Véronique Picard

Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels.

Dehydrated hereditary stomatocytosis (DHS) is a genetic condition with defective red blood cell (RBC) membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations inthe mechanically activated PIEZO1(FAM38A) ion channel were associated with DHS. 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 DHS cases, we identifythree novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for DHS. All the DHS-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 RBCs of DHS patients. Our findings also suggest a new role for mechanotransduction in RBC biology and pathophysiology.

A mutation in the Gardos channel is associated with hereditary xerocytosis

The Gardos channel is a Ca(2+)-sensitive, intermediate conductance, potassium selective channel expressed in several tissues including erythrocytes and pancreas. In normal erythrocytes, it is involved in cell volume modification. Here, we report the identification of a dominantly inherited mutation in the Gardos channel in 2 unrelated families and its association with chronic hemolysis and dehydrated cells, also referred to as hereditary xerocytosis (HX). The affected individuals present chronic anemia that varies in severity. Their red cells exhibit a panel of various shape abnormalities such as elliptocytes, hemighosts, schizocytes, and very rare stomatocytic cells. The missense mutation concerns a highly conserved residue among species, located in the region interacting with Calmodulin and responsible for the channel opening and the K(+) efflux. Using 2-microelectrode experiments on Xenopus oocytes and patch-clamp electrophysiology on HEK293 cells, we demonstrated that the mutated channel exhibits a higher activity and a higher Ca(2+) sensitivity compared with the wild-type (WT) channel. The mutated channel remains sensitive to inhibition suggesting that treatment of this type of HX by a specific inhibitor of the Gardos channel could be considered. The identification of a KCNN4 mutation associated with chronic hemolysis constitutes the first report of a human disease caused by a defect of the Gardos channel.

Development of a recombinant antithrombin variant as a potent antidote to fondaparinux and other heparin derivatives

Heparin derivative-based therapy has evolved from unfractionated heparin (UFH) to low-molecular-weight heparins (LMWHs) and now fondaparinux, a synthetic pentasaccharide. Contrary to UFH or LMWHs, fondaparinux is not neutralized by protamine sulfate, and no antidote is available to counteract bleeding disorders associated with overdosing. To make the use of fondaparinux safer, we developed an antithrombin (AT) variant as a potent antidote to heparin derivatives. This variant (AT-N135Q-Pro394) combines 2 mutations: substitution of Asn135 by a Gln to remove a glycosylation site and increase affinity for heparins, and the insertion of a Pro between Arg393 and Ser394 to abolish its anticoagulant activity. As expected, AT-N135Q-Pro394 anticoagulant activity was almost abolished, and it exhibited a 3-fold increase in fondaparinux affinity. AT-N135Q-Pro394 was shown to reverse fondaparinux overdosing in vitro in a dose-dependent manner through a competitive process with plasma AT for fondaparinux binding. This antidote effect was also observed in vivo: administration of AT-N135Q-Pro394 in 2.5-fold molar excess versus plasma AT neutralized 86% of the anti-Xa activity within 5 minutes in mice treated with fondaparinux. These results clearly demonstrate that AT-N135Q-Pro394 can reverse the anticoagulant activity of fondaparinux and thus could be used as an antidote for this drug.

Association between ABCB1 C3435T polymorphism and increased risk of cannabis dependence.

Prolonged cannabis use has a significant impact on health and well-being. Genetic factors are known to influence cannabis dependence, but few specific genetic markers have been identified. ABCB1 polymorphisms are known to modify drug pharmacokinetics but have yet to be studied for their role in generating and maintaining cannabis dependence. The objective of this study is to determine if ABCB1 C3435T polymorphism may represent an independent genetic marker for cannabis dependence risk. An open bi-centric association study was conducted in two French Addiction Centres. Caucasian patients diagnosed with isolated cannabis dependence were compared with healthy age-matched controls for socio-demographic, clinical and genetic data using chi-square test, Fishers exact test, or Mann-Whitney U test. Independent association between ABCB1 C3435T SNP marker and cannabis dependence was evaluated using multiple logistic regression analysis. Versus controls (n=40), patients with cannabis dependence (n=40) had a significantly higher 3435C allele frequency (62.5% versus 43.8% respectively, P=0.017) and CC genotype (50% versus 20%, P=0.005, OR=4.00 [1.50-10.60]). Multiple logistic regression analysis of the C3435T SNP and variables identified in univariate analyses indicated that the CC genotype was independently associated with cannabis dependence (P=0.045, OR=6.61 [1.05-46.58]). This is the first time a significant specific genetic marker has been shown in cannabis dependence. ABCB1 polymorphisms may alter Delta9THC distribution, its psychoactive effects and individual vulnerability to dependence. These results pave the way to a new pharmacogenetic hypothesis in cannabis dependence.

