Effectiveness of an information booklet regarding home based management on knowledge of caregivers of children with thalassemia

Background: Thalassemia is one of the most common inherited diseases worldwide. It is (mediterranean anemia, cooley's anemia) inborn hemolytic anemia characterized by a drop in the synthesis of hemoglobin. It produces hypochromic microcytic anemia due to imperfect hemoglobinization of RBCs, hemolysis, and futile erythropoiesis. It is due to mutations on chromosome 11 which leads to the synthesis of abnormal or absent beta globin chains. Recently there has been increasing cases of thalassemia worldwide. Hence, current study was planned to assess the knowledge of caregivers regarding homebased management of children with thalassemia.Methods: We assessed caregivers’ knowledge regarding home-based management of children with thalassemia in thalassemia day care department of pediatric KGMU Lucknow, using a self structured questionnaire. Statistical analysis was performed using SPSS software version 24.Results: The level of knowledge regarding pretest and posttest mean scores is 9.50 and 14.17 respectively. Paired t test calculated value was -9.808 which was much higher than the table value at p<0.05 level of significance.Conclusions: The study concluded that information booklet teaching was effective in improving the knowledge level of caregivers of children with thalassemia. More studies need to be done with larger sample size to empower cargivers regarding thalassemia.

Thalassemia: With the “Red” in the Bag Amid COVID-19 Reflections

This narrative piece written from a patient perspective angle who lives with a chronic condition—Beta thalassemia major (also known as Cooley’s Anemia), a serious, life-long, genetic blood disorder. The narrative portrays how the present situation of COVID-19 pandemic adds an extra layer to the challenges in the lives of people living with thalassemia.

Rescue of a Humanized Mouse Model of β-Thalassemia Major By Allogeneic Bone Marrow Transplantation in the Absence of Cytoreductive Conditioning

Introduction β-thalassemia is a heterogeneous group of inherited blood disorders marked by defects in β-globin chain production. β-thalassemia major, or Cooley's anemia (CA), is the most severe form of the disease and results in a complete absence of β-globin chains and thus lacks all the major adult hemoglobin (HbA) in erythroid cells. This results in ineffective erythropoiesis, severe anemia, and childhood death if untreated. Fortunately, allogeneic bone marrow transplantation (BMT) can cure CA patients; however, current therapy requires finding a suitable HLA-matched donor, potentially lethal myeloablative conditioning, and post-transplantation immunosuppression. Furthermore, BMT can result in severe adverse events such as death, graft rejection, infection, or graft-versus-host disease. Methods Our group has developed a humanized mouse model of CA by targeted gene replacement of the adult mouse α- and β-globin genes with human α-, γ-, and nonfunctional β0-globin genes. Newborn CA mice received anti-CD122 antibody on the second day of life and single intravenous injection of bone marrow cells on the third day of life. Animals were bled periodically to monitor donor red blood cell (RBC) chimerism by flow cytometry. After at least 5 months, animals were sacrificed for analysis of donor hematopoietic stem and progenitor (HSPC) chimerism as well as disease and treatment related pathology. Results Homozygous CA mice synthesize solely human fetal hemoglobin at birth and succumb to lethal anemia before weaning with a mean postnatal survival of two weeks. A single injection of bone marrow cells into CA pups results in stable, long-term hematopoietic chimerism that is capable of reconstituting greater than 90% of RBCs. Interestingly, at the HSPC level, donor chimerism was determined to be much lower with a range of <1% to 15%. This suggests there is a tremendous survival advantage of donor erythroid cells over those derived from the recipient. Transplanted animals are transfusion independent, fertile, and exhibit no evidenced of graft-versus-host disease. Conclusions In conclusion, we have developed a mouse model of β-thalassemia that expresses human globin chains and completes the developmental fetal-to-adult hemoglobin switch after birth. Furthermore, we have demonstrated rescue of these animals from lethal anemia by allogeneic BMT in the absence of cytoreductive conditioning. These data suggest that exploitation of the naivety of the newborn immune system provides a means to circumvent the need for toxic cyto-reductive conditioning. Based on the success of these studies, we have begun to test the hypothesis that perinatal BMT without cyto-reductive conditioning is capable of overcoming MHC-mismatch between graft and host. These studies pave the way for safer transplantation strategies in children with Cooley's anemia. Disclosures No relevant conflicts of interest to declare.

An HPFH Mutation in the γ-Globin Gene Promoter of Humanized Cooley's Anemia Mice Rescues Them from Perinatal Lethality and Renders Them Transfusion Independent

Cooley's Anemia (CA), β-thalassemia major, is a genetic disease caused by an impairment in β-globin protein synthesis. The resulting excess in α-globin chains causes the premature destruction of erythroid cells (ineffective erythropoiesis), anemia, and if left untreated, death within the first years of life. Several mutations in the promoters of the fetal γ-globin genes have been identified which impair the silencing of the fetal genes in adulthood, a condition termed hereditary persistence of fetal hemoglobin (HPFH). The amount of fetal hemoglobin (Hb F) expressed and the distribution within the RBC population (pancellular vs heterocellular), varies widely with different HPFH mutations. It has also been observed that thalassemia patients who co-inherit HPFH mutations that express higher levels of Hb F have milder disease symptoms. The purpose of this study is to determine whether the incorporation of a non-deletional HPFH mutation into the promoter of the human g-globin gene in a humanized mouse model of CA can rescue the animals from their perinatal lethality. Heterozygous humanized HPFH -175 mice express pancellular, high-levels of Hb F into adulthood. Homozygous HPFH -175 CA mice are rescued from their perinatal lethality, surviving solely on 100% human Hb F and are transfusion independent for life. This result is significant because it demonstrates that introduction of an HPFH mutation into the γ-globin gene promoter by gene editing may be a viable therapeutic option for CA patients in the future. Disclosures No relevant conflicts of interest to declare.

