Published online before print June 13, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: jmoldx.2008.080021v1 10/4/355    most recent Purchase Article View Shopping Cart Services 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 Citing Articles via Google Scholar Google Scholar Articles by Vaughn, C. P. Articles by Samowitz, W. S. Search for Related Content PubMed PubMed Citation Articles by Vaughn, C. P. Articles by Samowitz, W. S.

Journal of Molecular Diagnostics 2008, Vol. 10, No. 4
Copyright © 2008 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2008.080021

Confirmation of Single Exon Deletions in MLH1 and MSH2 Using Quantitative Polymerase Chain Reaction

Cecily P. Vaughn^*, Elaine Lyon^* and Wade S. Samowitz^*

From the ARUP Institute for Clinical and Experimental Pathology, * Salt Lake City; and the Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah

Deletions of one or more exons in the mismatch repair genes MLH1 and MSH2 have been implicated in a significant fraction of hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Multiplex ligation-dependent probe amplification (MLPA) detection of deletions of multiple sequential exons is widely accepted; however, there is concern over the reliability of MLPA results showing single exon deletions. Given the clinical implications of a diagnosis of Lynch syndrome, it is important to use an alternative technique to confirm single exon deletions. To verify single exon deletions, we developed a SYBR Green-based quantitative polymerase chain reaction (PCR) assay. Clinical DNA samples containing deletions in 33 of the 35 exons in MLH1 and MSH2, previously screened by MLPA, were evaluated by quantitative PCR. Gene dosage ratios were determined by both the relative standard curve method and by the 2^–C_T method. Deleted exons had gene dosage ratios of 0.4 to 0.6, while nondeleted exons exhibited ratios of 0.8–1.3. We found 100% concordance between the quantitative PCR and MLPA results, including confirmation of all single exon deletions. The 2^–C_T method was as accurate as using standard curves for the calculation of ratios. Single exon deletions in MLH1 and MSH2 can be verified using quantitative PCR. Assays using this method are simple to design and easy to perform, making them ideal for confirmatory testing.

This article has been cited by other articles:

				E. Lyon and C. T. Wittwer LightCycler Technology in Molecular Diagnostics J. Mol. Diagn., March 1, 2009; 11(2): 93 - 101. [Abstract] [Full Text] [PDF]