Copy-number variation genotyping of GSTT1 and GSTM1 gene deletions by real-time PCR.
BACKGROUND
Structural variation in the human genome is increasingly recognized as being highly prevalent and having relevance to common human diseases. Array-based comparative genome-hybridization technology can be used to determine copy-number variation (CNV) across entire genomes, and quantitative PCR (qPCR) can be used to validate de novo variation or assays of common CNV in disease-association studies. Analysis of large qPCR data sets can be complicated and time-consuming, however.


METHODS
We describe qPCR assays for GSTM1 (glutathione S-transferase mu 1) and GSTT1 (glutathione S-transferase theta 1) gene deletions that can genotype up to 192 samples in duplicate 5-microL reaction volumes in <2 h on the ABI Prism 7900HT Sequence Detection System. To streamline data handling and analysis of these CNVs by qPCR, we developed a novel interactive, macro-driven Microsoft Excel(R) spreadsheet. As proof of principle, we used our software to analyze CNV data for 1478 DNA samples from a family-based cohort.


RESULTS
With only 8 ng of DNA template, we assigned CNV genotypes (i.e., 2, 1, or 0 copies) to either 96% (GSTM1) or 91% (GSTT1) of all DNA samples in a single round of PCR amplification. Genotyping accuracy, as ascertained by familial inheritance, was >99.5%, and independent genotype assignments with replicate real-time PCR runs were 100% concordant.


CONCLUSIONS
The genotyping assay for GSTM1 and GSTT1 gene deletion is suitable for large genetic epidemiologic studies and is a highly effective analysis system that is readily adaptable to analysis of other CNVs. .
Rose-Zerilli
Matthew J
MJ
Respiratory Genetics Group, Human Genetics, Infection, Inflammation and Immunity, Public Health Sciences and Medical Statistics, Community Clinical Sciences Divisions, School of Medicine, University of Southampton, Southampton, UK.
Barton
Sheila J
SJ
Henderson
A John
AJ
Shaheen
Seif O
SO
Holloway
John W
JW
D017353:Gene Deletion; D018628:Gene Dosage; D005838:Genotype; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D012984:Software; D013997:Time Factors
D005982:Glutathione Transferase; C413545:glutathione S-transferase T1