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The genomic analysis brings a new piece to the molecular jigsaw of idiopathic erythrocytosis


Erythrocytosis is a clinical condition characterized by increased red cell mass, hemoglobin, and hematocrit values. A significant fraction of patients is described as having idiopathic erythrocytosis. We have previously demonstrated an association between erythrocytosis and the JAK2 GGCC_46/1 haplotype and CALR rs1049481_G allele. In the present study, we investigated genomic and clinical features of 80 erythrocytosis patients with the aim to provide useful information in clinical practice. Patients with idiopathic erythrocytosis could have a genomic germline background, eventually associated with somatic variants. Through association analysis, we show that male patients presenting with idiopathic erythrocytosis, and normal EPO levels could be the best candidates for the search for the JAK2 GGCC_46/1 haplotype and CALR rs1049481_G allele. Further studies are needed to confirm these findings and to depict detailed genomic and phenotypical characteristics of these patients.

To the editor,

Erythrocytosis is characterized by an erythrocyte count above the gender specific normal range and increased hemoglobin and hematocrit values [1]. Polycythemia vera (PV) accounts for most primary acquired erythrocytosis cases; the JAK2 V617F or JAK2 exon 12 variants are considered PV “driver” mutations. However, about 4% of PV cases lack a molecular marker [1, 2].

Although recent evidence has added useful information to define erythrocytosis [3, 4] a significant fraction of patients is described as affected by idiopathic erythrocytosis (IE), characterized by a genetic marker absence; the IE clinical management still represents an unmet need. We previously demonstrated an association between erythrocytosis and two single nucleotide polymorphisms (SNPs): JAK2 GGCC_46/1 and CALR rs1049481_G [5]. In this study, we investigated genomic and clinical features of a larger cohort of patients to unveil the IE molecular complexity (Additional file 1). Based on clinical and genomic data of a more extensive patient’s cohort, we suggest a hierarchical model in which male patients presenting with IE and normal erythropoietin (EPO) levels are the best candidates for the search for JAK2 and CALR SNPs. Furthermore, in this subset of patients, we identified additional mutations in genes commonly involved in clonal hematopoiesis (CH).

The JAK2 and CALR SNPs were genotyped in 80 cases (Additional file 2: Table S1) as previously described [5]. Fifty-three (66.3%) were positive and 27 (33.7%) negative for the JAK2 haplotype. Regarding CALR, 54 (67.5%) cases had at least one G allele.

The JAK2 SNP was associated with erythrocytosis, a significant difference in frequency being detected as compared to healthy European controls (p = 0.0011). The association was also demonstrated in terms of allelic frequency (p = 0.0019) and genotype distribution (p = 0.0035).

The simultaneous presence of both SNPs was observed in 38 (47.5%) cases compared to controls (137/503, 27.2%) (p = 0.0004). A significant association between SNPs and erythrocytosis was also observed in cases showing normal EPO (p = 0.0002).

Since both SNPs are in accordance with Hardy–Weinberg equilibrium in controls (p > 0.05), association analysis was performed between the SNPs investigated and erythrocytosis using the SNPassoc R package [6]. A significant association between JAK2 SNP and erythrocytosis risk was observed under the dominant model, with a 2.29-fold higher risk in people bearing at least one alternative allele compared to subjects having none (OR = 2.29; p = 0.0007576) (Table 1). Considering CALR, the presence of at least one G allele is associated with an increased risk under a log-additive model (0,1,2 G: OR = 1.37; p = 0.06609).

Table 1 Associations between JAK2 GGCC_46/1 haplotype and erythrocytosis cases

To improve the accuracy of the test, several covariates were incorporated; the association became stronger after adjustment for the presence of CALR rs1049481_G as a categorical variable, as well as gender, and EPO level (Table 1). The erythrocytosis risk is higher when the three covariates are introduced simultaneously (OR = 3.13, p = 0.000051; Table 1). Considering patients with normal EPO levels, all observed associations between JAK2 SNP and erythrocytosis under the dominant model were strengthened (with CALR rs1049481_G as covariate: OR = 2.75, p = 0.0001381; with gender: OR = 3.11, p = 0.0000522).

