Serge Pissard

Pathophysiology of sickle cell disease is mirrored by the red blood cell metabolome

Emerging metabolomic tools can now be used to establish metabolic signatures of specialized circulating hematopoietic cells in physiologic or pathologic conditions and in human hematologic diseases. To determine metabolomes of normal and sickle cell erythrocytes, we used an extraction method of erythrocytes metabolites coupled with a liquid chromatography-mass spectrometry-based metabolite profiling method. Comparison of these 2 metabolomes identified major changes in metabolites produced by (1) endogenous glycolysis characterized by accumulation of many glycolytic intermediates; (2) endogenous glutathione and ascorbate metabolisms characterized by accumulation of ascorbate metabolism intermediates, such as diketogulonic acid and decreased levels of both glutathione and glutathione disulfide; (3) membrane turnover, such as carnitine, or membrane transport characteristics, such as amino acids; and (4) exogenous arginine and NO metabolisms, such as spermine, spermidine, or citrulline. Finally, metabolomic analysis of young and old normal red blood cells indicates metabolites whose levels are directly related to sickle cell disease. These results show the relevance of metabolic profiling for the follow-up of sickle cell patients or other red blood cell diseases and pinpoint the importance of metabolomics to further depict the pathophysiology of human hematologic diseases.

Erythrocyte density in sickle cell syndromes is associated with specific clinical manifestations and hemolysis.

Dense, dehydrated red blood cells (DRBCs) are a characteristic feature of sickle-cell disease (SCD). DRBCs play a role in the pathophysiology of SCD acute and chronic organ damage because of heightened tendency to undergo polymerization and sickling because of their higher hemoglobin S concentration. Relations between red cell density (assessed with phthalate density-distribution profile method) and several hematologic, biochemical, genetic parameters, and clinical manifestations were studied in a large cohort of homozygous patients. The percentage of DRBCs was significantly higher in patients who experienced skin ulcers, priapism, or renal dysfunction. Presence of α-thalassemia deletions was associated with fewer DRBCs. A multivariable analysis model showed DRBCs to be positively associated with hemolytic parameters such as lactate dehydrogenase and bilirubin and negatively with fetal hemoglobin. The percentage of DRBCs decreased by 34% at 6 months of hydroxycarbamide (xydroxyurea) therapy. Thus, DRBCs are associated with specific clinical manifestations and biologic markers and may be a useful addition to the biologic and clinical evaluation of patients with SCD, because they can easily be measured in a hematocrit tube.

A genetic score for the prediction of beta-thalassemia severity.

Clinical and hematologic characteristics of beta(β)-thalassemia are determined by several factors resulting in a wide spectrum of severity. Phenotype modulators are: HBB mutations, HBA defects and fetal hemoglobin production modulators (HBG2:g.−158C>T polymorphism, HBS1L-MYB intergenic region and the BCL11A). We characterized 54 genetic variants at these five loci robustly associated with the amelioration of beta-thalassemia phenotype, to build a predictive score of severity using a representative cohort of 890 β-thalassemic patients. Using Cox proportional hazard analysis on a training set, we assessed the effect of these loci on the age at which patient started regular transfusions, built a Thalassemia Severity Score, and validated it on a testing set. Discriminatory power of the model was high (C-index=0.705; R2=0.343) and the validation conducted on the testing set confirmed its predictive accuracy with transfusion-free survival probability (P<0.001) and with transfusion dependency status (Area Under the Receiver Operating Characteristic Curve=0.774; P<0.001). Finally, an automatized on-line calculation of the score was made available at http://tss.unica.it. Besides the accurate assessment of genetic predictors effect, the present results could be helpful in the management of patients, both as a predictive score for screening and a standardized scale of severity to overcome the major-intermedia dichotomy and support clinical decisions.

