[Show abstract][Hide abstract] ABSTRACT: Dehydrated hereditary stomatocytosis is a genetic condition with defective red blood cell membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations in the mechanically activated PIEZO1 (FAM38A) ion channel were associated with dehydrated hereditary stomatocytosis. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated dehydrated hereditary stomatocytosis cases, we identify three novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for dehydrated hereditary stomatocytosis. All the dehydrated hereditary stomatocytosis-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in red blood cells of dehydrated hereditary stomatocytosis patients. Our findings also suggest a new role for mechanotransduction in red blood cell biology and pathophysiology.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pH(i)) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH(3) transport through the channel and NH(3) diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rh(null) lacking RhAG, we observed that alkalinization rate constant values decreased ∼50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer.
[Show abstract][Hide abstract] ABSTRACT: 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.
American Journal of Hematology 09/2011; 86(9):727-32. DOI:10.1002/ajh.22096 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Complete loss of protein 4.1R in red blood cell membrane is a very rare condition in humans. We here explore the third case. The morphological and biochemical observations suggested that the proband suffers from homozygous hereditary elliptocytosis. Both parents, who are consanguineous, have an elliptocytosis with no cell fragmentation, typical of a heterozygous 4.1R deficiency with a silent allele. A genomic deletion was found; it encompasses about 50 kb of genomic DNA, and suppresses the two key exons 2 and 4, which contain the two functional AUG translation initiation sites in erythroid and nonerythroid cells. The alternative first exons are intact, hence preserving the transcription potential of the altered gene. Extensive analysis of 4.1R transcripts revealed multiple splicing defects upstream of the deleted sequences. Importantly, we found that most of the transcripts generated from the altered gene are intercepted by the nonsense-mediated mRNA decay mechanism, suggesting that the massive degradation of the mRNA species jeopardizes the production of shortened but functional protein 4.1R from an alternative translation initiation site downstream of the deletion.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: We report two unusual presenting manifestations of Wiskott-Aldrich syndrome (WAS), recurrent acute hemorrhagic edema of infancy (AHEI); a form of cutaneous vasculitis and hyperostosis of the tibia. Though cutaneous vasculitis is known to occur in WAS, presentation in early infancy and as AHEI is extremely uncommon. Hyperostosis is not a well-recognized association in WAS; only three patients with this association have been previously reported. In our patient these two unusual manifestations preceded the onset of recurrent infections. Recognition of this rare presentation led us to an early diagnosis of WAS, associated with p.Glu31Lys mutation in the WAS protein.
[Show abstract][Hide abstract] ABSTRACT: Several pediatric patients showing symptoms consistent with the Wiskott-Aldrich syndrome (WAS) were referred to us and turned out to display the c.273+11dup change in the WAS gene. It consisted of the insertion of one C in an unusual tract of 7C near the intron 2 donor splicing site of the WAS gene. In the patients, non-synonymous WAS mutations were found twice only and one mutation was elucidated in RUNX1. In the absence of a non-synonymous mutation in the WAS gene, the c.273+11dup change affected neither the levels nor the sequence of WAS mRNA. In the presence of a non-synonymous WAS mutation, the c.273+11dup alteration failed to worsen the expected phenotype. Minor splicing abnormalities concerning exon 10 were observed both in WAS patients, and in healthy individuals carrying or not carrying the c.273+11dup. The c.273+11dup change was encountered four times in 107 normal male and female controls (172 alleles tested: 2.3%), and eight times in a series of 248 male patients (248 alleles tested: 3.2%). We conclude that the presence of the additional C in the WAS gene is a functionally neutral polymorphism.
European Journal Of Haematology 06/2011; 87(4):366-71. DOI:10.1111/j.1600-0609.2011.01674.x · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction
Nous avons étudié une famille originaire de Mayotte présentant une ovalocytose mélanésienne, trait caractérisé par la présence asymptomatique de nombreux ovalostomatocytes de rigidité accrue. Transmise selon le mode autosomique dominant, l'ovalocytose est la conséquence d'une unique délétion des codons 400-408 du gène SLC4A1 qui code pour la bande 3. Jamais décrite à l'état homozygote, l'ovalocytose est supposée non viable sous cette forme.
