Autosomal Dominant Hemolytic Anemia Research Articles

Proteome alterations in erythrocytes with PIEZO1 gain-of-function mutations.

Gain-of-function mutations in PIEZO1 cause dehydrated hereditary stomatocytosis (DHS) or hereditary xerocytosis, an autosomal dominant hemolytic anemia characterized by high reticulocyte count, a tendency to macrocytosis, and mild jaundice, as well as by other variably penetrant clinical features, such as perinatal edema, severe thromboembolic complications after splenectomy, and hepatic iron overload. PIEZO1 mutations in DHS lead to slowed inactivation kinetics of the ion channel and/or facilitation of channel opening in response to physiological stimuli. To characterize the alterations of red blood cell proteome in patients with mutated PIEZO1, we used a differential approach to compare the proteome of patients with DHS (16 patients from 13 unrelated ancestries) vs healthy individuals. We identified new components in the regulation of the complex landscape of erythrocytes ion and volume balance mediated by PIEZO1. Specifically, the main impaired processes in patients with DHS were ion homeostasis, transmembrane transport, regulation of vesicle-mediated transport, and the proteasomal catabolic process. Functional assays demonstrated coexpression of PIEZO1 and band 3 when PIEZO1 was activated. Moreover, the alteration of the vesicle-mediated transport was functionally demonstrated by an increased vesiculation rate in patients with DHS compared with healthy controls. This finding also provides an explanation of the pathogenetic mechanism underlying the increased thrombotic rate observed in these patients. Finally, the newly identified proteins, involved in the intracellular signaling pathways altered by PIEZO1 mutations, could be used in the future as potential druggable targets in DHS.

A novel PIEZO1 mutation in a patient with dehydrated hereditary stomatocytosis: a case report and a brief review of literature

BackgroundDehydrated hereditary stomatocytosis (DHS) or hereditary xerocytosis is a rare, autosomal dominant hemolytic anemia characterized by macrocytosis, presence of stomatocytes and dehydration of red blood cells (RBCs). The dehydration is caused by a defect in cellular cation content. The most frequent expression of the pathology is hemolytic well-compensated anemia with high reticulocyte count, a tendency to macrocytosis, increased mean corpuscular hemoglobin concentration (MCHC) and mild jaundice. We here describe a new mutation of PIEZO1 gene, the most frequent mutated gene in DHS, in a family affected by hereditary hemolytic anemia.Case presentationWe describe the case of a 12-years-old girl with well-compensated chronic hemolysis, increased MCHC and a father who had the same hematological characteristics. After excluding secondary causes of chronic hemolysis and enzymatic defects of the RBCs, microscopic observation of the peripheral blood smear, tests of RBC lysis, ektacytometry, SDS-PAGE and in last instance genetic analysis has been performed. This complex diagnostic workup identified a new variant in the PIEZO1 gene, never described in literature, causative of DHS. This pathogenetic variant was also detected in the father.ConclusionsThis case report highlights the importance of a correct and exhaustive diagnostic-workup in patients with clinical suspicious for hemolytic anemia in order to make a differential diagnosis. This is relevant for the management of these patients because splenectomy is contraindicated in DHS due to high thrombotic risk.

Gain-of-function mutations in PIEZO1 directly impair hepatic iron metabolism via the inhibition of the BMP/SMADs pathway.

Dehydrated hereditary stomatocytosis (DHS), or xerocytosis, is an autosomal dominant hemolytic anemia. Most patients with DHS carry mutations in the PIEZO1 gene encoding a mechanosensitive cation channel. We here demonstrate that patients with DHS have low levels of hepcidin and only a slight increase of ERFE, the erythroid negative regulator of hepcidin. We demonstrated that at the physiological level, PIEZO1 activation induced Ca2+ influx and suppression of HAMP expression in primary hepatocytes. In two hepatic cellular models expressing PIEZO1 WT and two PIEZO1 gain-of-function mutants (R2456H and R2488Q), we highlight altered expression of a few genes/proteins involved in iron metabolism. Mutant cells showed increased intracellular Ca2+ compared to WT, which was correlated to increased phosphorylation of ERK1/2, inhibition of the BMP-SMADs pathway, and suppression of HAMP transcription. Moreover, the HuH7 cells, treated with PD0325901, a potent inhibitor of ERK1/2 phosphorylation, reduced the phosphorylation of ERK1/2 with the consequent increased phosphorylation of SMAD1/5/8, confirming the link between the two pathways. Another "proof of concept" for the mechanism that links PIEZO1 to HAMP regulation was obtained by mimicking PIEZO1 activation by cell Ca2+ overload, by the Ca2+ ionophore A23187. There was strong down-regulation of HAMP gene expression after this Ca2+ overload. Finally, the inhibition of PIEZO1 by GsMTx4 leads to phenotype rescue. This is the first demonstration of a direct link between PIEZO1 and iron metabolism, which defines the channel as a new hepatic iron metabolism regulator and as a possible therapeutic target of iron overload in DHS and other iron-loading anemias.

