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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: clinchem.2009.126300v1 55/9/1711    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 Google Scholar Articles by Aceto, G. M. Articles by Curia, M. C. PubMed PubMed Citation Articles by Aceto, G. M. Articles by Curia, M. C.

Nonfluorescent Denaturing HPLC–Based Primer-Extension Method for Allele-Specific Expression: Application to Analysis of Mismatch Repair Genes

¹ Department of Human Movement Sciences, University "G. d'Annunzio," Chieti, Italy;² Center of Excellence on Aging, "G. d'Annunzio" University Foundation, Chieti, Italy;³ Department of Oncology and Neurosciences, University "G. d'Annunzio," Chieti, Italy;⁴ Department of Experimental Pathology and Microbiology, University of Messina, Messina, Italy;⁵ Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Tumori and IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy;⁶ Department of Medicine and Sciences of Aging, University "G. d'Annunzio," Chieti, Italy.

^aAddress correspondence to this author at: Department of Oncology and Neurosciences, University "G. d'Annunzio," via dei Vestini, 66100 Chieti, Italy. Fax +39-08713554110; e-mail cama{at}unich.it.

Background: Altered germline expression of genes may represent a powerful marker of genetic or epigenetic predisposition to cancer or other diseases.

Methods: We developed and validated a method of nonfluorescent primer extension that uses a single dideoxynucleotide and denaturing HPLC (DHPLC) to analyze the relative allele expression. We devised 5 independent assays for measuring allele-specific expression (ASE) to exploit different markers of mismatch repair genes MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)]. We initially confirmed method reproducibility with genomic DNA (gDNA) from individuals heterozygous for a frequent single-nucleotide polymorphism in the MLH1 gene. After this preliminary validation with gDNA, we confirmed assay reproducibility with cDNA templates from control individuals. Relative allele expression was estimated by comparing the heights of the peaks corresponding to the 2 alleles. Results obtained with gDNA templates were used to normalize cDNA results.

Results: With these DHPLC-based primer-extension assays, we detected and confirmed a 5-fold imbalance in MLH1 allele expression in a mutation-negative patient with hereditary nonpolyposis colorectal cancer and in another patient with a modest degree of imbalance in MLH1 expression. Among control individuals, the relative expression of MLH1 alleles displayed a narrow range of variation.

Conclusions: Independent DHPLC-based primer-extension assays for measuring and confirming ASE can be developed for different sequence variants of interest. This DHPLC application provides a cost-effective method for detecting ASE in cases for which conventional screening fails to detect pathogenic mutations in candidate genes and may be applicable for confirming ASE revealed by other methods, such as those used for transcriptome-wide analyses. .