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.

Biallelic ANGPT2 loss-of-function causes severe early-onset non-immune hydrops fetalis.

BACKGROUND: Hydrops fetalis, a pathological fluid accumulation in two or more body compartments, is aetiologically heterogeneous. We investigated a consanguineous family with recurrent pregnancy loss due to severe early-onset non-immune hydrops fetalis. METHODS AND RESULTS: Whole exome sequencing in four fetuses with hydrops fetalis revealed that they were homozygous for the angiopoietin-2 (ANGPT2) variant Chr8 (GRCh37/Hg19): 6385085T>C, NM_001147.2:c.557A>G. The substitution introduces a cryptic, exonic splice site predicted to result in loss of 10 nucleotides with subsequent shift in reading frame, leading to a premature stop codon. RNA analysis in the heterozygous parents demonstrated loss of detectable mutant allele, indicative of loss-of-function via nonsense-mediated mRNA decay. Serum ANGPT2 levels were reduced in the parents. In a pregnancy with a healthy, heterozygous child, transiently increased fetal nuchal translucency was noted. CONCLUSION: Pathogenic heterozygous ANGPT2 missense variants were recently shown to cause autosomal dominant primary lymphoedema. ANGPT2 is a ligand of the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) pathway. It is critical to the formation and remodelling of blood and lymphatic vessels and is involved in vessel maintenance. ANGPT2 knockout mice die from generalised lymphatic dysfunction. We show here that a homozygous pathogenic variant causes loss-of-function and results in severe early-onset hydrops fetalis. This is the first report of an autosomal recessive ANGPT2-related disorder in humans.

Evidence of in vivo exogen protein uptake by red blood cells: a putative therapeutic concept.

For some molecular players in red blood cells (RBCs), the functional indications and molecular evidence are discrepant. One such protein is transient receptor potential channel of canonical subfamily, member 6 (TRPC6). Transcriptome analysis of reticulocytes revealed the presence of TRPC6 in mouse RBCs and its absence in human RBCs. We transfused TRPC6 knockout RBCs into wild-type mice and performed functional tests. We observed the "rescue" of TRPC6 within 10 days; however, the "rescue" was slower in splenectomized mice. The latter finding led us to mimic the mechanical challenge with the cantilever of an atomic force microscope and simultaneously carry out imaging by confocal (3D) microscopy. We observed the strong interaction of RBCs with the opposed surface at around 200 pN and the formation of tethers. The results of both the transfusion experiments and the atomic force spectroscopy suggest mechanically stimulated protein transfer to RBCs as a protein source in the absence of the translational machinery. This protein transfer mechanism has the potential to be utilized in therapeutic contexts, especially for hereditary diseases involving RBCs, such as hereditary xerocytosis or Gárdos channelopathy.

Pathogenic variants in MDFIC cause recessive central conducting lymphatic anomaly with lymphedema.

Central conducting lymphatic anomaly (CCLA), characterized by the dysfunction of core collecting lymphatic vessels including the thoracic duct and cisterna chyli, and presenting as chylothorax, pleural effusions, chylous ascites, and lymphedema, is a severe disorder often resulting in fetal or perinatal demise. Although pathogenic variants in RAS/mitogen activated protein kinase (MAPK) signaling pathway components have been documented in some patients with CCLA, the genetic etiology of the disorder remains uncharacterized in most cases. Here, we identified biallelic pathogenic variants in MDFIC, encoding the MyoD family inhibitor domain containing protein, in seven individuals with CCLA from six independent families. Clinical manifestations of affected fetuses and children included nonimmune hydrops fetalis (NIHF), pleural and pericardial effusions, and lymphedema. Generation of a mouse model of human MDFIC truncation variants revealed that homozygous mutant mice died perinatally exhibiting chylothorax. The lymphatic vasculature of homozygous Mdfic mutant mice was profoundly mispatterned and exhibited major defects in lymphatic vessel valve development. Mechanistically, we determined that MDFIC controls collective cell migration, an important early event during the formation of lymphatic vessel valves, by regulating integrin ß1 activation and the interaction between lymphatic endothelial cells and their surrounding extracellular matrix. Our work identifies MDFIC variants underlying human lymphatic disease and reveals a crucial, previously unrecognized role for MDFIC in the lymphatic vasculature. Ultimately, understanding the genetic and mechanistic basis of CCLA will facilitate the development and implementation of new therapeutic approaches to effectively treat this complex disease.

