M.Sc.(Med.), Faculty of Health Sciences, University of the Witwatersrand, 2008 / Introduction
It is widely recognised that stroke is a multi-factorial disorder in which platelets
play a crucial role in thrombus formation resulting in ischaemic stroke. Platelet
adhesion and aggregation are initiated by the interaction of various platelet
glycoproteins (GP’s) such as GPIbα, which binds to von Willebrand Factor and
GPIIb/IIIa a fibrinogen receptor. Recent studies have shown that the GP’s are
polymorphic and the polymorphisms described within GPIbα such as Kozak-
5T/C, the variable number of tandem repeats (VNTR) and the Human Platelet
antigen 2 (HPA2), have been implicated in the development of stroke, while the
PIA polymorphism of GPIIb/IIIa was found to contribute to “aspirin resistance”.
Therefore, these polymorphisms may be potentially important for early detection
and early intervention and thus setting the need to provide for a high volume
genotype testing at health care centres. One of the most used techniques to
determine platelet function is platelet aggregometry. However, the major
disadvantages of platelet aggregation is that it is influenced by a number of
environmental factors and its access is limited to tertiary health centres. Platelet
aggregation measures the functional expression of platelets, which is known to
deteriorate over time. It is for this reason that new methods at molecular level
such as polymerase chain reaction (PCR) are needed to explore the role of
genotypic expressions, which are not influenced by environmental factors.
Currently, conventional PCR is used to detect platelet polymorphisms in the
research settings and has limitations as a routine diagnostic test. Furthermore, it
is time consuming and is prone to contamination. With the recent advances in
real-time PCR it is possible to genotype large sample batches rapidly without
compromising on the quality, accuracy and precision of results. This study aims
to adapt conventional PCR methodology onto a real-time platform for detecting
platelet polymorphisms that have been implicated in both stroke and aspirin
resistance.
Materials and methods
A total of 60 caucasian patients classified as having ischaemic stroke by virtue of
MRI and Doppler analysis from the Stroke Clinic at the Johannesburg Hospital
were enrolled for this study. Healthy caucasian individuals (38), age and gender
matched were enrolled as controls. DNA samples were extracted from all the
subjects and the prevalence of the Kozak –5T/C, HPA-2, VNTR and GPIIIa PIA
polymorphisms were determined first by using conventional PCR and then the
real-time LightCycler TM PCR method.
Results
The frequency of the unfavourable alleles ( the PIA2 allele for the GPIIIa PIA
polymorphism, the T allele for the Kozak –5T/C polymorphism, the B allele for the
HPA-2 polymorphism and the C allele for the VNTR polymorphism) of the
different GP’s were higher in the stroke patients when compared to the control
subjects but did not reach statistical significance. There was complete statistical
agreement between the results obtained for the conventional PCR as compared
to the results obtained for real-time PCR except for the VNTR polymorphism, due
to the difficulty in designing and the unavailability of probes for the real-time PCR
assay. However, it is important to note that adapting the real-time PCR as a new
methodology would greatly benefit both the patients and the clinicians by
providing early detection and the possibility of early therapeutic intervention.
Conclusion
Therefore in conclusion, it is possible to perform not only conventional PCR for
platelet polymorphism but also real-time PCR on a large scale without
compromising on the quality, accuracy and precision on platelet polymorphisms
that play a significant role in stroke and aspirin resistance. However, a larger
population based study needs to be performed to confirm the findings.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/7426 |
| Date | 09 November 2009 |
| Creators | Moodly, Sadhaseevan |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf, application/pdf |
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