The potential clinical impact of pre-emptive screening of multiple polymorphisms in gene-encoding DPD on patients candidate for fluoropyrimidine based-chemotherapy: An experience of the Northern Italy Cancer Centre.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 2567-2567
Author(s):  
Francesco Iachetta ◽  
Angela Damato ◽  
Candida Bonelli ◽  
Alessandra Romagnani ◽  
Maria Banzi ◽  
...  
Keyword(s):  
Standard Dose ◽  
Dihydropyrimidine Dehydrogenase ◽  
The Other ◽  
Severe Toxicity ◽  
Wild Type ◽  
Gene Encoding ◽  
Mutational Status ◽  
Key Enzyme ◽  
Life Threatening ◽  
Potential Clinical Impact

2567 Background: Dihydropyrimidine dehydrogenase (DPD) is a key enzyme in the metabolism of fluorouracil. Deleterious polymorphisms in gene-encoding DPD ( DPYD) results in a DPD deficiency that causes life-threatening toxicities when the standard dose of fluorouracil is used. DPYD*2A (IVS14+1G > A) is the most common single-nucleotide polymorphism (SNP) associated with critical DPD deficiency. At present, most of the evidence supports screening for at least 3 SNPs (DPYD*2A, c.2846 A > T, c.1679T > G). The aim of this study is to confirm that the detection of additional polymorphisms of DPYD could enhance prevention of fluoropyrimidine toxicity. Methods: In 2011, we began to screen DPYD*2A in patients candidate for fluoropyrimidine based-chemotherapy. As the first step of the evaluation, we selected all cases of DPYD*2A wild type, from 2011 to 2012, who developed CTC-NCI-V.3 toxicity ≥ G3. In these patients, we researched the other 3 SNPs (c.2846 A > T, c.1679T > G, c.2194C > A). Mutational status was analyzed with real Time PCR. Results: From 2011 to 2016 we pre-emptively screened DPD deficiency in 1,863 patients and 32 subjects (1.6%), with results mutated for DPYD*2A. As the first step of the evaluation, 548 subjects were assessed from 2011 to 2012. We found 7 patients who were carriers of the DPYD*2A mutation (1.27%). Of the 541 wild type cases, 114 presented toxicities ≥ G3. In this subgroup, 22 patients (19%) proved to be mutated for the other SNPs of DPYP, as reported in the table below. Conclusions: Preliminary data show that in 22 (19%) of 114 patients who presented severe toxicity which was not correlated with DPYD*2A, we found other polymorphisms of gene encoding DPD. Out of the 3 SNPs evaluated, c.2194 C > A proved to be the most frequent, although it is the polymorphism that is least known and least studied. Such results suggest that the evaluation of additional polymorphisms could enhance the prevention of fluoropyrimidine toxicity. The results are expected to be clarified further in the second step, which is ongoing. [Table: see text]

scholarly journals Formate-Dependent H2 Production by the Mesophilic Methanogen Methanococcus maripaludis

2008 ◽  
Vol 74 (21) ◽  
pp. 6584-6590 ◽  
Author(s):  
Boguslaw Lupa ◽  
Erik L. Hendrickson ◽  
John A. Leigh ◽  
William B. Whitman
Keyword(s):  
Dry Weight ◽  
Maximal Activity ◽  
Growth Conditions ◽  
H2 Production ◽  
Resting Cells ◽  
Wild Type ◽  
Methanococcus Maripaludis ◽  
Gene Encoding ◽  
Major Pathway ◽  
Key Enzyme

