Downregulation of glob1 suppresses pathogenesis of human neuronal tauopathies in Drosophila by regulating tau phosphorylation and ROS generation.

Human tauopathies represent a group of neurodegenerative disorders, characterized by abnormal hyperphosphorylation and aggregation of tau protein, which ultimately cause neurodegeneration. The aberrant tau hyperphosphorylation is mostly attributed to the kinases/phosphatases imbalance, which is majorly contributed by the generation of reactive oxygen species (ROS). Globin(s) represent a well-conserved group of proteins which are involved in O2 management, regulation of cellular ROS in different cell types. Similarly, Drosophila globin1 (a homologue of human globin) with its known roles in oxygen management and development of nervous system exhibits striking similarities with the mammalian neuroglobin. Several recent evidences support the hypothesis that neuroglobins are associated with Alzheimer's disease pathogenesis. We herein noted that targeted expression of human-tau induces the cellular level of Glob1 protein in Drosophila tauopathy models. Subsequently, RNAi mediated restored level of Glob1 restricts the pathogenic effect of human-tau by minimizing its hyperphosphorylation via GSK-3ß/p-Akt and p-JNK pathways. In addition, it also activates the Nrf2-keap1-ARE cascade to stabilize the tau-mediated increased level of ROS. These two parallel cellular events provide a significant rescue against human tau-mediated neurotoxicity in the fly models. For the first time we report a direct involvement of an oxygen sensing globin gene in tau etiology. In view of the fact that human genome encodes for the multiple Globin proteins including a nervous system specific neuroglobin; and therefore, our findings may pave the way to investigate if the conserved oxygen sensing globin gene(s) can be exploited in devising novel therapeutic strategies against tauopathies.

Possible Involvement of Nitric Oxide in Enhanced Liver Injury and Fibrogenesis during Cholestasis in Cytoglobin-deficient Mice.

This study clarified the role of Cygb, the fourth globin in mammals originally discovered in rat hepatic stellate cells (HSCs), in cholestatic liver disease. Bile duct ligation (BDL) augmented inflammatory reactions as revealed by increased infiltrating neutrophils, CD68+-macrophages, and chemokine expression in Cygb-/- mice. In these mice, impairment of bile canalicular indicated by the loss of CD10 expression, down-regulation of bile salt transporters, increased total bile acid, and massive apoptotic and necrotic hepatocytes occurred with the release of cytochrome c, activation of caspase 3, resulting in reduced animal survival compared to wild-type mice. In Cygb-/- mouse liver, all of NO metabolites and oxidative stress were increased. Treatment with NO inhibitor restrained all above phenotypes and restored CD10 expression in BDL Cygb-/- mice, while administration of NO donor aggravated liver damage in BDL-wild type mice to the same extent of BDL-Cygb-/- mice. N-acetylcysteine administration had a negligible effect in all groups. In mice of BDL for 1-3 weeks, expression of all fibrosis-related markers was significantly increased in Cygb-/- mice compared with wild-type mice. Thus, Cygb deficiency in HSCs enhances hepatocyte damage and inflammation in early phase and fibrosis development in late phase in mice subjected to BDL, presumably via altered NO metabolism.

Absence of cytoglobin promotes multiple organ abnormalities in aged mice.

Cytoglobin (Cygb) was identified in hepatic stellate cells (HSCs) and pericytes of all organs; however, the effects of Cygb on cellular functions remain unclear. Here, we report spontaneous and age-dependent malformations in multiple organs of Cygb(-/-) mice. Twenty-six percent of young Cygb(-/-) mice (<1 year old) showed heart hypertrophy, cystic disease in the kidney or ovary, loss of balance, liver fibrosis and lymphoma. Furthermore, 71.3% (82/115) of aged Cygb(-/-) mice (1-2 years old) exhibited abnormalities, such as heart hypertrophy and cancer development in multiple organs; by contrast, 5.8% (4/68) of aged wild-type (WT) mice had abnormalities (p < 0.0001). Interestingly, serum and urine analysis demonstrated that the concentration of nitric oxide metabolites increased significantly in Cygb(-/-) mice, resulting in an imbalance in the oxidative stress and antioxidant defence system that was reversed by N(G)-monomethyl-L-arginine treatment. A senescent phenotype and evidence of DNA damage were found in primary HSCs and the liver of aged Cygb(-/-) mice. Moreover, compared with HSC(+/+), HSC(-/-) showed high expression of Il-6 and chemokine mRNA when cocultured with mouse Hepa 1-6 cells. Thus, the absence of Cygb in pericytes provokes organ abnormalities, possibly via derangement of the nitric oxide and antioxidant defence system and through accelerated cellular senescence.

