Congenital Erythrocytosis (CE), also called congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. 3Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and generally results from mutations in the erythropoietin-receptor gene (EPOR).Secondary congenital erythrocytosis arises from conditions which cause tissue hypoxia thus resulting in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (genes HBB, HBA1 and HBA2), decreased production of 2,3-biphosphoglycerate due to mutations in the BPGM gene, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1 and EGLN1). Depending on the affected gene CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo.Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients.With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr-Euronet developed a comprehensive internet-based database focusing on the registration of clinical history, hematological, biochemical and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database (LOVD).

Pyruvate kinase deficiency (PKD) is the most common enzyme defect of glycolysis and an important cause of hereditary, nonspherocytic hemolytic anemia. The disease has a worldwide geographical distribution but there are no verified data regarding its frequency. Difficulties in the diagnostic workflow and interpretation of PK enzyme assay likely play a role. By the creation of a global PKD International Working Group in 2016, involving 24 experts from 20 Centers of Expertise we studied the current gaps in the diagnosis of PKD in order to establish diagnostic guidelines. By means of a detailed survey and subsequent discussions, multiple aspects of the diagnosis of PKD were evaluated and discussed by members of Expert Centers from Europe, USA, and Asia directly involved in diagnosis. Broad consensus was reached among the Centers on many clinical and technical aspects of the diagnosis of PKD. The results of this study are here presented as recommendations for the diagnosis of PKD and used to prepare a diagnostic algorithm. This information might be helpful for other Centers to deliver timely and appropriate diagnosis and to increase awareness in PKD.

IntroductionCongenital Erythrocytosis can be classified as primary, when the defect is intrinsic to the RBC progenitors and independent of the serum erythropoietin (Epo) concentration, or secondary, when the erythrocytosis is the result of an up-regulation of Epo production. Primary erythrocytosis is associated with mutations in the EPOR gene, secondary congenital erythrocytosis can de due to mutations that stabilize the hemoglobin in the oxygenated form or to mutations in the genes that control the transcriptional activation of the EPO gene -VHL, EGLN1, EPAS1. Material and MethodsWith the main objective of describing the etiology and molecular basis of congenital erythrocytosis we have studied 70 consecutive unrelated patients presenting with idiopathic erythrocytosis from our hematology clinic or referred from other centers. According to a study algorithm we have sequenced all the genes described as associated with congenital erythrocytosis. Results and DiscussionErythrocytosis molecular etiology was identify in 25 (36%) of the 70 subjects. High-affinity Hb variants were the most common cause, present in 20% of the cases. New mutations were identified in the JAK2, EPOR, VHL and EGLN1 genes. Conclusions High affinity hemoglobin variants are a very rare cause of secondary congenital erythrocytosis, but it seems likely that their incidence may be underestimated. Our experience shows that in erythrocytosis with a dominant inheritance and normal or inappropriate high Epo levels, the HBB and HBA genes should be the first to be studied. In spite of the seven genes known to be involved in congenital erythrocytosis, the majority of the cases have unknown etiology.

SummaryPyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.

We describe the second patient with anionic exchanger 1/band 3 null phenotype (band 3 null ), which was caused by a novel nonsense mutation c.1430C>A (p.Ser477X) in exon 12 of SLC4A1. We also update on the previous band 3 null patient, thereby elucidating the physiological implications of total loss of AE1/band 3. Besides transfusion-dependent severe hemolytic anemia and complete distal renal tubular acidosis, dyserythropoiesis was identified in the band 3 null patient, suggesting a role for band 3 in erythropoiesis. Moreover, we also, for the first time, report that long-term survival is possible in band 3 null patients.