Unravelling Genetic Mechanisms of Erythrocytosis: A Real-Life Experience from a Single Center
Authors: Gurnari Carmelo , Lombardi Annamaria , Cosi Elisabetta , Biagetti Giacomo , Buccisano Francesco , Franceschini Luca , Rizzo Manuela , Di Veroli Ambra , Maurillo Luca , Falconi Giulia , Fabiani Emiliano , Cantonetti Maria , Provenzano Ida , Nasso Daniela , Lo-Coco Francesco , Randi Maria Luigia , Voso Maria Teresa 
Published: October 2019

Abstract
Introduction
Erythrocytosis is characterized by persistently raised hemoglobin (HB) and hematocrit (Ht) levels. Differential diagnosis includes Polycythemia Vera (PV), secondary, hereditary (HE) and idiopathic erythrocytosis (IE). Recently, Randi et al. (Br J Haematol, 2018) demonstrated the recurrence of HFE single nucleotide variants (SNVs) in patients with IE, postulating a possible link between Hemochromatosis genes and erythrocytosis. The most frequent HFE SNVs are C282Y and H63D, reported at allele frequencies of about 13% and 4% in Caucasian countries. HE has also been associated with mutations in genes members of the complex pathway of "oxygen sensing" (EPOR, VHL, HIF-2a and PHD2). To date, 30 mutations have been described in the PHD2 gene, all localized in the catalytic domain, which impair binding to HIF-2a. The PHD2p.C127S variant is frequently observed in Tibetans with D4E in cis (overall prevalence of this haplotype is 88.6% at altitudes above 3000m), in linkage disequilibrium with other missense mutations (in particular HIF-2a/EPAS1). Surprisingly, the combination of PHD2/HIF-2a variants result in a gain-of-function effect that blunts the hypoxic response, providing a molecular mechanism for the observed protection of Tibetans from erythrocytosis at high altitude. In normoxic conditions and in low-landers, the PHD2p.C127S variant may lead to increased erythropoiesis as reported in the literature.
Patients and Methods
Twenty-three patients with erythrocytosis (21 males and 2 females with a median age of 56 years, range 18-76, Table 1) negative for JAK2 mutations (both V617F and exon 12 variants), and with a bone marrow histology not suggestive of a myeloproliferative syndrome were studied for secondary causes of erythrocytosis using an appropriate algorithm (EPO levels, chest and abdomen imaging, spirometry, venous p50 of HB, arterial blood gas analysis). Since all tests were negative, HE genes mutation analysis was carried out, using Sanger sequencing of EPOR exon 8, the VHL coding region, PHD2 exon 1-3 and HIF-2a exon 12. HFE SNVs were studied using allele-specific real-time PCR.
Results
Sequencing of HE genes identified 4 carriers of PHD2 variants in 23 patients (17%). One patient had a novel missense heterozygous mutation (PHD2p.I269N). The study of his kindred showed that his sister and one daughter have HE and both carried the same heterozygous mutation of the propositus. Furthermore, his father had died with a diagnosis of PV in the pre-JAK2 era. In contrast, the patient's brother and the other daughter, both with normal Hb and Hct, had wild-type PHD2. Of note, the propositus and his two daughters were heterozygous for H63D in the HFE gene. The patient's pedigree is illustrated in Figure 1. Interestingly, three further patients tested positive for the PHD2 missense heterozygous variant (PHD2p.C127S) previously reported in the Tibetan population, and whose role in patients with erythrocytosis is still unclear.
HFE SNVs real-time PCR revealed 7 carriers of the H63D, 1 of C282Y and 1 of S65C SNV in the HFE gene (about 40% of our cohort). Interestingly, two H63D heterozygous patients were a father and his son. They were all males with a median age of 48 years (range 18-64). The prevalence of HFE SNVs in our cohort of patients with erythrocytosis is higher than expected for H63D and S65C for Caucasians (30% vs 13% for H63D and 4.35 vs 1.5% for S65C), while the allele frequency of C282Y was similar to that of the general population. Finally, the patient with the S65C SNV also had the "Tibetan" PHD2 polymorphism.
Conclusions
In addition to previously known PHD2 gene alterations, we report here the occurrence in HE patients of a novel PHD2 mutation with an autosomal-dominant inheritance likely involved in disease pathogenesis. The milder phenotypic features of this patient's daughter and sister in terms of erythocytosis, may be explained by the childbearing age and the absence of H63D SNV, respectively. The increased prevalence of HFE SNVs in patients with IE may indicate an effect of impaired iron metabolism on erythropoiesis.
Our data show that the inclusion of HFE SNVs and oxygen pathway mutational analysis in the diagnostic algorithm of erythrocytosis may help to better define the genetic basis of erythrocytosis. Further studies -including the analysis of molecules involved in iron storage pathway- in larger IE patient cohorts are warranted in order to clarify the link between HFE gene and IE.

Disclosures
Voso: Celgene: Research Funding, Speakers Bureau.