A method for accurate detection of genomic microdeletions using real-time quantitative PCR

Rosanna Weksberg^*¹^,2 , Simon Hughes^*³ , Laura Moldovan¹ , Anne S Bassett⁴ , Eva WC Chow⁴ and Jeremy A Squire⁵

¹Program in Genetics and Genomic Biology, The Research Institute, The Hospital for Sick Children, Toronto, Canada

²Division of Clinical and Metabolic Genetics, Department of Paediatrics, The Hospital for Sick Children, Toronto, Canada

³Queen Mary's School of Medicine and Dentistry, London, UK

⁴Clinical Genetics Research Program, Centre for Addiction and Mental Health, and Department of Psychiatry, University of Toronto, Toronto, Canada

⁵Ontario Cancer Institute and Department of Laboratory Medicine, Pathology and Medical Biophysics, University of Toronto, Toronto, Canada

author email corresponding author email* Contributed equally

BMC Genomics 2005, 6:180doi:10.1186/1471-2164-6-180


Published:	13 December 2005

Abstract

Background

Quantitative Polymerase Chain Reaction (qPCR) is a well-established method for quantifying levels of gene expression, but has not been routinely applied to the detection of constitutional copy number alterations of human genomic DNA. Microdeletions or microduplications of the human genome are associated with a variety of genetic disorders. Although, clinical laboratories routinely use fluorescence in situ hybridization (FISH) to identify such cryptic genomic alterations, there remains a significant number of individuals in which constitutional genomic imbalance is suspected, based on clinical parameters, but cannot be readily detected using current cytogenetic techniques.

Results

In this study, a novel application for real-time qPCR is presented that can be used to reproducibly detect chromosomal microdeletions and microduplications. This approach was applied to DNA from a series of patient samples and controls to validate genomic copy number alteration at cytoband 22q11. The study group comprised 12 patients with clinical symptoms of chromosome 22q11 deletion syndrome (22q11DS), 1 patient trisomic for 22q11 and 4 normal controls. 6 of the patients (group 1) had known hemizygous deletions, as detected by standard diagnostic FISH, whilst the remaining 6 patients (group 2) were classified as 22q11DS negative using the clinical FISH assay. Screening of the patients and controls with a set of 10 real time qPCR primers, spanning the 22q11.2-deleted region and flanking sequence, confirmed the FISH assay results for all patients with 100% concordance. Moreover, this qPCR enabled a refinement of the region of deletion at 22q11. Analysis of DNA from chromosome 22 trisomic sample demonstrated genomic duplication within 22q11.

Conclusion

In this paper we present a qPCR approach for the detection of chromosomal microdeletions and microduplications. The strategic use of in silico modelling for qPCR primer design to avoid regions of repetitive DNA, whilst providing a level of genomic resolution greater than standard cytogenetic assays. The implementation of qPCR detection in clinical laboratories will address the need to replace complex, expensive and time consuming FISH screening to detect genomic microdeletions or duplications of clinical importance.