title: Establishment of a PCR analysis method for canine BRCA2.
abstract: BACKGROUND
  Mammary tumors are the most common tumor type in both human and canine females. In women, carriers of mutations in BRCA2, a tumor suppressor gene product, have a higher risk of breast cancer. Canine BRCA2 has also been suggested to have a relationship with mammary tumors. However, clearly deleterious BRCA2 mutations have not been identified in any canine mammary tumors, as appropriate methods to detect mutations or a consensus BRCA2 sequence have not been reported.
  
  
  RESULTS
  For amplification and sequencing of BRCA2, we designed 14 and 20 PCR primer sets corresponding to the BRCA2 open reading frame (ORF) and all 27 exons, respectively, including exon-intron boundaries of the canine BRCA2 regions, respectively. To define the consensus canine BRCA2 ORF sequence, we used established methods to sequence the full-length canine BRCA2 ORF sequence from two ovaries and a testis obtained from individual healthy mongrel dogs and partially sequence BRCA2 genomic sequences in 20-56 tumor-free dogs, each aged over 6 years. Subsequently, we compared these sequences and seven previously reported sequences, and defined the most common base sequences as the consensus canine BRCA2 ORF sequence. Moreover, we established a detection method for identifying splicing variants. Unexpectedly, we also identified novel splicing variants in normal testes during establishment of these methods.
  
  
  CONCLUSIONS
  The present analysis methods for determining the BRCA2 base sequence and for detecting BRCA2 splicing variants and the BRCA2 ORF consensus sequence are useful for better understanding the relationship between canine BRCA2 mutation status and cancer risk.
authors.affiliation: Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Kitasato University, Aomori 034-8628, Japan. yyoshika@vmas.kitasato-u.ac.jp
authors.forename: Yasunaga
authors.initials: Y
authors.lastname: Yoshikawa
authors.forename: Masami
authors.initials: M
authors.lastname: Morimatsu
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ochiai
authors.forename: Kento
authors.initials: K
authors.lastname: Okuda
authors.forename: Takahiro
authors.initials: T
authors.lastname: Taoda
authors.forename: Seishiro
authors.initials: S
authors.lastname: Chikazawa
authors.forename: Asako
authors.initials: A
authors.lastname: Shimamura
authors.forename: Toshinori
authors.initials: T
authors.lastname: Omi
authors.forename: Makoto
authors.initials: M
authors.lastname: Bonkobara
authors.forename: Koichi
authors.initials: K
authors.lastname: Orino
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Watanabe
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D024682:BRCA2 Protein / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods* / Q000662:veterinary; D004283:Dog Diseases / Q000175:diagnosis / Q000235:genetics; D004285:Dogs; D005260:Female; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D054643:INDEL Mutation; D008297:Male; D015674:Mammary Neoplasms, Animal / Q000175:diagnosis / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010053:Ovary / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013737:Testis / Q000378:metabolism
chemical_list: D024682:BRCA2 Protein