Cytoglobin deficiency promotes liver cancer development from hepatosteatosis through activation of the oxidative stress pathway.

This study was conducted to clarify the role of cytoglobin (Cygb), a globin expressed in hepatic stellate cells (HSCs), in the development of liver fibrosis and cancer in nonalcoholic steatohepatitis (NASH). Cygb expression was assessed in patients with NASH and hepatocellular carcinoma. Mouse NASH model was generated in Cygb-deficient (Cygb(-/-)) or wild-type (WT) mice by giving a choline-deficient amino acid-defined diet and, in some of them, macrophage deletion and N-acetyl cysteine treatment were used. Primary-cultured mouse HSCs isolated from WT (HSCs(Cygb-wild)) or Cygb(-/-) (HSCs(Cygb-null)) mice were characterized. As results, the expression of CYGB was reduced in patients with NASH and hepatocellular carcinoma. Choline-deficient amino acid treatment for 8 weeks induced prominent inflammation and fibrosis in Cygb(-/-) mice, which was inhibited by macrophage deletion. Surprisingly, at 32 weeks, despite no tumor formation in the WT mice, all Cygb(-/-) mice developed liver cancer, which was ameliorated by N-acetyl cysteine treatment. Altered expression of 31 genes involved in the metabolism of reactive oxygen species was notable in Cygb(-/-) mice. Both HSCs(Cygb-null) and Cygb siRNA-transfected-HSCs(Cygb-wild) exhibited the preactivation condition. Our findings provide important insights into the role that Cygb, expressed in HSCs during liver fibrosis, plays in cancer development with NASH.

Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion.

BACKGROUND: The molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. RESULTS: We compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding. CONCLUSIONS: Globin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA-Seq is highly reproducible within a large dynamic range of detection and provides an accurate estimation of RNA concentration in peripheral whole blood. High-throughput sequencing is thus a promising technology for whole blood transcriptomics and biomarker discovery.

Neuroglobin-deficiency exacerbates Hif1A and c-FOS response, but does not affect neuronal survival during severe hypoxia in vivo.

BACKGROUND: Neuroglobin (Ngb), a neuron-specific globin that binds oxygen in vitro, has been proposed to play a key role in neuronal survival following hypoxic and ischemic insults in the brain. Here we address whether Ngb is required for neuronal survival following acute and prolonged hypoxia in mice genetically Ngb-deficient (Ngb-null). Further, to evaluate whether the lack of Ngb has an effect on hypoxia-dependent gene regulation, we performed a transcriptome-wide analysis of differential gene expression using Affymetrix Mouse Gene 1.0 ST arrays. Differential expression was estimated by a novel data analysis approach, which applies non-parametric statistical inference directly to probe level measurements. PRINCIPAL FINDINGS: Ngb-null mice were born in expected ratios and were normal in overt appearance, home-cage behavior, reproduction and longevity. Ngb deficiency had no effect on the number of neurons, which stained positive for surrogate markers of endogenous Ngb-expressing neurons in the wild-type (wt) and Ngb-null mice after 48 hours hypoxia. However, an exacerbated hypoxia-dependent increase in the expression of c-FOS protein, an immediate early transcription factor reflecting neuronal activation, and increased expression of Hif1A mRNA were observed in Ngb-null mice. Large-scale gene expression analysis identified differential expression of the glycolytic pathway genes after acute hypoxia in Ngb-null mice, but not in the wts. Extensive hypoxia-dependent regulation of chromatin remodeling, mRNA processing and energy metabolism pathways was apparent in both genotypes. SIGNIFICANCE: According to these results, it appears unlikely that the loss of Ngb affects neuronal viability during hypoxia in vivo. Instead, Ngb-deficiency appears to enhance the hypoxia-dependent response of Hif1A and c-FOS protein while also altering the transcriptional regulation of the glycolytic pathway. Bioinformatic analysis of differential gene expression yielded novel predictions suggesting that chromatin remodeling and mRNA metabolism are among the key regulatory mechanisms when adapting to prolonged hypoxia.

