Characterization of a Newly Discovered Non‐Coding Variant in the <i>EPO</i> Gene Identified in Two Unrelated Italian Pedigrees With Erythrocytosis

Interpretation of JAK2-unmutated congenital or acquired erythrocytosis is challenging [1-8]. Hereditary erythrocytosis (HEs) includes a group of rare congenital diseases characterized by an excessive production of red cells [4, 5, 7]. HEs should be suspected in children and young adults, particularly in the case of a positive family history [4], or in older patients with a long history of otherwise unexplained erythrocytosis. HEs are often caused by high oxygen affinity hemoglobin variants (HOAV), or mutations in the oxygen-sensing pathway genes, as VHL (von Hippel–Lindau), EGLN1/PHD2 (prolyl hydroxylase domain-containing protein 2), EPAS1/HIF2A (Hypoxia-Inducible Factor 2α), and EPO (encoding for erythropoietin, EPO) [4, 5, 7, 9-11]. In other cases, mutations in genes such as BPGM (2,3-Bisphosphoglycerate Mutase) or PIEZ01 (piezo-type mechanosensitive ion channel component 1) are responsible for HEs [4, 5, 7, 12]. In these conditions, serum EPO levels are usually normal to elevated [4, 5, 7], while a low plasma EPO concentration suggests the presence of an EPOR (EPO receptor) mutation [13]. Even though the knowledge on the molecular basis of HEs is growing, many subjects without a defined genetic lesion fall into the diagnosis of idiopathic erythrocytosis [4]. Recently, Martin et al. [14] have identified three novel variants in noncoding regulatory regions of the EPO gene within six families with erythrocytosis. These heterozygous variants are in the main promoter (c.-252C>T) and in intron 1 (c.14-28T>C and c.14-26A>G) regions of the EPO gene [14]. Interestingly, subjects had serum EPO levels within the normal range [14]. This novel molecular entity comprises a newly described functionality: compared to healthy adults, circulating EPO shows a distinct pattern on isoelectric focusing (IEF), like that of premature newborns (in whom fetal liver is the main source of EPO) or to individuals with erythrocytosis related to hepatic pathologic features [14, 15]. As a consequence, it is suggested that these EPO variants could lead to an elevated expression of a hyperactive “hepatic-like” EPO with high responsiveness to hypoxia [14-16]. Here, we report six subjects belonging to two unrelated Italian families with erythrocytosis, carrying the EPO (c.14-28T>C) variant [14]. Patients consented to the study that was conducted in accordance with the principles of the Declaration of Helsinki. After excluding JAK2 mutations and considering their family history of erythrocytosis, the probands of the two families (subject IV-11 for Family 1 and II-2 for Family 2, Figure 1) were investigated with a clinical DNA exome by SureSelect CD Clinical Focused Exome (Agilent Technologies), sequenced on Illumina NextSeq 550/2000 platforms (Illumina, San Diego, USA), to study genes known to be implicated in HEs. Genes included in the analysis were: BPGM, CYB5R3, EGLN1, EGLN2, EGLN3, ENG, EPAS1, EPO, EPOR, GATA1, GFI1B, GSN, HBA1, HBA2, HBB, HIF1A, JAK2, KCNN4, KDM6A, MPL, PIEZO1, RUNX1, SLC30A10, SH2B3, SRC, TET2, THPO, WAS, VHL. Variants were called and annotated using Expert Variant Interpreter (eVai) software (enGenome), and their classification followed the criteria of the American College of Medical Genetics and Genomics (ACMG). Suspected causative EPO gene variants were confirmed in probands and later searched in available relatives by Sanger sequencing (SeqStudio Genetic Analyzer, Thermo Fisher Scientific). No additional variants in genes known to be associated with HEs have been detected in the available members of both families. Case report Family 1 (Figure 1A): in early 2025, a 38-year-old male (subject IV-11) was referred for further investigations on erythrocytosis, firstly identified in adolescence and treated since then with regular phlebotomies and antiplatelets therapy. Some members (n = 9) of his father's side of the family received phlebotomies on a regular basis and/or have erythrocytosis (Figure 1A). Main laboratory and clinical findings of the proband and relatives for which DNA samples were available (subjects III-5, III-6, III-7, IV-8, IV-9) are reported in Table 1. Clinical exome sequencing of the proband (IV-11) revealed the presence of the germline heterozygous c.14-28T>C variant located in intron 1 of the EPO gene [14]. The variant, that we previously considered variant of unknown significance (VUS)/likely benign on the basis of our in silico analysis, was reclassified as pathogenic following the report by Martin et al. [14]. This variant was also identified in the three available family members (III-6, III-7, IV-8) known to have erythrocytosis, while it was absent in subjects III-5 and IV-9, with a normal complete blood count, providing evidence that the mutation segregates with the phenotype in affected individuals (Figure 1A and Table 1). Case report Family 2 (Figure 1B): In 2010, a father and a daughter (subjects II-2 and III-2) were referred for suspected familial erythrocytosis. The proband (II-2) had been managed since 1996 with phlebotomies and antiplatelet therapy. Under this approach, he experienced two retinal vein thrombosis episodes in 2006 and 2008: at the time of those events, the patient had isolated erythrocytosis and hypertension. His daughter (III-2) was diagnosed with erythrocytosis since 2008, and she was managed since then with phlebotomies, without antiplatelet therapy. She had no prior vascular events, but her medical history included solid tumors, having previously been diagnosed with thyroid