Long-term follow up of subtotal splenectomy for hereditary spherocytosis: a single center study

To the editor: Red cells of patients with hereditary spherocytosis (HS) have a decreased surface-to-volume ratio,[1][1] leading to their trapping and destruction during their passage through the splenic cords.[2][2][⇓][3]-[4][4] Therefore, surgical total splenectomy (TS) by removing the main site

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

Testing for hereditary spherocytosis: a French experience

In a recent article, Bianchi et al. have compared several laboratory tests for diagnosis of hereditary spherocytosis (HS) by studying 150 HS patients.[1][1] They concluded that the association of the eosin-5-maleimide (EMA) binding test and the acidified glycerol lysis test (AGLT) could identify all

Effect of the ABCB1 3435C>T polymorphism on tacrolimus concentrations and dosage requirements in liver transplant recipients

PURPOSE The effect of ABCB1 3435C>T on tacrolimus concentrations in liver transplant recipients was studied. Tacrolimus is a substrate for P-glycoprotein, the product of the ABCB1 gene. To determine whether the ABCB1 single-nucleotide polymorphism (SNP) 3435C>T was associated with variation in the tacrolimus concentration:dose ratio (C:D) in 42 liver transplant recipients during three months after transplantation. METHODS Forty-two Caucasian patients who underwent an orthotopic liver transplantation from cadaveric donors received a basic immunosuppressive regimen containing tacrolimus and corticosteroids; mycophenolate mofetil was added in 18 cases. The SNP 3435C>T in exon 26 was investigated by MboI restriction-enzyme digestion, leading to the identification of CC, TT, or CT status at nucleotide 3435. Results obtained for the three genotypes were compared for each of three values: daily weight-adjusted tacrolimus dose, blood trough tacrolimus concentration, and C:D. RESULTS The wild-type genotype (3435CC) was observed in 10 patients (24%); 23 patients (55%) were heterozygous (3435CT) and 9 patients (21%) were homozygous for the mutation (3435TT). One to three days after liver transplantation, the mean +/- S.D. C:D was significantly higher in subjects homozygous for the mutation compared with subjects with the wild-type allele (236 +/- 119 ng . kg/mL . mg versus 104 +/- 74 ng . kg/mL . mg, respectively; p = 0.0167). Subjects with the heterozygous allele had an intermediate mean +/- S.D. C:D (131 +/- 108 ng . kg/mL . mg). One or three months after transplantation, no significant difference in the tacrolimus C:D was evident among the three groups. CONCLUSION The ABCB1 3435C>T polymorphism influenced the tacrolimus C:D in the first days after liver transplantation.

Non-immune Hemolysis: Diagnostic Considerations

Non-immune hemolytic anemia (NIHA) is characterized by positive routine hemolytic tests but negative anti-human immunoglobulin (Coombs) test. Hereditary non-immune hemolysis includes disorders of erythrocytic enzymes, membrane, hemoglobin (qualitative and quantitative disorders), as well as the rare hereditary forms of thrombotic microangiopathies. Acquired NIHA includes paroxysmal nocturnal hemolysis (PNH), infections, drug and metal intoxications with as a target red blood cells or endothelium of capillaries, the rare acquired forms of thalassemia or erythrocytic membrane disorders, and hemolysis secondary to a dysfunctioning artificial (prosthetic) cardiac valve. Identification of the specific cause of NIHA is sometimes difficult and requires not only a good knowledge of this entity but mainly a qualified specialized hematologic laboratory. An algorithm to be used in every new patient consulting for NIHA is proposed in the last part of this article.

Red blood cell Gardos channel (KCNN4):the essential determinant of erythrocyte dehydration in Hereditary Xerocytosis

Recent advances have been made in the identification of molecular determinants of the rare hemolytic disease, dehydrated hereditary stomatocytosis (DHSt), also called hereditary xerocytosis (HX). This well-known red blood cell (RBC) pathology is characterized by an abnormal cation leak resulting in KCl loss and red blood cell dehydration. It leads to cell fragility and hemolytic anemia. Two different genes have been linked to this phenotype: PIEZO1 and KCNN4 coding, respectively, for Piezo1, a non-selective cation channel activated by mechanical forces, and a calcium activated K channel (KCNN4) also known the Gardos channel in RBCs. So far, three different point mutations in KCNN4 have been linked to HX. 6 The mutated KCNN4 is over-activated in patient RBCs leading to an increased K loss and, hence, water loss and cell dehydration. About a dozen mutations in Piezo1 are linked to HX, and some of them have been shown to change the kinetics of channel gating. In normal RBCs, Piezo1 appears to be a major factor in cell response to mechanical stress (by controlling calcium influx), and there is a functional connection between Piezo1 and KCNN4 through the modification of intracellular calcium concentration. Our present study was designed to evaluate in HX the functional link between mutated Piezo1 and KCNN4 and to assess the efficiency of a KCNN4 blocker, Senicapoc, to treat HX whatever the molecular cause. Our study focused on three independent index cases with a typical HX clinical and biological phenotype (Online Supplementary Table S1 and Figure 1); two were unreported cases (patients 1 and 2), and one was previously described (patient 3). Patient 1 was a 35-year-old man presenting with undiagnosed compensated hemolytic anemia and iron overload. He was investigated due to an unexplained fatal hydrops history during his wife’s second pregnancy; his first son was well and unaffected. Patient 2 was a 38-year-old women investigated for undiagnosed compensated hemolytic anemia. PIEZO1 sequencing for patient 1 and 2 revealed two new missense mutations : a c.1792G>A mutation in exon 14