Matériels et Méthodes
Les globules rouges des parents ont été analysés par les tests classiques et par ektacytométrie et électrophorèse des protéines membranaires. L'étude des locus de globines et du gène SLC4A1 a été réalisée par PCR séquençage, la recherche de délétions d'alpha-thalassémie par GAP-PCR.
La grossesse est compliquée, à 24 SA, par un hydramnios associé à une anémie sévère, nécessitant une première transfusion in utero, puis une seconde 7 semaines plus tard. Celle-ci est suivie par une bradycardie sévère qui entraîne le déclenchement de l'accouchement par césarienne à 31 SA. L'hémolyse se poursuivant, des transfusions mensuelles sont nécessaires. A l'âge de trois mois, une acidose tubulaire rénale distale est mise en évidence et traitée par supplémentation en bicarbonates. A 16 mois, l'enfant suit un programme transfusionnel et se porte bien. Les parents sont asymptomatiques, néanmoins, l'étude de l'hémoglobine permet d'identifier un variant 'La Désirade' à l'état hétérozygote chez le père et un trait Hb S associée à une délétion alpha-3.7 homozygote chez la mère. L'enfant est porteur du variant 'La Désirade' à l'état hétérozygote et d'une délétion alpha - 3.7 à l'état hétérozygote, qui n'expliquent pas la sévérité de l'hémolyse. L'étude des globules rouges par ektacytométrie met en évidence une ovalocytose chez les parents. La délétion des codons 400-408 du gène SLC4A1 est présente à l'état hétérozygote chez chacun des parents et à l'état homozygote chez l'enfant.
Nous avons mis en évidence la présence à l'état homozygote de la délétion caractéristique de l'ovalocytose mélanésienne. Le phénotype du proband démontre la sévérité de l'affection, traduite par un tableau d'anasarque fœtal, puis par une anémie sévère associée à une acidose tubulaire rénale distale. Contre la notion établie, l'ovalocytose homozygote peut être viable sous réserve d'une prise en charge anténatale intensive.
Congrès de la Société Française d'Hématologie, Paris; 03/2011
[Show abstract][Hide abstract] ABSTRACT: Congenital dyserythropoietic anemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and by distinct morphological abnormalities of erythroblasts in the bone marrow. Characteristic morphological aberrations were the cornerstone of diagnosis, but following the identification of several causative genes, the molecular approach could represent a rapid tool for the identification of these conditions. This review presents advances in diagnosis and classification of CDAs.
The classification of CDAs has long been based on morphological features. Now, the discovery of some of the responsible genes allows reconsideration of part of the classification. The first CDA partly accounted for genetically has been CDA 1, through the discovery in 2002 of the gene responsible, CDAN1, encoding codanin-1. Recently, the dramatic identification of the genes responsible for CDA II, SEC23B, and for a hitherto unnamed CDA, KLF1, took place. SEC23B encodes SEC23B which is a component of the coated vesicles transiting from the endoplasmic reticulum to the cis compartment of the Golgi apparatus. A unique mutation in KLF1, which encodes the erythroid transcription factor KLF1, causes major ultrastructural abnormalities, the persistence of embryonic and fetal hemoglobins, and the absence of some red cell membrane proteins.
Studies of genotype-phenotype relationship, as has already been done for CDA II, will allow a more accurate prognosis. Identification of the responsible genes has opened new vistas for research on CDAs.
Current opinion in hematology 03/2011; 18(3):146-51. DOI:10.1097/MOH.0b013e32834521b0 · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report on a truncated α-spectrin chain, spectrin(Exeter), associated with ellipto-poikilocytosis. Analysis of erythrocyte membranes of affected individuals revealed a truncated α-spectrin chain with normal amounts of spectrin dimer. In the proband and her father, one haploid set of α-spectrin cDNA lacked exons 11 and 12, leading to partial deletion of repeats α4 and α5 (83 amino acids) of the α-spectrin chain. In one allele of genomic DNA, a 3567bp deletion starting in intron 10 and ending in intron 12 of the SPTA1 gene was found. The common polymorphic SPTA1 α(LELY) allele was found in trans to the SPTA1αExeter allele in the proband. The proband had inherited the SPTA1Exeter allele from her father and the αLELY allele from her healthy, asymptomatic mother. This is the first report of an interstitial deletion in the SPTA1 gene associated with ellipto-poikilocytosis.
