CML is a haematological malignancy that is characterized by the BCR-ABL fusion oncogene that encodes a constitutively active tyrosine kinase. The treatment of choice for CML is a tyrosine kinase inhibitor and molecular monitoring of patients forms an integral part of disease management. When the expected response to tyrosine kinase inhibitor is not achieved within internationally accepted time frames, acquired resistance to tyrosine kinase inhibitors is suspected. Acquired resistance to tyrosine kinase inhibitors is primarily due to mutations in the BCR-ABL kinase domain. Types of mutations include single base mutations, insertions, deletions as well as duplications. Characterization of these mutations is important for treatment, since the type and position of the mutation may have an effect on how the patient responds to treatment. Although several methods have been described for detecting mutations, DNA sequencing is mostly used. Sequencing is currently the only technique that can simultaneously detect single base mutations, insertions and deletions in the BCR-ABL kinase domain. However, sequencing is costly as some patient samples do not have mutations and the lack of response to treatment is due to non-compliance. Thus, a screening method to exclude samples without mutations would make mutational analysis more cost-effective. High resolution melting (HRM) is a relatively new technique that is being used to screen for mutations, prior to sequencing. HRM has recently been used to screen the region of BCR-ABL encoding for the kinase domain for single base mutations. However, it was unknown whether HRM could be used to identify insertions, deletions or duplications in the kinase domain. This study has shown that HRM can be used to screen for mutations including insertions, deletions and duplications the region of BCR-ABL encoding for the kinase domain, prior to sequencing. HRM was performed on 40 patient samples, 10 of which had confirmed mutations in BCR-ABL in the region of the kinase domain. Of the 10 samples with mutations, three had single base mutations, one with a previously described insertion, seven had novel deletion variants. Furthermore, HRM detected a tandem duplication of the kinase domain in two patient samples that was not previously been possible with sequencing. There was 100% congruency between the detection of mutations using HRM and sequencing results, indicating similar sensitivity. HRM proved successful to indicate the presence of deletion variants. However, the deletion variants were detected in the HRM region preceding the area affected by the deletion. It was confirmed that the detection of the deletion variants was due to the PCR extension of HRM 1 amplicon into the HRM area of the deletion. It has been suggested that the insertion, deletions and duplications detected in this study may result in acquired resistance to tyrosine kinase inhibitor. In conclusion, this was the first study to use high-resolution melting to detect insertions, deletions and duplications in the region of BCR-ABL encoding for the kinase domain, indicating the suitability of the assay for screening for mutations prior to sequencing.

In 2007, the first case of field resistance to Cry1Ab was reported in South Africa, which is a concern as it negates the benefit of this technology. It has been suggested that a major contributing factor to the development of resistance in the target insect was the lack of compliance by commercial farmers to plant refugia. However, another possible mechanism of resistance development is the production of sub-lethal doses of Cry1Ab that could have resulted in a high selective pressure for resistance alleles. Although there are studies that have determined levels of Cry1Ab in different tissue in MON810 maize, the available data is not complete, especially for important feeding tissue of B. fusca larvae, such as silk and cob sheath. In this study, a comprehensive analysis of levels of Cry1Ab within and between different tissue over the growing season was conducted, taking the effect of gene flow also into account. Field trials were performed over the 2008/2009 and 2009/2010 growing seasons under conventional farming practice. Gene flow was allowed to occur between IR and non-IR maize in the 2008/2009 growing season, and the F1 seed was planted in the 2009/2010 growing season. The levels of Cry1Ab were monitored over both growing seasons, including the F1 plants in the second season. Notably, this study was the first to determine levels of Cry1Ab in cob sheath, which is considered one of the primary food sources for B. fusca larvae. It was found that there was considerable variation in levels of Cry1Ab within and between different tissue over the growing season. The data for the majority of the sampling points was moderately to highly skewed, indicating the non-parametric range in variation of Cry1Ab levels. There was a significant difference in Cry1Ab production between the two growing seasons, which was attributed to the lower than average rainfall in the 2008/2009 growing season and a higher than average rainfall in the 2009/2010 growing