Variantes genéticas y actividad enzimática de la citidin desaminasa: relación con la toxicidad a la capecitabina y recomendación de ajuste de las dosis / Genetic variants and enzyme activity in citidin deaminase: Relationship with capecitabine toxicity and recommendation for dose adjustment

Objetivo: la capecitabina es un fármaco antineoplásico utilizado en el tratamiento del cáncer de mama y de colon que puede dar lugar a una toxicidad grave, llegando a ser mortal en algunos pacientes. La variabilidad interindividual de esta toxicidad es debida en gran medida a las variaciones genéticas en los genes diana y las enzimas de metabolismo de este fármaco, como la timidilato sintasa y la dihidropirimidina deshidrogenasa. La enzima citidin desaminasa (CDA), imprescindible en la activación de la capecitabina, también presenta diversas variantes asociadas con un mayor riesgo de toxicidad al tratamiento, aunque su papel como biomarcador aún no está claramente definido. Por ello, nuestro objetivo principal es estudiar la asociación entre la presencia de las variantes genéticas en el gen CDA, su actividad enzimática y el desarrollo de la toxicidad grave en los pacientes tratados con capecitabina, cuya dosis inicial se haya ajustado con base en el perfil genético del gen de la dihidropirimidina deshidrogenasa (DPYD). Método: estudio de cohortes observacional multicéntrico prospectivo, centrado en el análisis de la asociación genotipo-fenotipo de la enzima CDA. Tras la fase experimental, se desarrollará un algoritmo que permita determinar el ajuste necesario de las dosis para disminuir el riesgo de toxicidad del tratamiento en función del genotipo CDA, elaborando una guía clínica para la dosificación de la capecitabina en función de las variantes genéticas en DPYD y CDA. Con base en esta guía, se creará una herramienta bioinformática que genere el informe farmacoterapéutico de manera automática, facilitando la implementación del consejo farmacogenético en la práctica clínica. Esta herramienta proporcionará un gran respaldo en la toma de decisiones farmacoterapéuticas basadas en el perfil genético del paciente, incorporando la medicina de precisión en la rutina clínica. ... (AU)

Objective: Capecitabine, an antineoplastic drug used in the treatment of breast and colon cancer, can cause severe, even fatal toxicity in some patients. The interindividual variability of this toxicity is largely due to genetic variations in target genes and enzymes of metabolism of this drug, such as thymidylate synthase and dihydropyrimidine dehydrogenase. The enzyme cytidine deaminase (CDA), involved in the activation of capecitabine, also has several variants associated with an increased risk of toxicity to treatment, although its role as a biomarker is not yet clearly defined.Therefore, our main objective is to study the association between the presence of genetic variants in CDA gen, CDA enzymatic activity and the development of severe toxicity in patients treated with capecitabine whose initial dose was adjusted based on the genetic profile of the dihydropyrimidine dehydrogenase gen (DPYD). Method: Prospective multicenter observational cohort study, focused on the analysis of the genotype-phenotype association of the CDA enzyme.After the experimental phase, an algorithm will be developed to determine the dose adjustment needed to reduce the risk of treatment toxicity according to CDA genotype, developing a clinical guide for capecitabine dosing according to genetic variants in DPYD and CDA. Based on this guide, a Bioinformatics Tool will be created to generate the pharmacotherapeutic report automatically, facilitating the implementation of pharmacogenetic advice in clinical practice. This tool will be a great support in making pharmacotherapeutic decisions based on the patient's genetic profile, incorporating precision medicine into clinical routine. Once the usefulness of this tool has been validated, it will be offered free of charge to facilitate the implementation of pharmacogenetics in hospital centers and equitably benefit all patients on capecitabine treatment. (AU)

[Translated article] Genetic variants and enzyme activity in citidin deaminase: Relationship with capecitabine toxicity and recommendation for dose adjustment.