[Show abstract][Hide abstract] ABSTRACT: We describe a new approach to stabilize nonsense mRNA, based on the inhibition of the NMD mechanism, by combining cycloheximide-mediated inhibition of translation, and caffeine-mediated inhibition of UPF1 phosphorylation. This approach aimed to identify the impact of a 4.1R splicing mutation. This mutation is involved in a partial deficiency of 4.1R in the homozygous state in a patient with hereditary elliptocytosis and a moderated hemolytic anemia. We show that, in addition to two known minor shortened and stable spliceoforms, the mutation activates an intronic cryptic splice site, which results in a nonsense mRNA major isoform, targeted to degradation in intact cells by NMD. This accounts for the main cause of 4.1R partial deficiency. In a general perspective, blocking the NMD mechanism would help to identify a missing isoform, and pave the path for a molecular targeting strategy to circumvent a deleterious splicing pathway in favor of a therapeutic splicing pathway.
[Show abstract][Hide abstract] ABSTRACT: A female patient of Portuguese origin, who was born to consanguineous parents, presented with moderate anemia, mild jaundice and splenomegaly. Bone marrow aspiration showed an erythroid hyperplasia and binucleate erythroblasts, evoking a congenital dyserythropoietic anemia, type II (CDA II). Although microcytosis cast some doubt on the diagnosis, investigation was pursued along this line. The CDA II was finally ruled out as underglycosylation of band 3, remnants of endoplasmic reticulum cisternae and mutations in the SEC23B gene were all missing. On the other hand, analysis of the α-globin genes showed a base substitution at codon 125 (CTG→CGG) of the α2-globin gene, ascertaining a homozygosity for Hb Plasencia (HBA2:c.377T>G). This variant has an unstable α chain. In the absence of a thorough work-up, dyserythropoietic anemia associated with hemoglobin (Hb) variants having a moderately unstable α chain, may be mistaken for CDA II.
[Show abstract][Hide abstract] ABSTRACT: SEC23B gene encodes an essential component of the coat protein complex II (COPII)-coated vesicles. Mutations in this gene cause the vast majority the congenital dyserythropoietic anemia Type II (CDA II), a rare disorder resulting from impaired erythropoiesis. Here, we investigated 28 CDA II patients from 21 unrelated families enrolled in the CDA II International Registry. Overall, we found 19 novel variants [c.2270 A>C p.H757P; c.2149-2 A>G; c.1109+1 G>A; c.387(delG) p.L129LfsX26; c.1858 A>G p.M620V; c.1832 G>C p.R611P; c.1735 T>A p.Y579N; c.1254 T>G p.I418M; c.1015 C>T p.R339X; c.1603 C>T p.R535X; c.1654 C>T p.L552F; c.1307 C>T p.S436L; c.279+3 A>G; c. 2150(delC) p.A717VfsX7; c.1733 T>C p.L578P; c.1109+5 G>A; c.221+31 A>G; c.367 C>T p.R123X; c.1857_1859delCAT; p.I619del] in the homozygous or the compound heterozygous state. Homozygosity or compound heterozygosity for two nonsense mutations was never found. In four cases the sequencing analysis has failed to find two mutations. To discuss the putative functional consequences of missense mutations, computational analysis and sequence alignment were performed. Our data underscore the high allelic heterogeneity of CDA II, as the most of SEC23B variations are inherited as private mutations. In this mutation update, we also provided a tool to improve and facilitate the molecular diagnosis of CDA II by defining the frequency of mutations in each exon.
American Journal of Hematology 12/2010; 85(12):915-20. DOI:10.1002/ajh.21866 · 3.80 Impact Factor