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.

CDA Deficiency as a Possible Culprit for Life-Threatening Toxicities After Cytarabine Plus 6-Mercaptopurine Therapy: Pharmacogenetic Investigations

“…However, a rapid drop in white blood cells and neutrophils occurred after Ara-C intake and treatment was discontinued again until further pharmacogenetic informations were made available ( Figure 1). Next, our investigations focused on Ara-C as a culprit for the severe toxicities and we performed gene scanning by using a recently published high-resolution melting method on the gene coding for CDA, the liver enzyme responsible for the metabolism of cytidine-derivatives [11]. This method allowed us to rapidly detect any sequence variation among coding and exon-flanking regions of the CDA gene.…”

A common polymorphism in NR1H2 (LXRbeta) is associated with preeclampsia

“…Genomic DNA was extracted from 10 ml of peripheral maternal whole blood collected into tubes containing EDTA (ethylenediaminetetraacetic acid) and sodium chloride, as described elsewhere [ 26 ]. The SNPs chosen for study were genotyped, as previously described [ 27 ]. Briefly, 10 ng of genomic DNA was subjected to PCR (polymerase chain reaction) amplification with LightCycler ® 480 High-Resolution Melting Master Mix (Roche Diagnostics, Meylan, France), using a touchdown protocol as follows: initial denaturation at 95°C for 10 minutes then 50 cycles of denaturation at 95°C for 10 seconds; annealing at temperatures decreasing from 70°C to 60°C (step size: 0.5°C/cycle) for 20 seconds and elongation at 72°C for 20 seconds.…”

CDA Gene Polymorphisms and Enzyme Activity: Genotype–phenotype Relationship in an Italian–Caucasian Population

“…Notably, ethnicity markedly influences SNP frequencies [9,10]. The CDA*27Q allele shows Aim: To assess the distribution of CDA activity from whole blood of 142 healthy subjects, determining its main predictors among genetic (six CDA SNPs) and physiological factors (age and gender).…”