Detection of the calreticulin type 1 and type 2 mutations in a group of myeloproliferative neoplasm patients using molecular methods

Dissertation (MSc)--University of Pretoria, 2016.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.HaematologyMScUnrestricte