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Molecular analysis of germline and somatic mutation in adenomatous polyposis coli

Gayther, Simon Andrew.; (1994) Molecular analysis of germline and somatic mutation in adenomatous polyposis coli. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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The gene responsible for the dominantly inherited cancer syndrome adenomatous polyposis coli (APC) has recently been isolated. APC confers a predisposition to colorectal cancer on affected individuals and is characterised by the development of multiple adenomatous polyps throughout the colon and rectum during adolescence and early adulthood. In addition, frequent allele loss at chromosome 5q21-22 encompassing the APC gene region, in colorectal tumours from individuals with sporadic colorectal cancer has implicated the APC gene in the general molecular pathogenesis of colorectal cancer. The discovery of large, often cytogenetically visible alterations in APC patients was instrumental in localisation of the APC gene to chromosome 5q21-22 and in its eventual isolation. To identify APC patients potentially harbouring large alterations around the APC locus, a long range physical map encompassing approximately 3.5Mb was constructed using pulsed field gel electrophoresis and Southern blot hybridisation analysis with cDNA fragments representing APC and MCC genes and a tightly linked cosmid marker EF5.44, as hybridisation probes. This physical map was then used for comparative analysis of DNA derived from a panel of APC patients. Three common colorectal cancer cell lines and two individuals from a breast cancer susceptibility family with a large, cytogenetically characterised rearrangement on chromosome 5q were also analysed in a similar manner. Aberrant hybridisation patterns were observed in individuals from two unrelated families and were further characterised by fluorescence in situ hybridisation analysis using yeast artificial chromosome probes from a contig spanning the APC gene region. Patients which had been used in pulsed field gel electrophoresis and FISH analysis, together with a series of well documented APC familial cases and new mutation cases, were analysed for mutations within a 3kb region of the APC gene. Germline variants were detected using the polymerase chain reaction (PCR) combined with single-strand conformation polymorphism analysis (SSCP) and a heteroduplex mismatch assay. This was followed by direct sequencing of the critical regions to precisely characterise any mutations. APC patients were selected to represent a range of phenotypic criteria in an attempt to evaluate the relationship between the nature of the mutation detected and the disease phenotype. In addition similar analysis was performed on available tumour material from these individuals to search for somatic mutations within the same region of APC. Germline mutations were detected in 14 of 22 unrelated APC patients of which 10 were new mutation cases. Mutations detected in 7 (of 12) families allowed vertical transmission and segregation of the alteration with the disease to be demonstrated in 5 families. APC cases for whom the disease causing mutation was identified were assessed for the severity of the APC phenotype based on an early age at presentation or cancer development and the profusion of adenomatous polyps throughout the colorectum. This enabled correlation to be drawn between the position of the mutation and severe forms of APC. Colorectal tumours from four familial APC cases and two new mutation cases were also available for parallel analysis. APC alterations consisting of intragenic mutation, allelic loss or unique singlestrand/heteroduplex conformers were detected in 17 of 30 colorectal adenomas and one carcinoma. Finally, during the course of PCR-SSCP analysis three rare, non-disease causing variants and one common polymorphism were additionally characterised.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Molecular analysis of germline and somatic mutation in adenomatous polyposis coli
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Health and environmental sciences; Adenomatous polyposis coli
URI: https://discovery.ucl.ac.uk/id/eprint/10101761
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