Knockdown of cytoglobin expression sensitizes human glioma cells to radiation and oxidative stress.

Cytoglobin is a recently identified vertebrate globin whose functions include scavenging reactive oxygen and nitrosative species. In tumor cells, CYGB may function as a tumor suppressor gene. Here we show that knockdown of cytoglobin expression can sensitize human glioma cells to oxidative stress induced by chemical inhibitors of the electron transport chain and as well can increase cellular radiosensitivity. When treated with antimycin A, an inhibitor of the mitochondrial electron transport chain, cytoglobin-deficient cells showed significantly higher H2O2 levels, whereas H2O2 levels were significantly reduced in cytoglobin-overexpressing cells. In addition, cytoglobin knockdown significantly decreased the doubling time of glioma cell lines, consistent with a putative tumor suppressor function. These finding suggest that modulating cytoglobin levels may be a promising treatment strategy for sensitizing human glioma cells to oxidative stress that is induced by ionizing radiation, certain chemotherapies and ischemia-reperfusion.

Role of neuroglobin in regulating reactive oxygen species in the brain of the anoxia-tolerant turtle Trachemys scripta.

Neuroglobin (Ngb) is an oxygen binding heme protein found in nervous tissue with a yet unclear physiological and protective role in the hypoxia-sensitive mammalian brain. Here we utilized in vivo and in vitro studies to examine the role of Ngb in anoxic and post-anoxic neuronal survival in the freshwater turtle. We employed semiquantitative RT-PCR and western blotting to analyze Ngb mRNA and protein levels in turtle brain and neuronally enriched cultures. Ngb expression is strongly up-regulated by hypoxia and post-anoxia reoxygenation but increases only modestly in anoxia. The potential neuroprotective role of Ngb in this species was analyzed by knocking down Ngb using specific small interfering RNA. Ngb knockdown in neuronally enriched cell cultures resulted in significant increases in H(2)O(2) release compared to controls but no change in cell death. Cell survival may be linked to activation of other protective responses such as the extracellular regulated kinase transduction pathway, as phosphorylated extracellular regulated kinase levels in anoxia were significantly higher in Ngb knockdown cultures compared to controls. The greater expression of Ngb when reactive oxygen species are likely to be high, and the increased susceptibility of neurons to H(2)O(2) release and external oxidative stress in knockdown cultures, suggests a role for Ngb in reducing reactive oxygen species production or in detoxification, though it does not appear to be of primary importance in the anoxia tolerant turtle in the presence of compensatory survival mechanisms.

siRNA-mediated reduction of alpha-globin results in phenotypic improvements in beta-thalassemic cells.

beta-thalassemia is an inherited hemoglobinopathy caused by defective synthesis of the beta-globin chain of hemoglobin, leading to imbalanced globin chain synthesis. Excess alpha-globin precipitates in erythroid progenitor cells resulting in cell death, ineffective erythropoiesis and severe anemia. Decreased alpha-globin synthesis leads to milder symptoms, exemplified in individuals who co-inherit alpha- and beta-thalassemia. In this study, we investigated the feasibility of utilizing short-interfering RNA (siRNA) to mediate reductions in alpha-globin expression. A number of siRNA sequences targeting murine alpha-globin were tested in hemoglobinized murine erythroleukemic cells. One highly effective siRNA sequence (si-alpha 4) was identified and reduced alpha-globin by approximately 65% at both the RNA and the protein level. Electroporation of si-alpha 4 into murine thalassemic primary erythroid cultures restored alpha :beta-globin ratios to balanced wild-type levels and resulted in detectable phenotypic correction. These results indicate that siRNA-mediated reduction of alpha-globin has potential therapeutic applications in the treatment of beta-thalassemia.