PIEZO1 Hypomorphic Variants in Congenital Lymphatic Dysplasia Cause Shape and Hydration Alterations of Red Blood Cells.

PIEZO1 is a cation channel activated by mechanical force. It plays an important physiological role in several biological processes such as cardiovascular, renal, endothelial and hematopoietic systems. Two different diseases are associated with alteration in the DNA sequence of PIEZO1: (i) dehydrated hereditary stomatocytosis (DHS1, #194380), an autosomal dominant hemolytic anemia caused by gain-of-function mutations; (ii) lymphatic dysplasia with non-immune fetal hydrops (LMPH3, #616843), an autosomal recessive condition caused by biallelic loss-of-function mutations. We analyzed a 14-year-old boy affected by severe lymphatic dysplasia already present prenatally, with peripheral edema, hydrocele, and chylothoraces. By whole exome sequencing, we identified compound heterozygosity for PIEZO1, with one splicing and one deletion mutation, the latter causing the formation of a premature stop codon that leads to mRNA decay. The functional analysis of the erythrocytes of the patient highlighted altered hydration with the intracellular loss of the potassium content and structural abnormalities with anisopoikolocytosis and presence of both spherocytes and stomatocytes. This novel erythrocyte trait, sharing features with both hereditary spherocytosis and overhydrated hereditary stomatocytosis, complements the clinical features associated with loss-of-function mutations of PIEZO1 in the context of the generalized lymphatic dysplasia of LMPH3 type.

Evaluation of Phenotype-Genotype Correlation in Two Common PIEZO1 Mutations p.R2456H and p.L2495_E2495dup

