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Genexpressionsanalyse von Patienten mit Polycythaemia Rubra Vera mittels cDNA Microarrays
In this work, several attempts to elucidate the physiological role of the murine PRV-1 gene by whole mount in-situ hybridisation experiments did not yield reproducible results. Nevertheless, expression of the mPRV-1 mRNA could be demonstrated from embryonic day 6.5 on by RT-PCR experiments. In order to obtain a model system for physiological analysis of the PRV-1 protein, transgenic mouse strains were generated which express the human and the murine PRV-1 gene in their haematopoietic system. Expression of the transgene was demonstrated on mRNA as well as on protein level in the two transgenic founder lines for the human PRV-1 gene. Analysis of different blood parameters of these mice in a collaboration with the LMU in Munich revealed significant differences compared to wild-type littermates with respect to granulocyte counts and different factors involved in iron homeostasis. Overexpression of the PRV-1 mRNA, decreased c-Mpl protein levels on platelets and the formation of EECs are laboratory findings which can be used for differential diagnosis among the CMPDs. Nevertheless, none of these markers can define a single disease entity and none of them seems to represent the initial step in the
pathogenesis of PV. This led us to perform cDNA microarray analysis of PV patients in order to get a more global view of gene expression in this myeloproliferative disorder. Granulocyte RNAs from 40 polycythaemia vera (PV) and 12 secondary erythrocytosis (SE) patients were analysed. Peripheral granulocytes were chosen as the source of RNA, since these cells yield high amounts of RNA and arise clonally from the aberrant stem cell. In order to identify genes which are specifically altered in PV, RNA from purified granulocytes of 10 patients with SE and 10 PV patients was hybridised to a control RNA pool consisting of total granulocyte RNA from 50 healthy
controls. Of all the 7000 cDNAs investigated, 64 differentiate between patients with SE and PV in this training set (p<0.01). A hierarchical clustering analysis based on these genes resulted in a correct classification of the remaining 30 PV and 2 SE patients what indicates that these genes represent a unique gene expression pattern discriminating PV from SE.
Some of the genes which are differentially expressed in PV patients with respect to healthy controls could play a role in the context of PV by contributing to the malignant 136 phenotype, whereas others may rather represent secondary effects which are predominantly caused by the elevated numbers of circulating blood cells.
A large number of genes which show elevated RNA levels in PV patients are
regulated by the transcription factor SP1. This raises the hypothesis of a
transcriptional dysregulation in PV with respect to SP1 or other related transcription factors. Concerning the pathogenesis of PV elevated mRNA and protein levels could be demonstrated for the erythroid transcription factor NF-E2. NF-E2 seems to be of special interest as this factor has been described to promote the growth of EPOindependent colonies when overexpressed in primary haematopoietic progenitor cells (167) and therefore represents an extremely interesting candidate for the molecular etiology of PV. The characterization of 48 patients with essential thrombocythaemia (ET) with respect to the markers PRV-1 mRNA, c-Mpl protein, clonality and EEC-growth was also part of this work. As a result it turned out that elevated PRV-1 mRNA and decreased c-Mpl protein expression are acquired independently and do not define the same subgroup of ET patients. In contrast, clonal haematopoiesis and PRV-1 mRNA overexpression correlated in a cohort of 12 ET patients. The subgroup of ET
patients which show elevated PRV-1 mRNA levels has been shown to be at a higher
risk for thromboembolic complications as compared to those with normal PRV-1
levels. Therefore, this molecular marker could be a valuable tool in the risk
stratification procedure for ET patients.
cDNA microarray analysis of granulocyte RNA from one PRV-1 positive and one
PRV-1 negative ET patient was also performed. The PRV-1 positive patient showed
a PV-like gene expression signature. This, together with the recently published
finding that PRV-1 positive ETs will transform to PV in later stages of the disease supports the hypothesis of "masked"; PV in PRV-1 positive ET patients.
Die Arbeit befaßt sich mit der differentiellen Genexpression in Patienten mit Polycythaemia Rubra Vera (PV), einer seltenen hämatopoietischen Stammzellerkrankung. Die Genexpression dieser Patienten wird verglichen mit der von Patienten mit Sekundärer Erythrozytose (SE). Das erhaltene Genexpressionsprofil für die PV erlaubt eine klare Abgrenzung von der SE. Außerdem können Rückschlüsse auf eine mögliche molekulare Ursache gezogen werden.
Ein weiteres Projekt der Dissertation war die Generierung transgener Mäuse für das humane PRV-1 Gen, das bei Patienten mit PV auf RNA-Ebene deutlich überexprimiert ist.
|SWD-Schlagwörter:||Myeloproliferatives Syndrom , Differentielle Genexpression , Microarray , Transgene Tiere , Diagnose|
|Freie Schlagwörter (deutsch):||Polycythaemia Rubra Vera , Microarrays , Myeloproliferative Erkrankungen , Genexpression , Transgene Mäuse|
|Freie Schlagwörter (englisch):||Polycythaemia Rubra Vera , microarrays , myeloproliferative disorders , gene expression , transgenic mice|
|Fakultät:||Fakultät für Biologie|
|DDC-Sachgruppe:||Medizin und Gesundheit|
|Erstgutachter:||Neuhaus, Gunther (Prof. Dr.)|
|Tag der mündlichen Prüfung:||13.12.2004|