Advancing Diagnostic Accuracy and Lowering Idiopathic Erythrocytosis Rates through Next-Generation Sequencing

Absolute erythrocytosis is defined by an increase in red blood cells, hemoglobin and hematocrit. It can be primary, involving defects in erythroid progenitor cells, or secondary, stemming from issues outside the erythroid compartment. However, around 70% of cases lack a known cause. The current study aimed to identify potential genetic contributors to erythrocytosis and to assess the true prevalence of idiopathic erythrocytosis. Between 2018 and 2024, 55 patients with unexplained erythrocytosis, were clinically referred to investigate the underlying cause. All patients were negative for JAK2 V617F and exon 12 mutations and had no identified secondary causes of erythrocytosis. A NGS panel included BPGM, EGLN1, EPAS1, EPOR, GATA1, GELSOLIN, HBA1, HBA2, HBB, JAK2, MPL, RUNX1, SH2B3, SRC, THPO, VHL, and WAS, was conducted by the CRIMM laboratory at Careggi Hospital in Florence. All patients provided informed consent. DNA extraction was performed from granulocytes in approximately 12 ml of peripheral blood collected at the Hematology Department of Federico II University of Naples. The median age was 40 years (range 17–74), the median Hb and Hct were 17 g/L (range 15.5–19) and 51% (range 42–63), respectively. Most patients were male (51/55, 93%), which aligns with prior studies indicating that men are more frequently affected by JAK2-negative erythrocytosis. Among the 55 patients, 15 (27.3%) displayed six distinct gene variants. EGLN1 380G>C mutation was found in 4 patients, leading to the C127S protein change. One patient had EPAS1 c.466G>T mutation, resulting in the G156W protein change. One patient exhibited HBB c.371C>T mutation leading to a T124I protein change. Four patients showed various MPL mutations, such as 117G>T (K39N), 1610G>A (R537Q), and 209C>T (P70L). Two patients had SH2B3 mutations, in particular 1426C>T and 622G>C, resulting in L476F and E208Q protein changes, respectively. VHL 598C>T mutation was found in two patients, leading to an R200W protein change. One patient carried two different mutations: EGLN1 380G>C and MPL 1610G>A. All the identified mutations have been previously described in the literature, suggesting a potential pathogenic role in erythrocytosis. Spontaneous erythroid colony cultures, in the absence of exogenous EPO, were performed for the patients carrying mutation in the MPL gene, but no spontaneous erythroid colonies were observed. The remaining patients (40/55, 72.7%) without detectable mutations were consequently diagnosed as idiopathic erythrocytosis. This study explored the genetic landscape of 55 patients with unexplained erythrocytosis. A comprehensive NGS panel identified six different gene variants, demonstrating the NGS’s value in identifying genetic causes in this setting. However, the majority of patients had no mutations, indicating that idiopathic erythrocytosis remains a significant challenge in clinical practice. Our results highlight the complexity of erythrocytosis and the need for continued research to elucidate unknown causes.