Characterization of a novel deletion causing (deltabeta)0-thalassemia in a Thai family.

A novel deletion of the human beta-globin gene cluster associated with the increased level of fetal hemoglobin (Hb F) in adult life has been demonstrated in a Thai family. A Thai girl who was mistakenly diagnosed as beta-thalassemia/HbE is found to be the compound heterozygote of this mutation and Hb E. The heterozygous father had mild hypochromic and microcytic red blood cells and a high level of Hb F (23.2%). Polymorphic restriction sites in the beta-globin gene cluster identified the homozygous alleles, which localized the deletion region between the psibeta-globin and the 3' beta-globin genes. DNA polymerase that can amplify a long DNA template was employed to examine DNA fragment encompassing this deletion. A 11.3 kilobases (kb) of DNA deletion, beginning approximately 3.1 kb 5' to the delta-globin gene and end in the intron 2 of the beta-globin gene was detected. DNA analysis revealed that this is a case of (deltabeta)(0)-thalassemia with a novel mutation, which can lead to a mild form of beta-thalassemia upon interaction with Hb E.

[Beta(o)/beta(o) thalassemia with a mild phenotype]. / Thalassémie beta(o)/beta(o) avec phénotype "intermédiaire": un diagnostic rare... auquel il faut penser.

We report the case of a 30 years old patient of Algerian origin, presenting a beta-thalassemia major with a phenotype of intermediate severity. Its genotype is beta(o)/beta(o), leading to a complete absence of beta-globin synthesis. This genotype is usually responsible for major clinical complications and a severe anaemia requiring regular transfusions. However, the patient presents with a mild form of the disease and a moderate relatively well tolerated anaemia. This phenotype was found related to a high level of synthesis of foetal haemoglobin, dependent most probably on an homozygous state for the polymorphism (XmnI -158, C>T) in the promoter of the Ggamma gene. This observation shows that it is important to keep in mind that beta-thalassemia major may have a mild or intermediate phenotype because of polymorphisms of the beta locus.

Major erythrocyte membrane protein genes in EKLF-deficient mice.

OBJECTIVES: Mice deficient in the transcription factor erythroid Krüppel-like factor, KLF1 (EKLF) die approximately 14.5 days postcoitum of anemia, attributed to decreased expression of the beta-globin gene. The objectives of this study were to rescue EKLF-deficient embryos with mice expressing gamma-globin from beta-spectrin or ankyrin promoters and to characterize expression of the major erythrocyte membrane genes in EKLF-deficient cells. METHODS: Transgenic beta-spectrin/gamma-globin or ankyrin/gamma-globin mice were bred onto EKLF-deficient and wild-type backgrounds. Animals were genotyped, gamma-globin mRNA levels measured, and hemoglobin electrophoresis performed. Steady-state mRNA levels and transcriptional rates of the major erythrocyte membrane protein genes were assayed. RESULTS: beta-spectrin/gamma-globin or ankyrin/gamma-globin mice on EKLF-deficient and wild-type backgrounds had identical levels of gamma-globin mRNA, indicating EKLF-independence of these promoters. gamma-Globin expression improved globin chain imbalance, but hemolysis was not improved and no live-born EKLF-deficient/(A)gamma-globin mice were obtained. Circulating erythroid cells from EKLF-deficient/(A)gamma-globin embryos exhibited hemolysis reminiscent of that seen in patients with severe erythrocyte membrane defects. Levels of beta-spectrin, ankyrin, and band 3 mRNA, but not alpha-spectrin, were decreased in EKLF-deficient fetal liver RNA. In a run-on assay, levels of transcription of the ankyrin and band 3 genes were decreased in EKLF-deficient fetal liver nuclei. CONCLUSIONS: These results indicate that the EKLF-responsive regions of the ankyrin and beta-spectrin genes are outside their promoters and that EKLF is necessary for full transcriptional activity of the ankyrin and band 3 genes; the results also provide additional evidence that defects in addition to beta-globin deficiency, including an abnormal erythrocyte membrane, contribute to the anemia and embryonic lethality in EKLF-deficient mice.