season. The overall trend in Cry1Ab production was congruent with the pattern of target insect larval survival after feeding on different tissue as reported by Van Rensburg (2009). Based on these data we suggest that important insect feeding tissue, namely silk, cob sheath and cob, could be producing sub-lethal doses of Cry1Ab that may result in ineffective control of insect pests. It appears that the decline in Cry1Ab production at late growth stages, in conjunction with variable levels of Cry1Ab between different tissue, may compromise the high dose/refugia strategy, resulting in selective pressure for the evolution of resistance. The gene flow study determined that outcrossing between IR and non-IR maize adversely affects the level of Cry1Ab in F1 plants. The levels of Cry1Ab were significantly lower in F1 maize when compared to a commercial MON810 maize hybrid, possibly as a result of reduced fitness. These data support the observation of increased insect larvae damage to F1 plants, suggesting that F1 maize may produce sub-lethal doses of endotoxin, and consequently will not effectively control insect pests. The considerably lower expression of Cry1Ab in F1 plants is a consideration in respect to subsistence farming practice in Africa, where seed is saved or exchanged among farmers. We postulate that the introduction of IR maize in subsistence farming could promote the development of insect resistance if not managed correctly. In conclusion, the current study has determined that there is a wide range of level of Cry1Ab within and between different tissue over the growing season. Gene flow adversely affects Cry1Ab production, potentially due to reduced fitness of the F1 plants. These data support the observation of differential rates of larvae survival when feeding on different IR maize tissue. Finally, the study provides an important basis for understanding the potential role that variable levels of Cry1Ab may have had on the development of resistance in B. fusca in South Africa.

Endothelial microparticle reseach is currently a very novel and exciting topic in the field of haemosistasis and thrombosis. The role of microparticles in inflammatory and thrombotic disorders is however not fully understood. Dysfunction of endothelial cells is hypothesized to be a trigger of microparticle formation. In inflammatory disorders like sepsis and thrombotic disorders like atherosclerosis and thrombotic thrombocytopenic purpura, endothelial microparticle formation is altered and the numbers thereof may increase or decrease. It is not known if microparticles are the cause or the consequence of these disorders. To understand the role of endothelial microparticles in inflammation and thrombosis, the effect of inflammatory cytokines and coagulation stimuli was studied as well as combinations thereof on endothelial microparticle formation and on microparticle VWF and its regulating protease, ADAMTS-13 in HUVEC. In this study, the formation of microparticles in cultured human umbilical vein endothelial cells (HUVEC) was stimulated by different inflammatory agents: IL-6 (100 ng/ml), IL-8 (100 ng/ml) and TNF-α (100 ng/ml), coagulation stimuli: TF (2 U/ml) and thrombin (2 U/ml) and combinations thereof. The number of endothelial microparticles that formed was determined using flow cytometry. VWF and ADAMTS-13 levels of the microparticles were assessed by ELISAs and microparticle thrombin generation was measured by thrombin generation assays. VWF multimers were visualized by a Western Blot technique. IL-6 did not have any effect on HUVEC-derived microparticles due to the lack of the receptor for IL-6 on these cells. IL-8 only slightly increased effect on microparticle VWF and ADAMTS-13 levels. TNF-α had a significant effect on microparticle numbers and contributed to almost 80% of thrombin generated by the microparticles. It has however almost no effect on VWF levels. The coagulation stimulus TF, on the other hand, induced the highest increase in microparticle VWF levels and increased microparticle numbers impressively. Yet, it had no effect on the thrombin generation by the microparticles. TF in combination with TNF-α also induced an increase in microparticle VWF and a small decrease in ADAMTS-13 levels. So, TF may contribute to the increased VWF levels that are commonly found in TTP patients where inflammation and thrombosis occur. Interestingly, thrombin had a protective effect on the intact HUVEC by preventing microparticle formation. The combination stimuli of thrombin and inflammatory agents also had a protective effect on HUVEC. This highlighted the regulatory role of thrombin in intact endothelial cells and also the protection that it provides against thrombosis in extremely inflammatory environments. Endothelial microparticles can therefore be detrimental or beneficial, depending on the different stimuli and different environments. Inflammatory and coagulation stimuli may still pose a significant risk of clotting by altering microparticle quantity and content. This study contributes to understand the role that endothelial microparticles play in inflammation and thrombosis.

