Molecular genetic analysis of DNA alterations in Irish colorectal cancer

Colorectal cancer is the most common cause of death due to malignancy in nonsmokers in the western world. In 1995 there were 1,757 cases of colon cancer in Ireland. Most colon cancer is sporadic, however ten percent of cases occur where there is a previous family history of the disease. In an attempt to understand the tumorigenic pathway in Irish colon cancer patients, a number of genes associated with colorectal cancer development were analysed in Irish sporadic and HNPCC colon cancer patients. The hereditary forms of colon cancer include Familial adenomatous polyposis coli (FAP) and Hereditary Non-Polyposis Colon Cancer (HNPCC). Genetic analysis of the gene responsible for FAP, (the APC gene) has been previously performed on Irish families, however the genetic analysis of HNPCC families is limited. In an attempt to determine the mutation spectrum in Irish HNPCC pedigrees, the hMSH2 and hMLHl mismatch repair genes were screened in 18 Irish HNPCC families. Using SSCP analysis followed by DNA sequencing, five mutations were identified, four novel and a previously reported mutation. In families where a mutation was detected, younger asyptomatic members were screened for the presence of the predisposing mutation (where possible). Detection of mutations is particularly important for the identification of at risk individuals as the early diagnosis of cancer can vastly improve the prognosis. The sensitive and efficient detection of multiple different mutations and polymorphisms in DNA is of prime importance for genetic diagnosis and the identification of disease genes. A novel mutation detection technique has recently been developed in our laboratory. In order to assess the efficacy and application of the methodology in the analysis of cancer associated genes, a protocol for the analysis of the K-ras gene was developed and optimised. Matched normal and tumour DNA from twenty sporadic colon cancer patients was analysed for K-ras mutations using the Glycosylase Mediated Polymorphism Detection technique. Five mutations of the K-ras gene were detected using this technology. Sequencing analysis verified the presence of the mutations and SSCP analysis of the same samples did not identify any additional mutations. The GMPD technology proved to be highly sensitive, accurate and efficient in the identification of K-ras gene mutations. In order to investigate the role of the replication error phenomenon in Irish colon cancer, 3 polyA tract repeat loci were analysed. The repeat loci included a 10 bp intragenic repeat of the TGF-β-RII gene. TGF-β-RII is involved in the TGF-β epithelial cell growth pathway and mutation of the gene is thought to play a role in cell proliferation and tumorigenesis. Due to the presence of a repeat sequence within the gene, TGFB-RII defects are associated with tumours that display the replication error phenomenon. Analysis of the TGF-β-RII 10 bp repeat failed to identify mutations in any colon cancer patients. Analysis of the Bat26 and Bat 40 polyA repeat sequences in the sporadic and HNPCC families revealed that instability is associated with HNPCC tumours harbouring mismatch repair defects and with 20 % of sporadic colon cancer tumours. No correlation between K-ras gene mutations and the RER+ phenotype was detected in sporadic colon cancer tumours.

Genetic analysis of bacteriophages from clinical and environmental samples

Bacteriophages, viruses infecting bacteria, are uniformly present in any location where there are high numbers of bacteria, both in the external environment and the human body. Knowledge of their diversity is limited by the difficulty to culture the host species and by the lack of the universal marker gene present in all viruses. Metagenomics is a powerful tool that can be used to analyse viral communities in their natural environments. The aim of this study was to investigate diverse populations of uncultured viruses from clinical (a sputum of patient with cystic fibrosis, CF) and environmental samples (a sludge from a dairy food wastewater treatment plant) containing rich bacterial populations using genetic and metagenomic analyses. Metagenomic sequencing of viruses obtained from these samples revealed that the majority of the metagenomic reads (97-99%) were novel when compared to the NCBI protein database using BLAST. A large proportion of assembled contigs were assignable as novel phages or uncharacterised prophages, the next largest assignable group being single-stranded eukaryotic virus genomes. Sputum from a cystic fibrosis patient contained DNA typical of phages of bacteria that are traditionally involved in CF lung infections and other bacteria that are part of the normal oral flora. The only eukaryotic virus detected in the CF sputum was Torque Teno virus (TTV). A substantial number of assigned sequences from dairy wastewater could be affiliated with phages of bacteria that are typically found in the soil and aquatic environments, including wastewater. Eukaryotic viral sequences were dominated by plant pathogens from the Geminiviridae and Nanoviridae families, and animal pathogens from the Circoviridae family. Antibiotic resistance genes were detected in both metagenomes suggesting phages could be a source for transmissible antimicrobial resistance. Overall, diversity of viruses in the CF sputum was low, with 89 distinct viral genotypes predicted, and higher (409 genotypes) in the wastewater. Function-based screening of a metagenomic library constructed from DNA extracted from dairy food wastewater viruses revealed candidate promoter sequences that have ability to drive expression of GFP in a promoter-trap vector in Escherichia coli. The majority of the cloned DNA sequences selected by the assay were related to ssDNA circular eukaryotic viruses and phages which formed a minority of the metagenome assembly, and many lacked any significant homology to known database sequences. Natural diversity of bacteriophages in wastewater samples was also examined by PCR amplification of the major capsid protein sequences, conserved within T4-type bacteriophages from Myoviridae family. Phylogenetic analysis of capsid sequences revealed that dairy wastewater contained mainly diverse and uncharacterized phages, while some showed a high level of similarity with phages from geographically distant environments.

