Association Between Genetic Polymorphisms of MIR3142HG and the Risk of Steroid-induced Osteonecrosis of the Femoral Head in the Population of Northern China

Background: Steroid-induced osteonecrosis of the femoral head (ONFH) is aseptic necrosis of the femoral head caused by glucocorticoid use. Once necrotic femoral head necrosis occurs, it irreversibly affects the quality of life seriously. Studies have shown that the susceptibility to steroid-induced ONFH is likely to be related to the variation of miRNA coding genes. Therefore, this study aimed was to investigate the effect of MIR3142HG on steroid-induced ONFH.Methods: Agena MassARRAY was used to genotype MIR3142HG gene rs1582417, rs2431689, rs7727155 and rs17057846 in 199 patients and 506 healthy people. A genetic model and haplotype analysis were used to evaluate the relationship between the MIR3142HG polymorphism and the risk of steroid-induced ONFH. The odds ratio (OR) and 95% confidence intervals (CIs) were obtained through logistic regression to assess the influence of gene polymorphisms on the occurrence of steroid-induced ONFH.Results: The consequences show that rs7727115(OR=0.76,p=0.036) is a protective factor, it could reduce the risk of steroid-induced ONFH, rs1582417(OR=1.28, p=0.041) could increase the risk of steroid-induced ONFH. Stratified analysis , according to each clinical index shows that MIR3142HG TC-CC genotype facilitated the risk of steroid-induced ONFH in male ( p<0.05). In addition, rs2431689 is related to HDL-C(p=0.012) and ApoA1 (p=0.010) levels, and rs17057846 (p=0.024) is related to ApoB levels.The linkage analysis indicated that three SNPs (rs2431689, rs7727115 and rs17057846) in MIR3142HG with significant chain imbalance. In addition, haplotype “GGG” of MIR3142HG was found out is harmful for steroid-induced ONFH.Conclusion: Our results firstly confirm that the genetic polymorphism of MIR3142HG is associated with steroid-induced ONFH susceptibility in Chinese Han population.

Homozygous Expression of a Novel Senegalese-Type Partial Deletion of the Beta and Delta Genes Causes a Delta0 Beta+ Thalassemia.

Abstract 2128 Clinical phenotype in β-thalassemia syndromes is determined by the degree of chain imbalance. An increase in γ-globin production will compensate for the absent or deficient β-globin synthesis and will result in the amelioration of the chain imbalance, and hence an improvement in clinical features. The known genotypes of δβ-thalassemia are associated with an increase in Hb F production, which results in the amelioration of the clinical presentation. Most δβ-thalassemias result from deletions that remove the δ- and β-globin genes, (δβ)0 with a compensatory increase in γ-globin (Hb F) expression. We report an unusual case of homozygous δ0β+ thalassemia that provides interesting insights into increased γ-globin expression and the regulation of β-globin gene expression. An 8-year old boy of African ancestry presented with lifelong jaundice and pallor. He also experienced episodes of worsening symptoms. He exhibited frontal bossing, pale mucosa, scleral icterus, and moderate splenomegaly. He was known to have G6PD deficiency and was suspected of having additional erythrocyte pathology. The CBC revealed a Hb of 8.7, Hct 26.4, MCV 64.7, WBC 10,700, platelets 283,000, reticulocytes 2.2%, and total bilirubin 5.3. Hemoglobin analysis by HPLC and IEF revealed HbA 13.4%, Hb F 86.6%, and no additional components. Alpha thalassemia −3.7kb deletion was not detected. Globin chain analysis revealed α, β, Gγ and AγI chains. DNA analysis revealed a novel Senegalese-type deletion of the beta and delta genes, resulting in a delta0 beta+ thalassemia. The subject's parents who were both from the same small village in Niger had normal hematology values. Their hemoglobin analyses revealed Hb A 94. 8%, Hb A2 2.0%, Hb F 3.2% and Hb A 93.5%, Hb A2 2.1%, Hb F 4.5% in the father and mother, respectively. They were both heterozygous for the delta-beta deletion identified in their son. DNA analysis revealed a breakpoint in the delta gene at nucleotides 54755–54760 and a breakpoint in the beta gene at nucleotides 62153– 62158 [GenBank Ref ID: HUMHBB] with a 5 nucleotide “CAACA” bp region overlapping area. The subject, who is homozygous for the identified deletions, has a clinical phenotype of thalassemia intermedia. He has not yet required red cell transfusions. This is the first instance of a Senegalese-type deletion occurring in the homozygous state. The genotype provides insights into regulation of globin gene expression. While the ∼7 Kb deletion in the δβ-intergenic region may be responsible for the increased expression of the γ-globin gene similar to Hb Lepore deletions, the continued low level expression of the β-globin gene is most probably the result of the juxtaposition of the inefficient δ-globin promoter brought in the vicinity of the β-globin gene. Disclosures: No relevant conflicts of interest to declare.