ABSTRACT Methanococcus maripaludis, an H2- and formate-utilizing methanogen, produced H2 at high rates from formate. The rates and kinetics of H2 production depended upon the growth conditions, and H2 availability during growth was a major factor. Specific activities of resting cells grown with formate or H2 were 0.4 to 1.4 U�mg−1 (dry weight). H2 production in formate-grown cells followed Michaelis-Menten kinetics, and the concentration of formate required for half-maximal activity (Kf ) was 3.6 mM. In contrast, in H2-grown cells this process followed sigmoidal kinetics, and the Kf was 9 mM. A key enzyme for formate-dependent H2 production was formate dehydrogenase, Fdh. H2 production and growth were severely reduced in a mutant containing a deletion of the gene encoding the Fdh1 isozyme, indicating that it was the primary Fdh. In contrast, a mutant containing a deletion of the gene encoding the Fdh2 isozyme possessed near-wild-type activities, indicating that this isozyme did not play a major role. H2 production by a mutant containing a deletion of the coenzyme F420-reducing hydrogenase Fru was also severely reduced, suggesting that the major pathway of H2 production comprised Fdh1 and Fru. Because a Δfru-Δfrc mutant retained 10% of the wild-type activity, an additional pathway is present. Mutants possessing deletions of the gene encoding the F420-dependent methylene-H4MTP dehydrogenase (Mtd) or the H2-forming methylene-H4MTP dehydrogenase (Hmd) also possessed reduced activity, which suggested that this second pathway was comprised of Fdh1-Mtd-Hmd. In contrast to H2 production, the cellular rates of methanogenesis were unaffected in these mutants, which suggested that the observed H2 production was not a direct intermediate of methanogenesis. In conclusion, high rates of formate-dependent H2 production demonstrated the potential of M. maripaludis for the microbial production of H2 from formate.

Cost implications of reactive versus prospective testing for dihydropyrimidine dehydrogenase (DPD) deficiency in patients with colorectal cancer.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3627-3627
Author(s):  
Gul Ahmed ◽  
Jo O' Keeffe ◽  
Denise O Mullane ◽  
Alison Bransfield ◽  
Andrew Kenny ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Metastatic Disease ◽  
Standard Dose ◽  
Dihydropyrimidine Dehydrogenase ◽  
Severe Toxicity ◽  
First Line ◽  
Time Period ◽  
Cost Implications ◽  
The Cost ◽  
First Time

3627 Background: DPD is an enzyme encoded by the DPYD gene involved in the metabolism of the chemotherapy drug 5-fluorouracil (5FU) and the oral 5FU prodrug capecitabine. Patients (pts) with DPYD mutations are at risk of severe toxicities from standard dose 5FU, although they may safely receive lower dose therapy with careful monitoring and dose escalation. Methods: In this retrospective study we identified all pts starting 5FU-based chemotherapy for colorectal cancer (CRC) at our institution between Jan 1 2010 and Dec 31 2012. During this time DPD testing was usually performed in a reactive manner, typically for pts experiencing severe toxicities. We reviewed the charts of pts who tested positive for DPYD mutations and assessed the financial implications of their hospitalizations with toxicity. These costs were compared to the costs which would have incurred if all pts starting such therapy had been proactively tested. Results: A total of 134 pts started first-line 5FU-based chemotherapy for CRC over the study period, 66 in the adjuvant setting and 68 for metastatic disease. 31 pts had DPYD mutation testing performed. 6 tests (19% of those tested, 4.5% of the total population) revealed heterozygote DPYD mutations. 5 pts had already experienced severe treatment-related toxicity resulting in cessation of therapy, while one was tested prospectively and received chemotherapy with dose reduction ab initio. The total cost related to hospitalization with toxicity for these 5 pts was €155,083. At €177 per test, the cost to prospectively test all pts starting first-line 5FU-based therapy over the time period would have been €23,718 representing a saving of €131,365 through avoiding these admissions alone. 4 pts who tested positive for DPYD mutations were receiving adjuvant therapy and none restarted therapy following severe toxicity early in their therapy. 2 pts subsequently relapsed with metastatic disease. Conclusions: Prospective testing for DPYD mutations in pts with CRC starting 5FU-based therapy for the first time represents a considerable cost-saving opportunity, in addition to potentially avoiding prolonged hospitalization and morbidity for a sizeable minority of pts.

