Jean Delaunay

Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1

Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34(+) cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells.

A Dominant Mutation in the Gene Encoding the Erythroid Transcription Factor KLF1 Causes a Congenital Dyserythropoietic Anemia

The congenital dyserythropoietic anemias (CDAs) are inherited red blood cell disorders whose hallmarks are ineffective erythropoiesis, hemolysis, and morphological abnormalities of erythroblasts in bone marrow. We have identified a missense mutation in KLF1 of patients with a hitherto unclassified CDA. KLF1 is an erythroid transcription factor, and extensive studies in mouse models have shown that it plays a critical role in the expression of globin genes, but also in the expression of a wide spectrum of genes potentially essential for erythropoiesis. The unique features of this CDA confirm the key role of KLF1 during human erythroid differentiation. Furthermore, we show that the mutation has a dominant-negative effect on KLF1 transcriptional activity and unexpectedly abolishes the expression of the water channel AQP1 and the adhesion molecule CD44. Thus, the study of this disease-causing mutation in KLF1 provides further insights into the roles of this transcription factor during erythropoiesis in humans.

The monovalent cation leak in overhydrated stomatocytic red blood cells results from amino acid substitutions in the Rh-associated glycoprotein

Overhydrated hereditary stomatocytosis (OHSt) is a rare dominantly inherited hemolytic anemia characterized by a profuse membrane leak to monovalent cations. Here, we show that OHSt red cell membranes contain slightly reduced amounts of Rh-associated glycoprotein (RhAG), a putative gas channel protein. DNA analysis revealed that the OHSt patients have 1 of 2 heterozygous mutations (t182g, t194c) in RHAG that lead to substitutions of 2 highly conserved amino acids (Ile61Arg, Phe65Ser). Unexpectedly, expression of wild-type RhAG in Xenopus laevis oocytes induced a monovalent cation leak; expression of the mutant RhAG proteins induced a leak about 6 times greater than that in wild type. RhAG belongs to the ammonium transporter family of proteins that form pore-like structures. We have modeled RhAG on the homologous Nitrosomonas europaea Rh50 protein and shown that these mutations are likely to lead to an opening of the pore. Although the function of RhAG remains controversial, this first report of functional RhAG mutations supports a role for RhAG as a cation pore.

An Extreme Consequence of Splenectomy in Dehydrated Hereditary Stomatocytosis: Gradual Thrombo‐embolic Pulmonary Hypertension and Lung–Heart Transplantation

Dehydrated hereditary stomatocytosis (DHS) belongs to the heterogeneous class of hemolytic anemias with leaky red cell membranes. Splenectomy is a highly deleterious treatment, because it favors, with virtually no exception, the occurrence of thrombo‐embolic disease. We describe here the extreme case of a patient with DHS and an associated sickle cell trait. Splenectomy was carried out due to a splenic infarction that occurred during an airplane journey. About 12 years later, the patient noticed an exertional dyspnea, which gradually worsened to such a degree that she became severely incapacitated within 5 years. Eventually, the patient developed a cor pulmonale associated with chronic thrombo‐embolic pulmonary hypertension (CTEPH) and successfully underwent a heart–lung transplant operation. This case ranks as one of the most severe examples ever recorded of the effect that splenectomy may have in DHS patients. Nonetheless, it represents the first case to receive a heart–lung transplant.

Stomatin-deficient cryohydrocytosis results from mutations in SLC2A1: a novel form of GLUT1 deficiency syndrome

The hereditary stomatocytoses are a series of dominantly inherited hemolytic anemias in which the permeability of the erythrocyte membrane to monovalent cations is pathologically increased. The causative mutations for some forms of hereditary stomatocytosis have been found in the transporter protein genes, RHAG and SLC4A1. Glucose transporter 1 (glut1) deficiency syndromes (glut1DSs) result from mutations in SLC2A1, encoding glut1. Glut1 is the main glucose transporter in the mammalian blood-brain barrier, and glut1DSs are manifested by an array of neurologic symptoms. We have previously reported 2 cases of stomatin-deficient cryohydrocytosis (sdCHC), a rare form of stomatocytosis associated with a cold-induced cation leak, hemolytic anemia, and hepatosplenomegaly but also with cataracts, seizures, mental retardation, and movement disorder. We now show that sdCHC is associated with mutations in SLC2A1 that cause both loss of glucose transport and a cation leak, as shown by expression studies in Xenopus oocytes. On the basis of a 3-dimensional model of glut1, we propose potential mechanisms underlying the phenotypes of the 2 mutations found. We investigated the loss of stomatin during erythropoiesis and find this occurs during reticulocyte maturation and involves endocytosis. The molecular basis of the glut1DS, paroxysmal exercise-induced dyskinesia, and sdCHC phenotypes are compared and discussed.