Hereditary xerocytosis (HX) is a rare autosomal dominant hemolytic anemia caused by mutations in the mechanosensitive cation channel PIEZO1 or, less commonly, in the Ca2+-gated K+ channel KCNN4 (Gardos channel). It is a clinically heterogeneous condition, characterized by erythrocyte dehydration. As erythrocytes traverse narrow capillaries and sinusoids, PIEZO1 is thought to be activated by mechanical stimuli, leading to increased intracellular Ca2+ which then may activate KCNN4, causing K+ efflux and water loss. The p.R2456H and p.L2495_E2496dup PIEZO1 mutations are likely the most common of the PIEZO1 disease-causing mutations and both result in delayed channel inactivation (Glogowska et al., 2017, Blood 130:1845). The prolonged channel activity in response to mechanical stimulation is likely to contribute to erythrocyte dehydration.Ten patients with PIEZO1-associated HX (5 with p.R2456H and 5 with p.L2495_E2496dup PIEZO1 heterozygous mutations) had their diagnosis established utilizing a Next Generation sequencing panel for hereditary hemolytic and congenital dyserythropoietic anemias (HHA/CDA panel). These patients, along with family members who had similar clinical characteristics (a total of 8 patients with p.R2456H and 6 patients with p.L2495_E2496dup) ranging from 2 - 63 years of age, were enrolled in an IRB-approved study to explore the phenotype-genotype correlation of the disease. We collected patient and family histories and laboratory data and performed phenotypic testing including ektacytometry and erythrocyte cation determinations by atomic spectroscopy.Most of the patients in our cohort had compensated hemolysis without anemia at the time of evaluation and had not required any transfusions. All of the patients had elevated reticulocyte counts ranging from 5.7 to 15.7 %. Reticulocyte counts of individuals with the p.R2456H mutation were significantly higher than those with the p.L2495_E2496dup mutation (Student's t test, p=0.01). Some of the patients had splenomegaly, but it was not a universal finding. Although elevated MCHC is often considered a hallmark of HX, most of the individuals in this cohort had increased MCV and MCH but only high-normal MCHC. Varying percentages of hyper dense cells were detected in blood samples analyzed using an Advia Hematology System and stomatocytes were notable in most peripheral blood smears.Several complications have been associated with HX including hydrops fetalis, hemolytic episodes, thrombosis (especially after splenectomy), gallstones, and iron overload. One patient in our cohort had experienced ascites and a pleural effusion in the perinatal period and one was diagnosed with gallstones at age 11. Nearly all the patients had an increase in total bilirubin. Ferritin levels were increased in the majority of the patients tested after 15 years of age. The three patients older than 40 years of age had iron overload verified by T2* MRI imaging of the liver.HX presents several diagnostic challenges due to its heterogeneity. Two of the subjects in our study had been previously diagnosed with hereditary spherocytosis (HS) and one had the diagnosis of congenital dyserythropoietic anemia (CDA) prior to genetic evaluation. Misdiagnosis of individuals with HX as having HS is especially problematic since splenectomy is contraindicated in HX due to a greatly increased incidence of thromboembolic complications. Another diagnostic consideration concerns the use of osmotic fragility tests. Although osmotic fragility tests are frequently used to diagnose HX, ektacytometry provides greater accuracy. In the present study two patients who had ektacytometry results characteristic of HX had reportedly normal osmotic fragility tests in previous work-up. All individuals in our cohort with confirmed disease-causing mutations in PIEZO1 demonstrated the classic left shifted ektacytometry osmoscan indicative of erythrocyte dehydration. In addition, all individuals with these mutations demonstrated the expected decrease in erythrocyte potassium content not associated with a proportional increase in sodium. This illustrates the importance of specialized testing (ektacytometry and/or intracellular cation determinations) and the use of appropriately designed genetic panels to establish an accurate diagnosis in patients with hemolytic anemia of ambiguous phenotype, especially when splenectomy is being contemplated. DisclosuresQuinn:Amgen: Research Funding; Global Blood Therapeutics: Research Funding; Silver Lake Research Corporation: Research Funding.

Genetic Diseases of PIEZO1 and PIEZO2 Dysfunction.

Mutations in the genes encoding the mechanosensitive cation channels PIEZO1 andPIEZO2 are responsible for multiple hereditary human diseases. Loss-of-function mutations in the human PIEZO1 gene cause autosomal recessive congenital lymphatic dysplasia. Gain-of-function mutations in the human PIEZO1 gene cause the autosomal dominant hemolytic anemia, hereditary xerocytosis (also known as dehydrated stomatocytosis). Loss-of-function mutations in the human PIEZO2 gene cause an autosomal recessive syndrome of muscular atrophy with perinatal respiratory distress, arthrogryposis, and scoliosis. Gain-of-function mutations in the human PIEZO2 gene cause three clinical types of autosomal dominant distal arthrogryposis. This chapter will review the hereditary diseases caused by mutations in the PIEZO genesand will discuss additional physiological systems in which PIEZO channel dysfunction may contribute to human disease pathophysiology.

Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary xerocytosis

AbstractHereditary xerocytosis (HX, MIM 194380) is an autosomal dominant hemolytic anemia characterized by primary erythrocyte dehydration. Copy number analyses, linkage studies, and exome sequencing were used to identify novel mutations affecting PIEZO1, encoded by the FAM38A gene, in 2 multigenerational HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and cosegregation with the disease phenotype in all affected persons in both kindreds. All patients were heterozygous for FAM38A mutations, except for 3 patients predicted to be homozygous by clinical and physiologic studies who were also homozygous at the DNA level. The FAM38A mutations were both in residues highly conserved across species and within members of the Piezo family of proteins. PIEZO proteins are the recently identified pore-forming subunits of channels that mediate mechanotransduction in mammalian cells. FAM38A transcripts were identified in human erythroid cell mRNA, and discovery proteomics identified PIEZO1 peptides in human erythrocyte membranes. These findings, the first report of mutation in a mammalian mechanosensory transduction channel-associated with genetic disease, suggest that PIEZO proteins play an important role in maintaining erythrocyte volume homeostasis.