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.

Biological importance of podoplanin expression in chorionic villous stromal cells and its relationship to placental pathologies.

Podoplanin, a reliable marker of lymphatic endothelium, is a mucin-type transmembrane protein. Although the human placenta is devoid of a lymphatic system, chorionic villous stromal (CVS) cells express podoplanin. In this study, the pattern of podoplanin expression in normal and pathological placental tissues and the biological role of podoplanin were investigated. In total, 198 placental tissues belonging to 184 patients, seen at the Department of Pathology of Bulent Ecevit University Education and Research Hospital, Zonguldak, Turkey, were evaluated histopathologically and determined to meet the study criteria. The tissues were assigned to control, cisternal placental disorders, inflammation and hypoxic-ischemic pathology groups. Podoplanin expression in CVS cells was graded from 0 to 3 depending on the staining intensity, as determined by an immunohistochemical evaluation of chorionic villi in the most intensively stained tissue region. Podoplanin levels in control CVS cells increased in parallel with placental maturation, whereas in molar pregnancies podoplanin expression was lower than in control tissues. In the acute placental inflammation group, podoplanin immunoreactivity was similar to that in the control group, whereas in the preeclampsia group, podoplanin expression was higher than in all other groups. Our study showed an increase in podoplanin expression in CVS cells during pregnancy. In preeclamptic patients, the increase in podoplanin expression may be a response to hypoxic-ischemic conditions, whereas in molar pregnancies the decrease in podoplanin levels may cause villous swelling by disrupting intercellular fluid homeostasis.

Potential new approaches to the management of the Hb Bart's hydrops fetalis syndrome: the most severe form of α-thalassemia.

The α-thalassemia trait, associated with deletions removing both α-globin genes from 1 chromosome (genotype ζ αα/ζ--), is common throughout Southeast Asia. Consequently, many pregnancies in couples of Southeast Asian origin carry a 1 in 4 risk of producing a fetus inheriting no functional α-globin genes (ζ--/ζ--), leading to hemoglobin (Hb) Bart's hydrops fetalis syndrome (BHFS). Expression of the embryonic α-globin genes (ζ-globin) is normally limited to the early stages of primitive erythropoiesis, and so when the ζ-globin genes are silenced, at ∼6 weeks of gestation, there should be no α-like globin chains to pair with the fetal γ-globin chains of Hb, which consequently form nonfunctional tetramers (γ4) known as Hb Bart's. When deletions leave the ζ-globin gene intact, a low level of ζ-globin gene expression continues in definitive erythroid cells, producing small amounts of Hb Portland (ζ2γ2), a functional form of Hb that allows the fetus to survive up to the second or third trimester. Untreated, all affected individuals die at these stages of development. Prevention is therefore of paramount importance. With improvements in early diagnosis, intrauterine transfusion, and advanced perinatal care, there are now a small number of individuals with BHFS who have survived, with variable outcomes. A deeper understanding of the mechanism underlying the switch from ζ- to α-globin expression could enable persistence or reactivation of embryonic globin synthesis in definitive cells, thereby providing new therapeutic options for such patients.

Inherited hemolytic anemia: a possessive beginner's guide.

Significant advances have been made in diagnosis and clinical management of inherited red cell membrane disorders that result in hemolytic anemia. Membrane structural defects lead to hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), whereas altered membrane transport function accounts for hereditary xerocytosis (HX) and hereditary overhydrated stomatocytosis (OHS). The degrees of membrane loss and resultant increases in cell sphericity determine the severity of anemia in HS and HE, and splenectomy leads to amelioration of anemia by increasing the circulatory red cell life span. Alterations in cell volume as a result of disordered membrane cation permeability account for reduced life span red cells in HX and OHS. Importantly, splenectomy is not beneficial in these 2 membrane transport disorders and is not recommended because it is ineffective and may lead to an increased risk of life-threatening thrombosis. Rational approaches are now available for the diagnosis and management of these inherited red cell disorders, and these will be discussed in this review.

hCALCRL mutation causes autosomal recessive nonimmune hydrops fetalis with lymphatic dysplasia.

