Molecular Alterations in Familial Colorectal Cancer

............................................................................................................ 9 REVIEW OF THE LITERATURE...................................................................... 10 1. Cancer genetics and epigenetics.................................................................... 10 1.1 Tumour suppressor genes........................................................................... 10 1.1.1 DNA repair genes................................................................................ 11 1.2 Oncogenes ................................................................................................. 11 1.3 Epigenetic factors ...................................................................................... 11 2. Cancer susceptibility ..................................................................................... 12 3. Familial colorectal cancer.............................................................................. 13 3.1 Familial adenomatous polyposis, FAP ....................................................... 13 3.1.1 FAP modifier genes............................................................................. 14 3.2 Hereditary non-polyposis colorectal cancer, HNPCC ................................. 14 3.2.1 Amsterdam and Bethesda criteria ........................................................ 15 4. Genetic instability in colorectal cancer ......................................................... 16 4.1 Features of colorectal tumours arising through the CIN and MSI pathways 17 4.2 Adenoma-carcinoma sequence ................................................................... 17 4.3 Microsatellite instability pathway............................................................... 18 4.3.1 DNA mismatch repair, MMR .............................................................. 19 4.3.2 MSI target genes ................................................................................. 20 5. Prospects of CRC studies .............................................................................. 22 5.1 Candidate low-penetrance CRC susceptibility alleles ................................. 22 5.2 Identification of novel low-penetrance susceptibility alleles ....................... 23 AIMS OF THE STUDY ........................................................................................ 24 MATERIALS AND METHODS .......................................................................... 25 1. Colorectal cancer patients and DNA samples............................................... 25 1.1 Common regions of allelic imbalance (I).................................................... 25 1.2 Genetic analyses of AURKA (II)................................................................. 25 1.3 MLH3 mutation analysis (III)..................................................................... 26 1.4 Genetic analyses of SEMG1 and intergenic T9 repeats (IV) ....................... 26 2. Cancer cell lines (IV) ..................................................................................... 26 3. Analysis methods ........................................................................................... 26 3.1 Common regions of allelic imbalance (I).................................................... 26 3.1.1 Microsatellite marker analysis ............................................................. 27 3.1.2 Comparative genomic hybridization .................................................... 27 3.1.3 Quantitative PCR ................................................................................ 27 3.2 Genetic analyses of AURKA (II)................................................................. 27 3.2.1 AURKA sequencing............................................................................. 27 3.2.2 Amplification analysis by sequencing and fragment analysis............... 28 3.3 MLH3 mutation analysis (III)..................................................................... 28 3.3.1 MLH3 sequencing and denaturing high-performance liquid chromatography (dHPLC) analysis .............................................................. 28 3.4 Genetic analyses of SEMG1 and intergenic T9 repeats (IV) ....................... 28 3.4.1 SEMG1 sequencing and denaturing high-performance liquid chromatography (dHPLC) analysis .............................................................. 28 3.4.2 Evaluation of SEMG1 mRNA levels by quantitative PCR ................... 29 3.4.3 Evaluation of SEMG1 protein levels by Western blotting.................... 29 3.4.4 Sequencing of intergenic T9 repeats.................................................... 29 RESULTS .............................................................................................................. 30 1. Common regions of allelic imbalance (I) ...................................................... 30 2. Analyses of AURKA (II)................................................................................. 30 3. MLH3 mutation analysis (III) ....................................................................... 32 4. Genetic analyses of SEMG1 and intergenic T9 repeats (IV) ........................ 32 DISCUSSION ........................................................................................................ 34 1. Novel colorectal cancer susceptibility loci (I) ............................................... 34 2. AURKA, a novel low-penetrance susceptibility gene (II).............................. 35 3. Role of MLH3 in microsatellite-unstable colorectal cancer (III) ................. 37 4. High frequency of microsatellite mutations in SEMG1 and intergenic T9 repeats (IV) ........................................................................................................ 39 CONCLUSIONS AND FUTURE PROSPECTS.................................................. 42 ACKNOWLEDGEMENTS .................................................................................. 43 REFERENCES...................................................................................................... 45