Abstract
In Chuvash polycythemia, homozygosity for the 598 C->T mutation in the von Hippel-Lindau gene (VHL) leads to upregulation of hypoxia inducible factor-1a (HIF1a), a transcription factor that mediates cellular responses to hypoxia. This defect in the oxygen-sensing pathway causes increased expression of a broad range of hypoxia-regulated genes. Clinically, Chuvash polycythemia (CP) patients display not only erythrocytosis, but also premature mortality related to cerebrovascular and peripheral thrombotic events. As it is not clear that the thrombophilic nature of CP correlates with elevated hematocrit (Gordeuk et al, Blood103: 3924, 2004), we postulated that homocysteine may be a contributive factor, as preliminary data suggests that CP homozygotes have elevated plasma homocysteine levels (Sergueva, in preparation). Levels of homocysteine depend on its synthesis, involving S-adenosylmethionine, and its metabolism, either via remethylation to methionine, involving methylenetetrahydrofolate reductase (MTHFR), or via degradation by transsulfuration, involving cystathionine beta-synthase (CBS). Severe MTHFR and CBS deficiencies due to rare homozygous mutations lead to extremely high levels of serum homocysteine and are characterized clinically by a high incidence of thromboembolic complications, in addition to a wide range of other clinical symptoms. A recent microarray analysis that looked at the regulation of gene transcription by HIF-1a revealed that CBS and MTHFR gene expressions appear to be down regulated by hypoxia in endothelial cells (Manalo et al, Blood 105: 659, 2005). Downregulation of the genes responsible for homocysteine metabolism may therefore explain the elevated plasma homocysteine concentrations in CP. As hypoxia-regulated genes are often cell-type specific, we studied several types of easily accessible cells and detected expression of CBS and MTHFR in platelets, granulocytes, and EBV-immortalized lymphocytes in normal controls. In order to quantitate this expression, we used real-time RT-PCR and found no quantitative difference between EBV-immortalized lymphocytes in 4 homozygous CP patients, 3 heterozygote CP patients and 1 control. We then examined the peripheral blood from one CP patient and three controls. Although the numbers were small, the CP granulocytes and platelets showed decreased expression of MTHFR compared to controls, with decreased CBS expression seen in the CP granulocytes. These results suggest that the upregulation of HIF1a seen in CP patients might lead to decreased metabolism of homocysteine, which in turn, might contribute to the increased thromboembolic risk seen in CP. As these findings will need to be confirmed with a larger number of patients, we are currently in the process of collecting all accessible CP samples from the U.S. and from Chuvashia and the Italian island of Ischia (Perrotta et al, Blood 107: 514, 2006). Using the peripheral blood cells and in vitro expanded endothelial cells from these patients (Ingram et al, Blood104:2752, 2004), we hope to analyze the transcripts and enzyme activity of the genes involved in homocysteine synthesis and metabolism and to correlate these findings with CP plasma homocysteine levels.
The 677C>T variant in methylenetetrahydrofolate reductase causes morphological and functional cerebrovascular deficits in mice
