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Received on January 5, 2007
Accepted on April 26, 2007
Molecular Diagnostics and Genetics |
1 Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT
2 Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT, and ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT
3 Department of Mathematics, University of Utah, Salt Lake City, UT
* To whom correspondence should be addressed. E-mail: carl.wittwer{at}path.utah.edu.
Background: Heteroduplex scanning techniques usually detect all heterozygotes, including common variants not of clinical interest.
Methods: We conducted high-resolution melting analysis on the 24 exons of the ACVRL1 and ENG genes implicated in hereditary hemorrhagic telangiectasia (HHT). DNA from 13 controls and 19 patients was PCR amplified in the presence of LCGreen® I, and all 768 exons melted in an HR-1® instrument. We used 10 wild-type controls to identify common variants, and the remaining samples were blinded, amplified, and analyzed by melting curve normalization and overlay. Unlabeled probes characterized the sequence of common variants.
Results: Eleven common variants were associated with 8 of the 24 HHT exons, and 96% of normal samples contained at least 1 variant. As a result, the positive predictive value (PPV) of a heterozygous exon was low (31%), even in a population of predominantly HHT patients. However, all common variants produced unique amplicon melting curves that, when considered and eliminated, resulted in a PPV of 100%. In our blinded study, 3 of 19 heterozygous disease-causing variants were missed; however, 2 were clerical errors, and the remaining false negative would have been identified by difference analysis.
Conclusions: High-resolution melting analysis is a highly accurate heteroduplex scanning technique. With many exons, however, use of single-sample instruments may lead to clerical errors, and routine use of difference analysis is recommended. Common variants can be identified by their melting curve profiles and genotyped with unlabeled probes, greatly reducing the false-positive results common with scanning techniques.
The following articles in journals at HighWire Press have cited this article:
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  C. A. Milbury, J. Li, and G. M. Makrigiorgos COLD-PCR-Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations Clin. Chem., December 1, 2009; 55(12): 2130 - 2143. [Abstract] [Full Text] [PDF]
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 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]
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M. Pichler, M. Balic, E. Stadelmeyer, C. Ausch, M. Wild, C. Guelly, T. Bauernhofer, H. Samonigg, G. Hoefler, and N. Dandachi Evaluation of High-Resolution Melting Analysis as a Diagnostic Tool to Detect the BRAF V600E Mutation in Colorectal Tumors J. Mol. Diagn., March 1, 2009; 11(2): 140 - 147. [Abstract] [Full Text] [PDF]
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 L. S. Kristensen and A. Dobrovic Direct Genotyping of Single Nucleotide Polymorphisms in Methyl Metabolism Genes Using Probe-Free High-Resolution Melting Analysis Cancer Epidemiol. Biomarkers Prev., May 1, 2008; 17(5): 1240 - 1247. [Abstract] [Full Text] [PDF]
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 J.-H. Lin, C.-P. Tseng, Y.-J. Chen, C.-Y. Lin, S.-S. Chang, H.-S. Wu, and J.-C. Cheng Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High-Resolution Melting Analysis J. Clin. Microbiol., March 1, 2008; 46(3): 1090 - 1097. [Abstract] [Full Text] [PDF]
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M. T. Seipp, D. Pattison, J. D. Durtschi, M. Jama, K. V. Voelkerding, and C. T. Wittwer Quadruplex Genotyping of F5, F2, and MTHFR Variants in a Single Closed Tube by High-Resolution Amplicon Melting Clin. Chem., January 1, 2008; 54(1): 108 - 115. [Abstract] [Full Text] [PDF]
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J. Montgomery, C. T. Wittwer, J. O. Kent, and L. Zhou Scanning the Cystic Fibrosis Transmembrane Conductance Regulator Gene Using High-Resolution DNA Melting Analysis Clin. Chem., November 1, 2007; 53(11): 1891 - 1898. [Abstract] [Full Text] [PDF]
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