Erythrocytosis is a disease with a characteristically high red blood cell count (RBC count), high hematocrit and high levels of hemoglobin. We recognize primary and secondary erythrocytosis according to the location of the defect and acquired and congenital based on its origin. Most commonly, erythrocytosis is due to non-genetic external factors or other chronic illnesses. Rarely it originates form acquired of congenital gene variants. Familial erythrocytosis (ECYT) is a rare hereditary hematological disorder. It is diagnosed by screening nine genes involved in regulation of erythropoiesis (EPOR, EPO), homeostasis of oxygen detection (VHL, EGLN1, EPAS1) and affinity of hemoglobin for oxygen (HBB, HBA1, HBA2 in BPGM). More than 60 % of patients with suspected ECYT remain undiagnosed (idiopathic erythrocytosis), as the current diagnostic method fails to explain its genetic background. This indicates that there are other, not yet identified potential genes and molecular mechanisms with an important role in development of the disease.
In the first part of this work, we analysed databases STRING and Reactome in search of interaction partners of already known genes, involved in erythrocytosis. For every newly identified interaction partner we evaluated its potential contribution to development of erythrocytosis based on the number different target genes it interacted with. Genes GRB2, SOCS3 and JAK2 had the maximum number of interactions (4). Genes EP300, HIF1A, HIF3A, PLCG1, CBL, KIT, STAT5A and PTPN6 had the second most interactions (3).
In the second part we optimized the PCR method for amplification of the nucleotide sequence of exon 12 in HIF1A gene. We chose this gene based on the previous bioinformatic analysis, where HIF1A was one of the genes with the maximum number of experimentally determined interactions with target genes. In addition, analysis of literature showed that there was a link between specific gene variants of exon 12 HIF1A and symptoms of erythrocytosis. Optimal PCR reaction with a selected pair of oligonucleotide primers (HIF1A-int11F-1 in HIF1A-int12R-1) was without aditional reactants, with 65 °C annealing temperature, 15 s extention time and 30 cycles. The optimized PCR method could be a useful addition to the routine diagnostic procedure in Slovenia, that would enhance our ability to explain and treat this disease.