title: Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors.
abstract: OBJECTIVE
  To establish novel polymorphic markers for analysis of loss of heterozygosity (LOH), so as to study the possible involvement of BRCA2 in mammary tumors obtained from dogs.
  
  
  METHODS
  Blood samples, mammary gland specimens, or mammary tumors from 3 tumor-bearing dogs and 10 tumor-free dogs.
  
  
  METHODS
  Nucleotide sequence analysis was performed with a DNA autosequencer. Loss of heterozygosity analysis was performed for markers established in the present study. The expression level of canine BRCA2 was quantified by real-time PCR analysis.
  
  
  RESULTS
  3 novel microsatellite markers with high heterozygosity rates (> 50%) were established, and the previously reported marker for canine BRCA2 gene locus was improved. These markers were used for the analysis of DNA from formalin-fixed and paraffin-embedded samples. By use of these markers, LOH in canine BRCA2 was identified as a result of recombination. In mammary tumor DNA that corresponded to the LOH-positive dog, the level of canine BRCA2 expression was decreased compared with that of nonneoplastic mammary gland tissue; the open reading frame contained 4 missense variations, 1 insertion variation, and 1 silent variation, some of which were localized to functional domains.
  
  
  CONCLUSIONS
  3 novel polymorphic markers were developed for LOH analysis of canine BRCA2 and identified a dog with LOH with some variations in the functional domains. These markers could be useful for assessing the relevance of BRCA2 variation in mammary tumors of dogs.
authors.affiliation: Laboratory of Cytology and Histology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan.
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: Masashi
authors.initials: M
authors.lastname: Nagano
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Tomioka
authors.forename: Nobuo
authors.initials: N
authors.lastname: Sasaki
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Hashizume
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Iwanaga
mesh_terms: D000818:Animals; D024682:BRCA2 Protein / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics; D004283:Dog Diseases / Q000097:blood / Q000235:genetics*; D004285:Dogs; D005260:Female; D014644:Genetic Variation; D019656:Loss of Heterozygosity; D015674:Mammary Neoplasms, Animal / Q000097:blood / Q000235:genetics*; D016366:Open Reading Frames
chemical_list: D024682:BRCA2 Protein; D017931:DNA Primers; D018076:DNA, Complementary; D004273:DNA, Neoplasm