Investigation of the genetic susceptibility to osteoporosis in the Irish population

Osteoporosis is a complex skeletal disorder characterized by compromised bone strength. Variation in bone mineral density (BMD) is a contributing factor. The aim of this research as to select informative single nucleotide polymorphisms (SNPs) in potential candidate genes from loci suggestively linked to BMD variation for fine mapping. The gene regulated by oestrogen in breast cancer 1 (GREB1), located at 2p25.1, was selected. GREB1 transcription is initiated early in the oestrogen receptor alpha regulated pathway. There was significant association between GREB1_03 and BMD variation at the lumbar spine and femoral neck (FN) in the discovery cohort. Significant association was observed between GREB1_04 and FN BMD in the replication cohort. The development and differentiation enhancing factor 2, the integrin cytoplasmic domain associated protein 1 and A-disintegrin and metalloprotease 17 were selected due to their respective roles in cell mobility and adhesion. There was no linkage or association observed between the Chr2 cluster SNPs and BMD. Two factors in bone remodelling are the attraction of bone cell precursors and endocrine regulation of the process, primarily through the action of parathyroid hormone (PTH). The C-C chemokine receptor type 3 (CCR3) encodes a CC chemokine receptor expressed in osteoclast precursors. The PTH receptor type 1 (PTHR1) encodes a G-protein coupled receptor for PTH. Association was observed between CCR3 haplotypes and BMD variation at the FN. There was no linkage or association observed between PTHR1 SNPs and BMD variation. Population genetic studies with complex phenotypes endeavour to elucidate the traits genetic architecture. This study presents evidence of association between GREB1 and BMD variation and as such, introduces GREB1 as a novel gene target for osteoporosis genetics studies. It affirms that common genomic variants in PTHR1 are not associated with BMD variation in Caucasians and supports the evidence that CCR3 may be contributing to BMD variation

Integrated genetic analysis systems

The overall objective of this thesis is to integrate a number of micro/nanotechnologies into integrated cartridge type systems to implement such biochemical protocols. Instrumentation and systems were developed to interface such cartridge systems: (i) implementing microfluidic handling, (ii) executing thermal control during biochemical protocols and (iii) detection of biomolecules associated with inherited or infectious disease. This system implements biochemical protocols for DNA extraction, amplification and detection. A digital microfluidic chip (ElectroWetting on Dielectric) manipulated droplets of sample and reagent implementing sample preparation protocols. The cartridge system also integrated a planar magnetic microcoil device to generate local magnetic field gradients, manipulating magnetic beads. For hybridisation detection a fluorescence microarray, screening for mutations associated with CFTR gene is printed on a waveguide surface and integrated within the cartridge. A second cartridge system was developed to implement amplification and detection screening for DNA associated with disease-causing pathogens e.g. Escherichia coli. This system incorporates (i) elastomeric pinch valves isolating liquids during biochemical protocols and (ii) a silver nanoparticle microarray for fluorescent signal enhancement, using localized surface plasmon resonance. The microfluidic structures facilitated the sample and reagent to be loaded and moved between chambers with external heaters implementing thermal steps for nucleic acid amplification and detection. In a technique allowing probe DNA to be immobilised within a microfluidic system using (3D) hydrogel structures a prepolymer solution containing probe DNA was formulated and introduced into the microfluidic channel. Photo-polymerisation was undertaken forming 3D hydrogel structures attached to the microfluidic channel surface. The prepolymer material, poly-ethyleneglycol (PEG), was used to form hydrogel structures containing probe DNA. This hydrogel formulation process was fast compared to conventional biomolecule immobilization techniques and was also biocompatible with the immobilised biomolecules, as verified by on-chip hybridisation assays. This process allowed control over hydrogel height growth at the micron scale.

The biodiversity, control and genetic characterisation of the lactococcal 936 group phages

Phages belonging to the 936 group represent one of the most prevalent and frequently isolated phages in dairy fermentation processes using Lactococcus lactis as the primary starter culture. In recent years extensive research has been carried out to characterise this phage group at a genomic level in an effort to understand how the 936 group phages dominate this particular niche and cause regular problems during large scale milk fermentations. This thesis describes a large scale screening of industrial whey samples, leading to the isolation of forty three genetically different lactococcal phages. Using multiplex PCR, all phages were identified as members of the 936 group. The complete genome of thirty eight of these phages was determined using next generation sequencing technologies which identified several regions of divergence. These included the structural region surrounding the major tail protein, the replication region as well as the genes involved in phage DNA packing. For a number of phages the latter genomic region was found to harbour genes encoding putative orphan methyltransferases. Using small molecule real time (SMRT) sequencing and heterologous gene expression, the target motifs for several of these MTases were determined and subsequently shown to actively protect phage DNA from restriction endonuclease activity. Comparative analysis of the thirty eight phages with fifty two previously sequenced members of this group showed that the core genome consists of 28 genes, while the non-core genome was found to fluctuate irrespective of geographical location or time of isolation. This study highlights the continued need to perform large scale characterisation of the bacteriophage populations infecting industrial fermentation facilities in effort to further our understanding dairy phages and ways to control their proliferation.