Investigation of the mutational consequences of defective mismatch repair in humans

• Main Author: Bacon, Andrea Louise
• Published: University of Edinburgh 2002
• Subjects: 616.994
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.641185

One explanation why sequences are mutated frequently in MMR deficient colerectal cancer (CRC) is they are inherently prone to mutations. To investigate this hypothesis, MMR deficient cells (1b1-1260, 1b1-1261) derived from normal tissue (B-lymphocytes), have been utilised. A SP-PCR technique was employed and demonstrated that 1b1-1261 and 1b1-1260 exhibit microsatellite instability. At the D2S123 locus, the sequence of the constitutional allele itself is shown to be a determinant of instability in the absence of MMR activity. A combination of SP-PCR, allele cloning and restriction digest methods were employed to determine whether differences in mutation frequencies of the TGFBR2 and BAX genes in MMR deficient CRC, may be due to differential stability in the presence of MMR defects. MMR deficiency is shown to be associated with an excess of mutations at the poly(A)₁₀ tract of TGFBR2 in cells of a normal lineage. Surprisingly, at the poly(G)₈ tract of the BAX gene, an excess of mutations is not observed. The inherent stability of Wnt pathway gene sequences frequently mutated in MMR proficient CRC was addressed. Mutations of CTNNB1 and APC are observed in a small proportion of MMR deficient CRCs. However, despite rigorous mutation analysis of exon 3 of the CTNNB1 gene, there is no indication that this region is particularly prone to mutation consequent of MMR defects. Extensive analysis of inherent mutability was also performed for the APC gene. Interestingly, an unexpectedly high level of instability was detected in exon 15 in both normal and cancer cells, proficient in MMR. But in 1b1-1261 the instability was markedly lower suggestive of complex relationship between MMR defects and mutation frequency at this locus.