A Phase 2 Trial of HQK-1001 (sodium 2,2-dimethylbutyrate), an Oral Fetal Globin Gene Inducer, in HbE-Beta Thalassemia in Thailand

Abstract 251 Beta thalassemia intermedia (BTI) syndromes are characterized by globin chain imbalance, ineffective erythropoiesis, hemolytic anemia, and have no therapeutic approved for the underlying pathology. Higher fetal globin (HbF) expression can compensate for beta globin deficiency, reduce globin chain imbalance, and ameliorate phenotype within the same genotypes. HQK-1001 (HemaQuest Pharmaceuticals, San Diego, CA) is an orally bioavailable, non-cytotoxic, short-chain fatty acid derivative which induces fetal globin expression in multiple experimental models. In thalassemic erythroid progenitors in vitro, HQK-1001 prolongs STAT-5 phosphorylation and increases expression of the pro-survival protein Bcl-xL. In a Phase I/II dose-escalation trial in BTI, HQK-1001 administered at 10, 20, 30, and 40 mg/kg/day for 8 weeks was well–tolerated, demonstrated a t½ of 9–11 hours, and treatment resulted in an increase in HbF and total hemoglobin (Hgb), with best results observed at 20 mg/kg. This open-label Phase 2 trial evaluated HQK-1001 administered orally at 20 mg/kg once daily for 26 weeks in adult patients with HbE-β0 thalassemia (NCT01609595). Ten subjects were enrolled, ages 20–36 years, including 7/10 female and 5/10 splenectomized subjects. The beta globin molecular mutations of the subjects, in addition to Codon 26 (G-A), included Codon 41/42 (-TTCT), Codon 17 (A-T), Codon 110 (T-C), and IVS I-I (G-T). At the time of this analysis, 9 of 10 subjects have completed at least 20 weeks of dosing. HbF has increased in all subjects above baseline levels by a mean of 10% (range 4.3% to 20.9%) and total HbF has increased by a mean of 1.07 g/dL, (range 0.52–2.38 g/dL, with positive changes in total HbF observed in 9/10 subjects. Total Hgb has increased by >0.5 g/dL above baseline (mean of 1.27 g/dL, range 0.7 to 2.2 g/dL) in 3 subjects after 3 to 5 months of dosing. Treatment was well-tolerated. There have been no severe drug-related adverse events, no serious adverse events, and no clinically relevant laboratory abnormalities. One subject developed transient proteinuria and edema without changes in renal function 3 weeks following an episode of pharyngitis. This ongoing trial demonstrates consistent induction of fetal globin expression by HQK-1001 at a well-tolerated dose level in subjects with HbE-beta thalassemia. Disclosures: Fucharoen: HemaQuest Pharmaceuticals: Research Funding. Perrine:HemaQuest Pharmaceuticals: Equity Ownership, Patents & Royalties.

Zinc-finger nuclease-mediated correction of α-thalassemia in iPS cells

Induced pluripotent stem (iPS) cell technology holds vast promises for a cure to the hemoglobinopathies. Constructs and methods to safely insert therapeutic genes to correct the genetic defect need to be developed. Site-specific insertion is a very attractive method for gene therapy because the risks of insertional mutagenesis are eliminated provided that a “safe harbor” is identified, and because a single set of validated constructs can be used to correct a large variety of mutations simplifying eventual clinical use. We report here the correction of α-thalassemia major hydrops fetalis in transgene-free iPS cells using zinc finger–mediated insertion of a globin transgene in the AAVS1 site on human chromosome 19. Homozygous insertion of the best of the 4 constructs tested led to complete correction of globin chain imbalance in erythroid cells differentiated from the corrected iPS cells.

Methemoglobinemia and ascorbate deficiency in hemoglobin E β thalassemia: metabolic and clinical implications

During investigations of the phenotypic diversity of hemoglobin (Hb) E β thalassemia, a patient was encountered with persistently high levels of methemoglobin associated with a left-shift in the oxygen dissociation curve, profound ascorbate deficiency, and clinical features of scurvy; these abnormalities were corrected by treatment with vitamin C. Studies of erythropoietin production before and after treatment suggested that, as in an ascorbate-deficient murine model, the human hypoxia induction factor pathway is not totally dependent on ascorbate levels. A follow-up study of 45 patients with HbE β thalassemia showed that methemoglobin levels were significantly increased and that there was also a significant reduction in plasma ascorbate levels. Haptoglobin levels were significantly reduced, and the high frequency of the 2.2 haptoglobin genotype may place an additional pressure on ascorbate as a free-radical scavenger in this population. There was, in addition, a highly significant correlation between methemoglobin levels, splenectomy, and factors that modify the degree of globin-chain imbalance. Because methemoglobin levels are modified by several mechanisms and may play a role in both adaptation to anemia and vascular damage, there is a strong case for its further study in other forms of thalassemia and sickle-cell anemia, particularly when splenic function is defective.