BAND 3CEINGE (Gly796Arg) Mutation Causes Dehydrated Hereditary Stomatocytosis (DHS) with Dyserythropoietic Phenotype

Stomatocytosis is an inherited autosomal dominant hemolytic anemia and includes overhydrated hereditary stomatocytosis (OHS), dehydrated hereditary stomatocytosis (DHS), hereditary cryohydrocytosis (CHC) and familial pseudohyperkalemia (FP). Here, we report a novel variant of hereditary stomatocytosis due to a de-novo band 3 mutation due to G>A transition at nucleotide 2500 in exon 17 (p. G796R, band3CEINGE) associated with dyserythropoietic phenotype. This 43-years-old Caucasian female (II-2) with unrelated parents was admitted to our hospital for mild anemia evaluation. The patient was in good health until 7 years when she frequently experienced asthenia. Anemia was first recognized at the age of eighth years with presence of jaundice and hyperchromic urine, but she had never received blood transfusions. We observed a mild hypochromic macrocytic anemia with a hemoglobin level of 11.5 g/dL, a mean cell volume (MCV) of 110 fL, and a mean hemoglobin concentration (MCH) of 36.1 pg, the reticulocyte count was 64 × 103/μL. There was a typical hemolytic features: high levels of indirect bilirubin (3.48 mg/dL) and lactate dehydrogenase ( 567 U/l, v.n. 240– 480 U/l ) with negativity at direct and indirect Coomb's test. Spleen was enlarged and ultrasonography detected 15 cm of longitudinal size. She was cholecystectomized at the age of 14 years because of numerous symptomatic small stones. Serum iron, soluble transferrin receptor, serum ferritin and transferrin saturation levels were all increased, while the transferrin was in the normal range.Other blood tests including osmotic fragility with incubated and fresh erythrocytes, serum electrolytes, B12 and folate levels, erythrocyte enzyme levels, EMA test and Pink test were normal. Peripheral blood smear showed anisopoikilocytosis with rare stomatocytes and no spherocytes. Bone marrow aspirate showed remarkable dyserythropoiesis with increased number of erythroblasts and binucleate erythroblasts, basophilic erythroblasts with alterations, irregular nuclei maturation, intererythroblastic bridges and erythroblasts with basophilic stippling. She received since the age of 14 yrs a diagnosis for congenital dyserythropoietic anemia type I. Patients red cells showedincrease Na+ content and decrease K+ content;reduced Na-K pump activity andincreased Na-H exchange, NKCC cotransport and KCC cotransport activities.We then functionally characterized band 3 CEINGE in Xenopus oocytes, showing that the mutated band 3 is converted from anion exchanger (Cl−, HCO3 −) function to unregulated cation pathway for Na+ and K+. The mutated band 3 was also associated with increased tyrosine phosphorylation pattern of some red cell membrane proteins. During erythropoiesis band 3 protein is the last cytoskeletal protein to appear, thus the dyserythropoietic phenotype may be related to a possible role of the mutated band 3 in perturbation of cytoskeleton assembly in the late stage of erythropoiesis, allowing us to conclude for a new variant of stomatocytosis with dyserythropoietic phenotype.

Autosomal dominant hemolytic anemia and adenosine deaminase overproduction.

Paglia et al have described a kindred with an autosomal dominant hemolytic anemia characterized by a 70- to 100-fold elevation in erythrocyte adenosine deaminase (ADA) activity and decreased ATP pools (1, 2). The disorder appears to be limited to red blood cells (RBC), as B lymphoblast, granulocyte, and skin fibroblast ADA activities fall within the normal range. Kinetic and physicochemical properties of ADA partially purified from proband RBC are normal, suggesting that the enzyme contains no structural abnormalities (3).

Autosomal dominant hemolytic anemia characterized by ovalocytosis: A family study of seven involved members

A three generation family is described with an autosomal dominant hemolytic anemia present in seven members. No linkage could be established with Rh type or ability to taste phenylthiourea (PTC). The disease was characterized by the presence of full ovalocytes. In six of the seven members an uncompensated hemolytic process was present, due to an intrinsic red cell defect of unknown nature. Splenectomy was performed in five members, producing a clinical remission in every case. For reasons outlined, the term “idiopathic hemolytic ovalocytosis” was suggested as a descriptive term for the disease in this family and similar cases previously described.