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Warfarin e PEG-IFN/RBV: sviluppo di test farmacogenetici per la personalizzazione della terapia farmacologica
Get PDFThe aim of this study was the development and the validation of two pharmacogenetic tests for determination of the personalized dosage of drugs used in clinical settings (Warfarin and PEG-Interferone/Ribavirin) in which genetic testing is necessary and recommended.
Warfarin (Coumadin®) is a widely prescribed anticoagulant used for treatment and prevention of thrombotic disorders. Although highly efficacious, Warfarin’s narrow therapeutic index and wide inter-individual variability in dose response make its dosing difficult.
The standard procedure to define the personalized dose is an iterative process that can take weeks or months and exposes the patients to an increased risk of over- or underanticoagulation and thus to serious side effects like thromboembolism or bleeding. The anticoagulation effect of the Coumadin® drug therapy is influenced by combination of genetic and non-genetic factors , which account for more than half of the inter-individual variation in Warfarin doses required to achieve a therapeutic level of anticoagulation or therapeutic INR (Prothrombin International Normalized Ratio). Since 2011, the FDA requires the product label to include a recommended daily Warfarin dose (mg/day) for therapeutic INR, which is to be based on the genotypes of CYP2C9 and VKORC1. In addition, Genome-Wide Association Studies (GWAS) have confirmed that CYP2C9, VKORC1 and CYP4F2 are the main genetic factors determining the effective Warfarin dose in Caucasians. Therefore, knowledge of the genetic profile provides a means to assess the differential risk of adverse reactions and allows the personalization of the therapy, e.g. lowering the dosage for patients with the alleles CYP2C9 *2 and *3 or that are homozygous for the A-allele (A/A) of the rs9923231 polymorphism in the VKORC1 gene.
The goal of this study was to develop an innovative genotyping system assessing the gene polymorphisms that mainly determine the clinical response to Warfarin. Four polymorphisms were investigated: rs9923231 in the VKORC1 gene, rs1799853 and rs1057910 in the CYP2C9 gene and rs2108622 in the CYP4F2 gene. To evaluate already available systems and decide on the method to be used for the test we performed a benchmarking of the tests present in the market.
The genotyping is performed using a multiplex PCR in combination with a Reverse Line Blot hybridization assay (mPCR/RLB). To ensure specific amplification the test uses the SSP-PCR (Sequence Specific Primer PCR) approach. The high specificity of this PCR method was confirmed by direct sequencing. The test identifies and differentiates SNPs using ASO probes (Allele Specific Oligonucleotide) that recognize the different alleles. The hybrid formed between the amplified target DNA sequence and the complementary probes is detected by a colorimetric reaction using biotin-streptavidin and alkaline phosphatase.
In the first part of the study several parameters like assay temperature, typology of hybridization and washing solutions, incubation time and probe design were analyzed in order to assess their influence on the assay’s performance. Next, the amplification protocol was defined and the conditions of the RLB assay were set up. The kit prototype was then validated on 125 samples, which had been previously genotyped with a standard method (TaqMan® Drug Metabolism Genotyping Assay, Life Technologies), and the analytical sensitivity of the assay was determined. All tested samples were accurately genotyped (100% diagnostic specificity and sensitivity) and the method efficiently worked in a broad range of DNA concentrations. Sensitivity tests demonstrated that the method developed in this study, provided reliable results within a DNA concentration range of 1 ng/reaction to 500 ng/reaction. The shelf life of the reagents was determined to be at least twelve months. In addition, the assay was shown to work on/with the following combinations of probe support materials (a nylon membrane and a nitrocellulose blotting membrane) and PCR master mixes (three commercial available master mixes). A software was developed that automates and facilitates interpretation of the RLB (strip) patterns.
After completion, the system, GENEQUALITY AB WARFARIN TYPE cod. 04-74A-20 (AB ANALITICA), was notified to the Ministero della Salute and was CE IVD marked for commercialization in the European Community.
As this system is the only assay on the market that analyzes the CYP4F2 gene in combination with the CYP2C9 and the VKORC1 gene, it represents the most complete approach for determining the genetic factors influencing Warfarin dosage in Caucasians.
The Hepatitis C virus (HCV) infection is a major health problem with more than 170 million infected individuals worldwide (2-2.5% of the world’s population).
Approximately 30% of the infections are cleared spontaneously, whereas 40% of the patients develop a chronic infection that can lead to cirrhosis, hepatocellular carcinoma and end-stage liver disease. The current standard of care is a combination treatment with pegylated interferon (PEG-INF) and Ribavirin (RBV), but its efficacy and tolerability are limited. Many efforts have been made to identify the factors that influence host resistance to HCV infection and treatment outcome. Several Genome-Wide Association Studies (GWAS) have demonstrated independently that variations in the IL28B (Interleukin 28B) gene are strongly associated with spontaneous clearance of the virus, early viral kinetics and with the varying PEG-INF/RBV therapy response in different populations. Particularly, polymorphism IL28B rs12979860 influences the prospect of recovery from an HCV infection. Patients homozygous for allele rs12979860-C show a two- to threefold higher SVR (Sustained Virological Response) rate compared to persons with a non-CC genotype when undergoing Peg-INF/RBV treatment, have a higher probability of spontaneous clearance of the virus and, during therapy, display a stronger reduction of virus RNA levels, which is reflected in higher RVR (Rapid Virological Response) and EVR (Early Virological Response) rates.