Chronic myeloid leukaemia (CML) is a haematological malignancy characterised by the BCR-ABL fusion oncogene which encodes for a constitutively active tyrosine kinase. Imatinib mesylate is a tyrosine kinase inhibitor that has effectively been used in the treatment of CML. However, some individuals experience adverse drugs reactions (ADRs) to imatinib. One of the reasons for varied treatment response among individuals may be as a result of inter-individual differences in the metabolism of imatinib. Imatinib is metabolised by the drug metabolizing enzymes CYP3A4 and CYP3A5. Single nucleotide polymorphisms (SNPs) in CYP3A4 and CYP3A5 have been described, some of which have been associated with altered catalytic activity of these enzymes. SNPs in CYP3A4 and CYP3A5 may also impact the expression of these genes and result in a less favourable response to imatinib treatment. Patients with a decrease in catalytic activity of CYP3A4 and CYP3A5 may experience ADRs due to prolonged exposure to imatinib. On the other hand an increase in activity may lead to ineffective treatment as a result of increased clearance of the drug. Thus the aim of this study was to screen CYP3A4 and CYP3A5 for SNPs using high resolution melting curve analysis (HRM) and determine the impact of these SNPs on gene expression in CML patients treated with imatinib. A total of ten SNPs were detected in CYP3A4, of which two SNPs, namely A15619G and A15649T have not been previously described in literature. A15619G was a synonymous SNP while A15649T resulted in a change in amino acid from glutamine to a histidine. A total of four SNPs were detected in CYP3A5, of which one SNP namely, G7226A, had not previously been reported in literature and did not result in an amino acid change. Out of all the detected SNPs in CYP3A4, the G20338A SNP was statistically associated with the occurrence of ADRs but not with mRNA expression. The I369V SNP was statistically associated with increased CYP3A4 mRNA expression. None of the SNPs detected in CYP3A5 significantly affected mRNA expression. Expression of CYP3A4 and CYP3A5 was not dependent on ethnicity or gender, with the exception of CYP3A5 which showed a statistically significant difference between males and females. Currently a limited amount of literature exists regarding SNPs in CYP3A4 and CYP3A5 and CML treatment. Given the potential impact that SNPs can have on the CYP3A4 and CYP3A5 enzymes and therefore imatinib treatment, it is an important issue that needs to be investigated. Determining the potential impact of SNPs and differential gene expression of CYP3A4 and CYP3A5 is important as it may allow for more effective imatinib treatment.

Chronic myeloid leukaemia (CML) is a haematopoietic stem cell disorder characterised by the BCR-ABL fusion gene. The BCR-ABL fusion gene encodes a constitutively active BCR-ABL tyrosine kinase, which is the driving force of the malignancy. Otherwise fatal, the use of imatinib mesylate has proved highly effective in the treatment of this disease in up to 85% of CML patients. However, approximately 25% of CML patients appear to respond suboptimally or experience treatment failure with imatinib. Suboptimal response in CML patients has been attributed to inadequate BCR-ABL kinase inhibition as a result of reduced intracellular accumulation of imatinib in target leukemic cells. The cellular influx of imatinib is mediated by the influx transport protein, SLC22A1. Therefore, its activity is considered a clinical determinant of imatinib uptake, and hence patients response to therapy. A number of studies use levels of SLC22A1 mRNA as a measure of SLC22A1 activity. It has been reported that cells over expressing levels of SLC22A1 mRNA showed significantly increased uptake of imatinib, thus, suggesting that levels of SLC22A1 mRNA can be used as a measure of SLC22A1 activity. However, there is a concern that imatinib may affect SLC22A1 expression. This consideration, however, is based on two studies involving a limited patient cohort and although widely accepted, has not been proven conclusively. Should it be proven that imatinib does influence SLC22A1 expression, levels of SLC22A1 mRNA may not be a reliable indicator of SLC22A1 activity. It is therefore important to understand the effect of treatment with imatinib on SLC22A1 gene expression. The data from this study demonstrated that imatinib induces expression of SLC22A1 mRNA in a non-linear dose dependent manner. It was also observed that expression of SLC22A1 was not dependent on time of exposure to imatinib. These results explain the differential expression of SLC22A1 mRNA reported in CML patients on a standard dose of 400 mg/day of imatinib. The trough plasma levels of imatinib achieved between patients after 24 hours of exposure to the same dose of imatinib may vary owing to inter individual differences. Since SLC22A1 expression is dependent on plasma levels of imatinib, therefore, patients administered the same dose of imatinib may show differential expression of SLC22A1. These findings suggest that imatinib does affect SLC22A1 mRNA expression and that the change in SLC22A1 expression observed at any particular time is dependent on the intracellular levels of imatinib achieved in CML patients within 24 hours of exposure to the drug. One of the challenges in this study was the availability of suitably qualified SLC22A1 antibodies for use in the Taqman protein assay to quantify SLC22A1 protein. Antibodies used in the Taqman assay have to fulfil specific criteria and out of 55 commercially available antibodies, only three SLC22A1 antibodies met the minimum requirements for use in the assay. However, despite various efforts focused at optimising the assay, the range of the assay was very limited and hence it was not possible to quantify SLC22A1 protein. We hypothesize that one of the reasons for assay failure could be as a result of antibodies not binding to the target protein at the required spatial distance to facilitate amplification by real-time PCR. Since the antibodies used in the assay have not been epitope mapped, it is uncertain whether they fulfil this requirement. Future research will be aimed at antibody production for manufacturing SLC22A1 antibodies suitable for use in the Taqman protein assay to enable successful quantification of SLC22A1 protein. In conclusion, this is the first study which specifically aimed to investigate the influence of imatinib on SLC22A1 expression. This is also the first study to demonstrate that expression of SLC22A1 is not time dependent, but follows a nonlinear correlation to imatinib concentration. Although it would have been useful to investigate the effect of increasing levels of SLC22A1 mRNA on intracellular uptake of imatinib in K562 cells, unfortunately, the latter technique requires the use of radio-labelled imatinib and specialized equipment which made it a limiting factor for use in this study. While this study does not invalidate the use of levels of SLC22A1 mRNA as a prognostic marker for treatment outcome, these findings suggest that levels of SLC22A1 mRNA as a measure of SLC22A1 activity is only applicable to newly diagnosed imatinib naive or previously untreated CML patients.

Von Willebrand disease (VWD) is a common bleeding disorder caused by either quantitative (type1 and 3) or qualitative (type 2) defects of von Willebrand factor (VWF). The diagnosis of VWD usually requires a panel of tests. Several analyses therefore are required to diagnose VWD. These tests are also subjected to pitfalls and it is important to take the pitfalls in to consideration when diagnosing VWD. Despite all these tests, the diagnosis and classification of VWD often remains a challenge. Identification of mutations that cause functional defects of VWF (type 2 VWD) is needed to improve the diagnosis of the disease. Mutations that cause functional abnormalities of VWF occur mostly in exon 28 of the VWF gene. Exon 28 primarily encodes the platelet GPIb and collagen binding domains of VWF (A1 domains) and the ADAMTS13 cleavage domain (A2 domains). Recently, studies in industrialised countries have been conducted on finding mutations on exon 28 but none have been done on South African populations. In this study we searched for mutations in exon 28 of the VWF gene in 5 patients with functional defects of VWF in order to set up the method for genetic analysis of VWD. We used two patients with type 2M, two with type 2B and one with type 2A VWD in this study. The whole exon 28 was analysed in four specific fragments, using PCR with primers that mismatch the pseudogene. The mutations were identified by automatic sequencing of the different fragments. The following polymorphisms were detected. A silent SNP 4641T/C in all five patients, the SNP 4141A/G in three patients, a silent SNP 3795G/A in one patient and a novel silent SNP 4923G/A in another patient. It is important to note that we found a novel SNP in an African patient with type 2B VWD, since no polymorphisms reported in exon 28 were from African populations. Several studies have proven the importance of mutational analysis is solving laboratory diagnosis paradox. The mutations found in the patients with type 2 VWD confirm the diagnosis and validates the importance of molecular diagnosis in VWD. With this study, we have successfully implemented a method to detect mutations in exon 28 of the VWF gene.

