title: Simplifying the detection of MUTYH mutations by high resolution melting analysis.
abstract: BACKGROUND
  MUTYH-associated polyposis (MAP) is a disorder caused by bi-allelic germline MUTYH mutation, characterized by multiple colorectal adenomas. In order to identify mutations in MUTYH gene we applied High Resolution Melting (HRM) genotyping. HRM analysis is extensively employed as a scanning method for the detection of heterozygous mutations. Therefore, we applied HRM to show effectiveness in detecting homozygous mutations for these clinically important and frequent patients.
  
  
  METHODS
  In this study, we analyzed phenotype and genotype data from 82 patients, with multiple (> or = 10) synchronous (19/82) or metachronous (63/82) adenomas and negative APC study (except one case). Analysis was performed by HRM-PCR and direct sequencing, in order to identify mutations in MUTYH exons 7, 12 and 13, where the most prevalent mutations are located. In monoallelic mutation carriers, we evaluated entire MUTYH gene in search of another possible alteration. HRM-PCR was performed with strict conditions in several rounds: the first one to discriminate the heteroduplex patterns and homoduplex patterns and the next ones, in order to refine and confirm parameters. The genotypes obtained were correlated to phenotypic features (number of adenomas (synchronous or metachronous), colorectal cancer (CRC) and family history).
  
  
  RESULTS
  MUTYH germline mutations were found in 15.8% (13/82) of patients. The hot spots, Y179C (exon 7) and G396D (exon 13), were readily identified and other mutations were also detected. Each mutation had a reproducible melting profile by HRM, both heterozygous mutations and homozygous mutations. In our study of 82 patients, biallelic mutation is associated with being a carrier of >/=10 synchronous polyps (p = 0.05) and there is no association between biallelic mutation and CRC (p = 0.39) nor family history (p = 0.63). G338H non-pathogenic polymorphism (exon 12) was found in 23.1% (19/82) of patients. In all cases there was concordance between HRM (first and subsequent rounds) and sequencing data.
  
  
  CONCLUSIONS
  Here, we describe a screening method, HRM, for the detection of both heterozygous and homozygous mutations in the gene encoding MUTYH in selected samples of patients with phenotype of MAP. We refine the capabilities of HRM-PCR and apply it to a gene not yet analyzed by this tool. As clinical decisions will increasingly rely on molecular medicine, the power of identifying germline mutations must be continuously evaluated and improved.
authors.affiliation: Department of Molecular Biology, 12 De Octubre University Hospital, Madrid, E-28041, Spain. isabellv2004@yahoo.es
authors.forename: Isabel
authors.initials: I
authors.lastname: López-Villar
authors.forename: Rosa
authors.initials: R
authors.lastname: Ayala
authors.forename: Jan
authors.initials: J
authors.lastname: Wesselink
authors.forename: Juan Diego
authors.initials: JD
authors.lastname: Morillas
authors.forename: Elena
authors.initials: E
authors.lastname: López
authors.forename: José Carlos
authors.initials: JC
authors.lastname: Marín
authors.forename: José
authors.initials: J
authors.lastname: Díaz-Tasende
authors.forename: Sara
authors.initials: S
authors.lastname: González
authors.forename: Luis
authors.initials: L
authors.lastname: Robles
authors.forename: Joaquín
authors.initials: J
authors.lastname: Martínez-López
mesh_terms: D000236:Adenoma / Q000235:genetics* / Q000473:pathology; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D045647:DNA Glycosylases / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D020022:Genetic Predisposition to Disease; D005838:Genotype; D018095:Germ-Line Mutation / Q000235:genetics*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008297:Male; D008875:Middle Aged; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011379:Prognosis; D055815:Young Adult
chemical_list: D004273:DNA, Neoplasm; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase