Rapid Detection of the hOGG1 Ser326Cys polymorphism Using LightCycler Technology

Abstract

Human 8-oxoguanine DNA glycosylase 1 (hOGG1) plays an important role in the repair of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodGuo), one of the major constituents in DNA damage. A recent in vitro study showed that the hOGG1 326Cys polymorphism (rs1052133) exhibits reduced 8-oxodGuo repair activity. This study aimed to develop a LightCycler (LC) assay to analyze the C>G polymorphism (Ser326Cys) in exon 7 of the hOGG1 gene followed by validation of the method using DNA samples from 260 polycyclic aromatic hydrocarbons(PAH)-exposed workers with known 8-oxodGuo DNA-adduct values measured by HPLC. Twenty DNA samples were analyzed by a PCR-RFLP analysis with Fnu4H I to generate control DNA. LC melting curve analyses of the hOGG1 exon 7 PCR product were characteristic of the probes hybridized to the non-mutated Ser-type (CC) at 65°C, or to the Cys mutant (GG) at 59°C. The distribution in the population of PAH-exposed workers (N=260) was 58% (CC), 38%(CG), and 4% (GG). The minor G allele displayed a frequency of 23 %. The distribution of 8-oxodGuo adducts for the Ser326Cys variants of hOGG1 revealed geometric means (GM) of 5.83 (CC), 5.27 (CG), and 6.53 (GG) 8-oxodGuo adducts/10⁶dGuo. Corresponding GM values of current smokers were 5.7 (CC), 5.6 (CG) and 7.0 (GG) 8-oxodGuo adducts/10⁶ dGuo. The analysis of the Ser326Cys polymorphism in 260 DNA samples with this new LC assay revealed that this method is reliable for high throughput analysis of this key polymorphism in the hOGG1 gene.