High-resolution melting analysis of sequence variations in the cytidine deaminase gene (CDA) in patients with cancer treated with gemcitabine

Ther Drug Monit. 2010 Feb;32(1):53-60. doi: 10.1097/FTD.0b013e3181c77c1b.

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.

MeSH terms

  • Aged
  • Antimetabolites, Antineoplastic / adverse effects
  • Antimetabolites, Antineoplastic / pharmacokinetics
  • Antimetabolites, Antineoplastic / therapeutic use*
  • Base Sequence
  • Cytidine Deaminase / genetics*
  • Deoxycytidine / adverse effects
  • Deoxycytidine / analogs & derivatives*
  • Deoxycytidine / pharmacokinetics
  • Deoxycytidine / therapeutic use
  • Female
  • Genetic Variation
  • Genotype
  • Humans
  • Male
  • Mutation
  • Neoplasms / drug therapy
  • Neoplasms / genetics*
  • Polymerase Chain Reaction / methods
  • Polymorphism, Single Nucleotide
  • Reproducibility of Results
  • Transition Temperature

Substances

  • Antimetabolites, Antineoplastic
  • Deoxycytidine
  • gemcitabine
  • Cytidine Deaminase