High-throughput genotyping of copy number variation in Glutathione S-Transferases M1 and T1 using real-time PCR in 20,687 individuals

Characteristic for the genes encoding glutathione S-transferase (GST) M1 and GSTT1 is a null allele, suggested to increase susceptibility to chronic diseases. We report an optimized method for the determination of copy number variation (CNV) in GST genes. Real-time multiplex PCR reactions were optimized for quantification of GSTM1 and GSTT1 CNV using the Δ C t method, a fixed volume of diluted DNA, a total volume of 10 μL, 384-well formats, and single determinations of each sample. Consistent genotyping was obtained using DNA in a range of 0.41 ng to 100 ng. In a general population sample of 20,687 individuals the genotype frequencies were concordant with other methods used as standards. Throughput was 4600 genotypes per day at a reagent price of 0.5 euros per sample. This high-throughput, low cost method accurately determines CNV in the GST genes enabling reliable estimates of disease prediction in large epidemiological samples.