scholarly journals A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionineβ-Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
João B. Vicente ◽  
Henrique G. Colaço ◽  
Francesca Malagrinò ◽  
Paulo E. Santo ◽  
André Gutierres ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Redox Homeostasis ◽  
Crystallographic Structure ◽  
Wild Type ◽  
Gene Encoding ◽  
Cellular Redox ◽  
Transsulfuration Pathway ◽  
Key Enzyme ◽  
A Minor ◽  
Ferrous Heme

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5′-phosphate- (PLP-) dependent cystathionineβ-synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (H2S). CBS activity, contributing to cellular redox homeostasis, is positively regulated byS-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenicCBSmutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired H2S-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria.

scholarly journals DPYD Exome, mRNA Expression and Uracil Levels in Early Severe Toxicity to Fluoropyrimidines: An Extreme Phenotype Approach

2021 ◽  
Vol 11 (8) ◽  
pp. 792
Author(s):  
Priscila Villalvazo ◽  
Belén Marzal-Alfaro ◽  
Pilar García-Alfonso ◽  
José Luis Revuelta-Herrero ◽  
Fabienne Thomas ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Adverse Reactions ◽  
Dehydrogenase Deficiency ◽  
Dihydropyrimidine Dehydrogenase ◽  
Severe Toxicity ◽  
Exon Sequence ◽  
Extreme Phenotypes ◽  
Life Threatening ◽  
Early Toxicity

Dihydropyrimidine dehydrogenase deficiency is a major cause of severe fluoropyrimidine-induced toxicity and could lead to interruption of chemotherapy or life-threatening adverse reactions. This study aimed to characterize the DPYD exon sequence, mRNA expression and in vivo DPD activity by plasma uracil concentration. It was carried out in two groups of patients with extreme phenotypes (toxicity versus control) newly treated with a fluoropyrimidine, during the first three cycles of treatment. A novel nonsense gene variant (c.2197insA) was most likely responsible for fluoropyrimidine-induced toxicity in one patient, while neither DPYD mRNA expression nor plasma uracil concentration was globally associated with early toxicity. Our present work may help improve pharmacogenetic testing to avoid severe and undesirable adverse reactions to fluoropyrimidine treatment and it also supports the idea of looking beyond DPYD.

Upfront Genotyping of DPYD*2A to Individualize Fluoropyrimidine Therapy: A Safety and Cost Analysis

2016 ◽  
Vol 34 (3) ◽  
pp. 227-234 ◽  
Author(s):  
Maarten J. Deenen ◽  
Didier Meulendijks ◽  
Annemieke Cats ◽  
Marjolein K. Sechterberger ◽  
Johan L. Severens ◽  
...  
Keyword(s):  
Cost Analysis ◽  
Drug Exposure ◽  
Standard Dose ◽  
Variant Allele ◽  
Dose Titration ◽  
Wild Type ◽  
Adequate Treatment ◽  
Life Threatening ◽  
Grade 3 ◽  
Historical Controls

