title: High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene.
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.
authors.affiliation: Department of Clinical Pathophysiology, Medical Genetics Unit, University of Florence, Florence, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Tricarico
authors.forename: Francesca
authors.initials: F
authors.lastname: Crucianelli
authors.forename: Antonio
authors.initials: A
authors.lastname: Alvau
authors.forename: Claudio
authors.initials: C
authors.lastname: Orlando
authors.forename: Roberta
authors.initials: R
authors.lastname: Sestini
authors.forename: Francesco
authors.initials: F
authors.lastname: Tonelli
authors.forename: Rosa
authors.initials: R
authors.lastname: Valanzano
authors.forename: Maurizio
authors.initials: M
authors.lastname: Genuardi
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D045647:DNA Glycosylases / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D020022:Genetic Predisposition to Disease / Q000235:genetics; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D044483:Intestinal Polyposis / Q000235:genetics; D009691:Nucleic Acid Denaturation; D018807:Polymorphism, Single-Stranded Conformational
chemical_list: D004247:DNA; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase