Masters Degrees (Haematology and Cell Biology)
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Browsing Masters Degrees (Haematology and Cell Biology) by Author "De Kock, A."
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Item Open Access Mutational analysis of the Janus Kinase 2 gene in patients with polycythaemia vera, essential thrombocythaemia and primary myelofibrosis(University of the Free State, 2011-11) Goodyear, Quintin Clive; De Kock, A.; Viljoen, C. D.All the cells of blood arise from two lineages, the myeloid and the lymphoid lineage. The various cells of blood perform vital functions in the body. These cell counts are closely regulated by regulatory pathways. Mutations within genes that encode for the proteins involved in these pathways can occur. These mutations can cause uncontrolled proliferation of the cells. Myeloproliferative neoplasms are malignancies where there is an uncontrolled increase in the formation of the myeloid cells. The four classical neoplasms are polycythaemia vera, essential thrombocythaemia, primary myelofibrosis and chronic myeloid leukaemia. A mutation (V617F) in the tyrosine kinase, Janus kinase 2, has been found to be the cause of at least three of the classical MPNs. The mutation lies in the domain of the protein that controls its tyrosine kinase activity. The tyrosine kinase thus is constitutively active and causes proliferation of the myeloid cells. The V617F mutation lies in exon 14 and more recently several mutations have been described in the neighbouring exons encoding for the regulatory domain of the gene. Very few studies have been done on the other exons of the JAK 2 gene. In the study it was attempted to screen 15 MPN patients for mutations in the JAK 2 gene. Two different cell populations (lymphocytes and granulocytes) of each patient were screened. It was found that the cell purity was not sufficient in the study and better separation techniques are required for future studies. Only the granulocytes were used for the remainder of the study. High resolution melting curve analysis was used to screen the patients for mutations, however the data did not correlate with the sequencing results and it was decided to proceed with sequencing of all the samples. Seven of the 25 exons of the JAK 2 gene were successfully sequenced. The remaining exons could not be screened due to time constraints and complications such as multiple amplicon formation. Two previously reported single nucleotide polymorphisms were found in exons 11 and 15 in two patients. The clinical significance thereof is uncertain however, the patient whom had the SNP in exon 15 was negative for the V617F mutation and had a MPN. In exon 14 the V617F mutation was identified and the prevalence thereof correlates to that reported in literature. A novel SNP was found in exon 13 of a PV patient negative for the V617F mutation and the significance thereof is also uncertain. Additionally a novel inverse duplication consisting of at least of exon 13 was also identified. No mutations were identified in exons 10, 12, 16 and 17 of the JAK 2 gene. This was, to our knowledge, the first report in South Africa that found the prevalence of the V617F mutation in MPN patients correlating to the prevalence reported in literature. A novel SNP was identified in exon 13 and further studies are needed on the possible effect thereof. The previously reported SNPs found in exons 11 and 15 might be the cause of the formation of a MPN, however further research is needed. A novel duplication variant was also identified and this might be a possible splice variant. The study showed that the region between exons 10 and 15 in the JAK 2 gene is a mutational hotspot and further studies are needed to elucidate the effect thereof.Item Open Access Sequensing of exon 28 of Von Willebrand factor in five patients with type 2 Von Willebrand disease(University of the Free State, 2009-05) Mothabeng, Maliengoane Sylvia; Meiring, S. M.; De Kock, A.English: 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.