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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-06142013-152947 |
| Date | 14 June 2013 |
| Creators | Wienand, Kirsty |
| Contributors | Prof VJ Louw, Prof CD Viljoen |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-06142013-152947/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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