The study of canine mammary tumors with special reference to mutations in p53 tumor suppressor gene by PCR-SSCP
Dhaygude, Vitthal (2006) The study of canine mammary tumors with special reference to mutations in p53 tumor suppressor gene by PCR-SSCP. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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The present study entitled “The study of canine mammary tumors with special reference to mutations in p53 tumor suppressor gene by PCR-SSCP” was conducted to know the prevalence of canine neoplasms as well as mammary tumors by analyzing the data available at Department of Pathology, Veterinary College, Anand and to investigate the role of p53 gene in molecular pathogenesis of canine mammary tumors by mutational analysis using molecular technique PCR-SSCP. Epidemiological studies on canine neoplasms with special reference to mammary tumors were carried out by obtaining the autopsy/ biopsy records of last five years (January 2001- December 2004). Information like breed, age, sex and histopathological type was analyzed and classified year wise, age wise and breed wise to ascertain the susceptibility of the canine mammary tumors. For study of mutations in the p53 tumor suppressor gene a total of sixteen cases of spontaneously occurring canine mammary tumors were collected by personal visits at clinics, hospitals and Dept. of Surgery at Veterinary College, Anand as well as Bombay Veterinary College, Mumbai with detailed history and gross observations of individual case. The tumor tissues were collected in two sets of vials from each individual case; one in 10% neutral buffered formalin for histopathological examination and other in liquid nitrogen (- 196°C) for DNA extraction and subsequent PCR- SSCP studies for detection of mutations in Exon 5, Exon 6 and Exon 5 to 7 of p53 tumor suppressor gene using gene specific primers. During the period from January 2001 to December 2005, a total of 124 autopsy / biopsy samples of various canine neoplasms were recorded. Among these, tumors of skin and soft tissues were recorded highest 74 (59.67 %) followed by mammary gland tumors 43 (34.67 %), transmissible venereal granulomas 3 (2.4 %) and tumors each of ovary and testes 2 (1.6 %). Among different breeds of canines, these neoplasms were recorded highest in Alsatian 27.41 % followed by Pomeranian 21.77%, Labrador 12.90 %, Mongrels 12.10%, Doberman 8.87 %, Spitz 8.5 %, Great-Dane, Cocker Spaniel, Tibetan Mastiff each of 2.42 % and Daschounds 1.6 %. Majority of tumors (70.15%) were recorded between 5-12 years of age. Overall there were 67.7 % tumors of benign nature while remaining 32.3 % tumors were malignant. A total of 43 mammary gland tumors were recorded during the period January 2001 to December 2005. All the tumors were recorded in female dogs with near equal frequency during last five years. Among different age groups the canine mammary tumors were recorded highest in the age group of 5 to 8 years (41.9%) followed by 9 to 12 years (37.2%), 13 to 16 years (11.6%) and 0 to 4 years (9.3%). Majority of the neoplasms (79.1%) were recorded between 5 to 12 years of age. Alsatian 37.2 % was found to be most susceptible to develop mammary tumors followed by Pomeranian 25.6 %, Doberman 13.9 %, Labrador 9.3 %, Spitz 6.9 % and Mongrels 6.9 %. Histopathologically out of a total 43 mammary tumors 48.8% were of benign nature while remaining 51.2 % were malignant. Benign neoplastic conditions recorded were adenoma simple & papillary (4), fibroadenoma (5) and mixed tumors involving myoepithelial cells, bone, cartilage and fibrous connective tissue (12). Malignant neoplastic conditions were adenocarcinoma solid, tubular and papillary (16) and mixed malignant tumors involving bone, cartilage and fibrous connective tissue (6). Out of a total 16 cases of mammary tumors collected for study of p53 gene mutations; majority of dogs (13/16) developed mammary tumors between the age of 8 to 10 years. Only three were spayed previously while in other 13 cases ovariohysterectomy was done at the time of surgical removal of the tumor. There was variation in size of the mammary tumor ranging between 2 cm X 1.5cm X 1 cm to 21cm X 15cm X 14cm. Shape of the tumors varied from ovoid, elongated, rounded to irregularly nodular. Maximum tumors were hard and firm in consistency. Mammary glands of the right side were found affected more in comparison to left side. The fourth (caudal abdominal) and fifth (inguinal) pairs of mammary gland were found more susceptible to develop mammary tumor. Histopathologically all the 16 tumors revealed six conditions of benign nature while remaining ten were of malignant nature. The benign neoplastic conditions diagnosed were mixed benign tumor involving myoepithelial cells, cartilage, fibrous connective tissue and glandular epithelial cells (5) and fibroadenoma (1). The malignant neoplastic conditions were diagnosed in 10 cases. They were adenocarcinoma (solid and papillary) in nine cases and squamous cell carcinoma in one case. DNA was extracted from 15 out of total 16 tumor samples by proteinase K and phenol method. PCR amplification of exon 5 and exon 7 of p53 tumor suppressor gene was obtained in all the 15 samples with separate gene specific primers for respective regions. The amplification of exon 6 could not be made by PCR with different annealing temperatures of 50 0C, 55 0C, 60 0C and 660C. PCR using Hot start Taq Polymerase was also not successful. PCR- SSCP profile revealed aberrantly migrating bands suggestive of mutations in amplified exon 5 (codon 117-175) of p53 tumor suppressor gene in 20% cases of spontaneous canine mammary tumors. Sequencing in one case of papillary adenocarcinoma out of three with mutant bands revealed four mutations. These were represented as two silent mutations; one on codon No. 117 (CTC→ CTT), second on codon 137 (CCA→ CCG) as well as two missense mutations on codon 157 (ACC→ACA) and codon 158 (GAG→AAG) which resulted amino acid change from threonine to lysine and glutamine to lysine. All the above changes in nucleotides were heterozygous. Screening of amplified exon 5 (from codon 165) to exon 7 (upto codon 241) of p53 gene segment for detection of mutations with PCR-SSCP revealed similar band pattern for all samples suggesting no mutations when primer E 5-7 was used. The PCR-SSCP studies suggested that mutations in p53 gene might be invariably involved leading to its inactivation in canine mammary neoplastic condition.
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