Next generation sequencing (NGS) analysis was performed on 44 patients; 34/44 (77%) sequenced cases with the JAK2 haplotype showed at least one allele G of CALR rs1049481. Overall, 22 genetic variants affecting 7 genes (ASXL1, TET2, DNMT3A, JAK2, KIT, RUNX1, ANKRD26) were detected in 17/44 cases (38.6%) (Fig. 1A). ASXL1 was the most frequently mutated gene (6/44, 14%) (Fig. 1A, B). Two non-canonical JAK2 variants were identified (Additional file 3: Table S2), already described in few patients with haematologic neoplasms [7].

Fig. 1
figure 1

A Oncoprint visualization of all genetic variants identified by targeted NGS analysis in 44 erythrocytosis cases. SNP: single nucleotide polymorphism. B Maps of the mutations on linear proteins of the most mutated genes in all sequenced cases. Green dots stand for missense mutations, while black dots indicate frameshift mutations. The height of the bar depends on the number of cases bearing each variant. HARE-HTH: HB1, ASXL, restriction endonuclease HTH domain (12–83); ASXH: Asx homology domain (234–362); PHD: PHD domain of transcriptional enhancer, Asx (1480–1539); PWWP: Pro-Trp-Trp-Pro domain (291–374); DNA_methylase: C-5 cytosine-specific DNA methylase (634–767); Tet_JBP: Oxygenase domain of the 2OGFeDO superfamily (1290–1905). C Diagnostic approach to erythrocytosis patients. PV: polycythemia vera, BOM: bone marrow biopsy

Recent evidence suggests that germline predisposition factors could have a role in the development of myeloproliferative neoplasms [3, 8,9,10]. Based on the integration of genomic data, clinical features, and statistical methodology, we have attempted to refine the typical characteristics of patients presenting with IE. The median age of our patients with typical CH genes mutations was 52 years (only 2 patients were > 60 years). Therefore, such mutations cannot be attributed to an aging-related CH [11].

We hypothesize that a degree of genomic instability could create a “fertile ground” for the development of erythrocytosis, characterized by a high prevalence of additional mutations in typical CH genes. Furthermore, association analysis builds a sort of genomic hierarchy, prioritizing the presence of JAK2 GGCC_46/1 over the CALR rs1049481_G allele. Finally, male patients with IE and normal EPO levels are more likely to benefit from the analysis of both JAK2 and CALR SNPs to better define the challenging diagnostic process of IE (Fig. 1C). Further studies are needed to confirm these findings and to depict detailed characteristics of IE patients.

Availability of data and materials

The sequence data from this study have been submitted to the National Center for Biotechnology Information (NCBI) Short Read Archive ( under accession number PRJNA609847.



Polycythemia vera


Idiopathic erythrocytosis


Single nucleotide polymorphism




Clonal hematopoiesis


Next generation sequencing


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This work was supported by “Associazione Italiana contro le Leucemie (AIL)-BARI”.


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Authors and Affiliations



Conception and design of the study: AZ, FT and FA. Acquisition of data and/or analysis and interpretation of data: PO, AZ, FT, LA, CC, IR, CFM, NC, GT, AR, IA, EP, MRC, GS, PM and FA. Drafting of the manuscript: FA. All authors revised the manuscript for important intellectual content. All authors read and approved the final manuscript.

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Correspondence to Francesco Albano.

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The local ethics committee approved the study. Informed consent was obtained from all patients before study inclusion, in accordance with the Declaration of Helsinki. Patients' records/information were anonymized and de-identified before analysis.

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Consent for publication was obtained from patients before their enrolment in the present study.

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The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1.


Additional file 2

: Table S1. Biological and clinical characteristics of cases analyzed in the present study.

Additional file 3: Table S2.

Variants identified by NGS analysis in 44 erythrocytosis cases.

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Zagaria, A., Tarantini, F., Orsini, P. et al. The genomic analysis brings a new piece to the molecular jigsaw of idiopathic erythrocytosis. Exp Hematol Oncol 11, 47 (2022).

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  • Erythrocytosis
  • Myeloproliferative neoplasms
  • SNPs
  • JAK2
  • EPO