A novel Real Time PCR strategy to detect SOD3 SNP using LNA probes.

Extracellular superoxide dismutase (SOD3) is the primary enzymatic antioxidant defence of the vascular wall. The physiopathological role of SOD3 has been examined in vascular-related diseases, atherosclerosis, hypertension, diabetes, ischaemia-reperfusion injury, lung disease, various inflammatory conditions, and neurological diseases. An important single nucleotide polymorphism (SNP), nt.760 G>C of the SOD3 gene (rs#1799895) leads to the amino acid substitution Arg(213)Gly (R213G) in the center of the heparin-binding domain and consequently to a lowered affinity for the endothelium. This mutation, which occurs with a relatively high frequency in the population (4\% of Swedish, 3\% of Australian and 6\% of Japanese people), is associated with decreased tissue antioxidant defences and increased risk of ischaemic heart disease. The identification of patients carrying this mutation is therefore of great interest in order to highlight lowered antioxidant defences at a vascular level which could lead to increased susceptibility toward coronary artery disease and atherogenesis. Here we describe a method to detect the 760 G>C single nucleotide polymorphism based on Real Time PCR strategy using locked nucleic acid (LNA) probes. This technique, a modification of classic TaqMan probes SNP genotyping, amplifies and detects the mutation in a single reaction tube. Moreover, the implementation of LNA probes remarkably increases the specificity of the reaction. The proposed method enables unambigous and rapid discrimination of wild type and mutant genotype both in plasmid and genomic DNA samples. In light of the role of SOD3 polymorphism, the genotyping of 760 G>C mutant has important clinical implications. The proposed assay combines rapidity, high specificity, can be easily automated and overall reduces labor and cost of analyses. Moreover, identification of patients with lowered vascular antioxidant defences could address pharmacogenomical approaches to the therapy of cardiovascular diseases.