Purpose Fluoropyrimidines are frequently prescribed anticancer drugs. A polymorphism in the fluoropyrimidine metabolizing enzyme dihydropyrimidine dehydrogenase (DPD; ie, DPYD*2A) is strongly associated with fluoropyrimidine-induced severe and life-threatening toxicity. This study determined the feasibility, safety, and cost of DPYD*2A genotype–guided dosing. Patients and Methods Patients intended to be treated with fluoropyrimidine-based chemotherapy were prospectively genotyped for DPYD*2A before start of therapy. Variant allele carriers received an initial dose reduction of ≥ 50% followed by dose titration based on tolerance. Toxicity was the primary end point and was compared with historical controls (ie, DPYD*2A variant allele carriers receiving standard dose described in literature) and with DPYD*2A wild-type patients treated with the standard dose in this study. Secondary end points included a model-based cost analysis, as well as pharmacokinetic and DPD enzyme activity analyses. Results A total of 2,038 patients were prospectively screened for DPYD*2A, of whom 22 (1.1%) were heterozygous polymorphic. DPYD*2A variant allele carriers were treated with a median dose-intensity of 48% (range, 17% to 91%). The risk of grade ≥ 3 toxicity was thereby significantly reduced from 73% (95% CI, 58% to 85%) in historical controls (n = 48) to 28% (95% CI, 10% to 53%) by genotype-guided dosing (P < .001); drug-induced death was reduced from 10% to 0%. Adequate treatment of genotype-guided dosing was further demonstrated by a similar incidence of grade ≥ 3 toxicity compared with wild-type patients receiving the standard dose (23%; P = .64) and by similar systemic fluorouracil (active drug) exposure. Furthermore, average total treatment cost per patient was lower for screening (€2,772 [$3,767]) than for nonscreening (€2,817 [$3,828]), outweighing screening costs. Conclusion DPYD*2A is strongly associated with fluoropyrimidine-induced severe and life-threatening toxicity. DPYD*2A genotype–guided dosing results in adequate systemic drug exposure and significantly improves safety of fluoropyrimidine therapy for the individual patient. On a population level, upfront genotyping seemed cost saving.

scholarly journals Identifying a Novel DPYD Polymorphism Associated with Severe Toxicity to 5-FU Chemotherapy in a Saudi Patient

2019 ◽  
Vol 2019 ◽  
pp. 1-3
Author(s):  
Nedal Bukhari ◽  
Faisal Azam ◽  
Mohammed Alfawaz ◽  
Mohammed Zahrani
Keyword(s):  
Dose Reduction ◽  
Adjuvant Treatment ◽  
Standard Dose ◽  
Dihydropyrimidine Dehydrogenase ◽  
Rectal Adenocarcinoma ◽  
Severe Toxicity ◽  
First Case

Dihydropyrimidine dehydrogenase (DPD) is the major enzyme in the catabolism of 5-Fluorouracil (5-FU) and its prodrug capecitabine. We report a 65-year-old female with rectal adenocarcinoma who experienced severe toxicities secondary to standard dose 5-FU based chemotherapy. She was found to be heterozygous for rs371313778, c.2434G>A. This finding prompted restarting 5-FU at 50% dose reduction with further titration in subsequent cycles. We herein report the first case of rs371313778, c.2434G>A (p.Val812lle) DPYD polymorphism leading to severe 5-FU toxicities. The patient eventually completed a 6-month course of adjuvant treatment with modification of 5-FU dose.

An easy-to-handle DPD deficiency test in saliva to identify patients at high-risk for life-threatening toxicity due to fluoropyrimidine therapy.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e14019-e14019
Author(s):  
Gilberto Schwartsmann ◽  
Marina Venzon Antunes ◽  
Andres Galarza ◽  
Roberta Zilles Hahn ◽  
Suziane Raymundo ◽  
...  
Keyword(s):  
High Risk ◽  
Dihydropyrimidine Dehydrogenase ◽  
Metabolic Ratio ◽  
Sample Population ◽  
Allele Carrier ◽  
Severe Toxicity ◽  
Medical Oncologists ◽  
Risk Patients ◽  
Life Threatening ◽  
Grade 3

