Ribeiro Ml

Genetic basis of Congenital Erythrocytosis mutation update and online databases

Congenital erythrocytosis (CE), or 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. Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and results from mutations in the Epo receptor gene (EPOR). Secondary CE arises from conditions causing tissue hypoxia and results in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (HBB, HBA mutations), decreased production of 2,3‐bisphosphoglycerate due to BPGM mutations, 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 With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr‐Euronet developed a comprehensive Internet‐based database focusing on the registration of clinical history, hematological,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.

Molecular study of congenital erythrocytosis in 70 unrelated patients revealed a potential causal mutation in less than half of the cases (Where is/are the missing gene(s)?)

Congenital 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 upregulation of Epo production. Primary erythrocytosis is associated with mutations in the EPOR gene, secondary CE 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. Chuvash polycythemia, caused by mutations in VHL gene, shares features of both primary and secondary erythrocytosis, with increased Epo production but also hypersensitivity of progenitors to Epo.

Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells

Summary Pyruvate 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.

Detection of new pathogenic mutations in patients with congenital haemolytic anaemia using next-generation sequencing

Congenital haemolytic anaemia (CHA) refers to a group of genetically heterogeneous disorders, mainly caused by changes in genes encoding globin chains, cytoskeletal proteins and red cell enzymes, in which accurate diagnosis can be challenging with conventional techniques.

RhD variant caused by an in-frame triplet duplication in the RHD gene.

BACKGROUND: The RHD gene is highly polymorphic and a large number of D variants have already been detected. Several mechanisms are involved in the origin of D variants. In‐frame deletions, resulting in a single‐amino‐acid deletion, have been described associated with RhD and RhCE variants. No in‐frame duplications and/or insertions have been reported in the RH genes to date.

Transient neonatal cyanosis associated with a new Hb F variant: Hb F viseu.

Neonatal cyanosis in healthy newborns can be associated either with methemoglobin due to cytochrome b5 reductase deficiency or to M-hemoglobin, a group of hemoglobin variants resulting from mutations in the globin chain genes. We report the clinical case of a neonate with cyanosis and normal cardiac and respiratory function. At birth the hematological parameters were normal; however, the methemoglobinemia was 16%. Spontaneously, the cyanosis gradually decreased and by the fifth month of age the methemoglobin level was normal. A heterozygous G&ggr;-globin gene (HBG2) missense mutation 87 C-A (Leu28Met) was identified. His father, with a history of transfusion in the neonatal period, is heterozygous for the same mutation. This hemoglobin variant, not previously described, was called Hb F Viseu and is the sixth G&ggr;-chain variant reported in association with neonatal cyanosis.

Hb Vila Real [β36(C2)Pro→His]: A Newly Discovered High Oxygen Affinity Variant

A 15-year-old girl was referred to the hematology out patient clinic for diagnosis of erythrocytosis. She was asymptomatic and submitted to extensive investigations to exclude pulmonary, renal or cardiac disease. Her mother has undergone regular phlebotomies over the past 20 years for polycythemia, with an obstetric history of two miscarriages, a stillborn baby, and two normal children born by elective Caesarean section. The proband and her mother presented a high hemoglobin (Hb) level with normal erythrocyte indices and normal leukocyte and platelet counts. Hb isoelectrofocusing (IEF) did not show any abnormal band. Quantification of Hb A, and Hb F (1 ) was normal (Table I). On cation exchange high performance liquid chromatography (HPLC) a Hb variant eluted with normal Hb A,. Reversed phase HPLC showed a p chain variant with 36% of total p chains. DNA was isolated from peripheral blood leukocytes (2) and amplified by polymerase chain reaction (PCR) using previously described conditions (3). Localization ofthe mutation was done by single strand conformation analysis (SSCA) (4) of DNA fragments encompass-

Abnormal NK cell lymphocytosis detected after splenectomy: association with repeated infections, relapsing neutropenia, and persistent polyclonal B-cell proliferation.