Comprehensive functional annotation of 18 missense mutations found in suspected hemochromatosis type 4 patients

Hemochromatosis type 4 is a rare form of primary iron overload transmitted as an autosomal dominant trait caused by mutations in the gene encoding the iron transport protein ferroportin 1 (SLC40A1). SLC40A1 mutations fall into two functional categories (loss- versus gain-of-function) underlying two distinct clinical entities (hemochromatosis type 4A versus type 4B). However, the vast majority of SLC40A1 mutations are rare missense variations, with only a few showing strong evidence of causality. The present study reports the results of an integrated approach collecting genetic and phenotypic data from 44 suspected hemochromatosis type 4 patients, with comprehensive structural and functional annotations. Causality was demonstrated for 10 missense variants, showing a clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups of loss-of-function mutations were distinguished: one impairing cell-surface expression and one altering only iron egress. Additionally, a new gain-of-function mutation was identified, and the degradation of ferroportin on hepcidin binding was shown to probably depend on the integrity of a large extracellular loop outside of the hepcidin-binding domain. Eight further missense variations, on the other hand, were shown to have no discernible effects at either protein or RNA level; these were found in apparently isolated patients and were associated with a less severe phenotype. The present findings illustrate the importance of combining in silico and biochemical approaches to fully distinguish pathogenic SLC40A1 mutations from benign variants. This has profound implications for patient management.

A novel (ɛγδβ)°-thalassemia deletion associated with an α globin gene triplication leading to a severe transfusion dependant fetal thalassemic syndrome

(ɛγδβ)°-thalassemia is a very rare form of thalassemia only recognized in heterozygotes and caused by unique and sporadic deletions.[1][1]–[3][2] The α/non α–globin chain ratio is imbalanced like in β thalassemia patients but without elevation of HbF or HbA2.[4][3] They are characterized

Variability of hemoglobin F expression in hemoglobin EE disease: Hematological and molecular analysis

Although the molecular basis of variability of hemoglobin (Hb) F has been extensively examined in β-thalassemia and sickle cell diseases, less study has been done on Hb E disorder. To address the variability of Hb F expression in Hb EE disease, we have examined multiple single nucleotide polymorphisms (SNPs) in the β-globin gene cluster, BCL11A and HBS1L-MYB genes and determined their associations with Hb F levels in this syndrome. Study was done on 141 adult Thai individuals with homozygous Hb E. Hematological parameters were recorded and Hb F measured using Hb-HPLC analyzer. It was found in 26 cases that co-inheritance of α-thalassemia could lead to significant lower production of Hb F. Association of Hb F expression with the (G)γ-Xmn I polymorphism and other SNPs including rs2297339, rs2838513, rs4895441 and rs9399137 in HBS1L-MYB gene and rs4671393 and rs11886868 in BCL11A gene was therefore analyzed in the remaining 115 cases without α-thalassemia. It was found that 4 of these 7 SNPs including (G)γ-XmnI polymorphism (rs7482144), HBS1L-MYB (rs4895441) and (rs9399137) and BCL11A (rs4671393) were significantly associated with higher proportions of subjects with high Hb F (Hb F≥5%). The result demonstrated that multiple genetic modifying factors including T allele of (G)γ-XmnI polymorphism (rs7482144), G allele of HBS1L-MYB (rs489441), C allele of HBS1L-MYB (rs9399137) and C allele of BCL11A (rs4671393) are associated with increased Hb F and in combination could explain approximately 80% of the variation of Hb F in Hb EE disease in Thai population. Other genetic factors regulating Hb F expression in this common genetic disorder remains to be elucidated.

Evaluation of the free α-hemoglobin pool in red blood cells: a new test providing a scale of β-thalassemia severity.

β-Thalassemias are characterized by an imbalance of globin chains with an excess of α-chains which precipitates in erythroid precursors and red blood cells (RBCs) leading to inefficient erythropoiesis. The severity of the disease correlates with the amount of unpaired α-chains.Our goal was to develop a simple test for evaluation of the free α-hemoglobin pool present in RBC lysates. Alpha-Hemoglobin Stabilizing Protein (AHSP), the chaperone of α-Hb, was used to trap excess a-Hb. A recombinant GST-AHSP fusion protein was bound to an affinity micro-column and then incubated with hemolysates of patients. After washing, the α-Hb was quantified by spectrophotometry in the elution fraction. This assay was applied to 54 patients: 28 without apparent Hb disorder, 20 β-thalassemic and 6 α-thalassemic. The average value of free α-Hb pool was 93 ± 21 ppm (ng of free α-Hb per mg of Hb subunits)in patients without Hb disorder, while it varies from 119 to 1,756 ppm, in β-thalassemic patients and correlated with genotype. In contrast,the value of the free α-Hb pool was decreased in α-thalassemic patients (65 ± 26 ppm). This assay may help to characterize β-thalassemia phenotypes and to follow the evolution of the globin chain imbalance(α/β+γ ratio) in response to treatment.

