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Genome-wide association studies (GWASs) have identified loci for erythrocyte traits in primarily European ancestry populations. We conducted GWAS meta-analyses of six erythrocyte traits in 71,638 individuals from European, East Asian, and African ancestries using a Bayesian approach to account for heterogeneity in allelic effects and variation in the structure of linkage disequilibrium between ethnicities. We identified seven loci for erythrocyte traits including a locus (RBPMS/GTF2E2) associated with mean corpuscular hemoglobin and mean corpuscular volume. Statistical fine-mapping at this locus pointed to RBPMS at this locus and excluded nearby GTF2E2. Using zebrafish morpholino to evaluate loss of function, we observed a strong in vivo erythropoietic effect for RBPMS but not for GTF2E2, supporting the statistical fine-mapping at this locus and demonstrating that RBPMS is a regulator of erythropoiesis. Our findings show the utility of trans-ethnic GWASs for discovery and characterization of genetic loci influencing hematologic traits.

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

Introduction: Deletion or mutation of members of the spectrin gene family contributes to many neurologic and neuropsychiatric disorders. While each spectrinopathy may generate distinct neuropathology, the study of ßI spectrin's role (Sptb) in the brain has been hampered by the hematologic consequences of its loss. Methods: Jaundiced mice (ja/ja) that lack ßI spectrin suffer a rapidly fatal hemolytic anemia. We have used exchange transfusion of newborn ja/ja mice to blunt their hemolytic pathology, enabling an examination of ßI spectrin deficiency in the mature mouse brain by ultrastructural and biochemical analysis. Results: ßI spectrin is widely utilized throughout the brain as the ßIΣ2 isoform; it appears by postnatal day 8, and concentrates in the CA1,3 region of the hippocampus, dentate gyrus, cerebellar granule layer, cortical layer 2, medial habenula, and ventral thalamus. It is present in a subset of dendrites and absent in white matter. Without ßI spectrin there is a 20% reduction in postsynaptic density size in the granule layer of the cerebellum, a selective loss of ankyrinR in cerebellar granule neurons, and a reduction in the level of the postsynaptic adhesion molecule NCAM. While we find no substitution of another spectrin for ßI at dendrites or synapses, there is curiously enhanced ßIV spectrin expression in the ja/ja brain. Discussion: ßIΣ2 spectrin appears to be essential for refining postsynaptic structures through interactions with ankyrinR and NCAM. We speculate that it may play additional roles yet to be discovered. [ABSTRACT FROM AUTHOR]

Adducins are cytoskeletal actin-binding proteins (alpha, beta, gamma) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates alpha- and beta-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of alpha- and beta-adducin on glomerular function and disease using beta-adducin null mice, congenic substrains for alpha- and beta-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of beta-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, alpha-actinin, ZO-1, Fyn). The introgression of polymorphic MHS beta-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, alpha-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS alpha-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic beta-adducin (ADD2, 1797T, rs4984) with a significant interaction with alpha-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins.

Adducins are cytoskeletal actin-binding proteins (alpha, beta, gamma) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates alpha- and beta-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of alpha- and beta-adducin on glomerular function and disease using beta-adducin null mice, congenic substrains for alpha- and beta-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of beta-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, alpha-actinin, ZO-1, Fyn). The introgression of polymorphic MHS beta-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, alpha-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS alpha-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic beta-adducin (ADD2, 1797T, rs4984) with a significant interaction with alpha-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins.

ICAM-4, a newly characterized adhesion molecule, is expressed early in human erythropoiesis and functions as a ligand for binding a4b1 and aV integrin-expressing cells. Within the bone marrow, erythroblasts surround central macrophages forming erythroblastic islands. Evidence suggests that these islands are highly specialized subcompartments where cell adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. Since erythroblasts express a4b1 and ICAM-4 and macrophages exhibit aV, ICAM-4 is an attractive candidate for mediating cellular interactions within erythroblastic islands. To determine whether ICAM-4 binding properties are conserved across species, we first cloned and sequenced the murine homologue. The translated amino acid sequence showed 68% overall identity with human ICAM-4. Using recombinant murine ICAM-4 extracellular domains, we discovered that hematopoietic a4b1-expressing HEL cells and non-hematopoietic aV-expressing FLY cells adhered to mouse ICAM-4. Cell adhesion studies showed that FLY and HEL cells bound to mouse and human proteins with similar avidity. These data strongly suggest conservation of integrin-binding properties across species. Importantly, we characterized a novel second splice cDNA that would be predicted to encode an ICAM-4 isoform, lacking the membrane-spanning domain. Erythroblasts express both isoforms of ICAM-4. COS-7 cells transfected with GFP constructs of prototypic or novel ICAM-4 cDNA showed different cellular localization patterns. Moreover, analysis of tissue culture medium revealed that the novel ICAM-4 cDNA encodes a secreted protein. We postulate that secretion of this newly described isoform, ICAM-4S, may modulate binding of membrane-associated ICAM-4 and could thus play a critical regulatory role in erythroblast molecular attachments.

Introduction Mammalian platelets are small anucleated blood cells specialized to continuously monitor and preserve the integrity of the cardiovascular system (hemostasis) (1-3). Once released from megakaryocytes, they circulate for 10 [...], The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG- GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain [beta]III spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in [beta]III spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact [beta]III spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most [Spnb3.sup.-/-] animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluR[delta], IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of [alpha]II spectrin. It appears that [beta]III spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y. cytoskeleton | membrane | spinocerebellar ataxia type 5 | excitatory amino acid transporter 4 | Purkinje doi/10.1073/pnas.1001522107

Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms ([4.1.sup.-/-]; Shi TS et al. J Clin Invest 103: 331, 1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. [4.1.sup.-/-] mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of [4.1.sup.-/-] erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 [+ or -] 3.2 in [4.1.sup.-/-] vs. 9.8 [+ or -] 1.3 mmol/[10.sup.13] cell x h in control mice), with an abnormal dependence on osmolality ([EC.sub.50] = 417 [+ or -] 42 in [4.1.sup.-/-] vs. 460 [+ or -] 35 mosmol/kg[H.sub.2]O in control mice), suggestive of an upregulated functional state. While the affinity for internal protons was not altered ([K.sub.0.5] = 489.7 [+ or -] 0.7 vs. 537.0 [+ or -] 0.56 nM in control mice), the [V.sub.max] of the H-induced Na/H exchange activity was markedly elevated in [4.1.sup.-/-] erythrocytes ([V.sub.max] 91.47 [+ or -] 7.2 compared with 46.52 [+ or -] 5.4 mmol/[10.sup.13] cell x h in control mice). Na/H exchange activation by okadaic acid was absent in [4.1.sup.-/-] erythrocytes. Altogether, these results suggest that erythroid protein 4.1 plays a major role in volume regulation and physiologically down-regulates Na/H exchange in mouse erythrocytes. Upregulation of the Na/H exchange is an important contributor to the elevated cell Na content of [4.1.sup.-/-] erythrocytes. spherocytosis; cell Na; Na/H exchange