OBJECTIVE: Capecitabine, an antineoplastic drug used in the treatment of breast and colon cancer, can cause severe, even fatal toxicity in some patients. The interindividual variability of this toxicity is largely due to genetic variations in target genes and enzymes of metabolism of this drug, such as Thymidylate Synthase (TS) and Dihydropyrimidine Dehydrogenase (DPD). The enzyme Cytidine Deaminase (CDA), involved in the activation of capecitabine, also has several variants associated with an increased risk of toxicity to treatment, although its role as a biomarker is not yet clearly defined. Therefore, our main objective is to study the association between the presence of genetic variants in CDA gen, CDA enzymatic activity and the development of severe toxicity in patients treated with capecitabine whose initial dose was adjusted based on the genetic profile of the DPD gen (DPYD). METHOD: Prospective multicenter observational cohort study, focused on the analysis of the genotype-phenotype association of the CDA enzyme. After the experimental phase, an algorithm will be developed to determine the dose adjustment needed to reduce the risk of treatment toxicity according to CDA genotype, developing a Clinical Guide for capecitabine dosing according to genetic variants in DPYD and CDA. Based on this guide, a Bioinformatics Tool will be created to generate the pharmacotherapeutic report automatically, facilitating the implementation of pharmacogenetic advice in clinical practice. This tool will be a great support in making pharmacotherapeutic decisions based on the patient's genetic profile, incorporating precision medicine into clinical routine. Once the usefulness of this tool has been validated, it will be offered free of charge to facilitate the implementation of pharmacogenetics in hospital centers and equitably benefit all patients on capecitabine treatment.

Genetic variants and enzyme activity in citidin deaminase: Relationship with capecitabine toxicity and recommendation for dose adjustment. / Variantes genéticas y actividad enzimática de la citidin desaminasa: relación con la toxicidad a la capecitabina y recomendación de ajuste de las dosis.

OBJECTIVE: Capecitabine, an antineoplastic drug used in the treatment of breast and colon cancer, can cause severe, even fatal toxicity in some patients. The interindividual variability of this toxicity is largely due to genetic variations in target genes and enzymes of metabolism of this drug, such as thymidylate synthase and dihydropyrimidine dehydrogenase. The enzyme cytidine deaminase (CDA), involved in the activation of capecitabine, also has several variants associated with an increased risk of toxicity to treatment, although its role as a biomarker is not yet clearly defined. Therefore, our main objective is to study the association between the presence of genetic variants in CDA gen, CDA enzymatic activity and the development of severe toxicity in patients treated with capecitabine whose initial dose was adjusted based on the genetic profile of the dihydropyrimidine dehydrogenase gen (DPYD). METHOD: Prospective multicenter observational cohort study, focused on the analysis of the genotype-phenotype association of the CDA enzyme. After the experimental phase, an algorithm will be developed to determine the dose adjustment needed to reduce the risk of treatment toxicity according to CDA genotype, developing a clinical guide for capecitabine dosing according to genetic variants in DPYD and CDA. Based on this guide, a Bioinformatics Tool will be created to generate the pharmacotherapeutic report automatically, facilitating the implementation of pharmacogenetic advice in clinical practice. This tool will be a great support in making pharmacotherapeutic decisions based on the patient's genetic profile, incorporating precision medicine into clinical routine. Once the usefulness of this tool has been validated, it will be offered free of charge to facilitate the implementation of pharmacogenetics in hospital centers and equitably benefit all patients on capecitabine treatment.

Development, and Internal, and External Validation of a Scoring System to Predict 30-Day Mortality after Having a Traffic Accident Traveling by Private Car or Van: An Analysis of 164,790 Subjects and 79,664 Accidents.

Predictive factors for fatal traffic accidents have been determined, but not addressed collectively through a predictive model to help determine the probability of mortality and thereby ascertain key points for intervening and decreasing that probability. Data on all road traffic accidents with victims involving a private car or van occurring in Spain in 2015 (164,790 subjects and 79,664 accidents) were analyzed, evaluating 30-day mortality following the accident. As candidate predictors of mortality, variables associated with the accident (weekend, time, number of vehicles, road, brightness, and weather) associated with the vehicle (type and age of vehicle, and other types of vehicles in the accident) and associated with individuals (gender, age, seat belt, and position in the vehicle) were examined. The sample was divided into two groups. In one group, a logistic regression model adapted to a points system was constructed and internally validated, and in the other group the model was externally validated. The points system obtained good discrimination and calibration in both the internal and the external validation. Consequently, a simple tool is available to determine the risk of mortality following a traffic accident, which could be validated in other countries.