carcinoma and breast cancer. The proband reported that his brother (II-1) and his nephew (III-1) had a history of erythrocytosis, but no further details were available (Figure 1B). Main laboratory and clinical findings of the proband and his daughter (II-2 and III-2) are reported in Table 1. After the publication by Martin et al. [14] clinical exome sequencing of subject II-2 was performed on a stored DNA sample, revealing the presence of the heterozygous germline c.14-28T>C variant located in intron 1 of the EPO gene. This variant was also identified in subject III-2. DNA samples from other family members were not available for analysis. Although the advent of next-generation sequencing has provided a substantial advantage in the diagnostic approach to HEs and in the understanding of their pathogenesis, many variants remain VUS outside of specific functional assays or extensive family studies. As a result, a definitive diagnosis cannot be established in a considerable proportion of patients. This was the case for the c.14-28T>C variant, which we previously classified as likely benign or VUS with different in silico prediction tools. Conversely, Martin et al. [14] considered possible splicing defects, as the variant affects the splice branchpoint consensus, and SPiP (Splicing Pipeline Prediction) predicted a 69% likelihood of splicing impairment [14]. However, minigene and splicing reporter assays showed an effect in only around 1.5% of transcripts, weakening the hypothesis of a significant splicing impact [14]. The demonstration of a clear effect on transcriptional dysregulation [14] has made it possible to elucidate the basis of some cases of congenital erythrocytosis, unexplained to date. In this two-center experience, we were able to identify the newly discovered c.14-28T>C variant located in intron 1 [14] of the EPO gene in six subjects belonging to two unrelated Italian families presenting with erythrocytosis. Correlation between the presence of the heterozygous intron 1 variant of the EPO gene and erythrocytosis is supported by the absence of the former in two subjects of Family 1, presenting with a normal complete blood count. Martin et al. [14] suggested that the variants targeting the regulatory elements of the EPO gene could lead to a lower degree of fully terminated glycosylation and consequently to a lower stability of circulating EPO. In the original paper most of the patients had serum EPO levels within the normal range; conversely, in our series three out of six patients had low EPO. This underlines the limit of using serum EPO levels as a discriminatory parameter for investigating the possible causative gene responsible for a HE. To date, among HEs, a proven increased risk of thrombosis is known for homozygous loss-of-function VHL mutations (Chuvash erythrocytosis, CE) and for EPAS1/HIF2A gain-of-function mutations [17]. In a large cohort of patients affected by CE, phlebotomies were associated to higher rates of new thrombotic episodes, probably due to the resulting iron deficiency which leads to inhibition of PHD2, increased HIF-regulated prothrombotic factors and EPO [17]. Of note, only one of the affected subjects (Family 2, subject II-2) described in our cohort reported a history of thrombotic events while receiving antiplatelet therapy and regular phlebotomies. The remaining five patients did not experience thrombosis despite a long history of erythrocytosis and no regular treatment with phlebotomies and antiplatelets. For the time being, six families from the original report [14] and our two families having these EPO variants are available in the literature. Recognizing this new entity could inform physicians on the possible therapeutic approach to these cases and help to collect information on their thrombotic risk. In fact, the role and the timing of phlebotomies in non-polycythemia vera erythrocytosis is still not well defined [4, 18, 19]. In a retrospective study of 206 patients with secondary or idiopathic erythrocytosis, neither thrombosis nor mortality were affected by phlebotomy or persistent erythrocytosis [20] leaving the treatment goal an unmet need. Our results confirm that a more extensive analysis of the EPO gene in case of a suspected hereditary erythrocytosis will enable the establishment of a more accurate diagnosis of patients, regardless of serum EPO levels. An international effort on the identification of these families is much needed. B.M., E.R., and F.P. contributed to the conception of the work and wrote the manuscript. E.F. and P.B. were responsible for genetic analysis and detailed the methods part of the manuscript. All authors contributed to the acquisition of data, to revise the manuscript critically for important intellectual content, to approve the final version of the manuscript and are accountable for all aspects of the work in ensuring that questions related to the accuracy and integrity of any part have been investigated and resolved. This work has been supported by grants from Fondazione Alberto e Antonietta Matarelli, Milan, Italy [Omics in myelofibrosis] (B.M. and F.P.) and by Associazione Italiana per la Ricerca sul Cancro, IG 2021 ID 25703 (E.R.). This work is generated within the European Reference Network on Rare Hematological Diseases (ERN-EuroBloodNet), F.P.A. 739541. F.P., B.M., P.B., and E.F. are EuroBloodNet members. The study was consented by patients and conducted in accordance with the principles of the Declaration of Helsinki. The authors declare no conflicts of interest. The data analyzed during the current study is available by mailing the corresponding author on reasonable request.