Stem cell transplantation with S-59 photochemically treated T-cell add-backs to establish allochimerism in murine thalassemia.

Hematopoietic cell transplantation (HCT) from HLA-identical sibling donors has curative potential for beta-thalassemia. The probability of surviving free of thalassemia under these conditions is approximately 85%. The application of this therapy is limited because many patients lack an HLA-identical sibling donor. HLA-mismatched stem cell transplantation for thalassemia is severely restricted by graft rejection and the risks for graft-versus-host disease (GVHD). Thus, the development of a novel method that facilitates immunological tolerance and improves the safety of HLA-mismatched HCT would greatly expand the opportunity of HCT for thalassemia patients. We hypothesized that removal of T cells from the donor hematopoietic stem cell preparation and subsequent add-back after photochemical treatment with S-59, a psoralen, would promote and stabilize the engraftment and significantly reduce the risk of GVHD. This was tested in a MHC-mismatched HCT model of murine thalassemia. S-59-treated T cells were infused simultaneously with bone marrow-derived stem cells into mice with a heterozygous deletion of one beta-globin alleles that had been conditioned with a sublethal dose of total body irradiation. Mice that received treated T cells showed increased engraftment compared to those that did not receive T cells. T-cell treatment improved survival without GVHD compared to recipients that received untreated T cells. We conclude that photochemical treatment of T cells facilitates engraftment and minimizes GVHD in allo-HCT for murine thalassemia, and sets the stage for further development of such protocols for the treatment of patients with thalassemia.

Rescued mice with Hb E transgene-developed red cell changes similar to human beta-thalassemia/HbE disease.

A novel C57BL/6 transgenic murine model of HbE has been developed, and the heterotetrameric ((m)alpha2(h)beta(E)2) hemoglobin shows significant complementation of mild thalassemia phenotype in double heterozygous (beta(m+)beta(m-), beta(hE)) and homozygous knockout (beta(m-)beta(m-), beta(hE)) mice with 100% heterotetrameric hemoglobin. Lethal homozygous beta-thalassemic mice rescued by HbE transgenes mimic beta-thalassemia/HbE phenotype in human. Although anemia was not pronounced, other hematologic parameters were abnormally similar to beta-knockout mice. Flow cytometric study revealed a highly oxidative status in the red cells, but there were no marked changes in PS red cells and RBC vesicles. RBC life span and half-time of rescued red cells were shortened, indicating a rapid RBC destruction.

A 191-kb genomic fragment containing the human alpha-globin locus can rescue alpha-thalassemic mice.

A 191-kb human bacterial artificial chromosome (BAC) containing the human alpha-globin genomic locus was used to generate transgenic mice that express, exclusively, human alpha-globin ((hu)alpha-globin). Expression of (hu)alpha-globin reaches a level of 36% of that of endogenous mouse alpha-globin ((mu)alpha-globin) on a heterozygous mouse alpha-thalassemia background ((mu)alpha-globin knockout, (mu)alpha(+/-)). Hemizygous transgenic mice carrying the (hu)alpha-globin locus on a heterozygous knockout background ((hu)alpha(+/0), (mu)alpha(++/--)) demonstrated complementation of most hematologic parameters. By crossing (hu)alpha(+/0), (mu)alpha(++/--) mice, we were able to generate mice entirely dependent on (hu)alpha-globin synthesis. Breeding and fluorescent in situ hybridization studies demonstrate that only mice homozygous for the transgene were able to rescue embryonic lethal homozygous (mu)alpha-globin knockout embryos ((mu)alpha(--/--)). Adult rescued mice produce hemoglobin at levels similar to wild-type mice, with partial red cell complementation based on mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW) measurements. Significant erythrocythemia above wild-type levels seems to be the main compensatory mechanism for the normalization of the hemoglobin levels in the rescued animals. Our studies demonstrate that the (hu)alpha-globin locus in the 191-kb transgene contains all the necessary elements for the regulated expression of (hu)alpha-globin in transgenic mice. This animal model should be valuable for studying the mechanisms regulating (hu)alpha-globin production and for development of therapeutic strategies for beta-thalassemia based on downregulation of alpha-globin expression.

Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized, purified hematopoietic stem cells.

Although sickle cell anemia was the first hereditary disease to be understood at the molecular level, there is still no adequate long-term treatment. Allogeneic bone marrow transplantation is the only available cure, but this procedure is limited to a minority of patients with an available, histocompatible donor. Autologous transplantation of bone marrow stem cells that are transduced with a stably expressed, antisickling globin gene would benefit a majority of patients with sickle cell disease. Therefore, the development of a gene therapy protocol that corrects the disease in an animal model and is directly translatable to human patients is critical. A method is described in which unmobilized, highly purified bone marrow stem cells are transduced with a minimum amount of self-inactivating (SIN) lentiviral vector containing a potent antisickling beta-globin gene. These cells, which were transduced in the absence of cytokine stimulation, fully reconstitute irradiated recipients and correct the hemolytic anemia and organ pathology that characterize the disease in humans. The mean increase of hemoglobin concentration was 46 g/L (4.6 g/dL) and the average lentiviral copy number was 2.2; therefore, a 21-g/L /vector copy increase (2.1-g/dL) was achieved. This transduction protocol may be directly translatable to patients with sickle cell disease who cannot tolerate current bone marrow mobilization procedures and may not safely be exposed to large viral loads.

The degree of phenotypic correction of murine beta -thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number.

Increased fetal hemoglobin (HbF) levels diminish the clinical severity of beta-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell-targeted gene transfer of a gamma-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based gamma-globin vectors for expression in transduced erythroid cell lines. Compared with gamma-globin, oncoretroviral vectors containing either a beta-spectrin or beta-globin promoter and the alpha-globin HS40 element, a gamma-globin lentiviral vector utilizing the beta-globin promoter and elements from the beta-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human gamma-globin was observed with levels of vector-encoded gamma-globin mRNA ranging from 9% to 19% of total murine alpha-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of beta-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric m(alpha)(2)h(gamma)(2) molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. gamma-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a gamma-globin lentiviral vector for gene therapy of beta-thalassemia.

Hb G-San Josè variant levels correlate with alpha-thalassemia genotypes.

Hb G-San Josè or beta7(A4)Glu-->Gly has been reported in Southern Italian or Mexican families. We have studied four families from Sicily and Campania, Southern Italy. In six carriers, the hemoglobin variant level ranged from 32 to 38%. In four double heterozygotes for Hb G-San Josè and alpha-thalassemia the variant level showed a strong correlation with the alpha-thalassemia genotype. In fact, the variant level was 15% when interacting with the - (alpha)20.5/alphaalpha, 19.6% with the alphaalpha/alphaPoly Aalpha, and 24.8% with alphaalpha/alpha(-5) ntalpha genotypes. In two double heterozygotes for Hb G-San Josè and beta+ -IVS-I-6 (T-->C) the hemoglobin variant level was 67%. These data show that the reduced synthesis of alpha chains causes drastic reduction of probability to form Hb G-San Josè in favor of the formation of Hb A. Moreover, this reduction, (i) correlates with the type of alpha-thalassemia genotype and with the degree of the alpha chain deficiency, and (ii) is, most probably, more marked than the degree of alpha chain reduction. The minor affinity of the beta chain variant for the alpha chains associated with the reduced synthesis of the alpha chains is probably the principal cause of the variant hemoglobin reduction. Moreover, the rapid removal of the abnormal chains by proteolytic enzymes must have an essential role in order to reduce the chain variant pool. These conclusions are in agreement with the results obtained in reticulocyte and in vitro recombination experiments.