Not available

In the foregoing project, an investigation was made into the relative KIR gene frequencies of three South African cohorts. Playing an important part in innate immunity, KIR fill a vital gap between viral onsets and cell mediated humeral immunity. Being able to sense when cells are abnormal, NK cells possess the ability to destroy cells which show altered HLA molecules during KIR/HLA interaction. Ethnic cohorts that were investigated included African black, mixed ancestry and the Caucasian populations. From these individuals DNA material was extracted using a âsalting outâ method before SSP-PCR genotyping. Seventeen primer pairs were used in the identification of individual KIR genes. PCR products were electrophoresed against a molecular weight marker in order to verify the correct fragment size. Products were viewed on a UV light where observations were noted, and indicated as present or absent. Data was recorded onto a spreadsheet indicating the absence or presence of each particular gene. Tabulated results were used in the construction of graphs as well as Ï2 calculations. These graphs were used in the critical analysis of linkage disequilibrium as well as comparative analysis between the ethnic cohorts. Findings indicate that all framework genes are present in all cohorts. The Black African and mixed ancestry cohorts have not been genotyped for the KIR genes before. Investigation within non-framework genes revealed the identification of several new haplotypes, with the majority observed within the mixed ancestry cohort. Positive linkage disequilibrium was detected between 2DL2-2DS2 and 2DL5B-2DS5 for both the black African and Caucasian cohorts while 2DL1-2DL2 and 2DL5B- 2DS5 linkages were found in the mixed ancestry population. No negative linkages were observed for any of the three cohorts.

Chronic myelogenous leukaemia (CML) is a malignant clonal disorder that results in the uncontrolled production of white blood cells. This disease is a result of a reciprocal translocation of the long arms of chromosome 9 and 22 resulting in a shortened chromosome 22, harbouring the BCR-ABL fusion gene, known as the Philadelphia chromosome. The BCR-ABL oncogene encodes for a constitutively activated tyrosine kinase that interferes with normal cell differentiation and apoptosis. CML can be effectively treated with tyrosine kinase inhibitors, such as imatinib mesylate (GleevecÃ). However, some CML patients experience adverse drugs reactions (ADRs) to imatinib and cannot be treated at the recommended dose. There is a concern that lowering the dose of imatinib to reduce the side effects can result in the development of resistant cancer cells, and thus a cessation in treatment is rather recommended. Imatinib is metabolized by the cytochrome P450 enzyme, CYP3A4. However, if the ADRs were a result of decreased metabolic effect of CYP3A4, it would be possible to reduce the dose of imatinib without effecting efficacy. It is hypothesised that single nucleotide polymorphisms (SNPs) can alter the catalytic activity of the CYP3A4 enzyme. Thus a decrease in metabolic rate can result in ADRs due an increased exposure to the drug. Therefore, the aim of this study was to determine whether SNPs in the CYP3A4 gene are associated with ADRs from imatinib treatment. In this study, the DNA sequence of the CYP3A4 gene from 25 CML patients treated with imatinib were compared to a reference DNA sequence obtained from Genbank. The SNPs identified during this study was statistically analysed, and their association with the presence of ADRs was determined using the online statistics package, SNPator. A total of six SNPs were detected, I193I, T15871G, CYP3A4*1G, C23187T, I369V and G73239A. Of these, I369V and G73239A are novel and not described previously in literature. It was found that I369V resulted in an amino acid change, involving a substitution of isoleucine with valine. The remaining SNPs identified in this study were located in intron regions, with the exception of I193I which is a synonymous SNP. There is little information available on the frequency of SNPs located in introns, since these SNPs are generally regarded to have no impact on the expression or activity of a protein. However, in this study an SNP located in intron 10 was significantly associated with the presence ADRs. Current hypothesises suggest that intron SNPs could affect the expression levels of a protein by influencing the splicing efficiency of mRNA and subsequently translation efficacy. Future research needs to elaborate on the role of CYP3A4*1G on CYP3A4 expression as well as on the prevalence of other alleles identified in this study in South African populations.