Other studies reported an association of certain functional variants of the ITPA (Inosine Triphosphatase) protein with the reduction of hemoglobin during Peg-INF/RBV therapy. These allelic variants of the ITPA gene decrease the enzymatic activity of ITPA, thus protecting from RBV-induced anemia. The aim was to develop an innovative system to genotype the most significant genetic polymorphisms important for the prediction of treatment response and risk of adverse reactions in patients infected with HCV. Four polymorphisms were investigated in this study: rs12979860 and rs8099917 in the IL28B gene and rs7270101 and rs1127354 in the ITPA gene.
Human genomic DNA was extracted from peripheral blood using an automated system (EZ1, Qiagen). Genotyping of the genetic variants was performed combining multiplex PCR with a Reverse Line Blot hybridization assay (mPCR/RLB). For amplification (PCR) of the target sequences, biotinylated primers were used. The primers were designed and analyzed in silico using bioinformatics alignment tools like “blat” (UCSC) and ClustalW, excluding genomic sequences that contain SNPs, SINEs/LINEs or other repetitive elements and all primer binding sites that show a sequence similarity of more than 95% with other genomic sequences. Due to the fact that a sequence that is highly homologous to the target region in IL28B gene is located on the same chromosome 15.000 bp upstream, the SSP-PCR approach was adopted in order to ensure a high specificity of the amplification. To evaluate the efficacy of the SPP-PCR, the amplification products were analyzed by direct sequencing. Subsequently, the protocol for the multiplex amplification was set up. Probes recognizing the different alleles of each investigated SNP (two ASO, Allele Specific Oligonucleotide, per SNP) were designed and their specificity analyzed under varying experimental conditions. This allowed estimation of the effect of parameters like temperature and time of incubation on the assay performance. The analytical sensitivity of the assay was determined using a range of DNA concentrations between 0.5 ng/µl and 200 ng/µl. The diagnostic specificity of the test was assessed analyzing 160 samples of a known genotype.
The test provided a result within a DNA concentration of 1 ng/reaction to 400 ng/reaction and all tested samples were accurately genotyped. This underlines the reliability and specificity of the assay. The shelf life of the reagents was determined to be at least eight months and the compatibility of the assay with other amplification master mixes or probe supports (membranes) was tested. The system, GENEQUALITY IL28B-ITPA TYPE cod. 04-47A-20 (AB ANALITICA), was notified to the Ministero della Salute and CE IVD marked for commercialization in the European Community.
RLB is a convenient way to identify up to 8 targets in 20 individual specimens simultaneously. It is more flexible and less costly than DNA microarrays while providing the same specificity and sensitivity. The system developed in this study, GENEQUALITY IL28B-ITPA TYPE, code 04-47A-20 (AB ANALITICA), allows analysis of the most important host genetic factors influencing the efficacy of the Peg-INF/RBV therapy and determining the risk of RBV-induced anemia. It is the only assay on the market that analyzes ITPA and IL28B polymorphisms together and therefore represents the most complete approach for determining the genetic constitution in regard to the IL28B and the ITPA gene for personalized management of patients with Hepatitis C.
A genotype-phenotype correlation analysis was performed to evaluate the association between IL28B and ITPA polymorphisms and the therapy outcome in a sub-group of patients (N=52). Genetic analysis of the IL28B polymorphism rs12979860 revealed that 27% of the patients carried the C/C genotype, whereas 56% and 17% carried the C/T and the T/T genotype, respectively. The T/T genotype of the IL28B polymorphism rs8099917 was in 40% of patients, whereas 48% carried T/G and 12% the G/G.
No association was found between variants of the IL28B polymorphisms and SVR rates: 57% of patients with IL28B rs12979860-CC and 52% with IL28B rs8099917-TT achieved SVR and 43% and 48%, respectively, failed to clear the HCV virus.
For analysis of the influence of ITPA, the patients were subdivided into the following three groups: normal ITPase activity (100% ITPA), mild ITPase deficiency (60% ITPA- 60% ITPase activity) and moderate to severe ITPase deficiency (≤30% ITPA, ≤30% ITPase activity). These groups reflected the individual Composite ITPase Deficiency Variable based on the genotypes of the ITPA polymorphisms rs7270101 and rs1127354. Patients with normal ITPase activity were compared to the groups of patients with mild ITPase deficiency and moderate to severe ITPase deficiency by statistical analysis. The results showed that the predicted ITPase deficiency correlated to a reduction of median Hb levels at 4, 12 and 24 weeks of treatment. A statistically significant difference was observed between the groups with normal ITPase activity and moderate to severe ITPase deficiency (≤30% ITPA) at 4 and 24 weeks (Dunnett’s Multiple Comparison Test: T4, p < 0.0001; T24, p < 0.0451). This clearly shows that patients with an at least by 70% decreased ITPase activity are protected from anemia throughout the treatment
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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