A pilot study of subcutaneous decitabine in β-thalassemia intermedia

Ineffective erythropoiesis, the hallmark of β-thalassemia, is a result of α/non-α globin chain imbalance.1 One strategy to redress globin-chain imbalance is to induce γ-globin gene (HBG) expression. Repression of HBG in adult erythroid cells involves DNA methylation and other epigenetic changes. Therefore, the cytosine analog decitabine, which can deplete DNA methyltransferase 1 (DNMT1), can potentially activate HBG. In 5 patients with β-thalassemia intermedia, a dose and schedule of decitabine intended to deplete DNMT1 without causing significant cytotoxicity (0.2 mg/kg subcutaneous 2 times per week for 12 weeks) increased total hemoglobin from 7.88 ± 0.88 g/dL to 9.04 ± 0.77 g/dL (P = .004) and absolute fetal hemoglobin from 3.64 ± 1.13 g/dL to 4.29 ± 1.13 g/dL (P = .003). Significant favorable changes also occurred in indices of hemolysis and red blood cell densitometry. Consistent with a noncytotoxic, differentiation altering mechanism of action, the major side effect was an asymptomatic increase in platelet counts without erythrocyte micronucleus or VDJ recombination assay evidence of genotoxicity. This study was registered at www.clinicaltrials.gov as #NCT00661726.

Lentiviral Vector-Mediated Transfer of the Gamma-Globin Gene Into Normal and Beta-Thalassemic Human CD34+ Cells Results in Potentially Therapeutic Levels of Fetal Hemoglobin in Erythroid Progeny.

Abstract 3579 Poster Board III-516 Beta-thalassemia results from severely reduced or absent expression of the beta-globin chain of hemoglobin, leading to severe anemia which is dependent upon transfusion for survival. The pathophysiology centers on the alpha- to beta-globin chain imbalance, which results in precipitation of excess alpha chains. These precipitates caused oxidative stress, membrane damage, and apoptosis of erythroid precursors in the bone marrow (BM). Allogeneic stem cell transplant can be curative but is only available to individuals with a matched donor. It is well known that increased levels of fetal hemoglobin (α2γ2;HbF), such as in the case of hereditary persistence of fetal hemoglobin, ameliorate the clinical severity of beta-thalassemia. We therefore developed a self-inactivating (SIN), erythroid-specific, gamma-globin lentiviral vector with the goal of reducing the chain imbalance through the production of HbF in red blood cells following transduction and transplantation of autologous HSCs. The vector, termed V5m3, contains 3.1-kb of transcriptional regulatory sequences from the β-globin locus control region, a 130-bp beta-globin promoter regulating transcription of the gamma-globin gene, and 3′ untranslated sequences from the native beta-globin gene. We previously showed that this vector was effective in correcting mouse models of beta-thalassemia and sickle cell disease (SCD). We further modified V5m3 to include a 400-bp chicken HS4 insulator element. Mobilized peripheral blood (PB)- or steady state bone marrow (BM)-derived CD34+ cells from normal or thalassemic human donors were used to evaluate vector performance. A two-phase in vitro model of human erythropoiesis was used in which a seed population of CD34+ cells is first expanded and then differentiated into late stage erythroblasts over a two week period. Gene transfer is performed 48 hours after initiation of culture. In this model, we routinely observe a 1000-fold expansion in total cell numbers where the vast majority of maturing erythroid cells are late stage erythroblasts reflected by nearly complete enrichment for expression of transferrin receptor (CD71; ≥98%) and glycophorin A (CD235; ≥80%) and loss or reduced expression of CD34 and CD45. Erythroid cells generated from normal adult PB and BM CD34+ cells demonstrate an adult pattern of hemoglobin production (HbA>96%;HbF<2%), as measured by acetate gel electrophoresis and HPLC, making this model ideal to evaluate enhancement of HbF levels by gene transfer. Normal PB CD34+ cells from four independent donors were transduced with the gamma-globin vector (MOI=20) or a GFP control vector (MOI=5). Vector transduction had no effect on cell growth or differentiation as monitored by consistent increases in total cell numbers and the appearance of CD71 (transferrin receptor) and glycophorin A on most cells (≥98% and '80%, respectively). The GFP vector achieved an average transduction rate of 87+/−6% and erythroblasts expressed low levels of HbF (1.7+/−0.6). Gamma globin gene transfer with the V5m3 (N=2) and V5m3-400 (N=4) vectors resulted in HbF levels ranging from 6 to 25%, with an average vector copy number of 0.8 to 1.1. We next tested the V5m3-400 vector using BM CD34+ cells from two patients with beta-thalassemia major. High levels of gene transfer were obtained with both the GFP and the globin vector, as evidenced by bulk marking (74+/−6% GFP+) or PCR analysis of CFU for presence of the V5m3-400 vector (12/12 positive Exp 1); (18/20 Exp 2); (18/24 Exp 3). Again, gene transfer did not perturb erythroid differentiation as monitored by the appearance glycophorin A (88+/−9%; GFP control and 86+/−6%; V5m3-400) as well as cell morphology. Erythroblasts derived from GFP transduced cells had a mean HbF level of 26+/−5% whereas those derived from cells transduced with V5m3-400 demonstrated a 100% increase of HbF to 58+/−2% with an average vector copy number of 0.6-0.8. Importantly, cultures of cells transduced with the globin vector demonstrated an average of 30% reduction in apoptotic cells (10% tunnel+) cells compared to GFP transduced control cells (15% tunnel+), suggesting rescue of erythroblasts through correction of the globin chain imbalance. Our data show that potentially therapeutic levels of HbF in thalassemic erythroblasts can be obtained following gene transfer using a gamma-globin lentiviral vector. Disclosures: No relevant conflicts of interest to declare.

