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Open Access Technical advance

High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene

Rossella Tricarico1, Francesca Crucianelli12, Antonio Alvau13, Claudio Orlando4, Roberta Sestini1, Francesco Tonelli5, Rosa Valanzano5 and Maurizio Genuardi16*

Author Affiliations

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

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BMC Cancer 2011, 11:305  doi:10.1186/1471-2407-11-305

Published: 21 July 2011

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Keywords:
HRMA; MUTYH; colorectal cancer; polyposis; mutation