NPR1 paralogs of Arabidopsis and their role in salicylic acid perception.

Salicylic acid (SA) is responsible for certain plant defence responses and NON EXPRESSER OF PATHOGENESIS RELATED 1 (NPR1) is the master regulator of SA perception. In Arabidopsis thaliana there are five paralogs of NPR1. In this work we tested the role of these paralogs in SA perception by generating combinations of mutants and transgenics. NPR2 was the only paralog able to partially complement an npr1 mutant. The null npr2 reduces SA perception in combination with npr1 or other paralogs. NPR2 and NPR1 interacted in all the conditions tested, and NPR2 also interacted with other SA-related proteins as NPR1 does. The remaining paralogs behaved differently in SA perception, depending on the genetic background, and the expression of some of the genes induced by SA in an npr1 background was affected by the presence of the paralogs. NPR2 fits all the requirements of an SA receptor while the remaining paralogs also work as SA receptors with a strong hierarchy. According to the data presented here, the closer the gene is to NPR1, the more relevant its role in SA perception.

A 2'-O-Methyltransferase Responsible for Transfer RNA Anticodon Modification Is Pivotal for Resistance to Pseudomonas syringae DC3000 in Arabidopsis.

Transfer RNA (tRNA) is the most highly modified class of RNA species in all living organisms. Recent discoveries have revealed unprecedented complexity in the tRNA chemical structures, modification patterns, regulation, and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge of the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2'-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance susceptibility during infection with the virulent bacterial pathogen Pseudomonas syringae DC3000. Lack of such tRNA modification, as observed in scs9 mutants, specifically dampens plant resistance against DC3000 without compromising the activation of the salicylic acid signaling pathway or the resistance to other biotrophic pathogens. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective disease resistance in Arabidopsis toward DC3000 and, therefore, expands the repertoire of molecular components essential for an efficient disease resistance response.

ß-carbonic anhydrases play a role in salicylic acid perception in Arabidopsis.

The plant hormone salicylic acid (SA) is required for defense responses. NON EXPRESSER OF PATHOGENESIS RELATED 1 (NPR1) and NON RECOGNITION OF BTH-4 (NRB4) are required for the response to SA in Arabidopsis (Arabidopsis thaliana). Here, we isolated several interactors of NRB4 using yeast two-hybrid assays. Two of these interactors, ßCA1 and ßCA2, are ß-carbonic anhydrase family proteins. Since double mutant ßca1 ßca2 plants did not show any obvious phenotype, we investigated other ßCAs and found that NRB4 also interacts with ßCA3 and ßCA4. Moreover, several ßCAs interacted with NPR1 in yeast, including one that interacted in a SA-dependent manner. This interaction was abolished in loss-of-function alleles of NPR1. Interactions between ßCAs and both NRB4 and NPR1 were also detected in planta, with evidence for a triple interaction, NRB4-ßCA1-NPR1. The quintuple mutant ßca1 ßca2 ßca3 ßca4 ßca6 showed partial insensitivity to SA. These findings suggest that one of the functions of carbonic anhydrases is to modulate the perception of SA in plants.

Loss of a Conserved tRNA Anticodon Modification Perturbs Plant Immunity.

tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Despite their vastly different chemical structures and their presence in different tRNAs, occurring in different locations in tRNA, the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent discoveries have revealed unprecedented complexity in the modification patterns of tRNA, their regulation and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge on the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance and activation of defenses in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2´-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance during the course of infection with the bacterial pathogen Pseudomonas syringae DC3000, and lack of such tRNA modification, as observed in scs9 mutants, severely compromise plant immunity against the same pathogen without affecting the salicylic acid (SA) signaling pathway which regulates plant immune responses. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective immune response in Arabidopsis, and therefore expands the repertoire of molecular components essential for an efficient disease resistance response.

Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense.

Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6), a previously reported DBP1 interactor, and MAP kinase (MAPK) MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV), and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

A plant small polypeptide is a novel component of DNA-binding protein phosphatase 1-mediated resistance to plum pox virus in Arabidopsis.

