High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene
1 Department of Clinical Pathophysiology, Medical Genetics Unit, University of Florence, Florence, Italy
2 Department of Surgery, University of Siena, Siena, Italy
3 Current Address: Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, USA
4 Department of Clinical Pathophysiology, Clinical Biochemistry Unit, University of Florence, Florence, Italy
5 Department of Clinical Pathophysiology, Surgery Unit, University of Florence, Florence, Italy
6 Fiorgen Foudation for Pharmacogenomics, Sesto Fiorentino, Italy
BMC Cancer 2011, 11:305 doi:10.1186/1471-2407-11-305Published: 21 July 2011
MUTYH-associated polyposis (MAP) is an autosomal recessive form of intestinal polyposis predisposing to colorectal carcinoma. High resolution melting analysis (HRMA) is a mutation scanning method that allows detection of heterozygous sequence changes with high sensitivity, whereas homozygosity for a nucleotide change may not lead to significant curve shape or melting temperature changes compared to homozygous wild-type samples. Therefore, HRMA has been mainly applied to the detection of mutations associated with autosomal dominant or X-linked disorders, while applications to autosomal recessive conditions are less common.
MUTYH coding sequence and UTRs were analyzed by both HRMA and sequencing on 88 leukocyte genomic DNA samples. Twenty-six samples were also examined by SSCP. Experiments were performed both with and without mixing the test samples with wild-type DNA.
The results show that all MUTYH sequence variations, including G > C and A > T homozygous changes, can be reliably identified by HRMA when a condition of artificial heterozygosity is created by mixing test and reference DNA. HRMA had a sensitivity comparable to sequencing and higher than SSCP.
The availability of a rapid and inexpensive method for the identification of MUTYH sequence variants is relevant for the diagnosis of colorectal cancer susceptibility, since the MAP phenotype is highly variable.