Epidemiological studies of canine neoplasms with special reference to mutational analysis of p53 gene in canine mammary tumors by PCR-SSCP
Pawar, Sanjay J. (2006) Epidemiological studies of canine neoplasms with special reference to mutational analysis of p53 gene in canine mammary tumors by PCR-SSCP. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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Epidemiological studies of canine neoplasms with special reference to mutational analysis of p53 gene in canine mammary tumors by PCR-SSCP were carried out to know age wise, sex wise and breed wise incidence of neoplastic conditions in canine and to study the mutations in exon 4 and exon 8 of p53 gene segment in canine mammary tumors. The epidemiological study was conducted by analyzing available data of last ten years (1996-2005) of 175 specimens of canine neoplasms at Department of Pathology, Veterinary College, Anand and PCR-SSCP based mutational analysis p53 gene segment was carried out on 16 canine mammary tumor specimens collected at Department of Surgery, Veterinary College, Anand and various clinics during surgical removal. The mutations were further confirmed by direct sequencing of PCR products by using Gene specific primers. During last ten years (1996-2005), Department of Pathology, Veterinary College, Anand has received 175 specimens of canine neoplasms for histopathology/ biopsy examinations. Data analysis of 175 specimens revealed occurrence of neoplastic conditions more in female (65.14%) than male (34.85%) dogs. Highest risk of development of various tumors was observed at age group between 7-9 years of age followed by 10-12 years, 4-6 years and 0-3 years. Frequency of occurrence of neoplasms was observed more in Alsatian breed (25.71%) followed by Pomerian (22.85%), Doberman (12%), Labrador (10.28%) and Mongrel (6.85%). Benign neoplastic conditions were observed in 51.42% cases while malignant neoplasms were observed in 48.57 % cases. Rate of malignancy was noted higher in Alsatian, Pomerian and Doberman breed whereas, Labrador and Mongrel breed showed majority of benign cases. Age group between 4-12 years shows majority of malignant cases and mammary gland tumors in females was deciding factor. Maximum malignancy was observed in the age group between 7 to 9 years of age followed by 10 to 12 years. Adenocarcinoma was the most frequently observed neoplasm followed by fibroma, transmissible veneral granuloma, basal cell carcinoma and squamous cell carcinoma. Skin, mammary gland and genital organs were the most common sites for the neoplasms. The tumors of the muscles, bones, urinary tract and gastrointestinal tract showed relatively low incidence. Clinical diagnosis of 16 mammary gland tumors used for p53 gene mutational analysis showed more incidences in Alsatian breed followed by Pomerinian and Doberman. Majority of the mammary tumors were occurred in the age group between 7-10 years (14/16). All of the tumors were observed in adult bitches. Risk of mammary tumor was greater in non spayed bitches (13/16) as compared to spayed bitches (3/16) indicating that hormonal imbalance may be major risk factor. Gross morphological examination of mammary tumors revealed majority of the tumors as spherical, ovoid or round and were nodular with hard consistency, some of which were contained inflammatory exudate. Fourth and fifth pair of mammary glands were the common mammary tumors occurring lands. The size of tumor was ranging from peanut to coconut size measuring from 1 to 12cm in diameter. Histopathological examinations showed six conditions of benign nature while remaining 10 were of malignant nature. Among the benign tumors, mixed tumor involving myoepithelial cells, cartilage, fibrous connective tissue and glandular epithelial cells were in five cases with single case of fibroadenoma. In malignant neoplasms adenocarcinoma (solid and papillary) and squamous cell carcinoma observed in nine and one case respectively. The exon 4 and exon 8 of p53 tumor suppressor gene was investigated for mutations in 16 spontaneous canine mammary neoplasms using PCR-SSCP with direct sequence analysis of PCR products. Genomic DNA was successfully extracted from 15 out of 16 tissues. PCR fragments having expected size of 233 bp were obtained from all 15 DNA samples. But PCR failed to amplify the target sequence with primer exon 8 inspite of using appropriate PCR conditions. Fifteen samples were screened for mutation detection on exon 4 of the p53 tumor suppressor gene by PCR-SSCP. Analysis revealed that 5 (30.3%) of 15 canine mammary tumors displayed aberrantly migrating bands indicative of a p53 gene mutation. Variation in migration pattern in SSCP bands was obtained in sample No. 1 (adenocarcinoma), 5 (myoepithelioma), 7 (benign mixed), 13 (myoepithelioma) and 14 (papillary adenocarcinoma). Mutations detected by PCRSSCP were further confirmed by sequence analysis in 3/5 samples. Sequence analysis of 3 tumor samples revealed 2 mutations in exon 4. One missense mutation on codon 106 (GCC→GGC) was found in all three samples. Whereas Sample No. 13 presented one silent mutation at codon 58 (GAT→GAA). The silent mutation in sample No. 13 was due to heterozygosity at third nucleotide (T) on codon 58. Mutations were present on highly conserved domain of p53 tumor suppressor gene suggesting importance of the region in apoptosis. These results demonstrated the involvement of p53 gene mutations in the development of the canine mammary tumors.
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