DNA-binding protein phosphatases (DBPs) have been identified as a novel class of plant-specific regulatory factors playing a role in plant-virus interactions. NtDBP1 from tobacco (Nicotiana tabacum) was shown to participate in transcriptional regulation of gene expression in response to virus infection in compatible interactions, and AtDBP1, its closest relative in the model plant Arabidopsis (Arabidopsis thaliana), has recently been found to mediate susceptibility to potyvirus, one of the most speciose taxa of plant viruses. Here, we report on the identification of a novel family of highly conserved small polypeptides that interact with DBP1 proteins both in tobacco and Arabidopsis, which we have designated DBP-interacting protein 2 (DIP2). The interaction of AtDIP2 with AtDBP1 was demonstrated in vivo by bimolecular fluorescence complementation, and AtDIP2 was shown to functionally interfere with AtDBP1 in yeast. Furthermore, reducing AtDIP2 gene expression leads to increased susceptibility to the potyvirus Plum pox virus and to a lesser extent also to Turnip mosaic virus, whereas overexpression results in enhanced resistance. Therefore, we describe a novel family of conserved small polypeptides in plants and identify AtDIP2 as a novel host factor contributing to resistance to potyvirus in Arabidopsis.

Utilización de marcadores genéticos de la CYP2E1 en la valoración de riesgos de exposición laboral a estireno mediante control biológico / Use of genetic markers for CYPE1 in the assessment of risks through occupational exposure to styrene using biologic controls

Hemos estudiado la utilidad del genotipado y fenotipado de la enzima CYP2E1 para la evaluación del riesgo de exposición laboral a estireno, correlacionando los datos obtenidos con los indicadores biológicos habituales en programas de Salud Laboral (ácidos mandélico y fenilglioxílico, principales metabolitos urinarios del estireno). Se examinaron 49 trabajadores con exposición conocida a estireno y un grupo control, determinándose mARN de CYP2E1 en sangre y polimorfismos de la enzima en muestras de mucosa oral. Nuestros resultados muestran que el efecto combinado del fenotipo de CYP2E1 y del genotipo de los alelos CYP2E1*5B y CYP2E1*6 puede explicar en parte la variabilidad en la excreción urinaria de metabolitos del estireno. La técnica de obtención de material biológico a partir de la mucosa oral puede ser de interés en el ámbito laboral (AU)

We have studied the usefulness of geno and phenotyping of the enzyme CYP2E1 for the assessment of risk through occupational exposure to styrene and correlated the results achieved with the habitual biologic markers in Occupational Health programmes (mandelic and phenylglyoxylic acids, the main urinary metabolites of styrene). The study group comprised 49 workers with known exposure to styrene and a control group; CYP2E1 mRNA in blood and enzyme polymorphisms in samples of the oral mucosa were assessed. Our results show that the combined affects of CYP2E1 phenotype and of the CYP2E1*5B and CYP2E1*6 allele genotype may in part explain the variability in the urinary excretion of styrene metabolites. The sampling technique for biologic material from the oral mucosa may be useful in the occupational environment (AU)

14-3-3 mediates transcriptional regulation by modulating nucleocytoplasmic shuttling of tobacco DNA-binding protein phosphatase-1.

Tobacco DBP1 is the founding member of a novel class of plant transcription factors featuring sequence-specific DNA binding and protein phosphatase activity. To understand the mechanisms underlying the function of this family of transcriptional regulators, we have identified the tobacco 14-3-3 isoform G as the first protein interacting with a DBP factor. 14-3-3 recognition involves the N-terminal region of DBP1, which also supports the DNA binding activity attributed to DBP1. The relevance of this interaction is reinforced by its conservation in Arabidopsis plants, where the closest relative of DBP1 in this species also interacts with a homologous 14-3-3 protein through its N-terminal region. Furthermore, we show that in planta 14-3-3 G is directly involved in regulating DBP1 function by promoting nuclear export and subsequent cytoplasmic retention of DBP1 under conditions that in turn alleviate DBP1-mediated repression of target gene expression.