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

• Published in: BMC cancer Vol. 11; no. 1; p. 305
• Main Authors: Tricarico, Rossella, Crucianelli, Francesca, Alvau, Antonio, Orlando, Claudio, Sestini, Roberta, Tonelli, Francesco, Valanzano, Rosa, Genuardi, Maurizio
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 21.07.2011; BioMed Central; BMC
• Subjects: Colorectal cancer; Colorectal Neoplasms - genetics; DNA - chemistry; DNA - genetics; DNA Glycosylases - genetics; DNA Mutational Analysis - methods; Gene mutations; Genetic aspects; Genetic Predisposition to Disease - genetics; Genotype; Heterozygosis; Heterozygosity; Heterozygote; Homozygote; HRMA; Intestinal Polyposis - genetics; mutation; MUTYH; Nucleic Acid Denaturation; Physiological aspects; Polymorphism, Single-Stranded Conformational; polyposis; Technical Advance; Italy
• ISSN: 1471-2407; 1471-2407
• DOI: 10.1186/1471-2407-11-305

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.