Hereditary 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. IntroductionHereditary xerocytosis (also known as HX or dehydrated stomatocytosis, DHSt; OMIM 194380) is an autosomal dominant hemolytic anemia characterized by primary erythrocyte dehydration. 1 HX erythrocytes exhibit decreased total cation and potassium content that are not accompanied by a proportional net gain of sodium and water. HX patients typically exhibit mild to moderate, compensated hemolytic anemia. Erythrocyte mean corpuscular hemoglobin concentration is increased and erythrocyte osmotic fragility is decreased, both reflecting cellular dehydration.A locus for HX on chromosome 16 (16q23-q24) was first identified in a large, 3-generation Irish family. 2 This locus was refined to D16S511-16qter via study of 10 kindreds with variants of HX, pseudohyperkalemia, or nonimmune hydrops fetalis. 3,4 Recent studies of one of the original HX families from Rochester, NY, 5 and of a large HX family from Manitoba, Canada 6 confirmed the linkage of the disease phenotype to chromosome 16q, and refined the candidate region to 16q24.2-16qter, a 2.4 million-bp interval containing 51 known or predicted genes. 6 To identify the HXassociated genetic locus, we performed high-resolution single nucleotide polymorphism (SNP) typing and whole-exome sequencing on selected persons from both the New York and Canadian HX kindreds.In the refined candidate region, no regions of copy number variation were detected at 16q24.2-16qter. A large region of homozygosity was detected in this region in DNA from a presumed homozygote from the New York kindred. Exome sequencing identified novel mutations affecting PIEZO1 (encoded by the FAM38A gene) in both HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and cosegregation with the disease phenotype in all...
Summary Modulation of intracellular chloride concentration ([Cl−]i) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl− exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl−]I; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl−]i and have implications for control of cell volume and neuronal function.
Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf ؊/؊ ) mice die at day 14.5 of gestation from severe anemia. In this study, we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf ؊/؊ embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis, transcriptional profiling was performed with RNA from wild-type and Eklf ؊/؊ early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation, with the critical regulator of the cell cycle, E2f2, at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf ؊/؊ early erythroid progenitor cells, which showed a delay in the G 1 -to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier, EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation.
Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype.
Summary Modulation of intracellular chloride concentration ([Cl−] i ) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl − exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl − ] I ; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl − ] i and have implications for control of cell volume and neuronal function.
Key Points Mutations in the Gardos channel, encoded by the KCNN4 gene, were identified in individuals from 2 hereditary xerocytosis kindreds. These findings support recent data indicating the Gardos channel plays a role in normal erythrocyte volume homeostasis.
Plasmodium falciparum relies on anion channels activated in the erythrocyte membrane to ensure the transport of nutrients and waste products necessary for its replication and survival after invasion. The molecular identity of these anion channels, termed "new permeability pathways" is unknown, but their currents correspond to up-regulation of endogenous channels displaying complex gating and kinetics similar to those of ligand-gated channels. This report demonstrates that a peripheral-type benzodiazepine receptor, including the voltage dependent anion channel, is present in the human erythrocyte membrane. This receptor mediates the maxi-anion currents previously described in the erythrocyte membrane. Ligands that block this peripheral-type benzodiazepine receptor reduce membrane transport and conductance in P falciparum-infected erythrocytes. These ligands also inhibit in vitro intraerythrocytic growth of P falciparum. These data support the hypothesis that dormant peripheral-type benzodiazepine receptors become the "new permeability pathways" in infected erythrocytes after upregulation by P falciparum. These channels are obvious targets for selective inhibition in anti-malarial therapies, as well as potential routes for drug delivery in pharmacologic applications. (Blood. 2011; 118(8):2305-2312) IntroductionThe most severe form of malaria in humans is caused by parasite Plasmodium falciparum, infecting 225 million people and causing 781 000 deaths in 2009 (World Health Organization, 2010). Erythrocyte invasion by P falciparum provides the parasite access to a plentiful source of nutrients in a locale that is largely shielded from host immune defenses. After invasion, the invading parasite uses a variety of strategies to adapt to the intraerythrocytic environment. To ensure the transport of nutrients and waste products necessary for its replication and survival, P falciparum relies on broad specificity anion channels activated in the erythrocyte membrane after invasion. 1 Initially, this transport was attributed to "new" permeability pathways (NPPs) 2 exported by the parasite to the host membrane. 3 However, later studies revealed that the current is because of up-regulation of endogenous channels 4 and that the diversity of anion channel activities recorded in these studies correspond to different kinetic modalities of a unique type of maxi-anion channel. 5 This channel displays complex gating and kinetics similar to those of ligand-gated channels. 5 Anions are transported through the human erythrocyte membrane by a 2-component system: a large electroneutral exchanger mediated by band 3 and a 4 orders of magnitude smaller electrogenic component estimated at approximately 10 S/cm 2 corresponding presumably to a small number of channels. 6 Remarkably, the molecular identification and characterization of this conductive pathway has not yet been achieved. Neither genomic nor proteomic studies have provided meaningful clues to the composition of this pathway. 7 Considering the small amount of protein a few hundred c...
Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function, but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription, dynamic chromatin architecture, transcription factor binding, and genomic organization in regulation of erythrocyte membrane protein genes, we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly, most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites, located primarily in introns. Cooccupancy with FOG-1, SCL, and MTA-2 was found at all regions of GATA-1 binding, with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4, GATA-1, NF-E2, FOG-1, SCL, and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia.The erythrocyte membrane is a multifunctional, complex structure that provides the red cell the deformability and stability required to withstand its travels through macro-and microcirculation. It plays critical roles in erythropoiesis, including responding to erythropoietin, importing iron required for hemoglobin synthesis, and regulating cellular metabolism. Qualitative and quantitative disorders of erythrocyte membrane proteins have been associated with inherited abnormalities of red cell shape, including hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis syndromes (65, 103). Despite biochemical and genetic linkage to specific erythrocyte membrane protein genes, e.g., ankyrin-1, ␣-or -spectrin, and band 3, mutations are found in the coding exons and promoter regions of only ϳ75% of cases studied. This suggests that the disease-causing mutation is located in critical regulatory regions outside the promoters and exons in a quarter of cases.Most erythrocyte membrane protein genes are large, comprised of Ͼ25 exons. They encode numerous diverse and complex isoforms, frequently generated by alternate splicing, alternate promoter usage, or alternate polyadenylation (18). In many cases, alternate promoters direct combinations of exons encoding diverse tissue-specific, cell type-specific, developmental-stage-specific, and/or differentiation stage-specific isoforms (6, 12, 13, 19, 21-24, 44, 52, 62, 78, 86, 108, 112-114). As such, erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and fu...
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