Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors.
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.
Yoshikawa
Yasunaga
Y
Laboratory of Cytology and Histology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan.
Morimatsu
Masami
M
Ochiai
Kazuhiko
K
Nagano
Masashi
M
Tomioka
Yukiko
Y
Sasaki
Nobuo
N
Hashizume
Kazuyoshi
K
Iwanaga
Toshihiko
T
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
D024682:BRCA2 Protein; D017931:DNA Primers; D018076:DNA, Complementary; D004273:DNA, Neoplasm