Benetti, Andrea

Bertozzi, Irene

Biagetti, Giacomo

Ceolotto, Giulio

Polese, Francesca

Randi, Maria Luigia

Regazzo, Daniela

English

Archivio istituzionale della ricerca - Università di Padova

Genes & Diseases (2024) 11, 561e563Available online at www.sciencedirect.comScienceDirectjournal homepage: www.keaipubl ishing.com/en/ journals /genes-diseasesRAPID COMMUNICATIONTargeted NGS analysis reveals a complexgenetic background of idiopathicerythrocytosis in a large Venetian familyAbsolute erythrocytosis, due to an increased production ofred blood cells, becomes manifest with hemoglobin (Hb)levels above 165 or 160 g/L or with a hematocrit (HCT)above 51% and 48% in males and females, respectively.1 Aminority of patients have polycythemia vera (PV); acquiredsecondary erythrocytosis frequently occurs due to appro-priate or inappropriate erythropoietin (EPO) excess, whilecongenital secondary erythrocytosis may derive from ge-netic defects causing tissue hypoxia. Hereditary eryth-rocytosis occurs in patients with genetic mutations of theoxygen-sensing pathway (VHL, PHD2, HIF-1-alpha) or theerythropoietin receptor (EPOR) genes.2 Recently, HFE genevariants have been described in sporadic erythrocytoses.3In a high proportion (about 70%) of patients with eryth-rocytosis, a specific etiology remains elusive despiteextensive testing, and the diagnosis of idiopathic eryth-rocytosis (IE) is ruled out.1A targeted NGS panel for patients with unexplainederythrocytosis was set up. The panel included the codingsequence of ASXL1, BPGM, EGLN1, EPAS1, HAMP, FTL, HFE,HFE2, JAK2, SLC11A2, SLC40A1, TFR2, VHL, FTH1, and EPORgenes involved or suspected to be involved in eryth-rocytosis. The identified variants were confirmed by directSanger sequencing.We report here the molecular characteristics of a largeVenetian family with some cases of erythrocytosis. Thegenealogic tree of the family is displayed in Figure 1. Thepatients’ main clinical and laboratory data are reported inTable S1.The proband was a 24-year-old man who had sufferedlymphoblastic acute leukemia (LAL) when he was 18 yearsold and had recovered with chemotherapy. Five years afterthe end of treatment, erythrocytosis (Hb Z 173 g/L,HT Z 50%) was identified with no significant alterations inPeer review under responsibility of Chongqing MedicalUniversity.https://doi.org/10.1016/j.gendis.2023.03.0302352-3042/ª 2023 The Authors. Publishing services by Elsevier B.V. onarticle under the CC BY-NC-ND license (http://creativecommons.org/lithe number of leucocytes and platelets and was associatedwith high serum ferritin (1421 ug/L). The JAK2V617F andrarer JAK2 exon 12 mutations were searched but not found.The patient had no signs or symptoms of erythrocytosis andphlebotomies were performed to reduce ferritin levels.Recently, the patient developed MDS/MPN-u, and a matchedunrelated donor (MUD) hematopoietic stem cell trans-plantation was performed. The NGS study showed thattogether with somatic ASXL1R1068* mutation (c.3202C > T,NM_015338), the patient carried germinal EGLN1C127S(c.380G > C, NM_022051) and JAK2L393V (c.1177C > G,NM_004972) variants. The ASXL1 variants are consideredpathogenic and responsible for epigenetic modificationslikely to be one of the first events in the tumorigenesisprocess; it is found in chronic myelomonocytic leukemias,atypical chronic myeloid leukemias, and unclassified mye-lodysplastic syndromes/myeloid neoplasms. While JAK2 so-matic mutations are well-known driver mutations in PV andother myeloproliferative neoplasms (MPN), the significanceof germinal modifications of JAK2 remains to be elucidated.The JAK2L393V germinal variant has been previouslydescribed in one patient who had documented normal RBCcounts for decades before overt PV became manifest.4 Thefunctional significance of this variant and its putative pre-disposing role to PV remain unclear, though its potential topredispose to malignancy is suspected, as the germline JAK2variant is frequently found in diffuse large B-cell tumors.The personal history of the proband revealed that hisfather (III.2) was affected by PV, confirmed by the presenceof the JAK2V617F somatic mutation (c.1849G > T,NM_004972). He carried also the homozygous EGLN1C127Svariant but no relations between EGLN1C127S and bloodmalignancies have been reported to date. No alterations inepigenetic genes were observed.Given the presence of genetic alterations linked to he-reditary erythrocytosis in the proband and his father, westudied other 14 subjects of the family founding somebehalf of KeAi Communications Co., Ltd. This is an open accesscenses/by-nc-nd/4.0/).Figure 1 Detailed description of the Venetian family with erythrocytosis. (A) Kindred of our Venetian. (B) Main clinical andhematologic data of the studied subjects. (C) Sanger sequencing of the most representative variant. Hb, hemoglobin; HT, he-matocrit; LAL, lymphocytic acute leukemia; MDS/MF, myelodysplastic syndromes/myeloid neoplasm; NHL, non-Hodgkin lymphoma;Plts, platelets at the molecular study time; PV, polycythemia vera; WBC, white blood cell; y, years.562 Rapid Communicationmolecular alteration in 12 of them. Elevated or borderlinehigh Hb and HT levels were found in 3 males (IV.5, IV.6, andIV.9), and an associated ruddy complexion was observed in2 of them. None of the evaluated females had eryth-rocytosis. In this respect, it is known that IE is more com-mon in males and the present data suggest that females’genetic pattern could be transmitted to