We report the case of a boy with hereditary spherocytosis who presented with mild microcytic hypochromic anemia and recurrent leg ulcers that had been present since childhood. Chronic natural killer (NK) cell and B-cell lymphocytosis was detected 1 year after therapeutic splenectomy during investigation of recurrent episodes of neutropenia and persistent lymphocytosis. NK cells proved to be abnormal at immunophenotyping studies, and B-cells were polyclonal and displayed a normal immunophenotype. Genotypic analysis of T-cell receptor (TCR)-β and TCR-γ genes showed a germ-line pattern. The clinical course of this patient was characterized by multiple pulmonary infections and amygdalitis. We discuss the potential roles of persistent immune stimulation due to chronic hemolysis and severe leg ulcers and of splenectomy in the origin of NK cell lymphocytosis and the relationship between NK cells and recurrent infections, relapsing neutropenia, and polyclonal B-cell response.Int J Hematol. 2002;75:484–488.

Primary familial congenital erythrocytosis: two novel EPOR mutations found in Spain

Sir, Congenital erythrocytosis is a rare disorder where patients present with high hemoglobin levels without a secondary cause. Knowledge about the molecular mechanisms involved in familial erythrocytosis has improved in recent years [1]. Taking into account the hematological parameters, familial history and serum erythropoietin (Epo) levels, it is possible to establish a diagnostic algorithm [2]. Low serum Epo level with a strong familial history can be due to mutations in Epo receptor gene (EPOR), known as Primary familial congenital polycythemia (PFCP) [3]. Identification of the causal mutations in congenital erythrocytosis helps to establish the clinical diagnosis and treatment; however, cases with mutations identified are sparse, and the underlying molecular defect remains unknown in the great majority of described cases. We studied two different cases from Spain referred to our laboratory for investigation of erythrocytosis with normal leukocyte and platelet counts and no splenomegaly. All secondary causes causing increased hemoglobin levels were already excluded as well as the JAK2 V617F and exon12 mutations. All subjects studied gave their informed consent. Case 1 Three members of the same family, belonging to three different subsequent generations, from La Coru~ na, Spain. The propositus is a 69-year-old man under regular phlebotomy treatment, diagnosed 14 years ago with idiopatic erythrocytosis. His son (42 years old) and his grandnephew (3 years old), presented also with high hemoglobin levels (Hb 18.9 and 20.0 g/dL, respectively) All of them have serum Epo bellow 2.0 mU/mL (Normal 3–34 mU/mL). As the Epo levels were low, and the family history suggests a dominant inheritance pattern, we sequenced the EPOR gene and found a novel heterozygous frameshift mutation due to the deletion of two nucleotides (c.1311_1312delTC) (Figure 1a). This mutation results in a premature termination at codon 443, which predicts the production of an EPOR molecule truncated for 66 amino acids at the C-terminal end of the protein (p.Pro438Metfs*6) (Figure 1c). Case 2 A 73-year-old male from La Coru~ na, Spain, presented with Hb18,9 g/dL; hematocrit (Hct) 58%. Clinical examination was unremarkable, with no signs or symptoms of respiratory insufficiency. Serum erythropoietin level was markedly reduced (2.9 mIU/mL). Neither children nor other family members were available to be studied.

Gene symbol: NT5C3. Disease: haemolytic anemia.

Department of Neurological and Behavioural Sciences, University of Siena, Policlinico Le Scotte, Viale Bracci 2, 53100 Siena, Italy, email: bianchi22@unisi.it, Tel.: +39-057-7585760, Fax: +39-057-740327 Gross insertions Accession Number Description Hgi0501 In two unrelated Portuguese patients with pyrimidine 5¢-nucleotidase (P5¢N)deficiency we have found the insertion of an Alu element within the exon9 of the structural gene for type-I isoform of P5’N, between nucleotides 743-744. The new Alu element share 98.2 identity with AluYa5 and AluYa1 subfamilies. RT-PCR analysis showed that the Alu insertion leads to the skipping of the exon9 in the mRNA transcript