Molecular Pathology of the hMSH2 Mutator Gene and Its Transcripts in Patients With Colorectal Cancer in the West of Scotland

Davoodi-Semiromi, Abdoreza (1998) Molecular Pathology of the hMSH2 Mutator Gene and Its Transcripts in Patients With Colorectal Cancer in the West of Scotland. PhD thesis, University of Glasgow.

Abstract

Colorectal cancer (CRC) is the second or third most common cancer in Western countries, and its incidence is rising. Among the hereditary forms Familial Adenomatous Polyposis (FAP) is a rare, dominantly inherited disease which is caused by germ-line mutations of the Adenmatous Polyposis Colorectal Cancer (APC) gene. A second form of CRC that shows familial aggregation is Hereditary Non-Polyposis Colorectal Cancer (HNPCC). The main aims of this study were: (i) to assess microsatellite instability in the tumour of patients with colorectal carcinoma and (ii) to identify germ-line mutations in two mismatch repair genes, using a variety of techniques. To do this, in two groups of unselected patients, 30 pairs of normal/tumour DNAs and 47 RNA samples were made available for microsatellite instability studies and mutation analysis, respectively. Nine polymorphic markers were chosen for microsatellite instability analysis including: D13S160, D2S119, D8S282, D18S34, 635/636, D2S123, LPLCA- A, D2S378, LPLCA, B. Using this approach, 30% (9/30) of the tumours exhibited instability at one or more loci (RER+). There was no significant difference between age and sex in patients with or without microsatellite instability. Using the reverse transcriptase polymerase chain reaction (RT-PCR), the entire coding sequence of the hMSH2 gene was amplified in either two or three overlapping segments. By employing RNA and DNA based techniques a number of germ-line mutations were found in the hMSH2 and hLMH1 genes. The mutations included: one novel splice site mutation responsible for exon 15 skipping, two novel deletions of exons 2-6 and 2-8 from the mRNA transcripts, four different mRNA deletions at different sites in the hMSH2 gene, one missense mutation at exon 6 and three intronic mutations at positions +9, -10 and +19 in intron 1, 11, and 14 respectively. A variation of the published sequence was also found at 3' end of the untranslated region in the hMSH2 gene. None of the families in this study were large enough for linkage analysis. In one HNPCC family, one splice site mutation was found at position +5 in intron 15. This resulted in exon 15 skipping from transcripts and produced a truncated hMSH2 protein. This germ-line mutation was present in 4 affected members of the family, but was not present in the healthy members of the family or in 177 other colorectal cancer patients. The individual who bears this germ-line mutation showed a RER+ phenotype in the tumour sample. A patient whose tumour was RER+ had a deletion of exons 2 to 6 inclusive which resulted in an out-of- frame deletion of codons 71-359. This deletion created a premature termination codon within exon 7 at nucleotide position +3 from the splice site. No genomic mutations were found which could account for the exon skipping. No other member of the family was available for further investigation. A patient with a tumour showing the RER+ phenotype had a deletion of exons 2 through exon 8 inclusive which resulted in an out-of- frame deletion of codons 71-466. This deletion also created a premature termination codon within exon 9 at position +12 from the splice site. No genomic alterations were found which could account for the exon skipping. This individual was the only member of the family available for investigation. Four different partial mRNA deletions in different exons were found. Three of them produced premature termination codons. No alteration could be found by sequencing the genomic splice sites, or in the corresponding exons. One of the partial deletions was found in four unrelated individuals. The breakage points of the aberrant mRNAs were not located at the splice site junctions in any case. In order to clarify whether or not these partial mRNAs deletions are normal, RT-PCR from 20 healthy individuals was carried out. The whole coding sequences of the hMSH2 gene were amplified in three overlapping fragments and no extra bands were observed. These results indicate that none of the mRNA deletions reported in this study are common normal variations. A T to C transition was found in a short polypyrimidine tract in intron 12 at nucleotide position -6. Particularly, short poylypyrimidine tracts at the 3' end of the intron have been shown to effect mRNA splicing (Roscigno et al. 1993), but this mutation had no affect on the mRNA splicing process, since no short transcript was found. It was, however, found in 15% of the normal population when 86 normal chromosomes were tested. A G to A transition was also found at codon 322 in exon 6 which changes glycine to asparatic acid. Since glycine is a non- polar neutral amino acid and asparatic acid is an acidic one, it is likely that the polarity of the protein will be changed.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J M Connor
• Keywords: Genetics, Molecular biology, Oncology
• Date of Award: 1998
• Depositing User: Enlighten Team
• Unique ID: glathesis:1998-75416
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 20:11
• Last Modified: 19 Nov 2019 20:11
• URI: https://theses.gla.ac.uk/id/eprint/75416