title: High-resolution melting analysis of sequence variations in the cytidine deaminase gene (CDA) in patients with cancer treated with gemcitabine.
abstract: Gemcitabine (2',2'-difluorodeoxycytidine) is a major antimetabolite cytotoxic drug with a wide spectrum of activity against solid tumors. Hepatic elimination of gemcitabine depends on a catabolic pathway through a deamination step driven by the enzyme cytidine deaminase (CDA). Severe hematologic toxicity to gemcitabine was reported in patients harboring genetic polymorphisms in CDA gene. High-resolution melting (HRM) analysis of polymerase chain reaction amplicon emerges today as a powerful technique for both genotyping and gene scanning strategies. In this study, 46 DNA samples from gemcitabine-treated patients were subjected to HRM analysis on a LightCycler 480 platform. Residual serum CDA activity was assayed as a surrogate marker for the overall functionality of this enzyme. Genotyping of three well-described single nucleotide polymorphisms in coding region (c.79A>C, c.208G>A and c.435C>T) was successfully achieved by HRM analysis of small polymerase chain reaction fragments, whereas unknown single nucleotide polymorphisms were searched by a gene scanning strategy with longer amplicons (up to 622 bp). The gene scanning strategy allowed us to find a new intronic mutation c.246+37G>A in a female patient displaying marked CDA deficiency and who had an extreme toxic reaction with a fatal outcome to gemcitabine treatment. Our work demonstrates that HRM-based methods, owing to their simplicity, reliability, and speed, are useful tools for diagnosis of CDA deficiency and could be of interest for personalized medicine.
authors.affiliation: Laboratoire de Biochimie, Centre Hospitalier Universitaire de Nîmes, Nîmes, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Raynal
authors.forename: Joseph
authors.initials: J
authors.lastname: Ciccolini
authors.forename: Cédric
authors.initials: C
authors.lastname: Mercier
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Boyer
authors.forename: Anne
authors.initials: A
authors.lastname: Polge
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lallemant
authors.forename: Kévin
authors.initials: K
authors.lastname: Mouzat
authors.forename: Serge
authors.initials: S
authors.lastname: Lumbroso
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Brouillet
authors.forename: Alexandre
authors.initials: A
authors.lastname: Evrard
mesh_terms: D000368:Aged; D000964:Antimetabolites, Antineoplastic / Q000009:adverse effects / Q000493:pharmacokinetics / Q000627:therapeutic use*; D001483:Base Sequence; D003564:Cytidine Deaminase / Q000235:genetics*; D003841:Deoxycytidine / Q000009:adverse effects / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000627:therapeutic use; D005260:Female; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D008297:Male; D009154:Mutation; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D044366:Transition Temperature; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D003841:Deoxycytidine; D003564:Cytidine Deaminase; D000093542:Gemcitabine