cancer in humans. [ABSTRACT FROM AUTHOR]

The biologically complex configurations paired of methimazole (MET) anti-thyroid drug with each of the three nucleic acid bases of single ring based on pyrimidine (uracil (U), thymine (T) and cytosine (C)) have been studied at different levels of density functional theory. Twelve double H-bonded complexes were proposed and designed stand on the data obtained from electrostatic potential (ESP) maps of the studied nucleic acid bases and MET drug. The interaction energies of the optimized structures of all three set of complexes (MET-U, MET-T and MET-C) were computed using M062X/AUG–cc–pVDZ computational level and then corrected using M062X–GD3 functional with the same basis sets. The corrected interaction energies for the of MET-U, MET-T and MET-C complex series range from −10.89 to −17.52, −11.31 to −17.62, and −12.33 to −19.54 kcal mol−1, respectively. The highest interaction energy has been calculated between C nucleobase and the MET drug. Structural geometries of the U, T and C nucleobases as well as MET drug show that they can act as H-bond donor and H-bond acceptor simultaneously. The existence, strength and nature of the hydrogen bond interactions in the complex were confirmed and investigated using vibrational frequency, natural bond orbital (NBO) and non-covalent interactions (NCI) analysis and also with quantum theory of atoms in molecules (QTAIM) analysis at M062X/AUG–cc–pVDZ level of theory. In the most stable complexes of each series, charge transfer occurs (CT) from MET drug to the U, T and C nucleobases. Analysis of the electron density property shows that the N1− H7···Sʹ6 hydrogen bond nature in the most stable MET-UR1R′1, MET-TR1R′1 and MET-CR1R′1 complexes is slightly covalent and the nature of other hydrogen bonds in three series of studied complexes is electrostatic. [ABSTRACT FROM AUTHOR]

X-ray photoelectron spectroscopy was used to analyze commercially available powders of thymine, uracil, cytosine, guanine, adenine, as well as DNA isolated from salmon testes and RNA from torula yeast. The powders were pressed onto a sample holder and analyzed as received from the manufacturer. Survey spectra and high-resolution spectra of O 1s, N 1s, C 1s of all nucleobases are reported, including a small Na 1s contamination in cytosine. Spectra of DNA and RNA are also included for comparison. Furthermore, the presented data are compared with previously published results, as well as theoretical values, and differences are discussed. [ABSTRACT FROM AUTHOR]