Globally, South Africa is the eighth largest producer of GM crops and also imports GM food. In addition to the promise of increased agricultural production, the introduction of GM crops is also having an impact on society in terms of consumer acceptance and trade. As a result, most countries manage GMOs in terms of development, use and application as well as require mandatory GM labelling for consumer preference. With an increase in GM developments, monitoring the food chain in terms of GM labelling and unapproved GM events will continue to pose a regulatory challenge. The aims of this thesis were the following: 1. To determine the uptake of GM food into the food chain; 2. To study the application of voluntary GM labelling; 3. To investigate the impact of mandatory GM labelling; and 4. To establish a monitoring system to detect illegal GMOs in South Africa. Until 2005 it was assumed that there were only low levels of GM crop in the food chain, based on production volumes. However, results from this thesis have shown that 76% of food products tested positive for the presence of GM in 2005. There was also no consideration of mandatory GM labelling as it was thought that voluntary GM labelling was successfully being applied in South Africa. Despite this, 31% of products labelled to indicate an absence of GM, such as âGMO freeâ, ânon-GMâ and âorganicâ, contained genetic modification above 1%, and 20% of these contained more than 5% genetic modification. These results demonstrated the extent of GM in the food chain in South Africa and highlighted the fact that voluntary GM labelling does not protect consumers against misleading claims. In 2008, the Consumer Protection Act mandated the labelling of GM in food products and ingredients. However, there was a lot of uncertainty as to how this would impact the food industry. The subsequent research on the impact of mandatory GM labelling in South Africa determined that 67% of maize and 54% of soybean products will have to be labelled for GM content. In addition to this, GM was also detected in 50% of products labelled to indicate an absence of GM. Furthermore, results indicated that the use of either a 1% or 5% threshold does not make a considerable difference in terms of the number of products implicated. The use of the term âmay contain genetic modificationâ as suggested by draft regulations to the Consumer Protection Act may provide a cost effective manner in which GM labelling can be applied in a developing country similar to South Africa, as it would reduce costs in terms of GM detection. The draft regulations for the Consumer Protection Act also make provision to indicate the absence of GM below a threshold that does not included terminology such as âGMO freeâ or ânon-GMâ. Furthermore, the draft regulations do not require third party verification and compliance will mainly be self-regulating. The implication of this is that consumers or consumer groups will become responsible for policing the application of GM labelling in South Africa. Finally, this thesis presents a GM monitoring scheme for unapproved GMOs, that have not been proven safe for human health and/or the environment. The scheme has the advantage of being cost effective and can be applied to the regulatory situation in any country, taking approved GM events into consideration. The scheme was applied to off-the-shelf food products in South Africa to determine the presence of illegal GMOs. Even though no unapproved GM events were detected, a potential illegal import of GM soybean event A2704-12 was found. It was also found that an approved GM soybean event was comingled with rice and wheat products, although not indicated in the ingredients. The research emanating from this thesis has contributed to inform discussions that have resulted in the inclusion of mandatory GM labelling in the Consumer Protection Act 68 of 2008. It is hoped that the research on the application of mandatory GM labelling and the monitoring for unapproved GM events in the food chain will have a similar impact on the regulatory system in South Africa.