Homozygous Southeast Asian ovalocytosis is a severe dyserythropoietic anemia associated with distal renal tubular acidosis.

To the editor: Southeast Asian ovalocytosis (SAO) is caused by a heterozygous 27-nucleotide deletion in SLC4A1 coding for band 3, the anion-exchange protein of the red cell membrane.[1][1][⇓][2]-[3][3] This asymptomatic dominant trait is considered as a host genetic adaptation to malaria in

Characterization of Two Unique α-Globin Gene Cluster Deletions Causing α-Thalassemia in Israeli Arabs

Abstract The molecular basis of α-thalassemia (α-thal) is complex. The use of multiplex ligation-dependent probe amplification (MLPA) has offered the possibility of identifying more gene deletions causing α-thal. Our objective was to determine the molecular basis of two patients with Hb H (β4) disease. By using MLPA in combination with comparative genomic hybridization (CGH) we identified two novel α-globin gene cluster deletions: a 30 kb deletion (patient 1) we refer to as – –JAL and a large 216 kb deletion (patient 2) we refer to as – –LOD. Patient 1 was a compound heterozygote for – –JAL and –α3.7 (rightward deletion). Twelve family members of patient 1 carrying the – –JAL deletion were available for evaluation: five with – –JAL/–α3.7, four with – –JAL/αHph Iα and three with – –JAL/αα. Their clinical picture of compound heterozygosity was compatible with moderate Hb H disease. In patient 2 (– –LOD/–α3.7), no additional symptoms were present despite the heterozygous deletion of seven known genes, three non coding RNAs (ncRNAs), four unknown genes and two pseudo genes. Further analysis of more patients with α-thal deletions will have implications for genetic counseling and appropriate therapy.

Augmentation de l’hémoglobine A2 au cours du pseudoxanthome élastique

Resume Introduction Le pseudoxanthome elastique (PXE)est habituellement associe a des mutations du gene ABCC6 .Un phenotype PXE a ete decrit chez des malades grecs et italiens souffrant d ’une thalassemie beta mais n ‘ayant pas de mutations de ABCC6 .Nous avons voulu determiner la frequence de la thalassemie beta dans une cohorte de malades francais ayant un PXE. Malades et methodes Cinquante malades atteints de PXE ont ete inclusdans l ’etude.Les examens biologiques comprenaient une electrophorese de l ’hemoglobine,une etude du gene ABCC6 et dans certains cas, une etude du gene beta-globine . Resultats Aucune thalassemie beta n ’ a ete diagnostiquee dans cette cohorte de malades francais atteints de PXE.En revanche,20 p.100 d ’entre eux avaient une augmentation significative mais isolee (sans anemie microcytaire)de l ’hemoglobine A2 (HbA2). Les comparaisons statistiques ne montraient pas de differences d ’origine geographique ou de gravite du PXE entre les malades ayant une HbA2 elevee et ceux ayant une HbA2 normale,sauf en ce qui concerne la severite de l ’atteinte cutanee.L ’etude du gene beta-globine ne mettait en evidence une mutation que chez les deux malades ayant l ’HbA2 la plus elevee.Un digenisme ABCC6 + beta-globine etait ecarte dans la genese du PXE. Discussion Le phenotypePXE observe chez certains thalassemiques beta parait etre en rapport avec une modification epigenetique de la transcription de ABCC6 et dependant specifiquement du locus beta-globine .L ’augmentation isolee de HbA2 constitue vraisemblablement un signe biologique accompagnant le PXE.La encore une interaction fonctionnelle epigenetique est suspectee entre ABCC6 et le locus beta-globine .Toutefois ces interactions reciproques sont manifestement desequilibrees puisque la modification de transcription de ABCC6 au cours de la thalassemie beta est responsable d ’un phenotype PXE,alors que l ’augmentation de HbA2 au cours du PXE ne s ’accompagne d ’aucune traduction clinique.