title: Detection of APC gene deletions using quantitative multiplex PCR of short fluorescent fragments.
abstract: BACKGROUND
  approximately 20% of classic familial adenomatous polyposis (FAP) cases and 70% to 80% of attenuated FAP (AFAP) cases are negative for the APC/MUTYH point mutation. Quantitative multiplex PCR of short fluorescent fragments (QMPSF), a technique for detecting copy number alterations, has been successfully applied to several cancer syndrome genes. We used QMPSF for the APC gene to screen FAP APC/MUTYH mutation-negative families to improve their diagnostic surveillance.
  
  
  METHODS
  we set up and validated APC-gene QMPSF using 23 negative and 1 positive control and examined 45 (13 FAP and 32 AFAP) unrelated members of APC/MUTYH mutation-negative families for copy number alterations. We confirmed the results using multiplex ligation-dependent probe amplification (MLPA). We used different approaches such as sequencing, quantitative real time-PCR (QRT-PCR), and fluorescence in situ hybridization (FISH) to further characterize the identified deletions.
  
  
  RESULTS
  APC QMPSF was capable of detecting deletions with an acceptable variability, as shown by mean values (SD) of allele dosage for the deleted control obtained from intra- and interexperimental replicates [0.52 (0.05) and 0.45 (0.10)]. We detected 3 gross deletions in 13 (23%) of the classic FAP cases analyzed (1 complete gene deletion and 2 partial deletions encompassing exons 9 and 10 and exons 11-15, respectively). No rearrangements were detected in the 32 AFAP cases.
  
  
  CONCLUSIONS
  QMPSF is able to detect rearrangements of the APC gene. Our findings highlight the importance of using a copy number alteration methodology as a first step in the routine genetic testing of FAP families in the clinical setting.
authors.affiliation: Translational Research Laboratory, IDIBELL-Institut Català d'Oncologia, Barcelona, Spain.
authors.forename: Ester
authors.initials: E
authors.lastname: Castellsagué
authors.forename: Sara
authors.initials: S
authors.lastname: González
authors.forename: Marga
authors.initials: M
authors.lastname: Nadal
authors.forename: Olga
authors.initials: O
authors.lastname: Campos
authors.forename: Elisabet
authors.initials: E
authors.lastname: Guinó
authors.forename: Miguel
authors.initials: M
authors.lastname: Urioste
authors.forename: Ignacio
authors.initials: I
authors.lastname: Blanco
authors.forename: Thierry
authors.initials: T
authors.lastname: Frebourg
authors.forename: Gabriel
authors.initials: G
authors.lastname: Capellá
mesh_terms: D011125:Adenomatous Polyposis Coli / Q000235:genetics; D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics*; D001483:Base Sequence; D045647:DNA Glycosylases / Q000235:genetics; D005091:Exons; D005456:Fluorescent Dyes; D017353:Gene Deletion; D018628:Gene Dosage; D017491:Genes, APC; D006579:Heterozygote; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D025601:Adenomatous Polyposis Coli Protein; D005456:Fluorescent Dyes; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase