A multiplex real-time PCR method for detection of GSTM1 and GSTT1 copy numbers

• Published in: Clinical biochemistry 2009-04, Vol.42 (6), p.500-509
• Main Authors: Timofeeva, Maria, Jäger, Birgit, Rosenberger, Albert, Sauter, Wiebke, Wichmann, Heinz-Erich, Bickeböller, Heike, Risch, Angela
• Format: Article
• Language: English
• Subjects: Algorithms; Blotting, Southern; Copy number polymorphism; DNA - analysis; Gene Deletion; Gene Dosage; Gene Duplication; Gene Frequency; Genetic Testing - methods; Glutathione Transferase - analysis; Glutathione Transferase - genetics; Glutathione-S-transferase M1; Glutathione-S-transferase T1; Humans; Polymorphism, Genetic; Real time PCR quantification; Reverse Transcriptase Polymerase Chain Reaction - methods; Sensitivity and Specificity
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2008.12.011

Deletion polymorphisms of Glutathione-S-transferase (GST) M1 and T1 are considered risk factors for various diseases. However, most previous studies only distinguished “null” and “non-null” genotypes. Our aim was to develop a reliable, high-throughput GSTM1/T1 genotyping method able to determine allele copy numbers. We developed a multiplex real time PCR method to distinguish between heterozygous (1/0) and homozygous (1/1) GSTM1 and GSTT1 genotypes. The principle of relative quantification was applied and an expectation–maximisation (EM) algorithm was developed to assign one of 3 possible genotypes: 1/1, 1/0 or 0/0 for each of the two genes. 1320 Caucasians were genotyped using the newly developed method. The observed genotype distributions did not deviate from the expected and were in Hardy–Weinberg equilibrium. GSTM1 duplication was detected in one sample. This new semiquantitative genotyping method is a sensitive and promising tool for large-scale molecular epidemiological and clinical studies.