Baharuddin, Puteri Jamilatul Noor Megat; (1997) Molecular cytogenetic analysis applied to multiple endocrine neoplasia type 1 and colorectal carcinogenesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The thesis covers two main topics. Karyotypic and molecular cytogenetic analysis of a series of lymphoblastoid cell lines from affected members of families segregating for the autosomal dominant condition, multiple endocrine neoplasia type 1 (MEN1) were carried out. Cytogenetic investigation of 42 MEN1 cell lines showed no observable chromosomal abnormalities involving the region of the MEN1 gene at 11q13. DNA extraction from 4 yeast artificial chromosome (YAC) clones flanking the region of the MEN1 gene, 32AF1, 9FF1, 17IC4 and 16IC1 was performed for the generation of suitable DNA probes. Fluoresecence in situ hybridisation analysis (FISH) using a combination of 2 YAC DNAs, 9FT1 and 17IC4 as probes on interphase nuclei and metaphase chromosomes in 47 MEN1 cell lines showed no detectable microdeletions in the region of the MEN1 gene. The second topic concerns aspects of colorectal carcinogenesis. Detailed cytogenetic analysis in four neoplastic colonic cell lines, AA/C1/SB/10C (transformed adenoma), JW2/33 F31 and LIM 1215 (hereditary carcinoma) and LIM 1899 (sporadic carcinoma) showed diverse chromosomal rearrangements. Single colour FISH using chromosome paints and specific centromeric DNA probes confirmed the origin of the chromosome 18 rearrangements in the transformed adenoma line. For the detection of the DCC (Deleted in Colorectal Carcinoma) gene, FISH analysis using a panel of 7 YAC clones (yA96E4, y36IB10, y26CB8, y39CG7, yl3HE1, y40DH1 and y12FC12) from a contig flanking the DCC gene at 18q21, showed 2 copies of chromosome 18 in the carcinoma lines. The AA/Cl/SB/10C cell line showed evidence of chromosome rearrangement and loss involving chromosome 18. A molecular approach using the polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) analysis with 3 microsatellite markers on 18q (D18S61), and two for the DCC gene (DCC1 and DCC2), showed intragenic DCC deletion in the AA/C1/SB/10C cell line. Molecular cytogenetic analysis of sporadic colorectal carcinomas from 20 patients, showed 1/20 case (5%) loss of heterozygosity (LOH) of 18q or loss of the DCC gene by interphase FISH on fixed normal and tumour smears. The microsatellite marker, D18S61, distal to the DCC gene locus was able to detect LOH of 18q in 2/20 cases (10%) and replication error (RER) in an additional 3/20 cases (15%) by the PCR-SSCP method. Using another marker, D18S8-M2 which detects an Msp1 polymorphic site within the DCC gene, LOH on 18q was found in a further single case 1/20 (5%) by restriction fragment length polymorphism (RFLP) analysis. Using markers proximal to the DCC locus, D18S46 and D18S474, LOH on 18q were not detected. These molecular changes (LOH and RER) were found in the stage II or Duke's B tumours and this association will provide information on the potential use of chromosome 18q loss as a prognostic marker in non-metastatic colorectal cancer.

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