their progeny whomay eventually develop IE, particularly if of male gender.This could be the case of patients III.8 and IV.6.Neither subjects carrying gene variants nor those withoutalterations had a history of thrombotic complications. Yet,the thrombotic risk in IE is higher than that of the generalpopulation (1.5/100 patients/year),5 and mainly patientswith overt erythrocytosis need to be adequately followed toprevent the occurrence of cardiovascular events.Four patients had developed malignancies: one chroniclymphocytic leukemia (II.4), one kidney tumor (III.5), onenon-Hodgkin lymphoma (NHL) (III.4), and one colon cancer(II.5). Among these 4 patients, we could study only patientsIII.4 and II.5; both carried the EGLN1C127S variant as 4 otherasymptomatic family members (II.7, III.11, IV.1, IV.3, IV.5).Thus, the EGLN1C127S gene variant was the most common inour kindred. Such mutation is typical of Tibetans and pro-tects them against erythrocytosis, hypoventilation, andpulmonary hypertension occurring at high altitudes. At nor-moxic conditions and in low-landers, the EGLN1C127S variantis present in 15%e30% of non-Tibetan controls, though itsfunction remains unclear. This variant is relatively commonin North-East of Italy and it may represent a genetic inher-itance from the barbarian invasions of people coming fromthe remote areas of central Asia that, once arrived in ourneighborhood, intermixed with the resident population.The germ line JAK2L393V variant was found in proband’smother (III.1) and sister (IV.1) who had also EGLN1C127S.These patients are being closely followed up so that theeventual appearance of PV is promptly recognized.Four patients (III.8, IV.6) carried the EPAS1T766P(c.2296A > C, NM_001430) variant, which in two cases (II.7,IV.5) was associated with the above-mentionedEGLLN1C127S. Patient IV.6 had very high Ht (57.1%) and Hb(195 g/L) levels. The EPAS1T766P variant is often consid-ered benign or of uncertain significance, though occasion-ally has been found to be associated withpheochromocytoma and/or paraganglioma (PPGL).The HFEH63D variant (c.187C > G, NM_000410), presentin a single ruddy member (IV.9) of the family, seems not toinfluence the normal activity of hepcidin but it could play arole in patients with increased hematocrit levels. Finally,the EPORG46E (c.137G > A, NM_000121) variant wasobserved in an acquired relative (IV.6). The alteration waspossibly inherited from his father who used leeches to treathis condition. Such a variant is considered benign and, atany rate, only of accessory relevance in the study of thisfamily.In conclusion, the NGS study of this family shows thefrequent coexistence of multiple mutations of genesinvolved in erythrocytosis and provides a valuable approachto identifying and eventually clarifying the role of complexmultiple molecular mutations occurring in erythrocytosis.Moreover, NGS studies allow identifying specific asymp-tomatic subjects with erythrocytosis, as they deserve to becarefully followed up to prevent the potential cardiovas-cular complications of this disease.Author contributionsAll authors participated in the clinical diagnosis of the pa-tients and sample testing. IB and MLR performed theRapid Communication 563research, analyzed the data, and wrote the paper. AB andGC prompted the NGS panel and performed NGS evalua-tions. DR performed molecular biology assays to identifymutations. FP and BG followed the patients and collectedtheir data. All authors revised the paper.Conflict of interestsThe authors have no conflict of interests to declare.FundingThe paper was partially funded by the Dotazione OrdinariaRicerca (DOR) - University of Padua.Appendix A. Supplementary dataSupplementary data to this article can be found online athttps://doi.org/10.1016/j.gendis.2023.03.030.References1. Gangat N, Szuber N, Pardanani A, et al. JAK2 unmutatederythrocytosis: current diagnostic approach and therapeuticviews. Leukemia. 2021;35(8):2166e2181.2. Bento C, Percy MJ, Gardie B, et al. Genetic basis of congenitalerythrocytosis: mutation update and online databases. HumMutat. 2014;35(1):15e26.3. Biagetti G, Catherwood M, Robson N, et al. HFE mutations inidiopathic erythrocytosis. Br J Haematol. 2018;181(2):270e272.4. Lanikova L, Babosova O, Swierczek S, et al. Coexistence of gain-of-function JAK2 germ line mutations with JAK2V617F in poly-cythemia vera. Blood. 2016;128(18):2266e2270.5. Bertozzi I, Ruggeri M, Nichele I, et al. Thrombotic and hemor-rhagic complications in idiopathic erythrocytosis. Am J Hema-tol. 2017;92(11):E639eE641.Irene Bertozzi a, Andrea Benetti a, Daniela Regazzo a,Francesca Polese b, Giacomo Biagetti a, Giulio Ceolotto c,Maria Luigia Randi a,*a First Medical Clinic, University of Padua Department ofMedicine e DIMED, Via Giustiniani 2, Padova 35123, ItalybTransfusion Medicine Department, Ospedale dell’Angelo -Venezia Mestre, Via Paccagnella 1, Venice 30174, Italyc Emergency Medicine, University of Padua Department ofMedicine e DIMED, Via Giustiniani 2, Padova 35123, Italy*Corresponding author.E-mail address: marialuigia.randi@unipd.it (M.L. Randi)25 November 2022Available online 18 May 2023

Targeted NGS analysis reveals a complex genetic background of idiopathic erythrocytosis in a large Venetian family

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Targeted NGS analysis reveals a complex genetic background of idiopathic erythrocytosis in a large Venetian family

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