e14019 Background: Severe dihydropyrimidine dehydrogenase (DPD) deficiency can be lethal in 0.5-3.0% of patients receiving fluoropyrimidines. Unfortunately, there is no routine test in medical practice to identify high-risk patients. Here, we evaluated the use of plasma and saliva uracil (U) to dihydrouracil (UH2) metabolic ratio and DPYD genotyping, as a means to identify patients with DPD deficiency and fluoropyrimidine toxicity. In addition, we report on a functional test using UH2/U metabolic ratio in dried saliva spots (DSS). Methods: Prior to fluoropyrimidine therapy, plasma and saliva samples were obtained from 60 patients with GI cancer. U and UH2 levels were measured by LC-MS/MS in plasma and saliva. Patients were also genotyped for DPYD (*7/*2A/*13/Y186C). WHO grading were used to report treatment toxicity. Results: In 21 patients (35%) toxicity was documented. For those, no variant allele carrier for DPYD was identified. The UH2/U metabolic ratios were 0.1-26.7 in plasma and 0.1-24.0 in saliva, with a higher correlation with toxicity grade in saliva as compared to plasma (rs 0.52 vs 0.28). Median metabolic ratios were lower in patients with severe toxicity as compared to those with no toxicity (0.59 vs 2.83 saliva; 1.62 vs 6.75 plasma, P < 0.01). A cut-off of 1.16 for the salivary UH2/U ratio was set (AUC 0.84) with 86% sensitivity and 77% specificity for the identification of grade 3-4 toxicity. A plasma cut-off of 4.0 (AUC 0.75) revealed a 71% sensitivity and 76% specificity. Moreover, saliva of 21 patients were applied to filter paper to obtain DSS and sent to the laboratory by regular mail. U and UH2 were stable in DSS stored at 45°C up to 7 days. In this set of patients, grade 3-4 toxicity was documented in 3/21 cases (14%), all three cases had metabolic ratios below 1.16 in DSS, confirming our prior results. Conclusions: DPYD genotyping failed to identify severe DPD deficiency, but the UH2/U metabolic ratios in saliva showed enough sensitivity and specificity to deserve further evaluation. DSS samples allowed medical oncologists working at distant sites to send us samples by post, with results available within a week. This test is being validated in a larger sample population.

scholarly journals Two Chalcone Synthase Isozymes Participate Redundantly in UV-Induced Sakuranetin Synthesis in Rice

2020 ◽  
Vol 21 (11) ◽  
pp. 3777
Author(s):  
Hye Lin Park ◽  
Youngchul Yoo ◽  
Seong Hee Bhoo ◽  
Tae-Hoon Lee ◽  
Sang-Won Lee ◽  
...  
Keyword(s):  
Chalcone Synthase ◽  
Developmental Stages ◽  
Rice Genome ◽  
The Other ◽  
Wild Type ◽  
Flavonoid Pathway ◽  
Key Enzyme ◽  
In The Wild ◽  
Bona Fide ◽  
Biochemical Analyses

Chalcone synthase (CHS) is a key enzyme in the flavonoid pathway, participating in the production of phenolic phytoalexins. The rice genome contains 31 CHS family genes (OsCHSs). The molecular characterization of OsCHSs suggests that OsCHS8 and OsCHS24 belong in the bona fide CHSs, while the other members are categorized in the non-CHS group of type III polyketide synthases (PKSs). Biochemical analyses of recombinant OsCHSs also showed that OsCHS24 and OsCHS8 catalyze the formation of naringenin chalcone from p-coumaroyl-CoA and malonyl-CoA, while the other OsCHSs had no detectable CHS activity. OsCHS24 is kinetically more efficient than OsCHS8. Of the OsCHSs, OsCHS24 also showed the highest expression levels in different tissues and developmental stages, suggesting that it is the major CHS isoform in rice. In oschs24 mutant leaves, sakuranetin content decreased to 64.6% and 80.2% of those in wild-type leaves at 2 and 4 days after UV irradiation, respectively, even though OsCHS24 expression was mostly suppressed. Instead, the OsCHS8 expression was markedly increased in the oschs24 mutant under UV stress conditions compared to that in the wild-type, which likely supports the UV-induced production of sakuranetin in oschs24. These results suggest that OsCHS24 acts as the main CHS isozyme and OsCHS8 redundantly contributes to the UV-induced production of sakuranetin in rice leaves.

scholarly journals Localization of the Bacillus subtilis murB Gene within the dcw Cluster Is Important for Growth and Sporulation

2006 ◽  
Vol 188 (5) ◽  
pp. 1721-1732 ◽  
Author(s):  
Gonçalo Real ◽  
Adriano O. Henriques
Keyword(s):  
Growth Rate ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Ectopic Expression ◽  
Normal Growth ◽  
Wild Type ◽  
Extra Copy ◽  
Gene Encoding ◽  
Key Enzyme ◽  
High Concentrations