Missense mutations in the ABCB6 transporter cause dominant familialpseudohyperkalemia

Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by increased serum [K+] in whole blood stored at or below room temperature, without additional hematological abnormalities. Functional gene mapping and sequencing analysis of the candidate genes within the 2q35–q36 critical interval identified—in 20 affected individuals among three multigenerational FP families—two novel heterozygous missense mutations in the ABCB6 gene that cosegregated with disease phenotype. The two genomic substitutions altered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that, in erythrocyte membranes, bears the Langereis blood group antigen system. The ABCB6 R375Q mutation did not alter the levels of mRNA or protein, or protein localization in mature erythrocytes or erythroid precursor cells, but it is predicted to modestly alter protein structure. ABCB6 mRNA and protein levels increase during in vitro erythroid differentiation of CD34+ erythroid precursors and the erythroleukemia cell lines HEL and K562. These data suggest that the two missense mutations in residue 375 of the ABCB6 polypeptide found in affected individuals of families with chromosome 2‐linked FP could contribute to the red cell K+ leak characteristic of this condition. Am. J. Hematol. 2013.

Role of the interaction between Lu/BCAM and the spectrin-based membrane skeleton in the increased adhesion of hereditary spherocytosis red cells to laminin

Lu/BCAM, the unique erythroid receptor for laminin 511/521, interacts with the erythrocyte membrane skeleton through spectrin binding. It has been reported that Hereditary Spherocytosis red blood cells (HS RBC) exhibit increased adhesion to laminin. We investigated the role of Lu/BCAM–spectrin interaction in the RBC adhesion properties of 2 splenectomised HS patients characterized by 40% spectrin deficiency. Under physiological flow conditions, HS RBC exhibited an exaggerated adhesion to laminin that was completely abolished by soluble Lu/BCAM. Triton extraction experiments revealed that a greater fraction of Lu/BCAM was unlinked to the membrane skeleton of HS RBC, as compared to normal RBC. Disruption of the spectrin interaction site in Lu/BCAM expressed in the transfected K562 cell line resulted in a weakened interaction to the skeleton and an enhanced interaction to laminin. These results demonstrated that the adhesion of HS RBC to laminin was mediated by Lu/BCAM and that its interaction with the spectrin‐based skeleton negatively regulated cell adhesion to laminin. Finally, the results of this study strongly suggest that the reinforced adhesiveness of spectrin‐deficient HS RBC to laminin is partly brought about by an impaired interaction between Lu/BCAM and the membrane skeleton.

Two founder mutations in the SEC23B gene account for the relatively high frequency of CDA II in the Italian population

Congenital Dyserythropoietic Anemia type II is an autosomal recessive disorder characterized by unique abnormalities in the differentiation of cells of the erythroid lineage. The vast majority of CDA II cases result from mutations in the SEC23B gene. To date, 53 different causative mutations have been reported in 86 unrelated cases (from the CDA II European Registry), 47 of them Italian. We have now identified SEC23B mutations in 23 additional patients, 17 Italians and 6 non‐Italian Europeans. The relative allelic frequency of the mutations was then reassessed in a total of 64 Italian and 45 non‐Italian unrelated patients. Two mutations, E109K and R14W, account for over one‐half of the cases of CDA II in Italy. Whereas the relative frequency of E109K is similar in Italy and in the rest of Europe (and is also prevalent in Moroccan Jews), the relative frequency of R14W is significantly higher in Italy (26.3% vs. 10.7%). By haplotype analysis we demonstrated that both are founder mutations in the Italian population. By using the DMLE+ program our estimate for the age of the E109K mutation in Italian population is ≈2,200 years; whereas for the R14W mutation it is ≈3,000 years. We hypothesize that E109K may have originated in the Middle East and may have spread in the heyday of the Roman Empire. Instead, R14W may have originated in Southern Italy. The relatively high frequency of the R14W mutation may account for the known increased prevalence of CDA II in Italy. Am. J. Hematol. 86:727–732, 2011.

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

To the editor:nnSoutheast 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

Southeast Asian ovalocytosis and a sickle cell trait in a young patient with sudden retinal stroke: a fortuitous association?

We report a case of retinal stroke in a patient from the Comoros Islands with both sickle cell trait and Southeast Asian ovalocytosis (SAO). Southeast Asian ovalocytosis is a dominantly inherited trait, frequent in Southeast Asia, caused by a 27 nucleotide deletion in the SLC4A1 gene that encodes band 3, leading to a decreased anion exchange but an increased cation leak across the erythrocyte membrane. We hypothesized that the red cell dehydration that can be induced by this cation leak can facilitate polymerization of Hb S [β6(A3)Gluu2009→Val, GAG>GTG]. Southeast Asian ovalocytosis could then be a risk factor for rare microvascular complications in sickle cell trait.