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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: clinchem.2008.120105v1 55/9/1680    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Google Scholar Articles by Rose-Zerilli, M. J. Articles by Holloway, J. W. PubMed PubMed Citation Articles by Rose-Zerilli, M. J. Articles by Holloway, J. W.

Copy-Number Variation Genotyping of GSTT1 and GSTM1 Gene Deletions by Real-Time PCR

¹ Respiratory Genetics Group, Human Genetics, ² Infection, Inflammation and Immunity, and ³ Public Health Sciences and Medical Statistics, Community Clinical Sciences Divisions, School of Medicine, University of Southampton, Southampton, UK; ⁴ Department of Community-Based Medicine, University of Bristol, Bristol, UK; ⁵ The National Heart and Lung Institute, Imperial College London, London, UK.

^aAddress correspondence to this author at: Respiratory Genetics Group, Human Genetics Division, University of Southampton, Duthie Bldg. (MP 808), Southampton General Hospital, Tremona Rd., Southampton SO16 6YD, UK. Fax +02380-794264; e-mail mjrz{at}soton.ac.uk.

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-µL 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® 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. .

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				C. Minelli, R. Granell, R. Newson, M. J Rose-Zerilli, M. Torrent, S. M Ring, J. W Holloway, S. O Shaheen, and J. A Henderson Glutathione-S-transferase genes and asthma phenotypes: a Human Genome Epidemiology (HuGE) systematic review and meta-analysis including unpublished data Int. J. Epidemiol., January 4, 2010; (2010) dyp337v2. [Abstract] [Full Text] [PDF]