We report the first case of nonimmune hydrops fetalis (NIHF) associated with a recessive, in-frame deletion of V205 in the G protein-coupled receptor, Calcitonin Receptor-Like Receptor (hCALCRL). Homozygosity results in fetal demise from hydrops fetalis, while heterozygosity in females is associated with spontaneous miscarriage and subfertility. Using molecular dynamic modeling and in vitro biochemical assays, we show that the hCLR(V205del) mutant results in misfolding of the first extracellular loop, reducing association with its requisite receptor chaperone, receptor activity modifying protein (RAMP), translocation to the plasma membrane and signaling. Using three independent genetic mouse models we establish that the adrenomedullin-CLR-RAMP2 axis is both necessary and sufficient for driving lymphatic vascular proliferation. Genetic ablation of either lymphatic endothelial Calcrl or nonendothelial Ramp2 leads to severe NIHF with embryonic demise and placental pathologies, similar to that observed in humans. Our results highlight a novel candidate gene for human congenital NIHF and provide structure-function insights of this signaling axis for human physiology.

Inherited or acquired modifiers of iron status may dramatically affect the phenotype in dehydrated hereditary stomatocytosis.

Severe iron overload is frequent in dehydrated hereditary stomatocytosis (DHSt) despite well-compensated hemolysis and no or little transfusion requirement. We investigated 4 patients with proven DHSt, in whom the degree of hemolysis was closely related to iron status. Genetic modifiers increasing iron stores (HFE:pCys282Tyr, HAMP:c-153C>T mutations) were accompanied with high liver iron concentrations and increased hemolysis, whereas therapeutic phlebotomies alleviated the hemolytic phenotype. There were no manifestations of hemolysis in one patient with low iron stores. Hemolysis reappeared when iron supplementation was given. The search for genetic or acquired modifiers of iron status and the modulation of iron stores may help in the management of these patients.

Metabolic causes of nonimmune hydrops fetalis: A next-generation sequencing panel as a first-line investigation.

PURPOSES: Hydrops fetalis is a life-threatening fetal condition, and 85% of all cases are classified as nonimmune hydrops fetalis (NIHF). Up to 15% of NIHF cases may be due to inborn errors of metabolism (IEM), but a large proportion of cases linked to metabolic disorders remains undiagnosed. This lack of diagnosis may be related to the limitations of conventional biological procedures, which involve sequential investigations and require multiple samples and steps. In addition, this approach is time consuming. We have developed a next-generation sequencing (NGS) panel to investigate metabolic causes of NIHF, ascites, and polyhydramnios associated to another fetal abnormality. METHODS: The hydrops fetalis (HydFet) panel was designed to cover the coding regions and flanking intronic sequences of 41 genes. A retrospective study of amniotic fluid samples from 40 subjects was conducted. A prospective study was subsequently initiated, and six samples were analyzed using the NGS panel. RESULTS: Five IEM diagnoses were made using the HydFet panel (Niemann-Pick type C (NPC), Barth syndrome, HNF1Β deficiency, GM1 gangliosidosis, and Gaucher disease). This analysis also allowed the identification of 8p sequence triplication in an additional case. CONCLUSION: NGS combined with robust bioinformatics analyses is a useful tool for identifying the causative variants of NIHF. Subsequent functional characterization of the protein encoded by the altered gene and morphological studies may confirm the diagnosis. This paradigm shift allows a significant improvement of IEM diagnosis in NIHF.

Novel mechanisms of PIEZO1 dysfunction in hereditary xerocytosis.

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 PIEZO1 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.

Red cell membrane disorders.

Significant advances have been made in our understanding of the structural basis for altered cell function in various inherited red cell membrane disorders with reduced red cell survival and resulting hemolytic anemia. The current review summarizes these advances as they relate to defining the molecular and structural basis for disorders involving altered membrane structural organization (hereditary spherocytosis [HS] and hereditary elliptocytosis [HE]) and altered membrane transport function (hereditary overhydrated stomatocytosis and hereditary xerocytosis). Mutations in genes encoding membrane proteins that account for these distinct red cell phenotypes have been identified. These molecular insights have led to improved understanding of the structural basis for altered membrane function in these disorders. Weakening of vertical linkage between the lipid bilayer and spectrin-based membrane skeleton leads to membrane loss in HS. In contrast, weakening of lateral linkages among different skeletal proteins leads to membrane fragmentation and decreased surface area in HE. The degrees of membrane loss and resultant increases in cell sphericity determine the severity of anemia in these two disorders. Splenectomy leads to amelioration of anemia by increasing the circulatory red cell life span of spherocytic red cells that are normally sequestered by the spleen. Disordered membrane cation permeability and resultant increase or decrease in red cell volume account for altered cellular deformability of hereditary overhydrated stomatocytosis and hereditary xerocytosis, respectively. Importantly, splenectomy is not beneficial in these two membrane transport disorders and in fact contraindicated due to severe postsplenectomy thrombotic complications.

