Theses and Dissertations (Haematology)

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    Detection of the calreticulin type 1 and type 2 mutations in a group of myeloproliferative neoplasm patients using molecular methods
    (University of Pretoria, 2016) Van Zyl, Walda B.; Prinsloo, A. (Adri); louisedutoit@yahoo.com; Du Toit, Louise de Villiers
    The myeloproliferative neoplasms (MPN), formerly referred to as chronic myeloproliferative disorders, are a group of haematopoietic stem cell disorders that are characterized by clonal proliferation of one or more mature myeloid lineages. The group of disorders, now classified as the classic MPNs, include chronic myeloid leukaemia (CML), as well as the Philadelphia (Ph) chromosome-negative MPNs: polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) or myelofibrosis (MF). In 2013, somatic mutations at exon 9 of CALR, the gene that encodes for calreticulin, were discovered through whole-exome sequencing and targeted re-sequencing in patients with MPNs. Among these mutations, more than 80% of CALR mutated patients possessed one of only two mutation types: type 1 (a 52-bp deletion) and type 2 (a 5-bp insertion). In MPNs, patients positive for a mutation in their calreticulin gene have shown milder symptoms and an overall better survival rate than patients who had one of the other common phenotypic driver mutations i.e. JAK2 or MPL. Determining which driver mutation is responsible for the manifestation of the MPN is therefore important in monitoring the treatment and prognosis of the patient. In the South African sector, a simple, efficient, cost effective and sensitive detection method for testing for CALR type 1 and type 2 mutations is necessary to fill the current diagnostic gap regarding the diagnosis of MPNs. This study aimed to evaluate the use of conventional PCR followed by restriction enzyme digestion, confirmation of the PCR results by nucleotide sequencing, as well as real-time PCR (qPCR) to detect these mutations. A conventional PCR commercial kit was also compared to that of the developed conventional PCR. For this study 24 blood samples were collected from the tertiary hospital haematology clinic, 24 archived DNA samples from a colleague's previous JAK2 mutation study were used and two DNA samples were obtained from a private pathology laboratory. A total of 50 samples were thus analysed. The newly developed in-house conventional PCR was successful in detecting the CALR type 1 and type 2 mutations in patients and the results were confirmed by Sanger sequencing. The results obtained using the commercial conventional PCR kit corresponded 100% with the results obtained by the in-house conventional PCR assay, followed by restriction enzyme digestion and finally confirmation of results by nucleotide sequencing analysis. The kit was, however, much more expensive than the in-house PCR assay. The qPCR using SYBR Green as well as the qPCR using probes were unable to detect the type 1 and type 2 mutations and further troubleshooting was not possible due to budgetary constraints. Overall, 3/50 patients contained the type 1 mutation and 2/50 contained the type 2 mutation, indicating the possibility of a low prevalence for the CALR type 1 and type 2 mutations in the South African population.
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    Detection of the Janus kinase 2 V617F mutation using molecular methods
    (University of Pretoria, 2016) Potgieter, Johan J.C.; Kock, Martha Magdalena; tshiphiri.netshidziv@nhls.ac.za; Senamela, Tshiphiri
    In 2005, a mutation located at exon 14 of the Janus Kinase gene on chromosome 9 was discovered in patients with Polycythaenia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF). The mutation (JAK2 V617F/G1849T) causes valine to be substituted by phenylalanine at codon 617. As a result the World Health Organisation (WHO) revised the diagnostic criteria of myeloproliferative neoplasms (MPN) in 2008 to include the detection of the JAK2 V617F mutation as a major diagnostic criterion for PV, ET and PMF. Molecular assays with high sensitivity and specificity should be offered by diagnostic laboratories for this purpose. To comply with these requirements, commercial and in-house assays that offer different sensitivity and specificity levels have been developed. In addition to the performance characteristics of diagnostic assays used to detect the JAK2 V617F mutation, associated cost remains an important factor to consider when selecting the assay that is best suited to a particular laboratory. Commercial diagnostic kits are expensive which led to the development of more cost-effective in-house assays for use in routine diagnostic laboratories. The purpose of this study was to develop real time polymerase chain reaction (PCR) assays for the detection of the JAK2 V617F mutation that could be used in diagnostic laboratories. Primers and locked nucleic acid (LNA) probes were designed. DNA was extracted from 60 fresh peripheral blood specimens. Allele specific PCR (AS-PCR) was performed on 59 DNA samples while direct sequencing and real time PCR assays were performed on all 60 samples. Performance of the different molecular assays was compared. In addition, the analytical sensitivity of the LNA real time PCR assay was determined by processing specimens comprising serial dilution of homozygous mutant in wild type DNA. Allele specific PCR identified 26 of the 59 as positive for the JAK2 V617F mutation. The LNA real time PCR assay and direct sequencing both showed 24 of 60 samples to harbour the mutation. Diagnostic sensitivity and specificity of the developed real time PCR assay and sequencing assay was 88% and 100%, respectively. There was 100% agreement between the real time PCR and sequencing assays. Agreement between real time PCR and AS-PCR was calculated to be 94% with kappa value of 0.89. The developed real time PCR assay showed acceptable performance when assessed against the comparative AS-PCR method. In addition, analytical sensitivity of 0.1% was demonstrated for this assay. The findings confirm the suitability of the developed LNA probe real time PCR assay to detect the JAK2 V617F mutation in a clinical laboratory.