Pathophysiology of β Thalassemia—A Guide to Molecular Therapies

The central mechanism underlying the pathophysiology of the β thalassemias can be related to the deleterious effects of imbalanced globin chain synthesis on erythroid maturation and survival. An imbalance of the α/non-α globin chains leads to an excess of unmatched α globin which precipitates out, damaging membrane structures leading to accelerated apoptosis and premature destruction of the erythroid precursors in the bone marrow (ineffective erythropoiesis). Close observation of the genotype/phenotype relationships confirms the pathophysiological mechanism and provides clues to molecular therapies, all of which aim to reduce the α/non-α chain imbalance. They include inheritance of the milder forms of β thalassemia, co-inheritance of α thalassemia, or genetic factors (quantitative trait loci, QTLs) for increasing γ globin expression. Currently, the most promising molecular therapeutic approaches include increasing β globin gene expression by stem cell gene therapy and increasing γ globin expression using pharmacological agents or by transduction of the γ globin genes.

Differential Regulatory and Compensatory Responses in Hematopoiesis/Erythropoiesis in α- and β-Globin Hemizygous Mice

Characterization of hematopoiesis/erythropoiesis in thalassemias from multipotent primitive cells to mature erythrocytes is of fundamental importance and clinical relevance. We investigated this process in α- and β-globin hemizygous mice, lacking the two adult tandemly organized genes from either the α- or β-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, β-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for α-globin hemizygous mice. In contrast, however, β-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of β-globin hemizygous mice showed more severely impaired maturation from the basophilic to orthochromatophilic erythroblasts and substantial loss of these late precursors probably as a consequence of a greater susceptibility to an excess of free α-chain than β-chain. Hence, only erythroid precursors exhibiting stochastically moderate chain imbalance would escape death and mature to reticulocyte/RBC stage, leading to survival and minimal loss of reticulocytes in the β-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in β-globin hemizygous mice, a dynamic compensatory hematopoiesis was observed at earlier differentiation stage as evidenced by a significant increase of erythroid progenitors (erythroid colony-forming units ∼100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units ∼7-fold). This early compensatory mechanism was less pronounced in α-globin hemizygous mice. The expansion of multipotent primitive and potentially stem cell populations, taken together with ineffective erythropoiesis and increased reticulocyte/RBC destruction could confer major cumulative advantage for gene targeting/bone marrow transplantation. Therefore, this study not only corroborated the strong potential effectiveness of transplantation for thalassemic hematopoietic therapy but also demonstrated the existence of a differential regulatory response for α- and β-thalassemia.

Deletion of the α-globin gene cluster as a cause of acquired α-thalassemia in myelodysplastic syndrome

Rarely, myelodysplastic syndrome (MDS) is complicated by an acquired form of α-thalassemia (α-thalassemia in myelodysplastic syndrome [ATMDS]) characterized by hypochromic, microcytic, anisopoikilocytic red blood cells with hemoglobin H (HbH) inclusions. Acquired mutations in ATRX, a chromatin remodeling gene, have recently been found in 12 patients with typical features of ATMDS, though they have not been detected in MDS patients with similar red blood cell findings but little HbH. The α-globin genes themselves have appeared normal in all ATMDS patients studied to date. Here we characterize the molecular defect in a unique MDS patient with rare HbH inclusions in which an abnormal clone lost a greater than 1.9-Mb segment of the telomeric region of the short arm of one allele of chromosome 16, including both α-globin genes. Red blood cell changes associated with this acquired somatic genotype (––/αα) are surprisingly severe, demonstrating that a minor globin chain imbalance may be unexpectedly deleterious during the abnormal erythropoiesis that occurs in the context of MDS.