ABSTRACT The Bacillus subtilis murB gene, encoding UDP-N-acetylenolpyruvoylglucosamine reductase, a key enzyme in the peptidoglycan (PG) biosynthetic pathway, is embedded in the dcw (for “division and cell wall”) cluster immediately upstream of divIB. Previous attempts to inactivate murB were unsuccessful, suggesting its essentiality. Here we show that the cell morphology, growth rate, and resistance to cell wall-active antibiotics of murB conditional mutants is a function of the expression level of murB. In one mutant, in which murB was insertionally inactivated in a merodiploid bearing a second xylose-inducible PxylA-murB allele, DivIB levels were reduced and a normal growth rate was achieved only if MurB levels were threefold that of the wild-type strain. However, expression of an extra copy of divIB restored normal growth at wild-type levels of MurB. In contrast, DivIB levels were normal in a second mutant containing an in-frame deletion of murB (ΔmurB) in the presence of the PxylA-murB gene. Furthermore, this strain grew normally with wild-type levels of MurB. During sporulation, the levels of MurB were highest at the time of synthesis of the spore cortex PG. Interestingly, the ΔmurB PxylA-murB mutant did not sporulate efficiently even at high concentrations of inducer. Since high levels of inducer did not interfere with sporulation of a murB + PxylA-murB strain, it appears that ectopic expression of murB fails to support efficient sporulation. These data suggest that coordinate expression of divIB and murB is important for growth and sporulation. The genetic context of the murB gene within the dcw cluster is unique to the Bacillus group and, taken together with our data, suggests that in these species it contributes to the optimal expression of cell division and PG biosynthetic functions during both vegetative growth and spore development.

scholarly journals Requirement of Purine and Pyrimidine Synthesis for Colonization of the Mouse Intestine by Escherichia coli

2010 ◽  
Vol 76 (15) ◽  
pp. 5181-5187 ◽  
Author(s):  
Jacqueline Vogel-Scheel ◽  
Carl Alpert ◽  
Wolfram Engst ◽  
Gunnar Loh ◽  
Michael Blaut
Keyword(s):  
Escherichia Coli ◽  
Differentially Expressed ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Pyrimidine Synthesis ◽  
Key Enzyme ◽  
Mouse Intestine ◽  
Gnotobiotic Mice

ABSTRACT To study the adaptation of an intestinal bacterium to its natural environment, germfree mice were associated with commensal Escherichia coli MG1655. Two-dimensional gel electrophoresis was used to identify proteins differentially expressed in E. coli MG1655 collected from either cecal contents or anaerobic in vitro cultures. Fourteen differentially expressed proteins (>3-fold; P < 0.05) were identified, nine of which were upregulated in cecal versus in vitro-grown E. coli. Four of these proteins were investigated further for their role in gut colonization. After deletion of the corresponding genes, the resulting E. coli mutants were tested for their ability to colonize the intestines of gnotobiotic mice in competition with the wild-type strain. A mutant devoid of ydjG, which encodes a putative NADH-dependent methylglyoxal reductase, reached a 1.2-log-lower cecal concentration than the wild type. Deletion of the nanA gene encoding N-acetylneuraminate lyase affected the colonization and persistence of E. coli in the intestines of the gnotobiotic mice only slightly. A mutant devoid of 5′-phosphoribosyl 4-(N-succinocarboxamide)-5-aminoimidazole synthase, a key enzyme of purine synthesis, displayed intestinal cell counts >4 logs lower than those of the wild type. Deletion of the gene encoding aspartate carbamoyltransferase, a key enzyme of pyrimidine synthesis, even resulted in the washout of the corresponding mutant from the mouse intestinal tract. These findings indicate that E. coli needs to synthesize purines and pyrimidines to successfully colonize the mouse intestine.

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