Nonfluorescent denaturing HPLC-based primer-extension method for allele-specific expression: application to analysis of mismatch repair genes.
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. .
Aceto
Gitana M
GM
Department of Human Movement Sciences, University G d'Annunzio, Chieti, Italy.
De Lellis
Laura
L
Catalano
Teresa
T
Veschi
Serena
S
Radice
Paolo
P
Di Iorio
Angelo
A
Mariani-Costantini
Renato
R
Cama
Alessandro
A
Curia
Maria Cristina
MC
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis* / Q000235:genetics; D000483:Alleles; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D053843:DNA Mismatch Repair; D017931:DNA Primers / Q000032:analysis* / Q000235:genetics; D005456:Fluorescent Dyes; D015870:Gene Expression; D006579:Heterozygote; D006801:Humans; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000032:analysis* / Q000235:genetics; D009687:Nuclear Proteins / Q000032:analysis* / Q000235:genetics; D009691:Nucleic Acid Denaturation
D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; D005456:Fluorescent Dyes; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein