Altered Blood Gene Transcription in Chuvash Polycythemia and Its Cell Lineage Specificity

Chuvash polycythemia (CP) is a monogenic disorder characterized by an upregulated hypoxic response at normoxia. Homozygosity for the VHL^R200W mutation leads to decreased degradation of the a subunits of hypoxia inducible factor (HIF)-1 and HIF-2 by the hypomorphic variant of VHL, the principal negative regulator of HIFs. An array of HIF-regulated genes, including the principal regulators of erythropoiesis and iron metabolism, have altered expression. Previous studies in CP using peripheral blood mononuclear cells (PBMCs), a heterogeneous mixture of cells, identified significant gene expression differences from wild type controls, but the cell linage specificity of these hypoxia-regulated genes remains unknown. In this study, we systematically analyzed gene expression by unbiased deep RNA sequencing in purified reticulocytes, granulocytes and platelets of CP and control individuals living at the same altitude of ~200 meters.

Thirty-one samples passed quality control: reticulocytes from 10 individuals (5 VHL^R200W homozygotes and 5 wild type controls), platelets from 7 individuals (3 VHL homozygotes and 4 controls) and granulocytes from 14 individuals (5 VHL homozygotes, 1 heterozygote and 8 controls). The samples were analyzed for expression differences (VHL homozygote/heterozygote versus wild type) in each cell type. We found abundant gene expression differences in these three cell types. The differential genes detected in the three cell types showed no more overlap than expected by random (Binomial test P=1 for all pairings of the three cell types), suggesting cell lineage specificity of hypoxic gene expression in CP.

At 5% false discovery rate (FDR, i.e., <5 false positives in 100 detected genes), 737 of 7238 analyzed genes (10%) were altered in the reticulocytes of VHL^R200W homozygotes, 271 up-regulated and 466 down-regulated. The up-regulated genes were enriched in pathways of "Telomere maintenance", "Oxidative phosphorylation", "Parkinson's disease", "Ribosome", "Systemic lupus erythematosis", "Apoptosis", "Influenza Infection", "Metabolism of proteins", "Huntington's disease", and "Integration of energy metabolism". The down-regulated genes were enriched in pathways of "Cell cycle" and "Ubiquitin mediated proteolysis".

At 5% FDR, 3646 of 12,334 analyzed genes (30%) were differentially expressed in the platelets of VHL^R200W homozygotes, 1830 up-regulated and 1816 down-regulated. The up-regulated genes were enriched in pathways of "Lysosome" and "Signaling in immune system". The down-regulated genes were enriched in pathways of "Hemostasis" and "Opioid signaling".

At 5% FDR, 3423 of 11,274 analyzed genes (30%) were differentially expressed in the granulocytes of VHL^R200W homozygotes, 1490 up-regulated and 1933 down-regulated. The up-regulated genes were enriched in pathways of "Gene Expression", "Metabolism of nucleotides", "Metabolism of proteins", and "Aminoacyl-tRNA biosynthesis". The down-regulated genes were highly enriched in immune pathways including "Chemokine signaling pathway", "Fc gamma R-mediated phagocytosis", "Endocytosis", "Neurotrophin signaling pathway", "B cell receptor signaling pathway", "Fc epsilon RI signaling pathway", as well as several cancer-related pathways.

The relative abundance of alternative transcript isoforms differed in VHL^R200W homozygotes relative to wild type controls for many genes in these three blood lineages indicating a role for HIFs in regulation of mRNA processing. At 1% FDR, 3121 of 12,514 analyzed genes (25%) in platelets, 233 of 7342 analyzed genes (3%) in reticulocytes, and 224 of 11,306 analyzed genes (2%) in granulocytes contained alternative exon(s) in VHL^R200W homozygotes compared to wild type controls.

In conclusion, we report marked gene expression variation in three blood cell lineages from individuals with CP, the first described disorder of congenital augmentation of hypoxia sensing. Dysregulated expression of genes not known to be transcriptionally regulated by HIFs may be due to the well-known but poorly defined effects of HIFs on epigenetic regulation of transcription. Our results demonstrate extensive cell lineage specificity in blood gene expression variations induced by augmented signaling of HIFs caused by the VHL^R200W mutation. This provides novel insights to our understanding of clinical complications in CP and more broadly of hypoxic gene regulation.