The Molecular Pathologies of BRCA1 in Ovarian Cancer Patients From the West of Scotland

Dursun, Ahmet (2001) The Molecular Pathologies of BRCA1 in Ovarian Cancer Patients From the West of Scotland. PhD thesis, University of Glasgow.

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Abstract

Ovarian cancer is one of the most common female malignancies and is the forth leading cause of cancer death in the western world. It is estimated that 3-13% of all ovarian cancers are familial and the tumour suppressor gene, BRCA1, is responsible for almost 90% of all these familial cases. BRCA1 is a tumour suppressor gene which spans 100 kb on chromosome 17q21 and contains 24 exons. The 7.8 kb mRNA encodes a protein of 1863 amino acids. The structure of BRCA1 is not strikingly homologous to other known molecules apart from the presence of a ring zinc finger domain and a 10 amino acid granin motif which are thought to be significant due to fact that they both are conserved in humans and other species studied. The overall aims of this study were to screen and characterise mutations in the BRCA1 gene using genomic DNA obtained from ovarian cancer patients from the West of Scotland. As a result of the information obtained from this study, genetic counselling would be possible for those who have inherited the mutations and for those who have a high risk of developing the same condition. In this study, 48 ovarian cancer patients with a family history of breast and/or ovarian cancer were screened for germline mutations in the BRCA1 gene. The strategy for germline mutation analysis involved the initial amplification of the BRCA1 gene by polymerase chain reaction (PCR) using genomic DNA as a template. After initial amplification, the product was electrophoresed in an 1% agarose gel in order to check for any abnormal alteration in size, quality and quantity of product. If no alteration was identified, further screening of the gene was carried out using the single strand conformational analysis (SSCP) and the protein truncation test (PTT). Any differences in the band pattern, when compared with negative controls (in both techniques) were fully characterised by automated DNA sequencing analysis. Using the strategy described above, 5 different alterations were detected on analysis of BRCA1 coding sequences. Of these, two were novel missense mutations, two were polymorphisms and one was an unclassified variant. The first novel missense mutation was g.3771 A>T, changing serine to cysteine at amino acid position 1218. This mutation is in the granin site at the amino acid positions 1214-1223 and the leucine zipper at amino acid positions 1209-1230. It is well known that cysteine has a very important role in stabilising the three-dimensional protein structure. Two cysteine residues in different parts of the polypeptide chain but adjacent in the three-dimensional structure of a protein can be oxidised to form a disulfide bridge. Disulfide bridges usually occur among secreted proteins and make them less susceptible to degradation. The second novel missense mutation was g.1380 G>A, changing glutamic acid to lysine at the amino acid position 421. This mutation's effect on the BRCA1 protein is unknown. However, this amino acid is conserved in the murine BRCA1 sequence. Two different polymorphisms were also detected in BRCA1 coding sequence. Patient no 28 is thought to be homozygous for polymorphism g.3667 A>G was also checked for possible large deletion within the exon 11. The first polymorphism, g.3667 A>G, was found in four out of forty-eight patients and the second polymorphism, g.4427 T>C, was found in six out of forty-eight patients. These two polymorphisms are the most frequently found in ovarian cancer cases and the normal population. Although, the polymorphisms are generally thought to be a variation in the gene sequence, histopathologicai studies suggest that the aggressiveness of tumour progress, histological type and survival rate may be related to the host variation. In order to establish the normal population frequency, one hundred samples from normal population was also screened. Six out of one hundred for polymorphism g.3667 A>G and 10 out of 100 for polymorphism g.4427 T>C were found positive for these polymorphisms. An insertion of 12 bp of DNA was also found in intron 20 (5'- GTATTCCACTCC-3') at IVS20+60. Three studies suggested that this variant is not present in any of the subject in the control population analysed. It was also reported that this variant did not modify the correct splicing of exon 19-21 by analysing the cDNA obtained from the patient's peripheral blood. It is possible that a relatively large insertion in a region of non-repetitive DNA might affect the kinetics of splicing and result in a lower level of normal mRNA from the mutant allele.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: G Lanyon
Keywords: Genetics, Oncology
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-76086
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:51
Last Modified: 19 Nov 2019 16:51
URI: http://theses.gla.ac.uk/id/eprint/76086

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