EPHB4 kinase-inactivating mutations cause autosomal dominant lymphatic-related hydrops fetalis.

Hydrops fetalis describes fluid accumulation in at least 2 fetal compartments, including abdominal cavities, pleura, and pericardium, or in body tissue. The majority of hydrops fetalis cases are nonimmune conditions that present with generalized edema of the fetus, and approximately 15% of these nonimmune cases result from a lymphatic abnormality. Here, we have identified an autosomal dominant, inherited form of lymphatic-related (nonimmune) hydrops fetalis (LRHF). Independent exome sequencing projects on 2 families with a history of in utero and neonatal deaths associated with nonimmune hydrops fetalis uncovered 2 heterozygous missense variants in the gene encoding Eph receptor B4 (EPHB4). Biochemical analysis determined that the mutant EPHB4 proteins are devoid of tyrosine kinase activity, indicating that loss of EPHB4 signaling contributes to LRHF pathogenesis. Further, inactivation of Ephb4 in lymphatic endothelial cells of developing mouse embryos led to defective lymphovenous valve formation and consequent subcutaneous edema. Together, these findings identify EPHB4 as a critical regulator of early lymphatic vascular development and demonstrate that mutations in the gene can cause an autosomal dominant form of LRHF that is associated with a high mortality rate.

Impaired PIEZO1 function in patients with a novel autosomal recessive congenital lymphatic dysplasia.

Piezo1 ion channels are mediators of mechanotransduction in several cell types including the vascular endothelium, renal tubular cells and erythrocytes. Gain-of-function mutations in PIEZO1 cause an autosomal dominant haemolytic anaemia in humans called dehydrated hereditary stomatocytosis. However, the phenotypic consequence of PIEZO1 loss of function in humans has not previously been documented. Here we discover a novel role of this channel in the lymphatic system. Through whole-exome sequencing, we identify biallelic mutations in PIEZO1 (a splicing variant leading to early truncation and a non-synonymous missense variant) in a pair of siblings affected with persistent lymphoedema caused by congenital lymphatic dysplasia. Analysis of patients' erythrocytes as well as studies in a heterologous system reveal greatly attenuated PIEZO1 function in affected alleles. Our results delineate a novel clinical category of PIEZO1-associated hereditary lymphoedema.

[Clinicopathologic features of fetal nuchal cystic hygroma: report of 40 cases].

OBJECTIVE: To study the pathogenesis, pathologic features and prognosis of fetal nuchal cystic hygroma. METHODS: Forty autopsied cases of fetal nuchal cystic hygroma were collected during January 2003 to December 2012. The clinical history, pathologic changes and immunohistochemical (EnVision method) findings were reviewed, and the pathogenesis and pathologic characteristics were analyzed. RESULTS: Of the 40 cases, 16 (40.0%) showed single malformation and 24 (60.0%) were associated with multiple malformations in other organs and/or systems.Nineteen cases were septated and 21 were not. The associated malformations occurred in the respiratory system, skeletal system and urinary system.In the cases of combined malformations of umbilical cord, 3 were single umbilical artery malformations and 1 was torsion and stricture of umbilical cord.Four cases had chromosomal analysis, and all were trisomy-21. CONCLUSIONS: Fetal nuchal cystic hygroma is a rare disease. The etiology is unknown, but it is not neoplastic.Lymphangioma is divided into 3 types:capillary lymphangioma, cavernous lymphangioma and cystic hygroma according to their expansile growth pattern. The overall prognosis is determined by any co-existing chromosomal anomalies, associated malformations and the time of diagnosis of the cystic hygroma.