A Cell-Based High-Throughput Screen For Novel Inducers Of Fetal Hemoglobin For Treatment Of Sickle Cell Disease, Cooley’s Anemia and β-Thalassemias

Decades of research has established that the most effective treatment for sickle cell disease (SCD) and Cooley’s anemia is increased fetal hemoglobin (HbF). Certain β-thalassemias may also benefit from fetal hemoglobin induction. Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the most widely available treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Identification of a drug specific for inducing or reactivating γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. An assay system developed in our lab to understand the mechanisms underlying the γ- to β-globin gene expression switch during development allowed us to generate a cell-based assay that was adapted for a HTS of 121,085 compounds from the libraries of the KU-HTS Laboratory (Prestwick, MicroSource, CMLD, Chembridge and ChemDiv compound libraries) and LCGC (OCL compound library). Transgenic mice were produced using a modified 213 Kb human β-globin locus yeast artificial chromosome (β-YAC). Two gene fusions were introduced into the β-YAC via homologous recombination in the host yeast, firefly luciferase was fused to the Aγ-globin promoter and Renilla luciferase was fused to the β-globin promoter. The resultant YAC was microinjected into fertilized mouse oocytes to produce transgenic mice. We used these mice to derive chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) containing the γ-luc/β-luc β-YAC, which were employed in the HTS. We identified 232 primary screen actives that induced γ-globin 2-fold or higher. A 4-assay, 10-point dose-response secondary screen using the same CID-dependent γ-luc/β-luc β-YAC BMCs reconfirmed that 211 of these active compounds induced γ-globin ≥2-fold with minimal or no β-globin induction, minimal cytotoxicity and did not directly inhibit purified luciferase enzyme. Additional secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the characteristics of seven of these 233 hits that were cherry-picked for further analysis. Four of the compounds were particularly promising, numbers 7, 42, 87 and 208. In CID-dependent wild-type β-YAC BMCs using the optimal dose for each compound, γ-globin mRNA induction ranged from 3- to 42-fold compared to 10-fold with sodium butyrate as measured by real-time qRT-PCR; F-cells ranged from 9.9-29.9% compared to 0.7% untreated and 15.9% treated with sodium butyrate as measured by flow cytometry. In human primary erythroid progenitor cells, the mRNA change was 1.6- to 3-fold compared to 1.75-fold with sodium butyrate and F-cells ranged from 9.1-29% compared to 5.7% untreated and 39.4% treated with sodium butyrate. Lead compounds will be tested in a pre-clinical β-YAC transgenic mouse model to determine their ability to induce HbF in vivo to aid development of these compounds for future clinical applications in hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

Study of Two Subjects with Absence of Severe Anemia with Homozygous β0 Thalassemia Due to Disrupted γ-to-β Switch

Abstract 5172 Sickle cell disease (SCD) and β thalassemia due to defects of the HBB (β globin gene) are among the most common inherited genetic disorders. At birth, there is a switch of γ globin transcription to β and d, with replacement of HbF by HbA and HbA2 virtually completed by six months of age. At that time, serious inherited disorders of the β gene, such as sickle cell disease and Cooley's anemia (homozygosity for β0 thalassemia mutations), become clinically apparent. Cooley's anemia is a life-threatening disorder wherein, in most patients, chronic transfusions or bone marrow transplantations are needed to sustain life. Rare patients with homozygosity or compound heterozygosity for β0 have no or only mild anemia. These patients maintain a high level of γ globin synthesis, apparently from a disrupted γ-to-β switch, thus attenuating their disease state. Recent work has demonstrated that BCL11A plays an important role in the suppression of γ-globin expression, as do polymorphisms of the gene that remain to be fully elucidated at a functional level. We recruited two unrelated subjects with homozygous β0 thalassemia mutations with no or only mild anemia (Patient #1, IVS2+1 G>A; Hb 14.2 Gm%; 97.2% HbF, 2.8% HbA2, Patient #2, IVS 2 G-T; Hb 11.2 Gm%; 92/5% HbF, 6.8% HbA2, 0.7% HbA). We sequenced transcripts and genomic loci of BCL11A from these patients. No mutations or splicing variants on transcripts were found. However, when the ≂f102 kb of genomic material from patient #1 was sequenced, 5 single nucleotide changes at intron II were found (2 known and 3 previously unpublished), while no genomic changes were found in patient #2. We then performed in vitro erythroid expansion from peripheral blood utilizing high erythropoietin concentrations and analyzed the cell proliferation and expression of globin and BCL11A genes. Interestingly, detectable amounts of β-globin transcripts were present in both patients during expansion, although protein levels were not detectable by the conventional HPLC method, probably due to limited sensitivity of this assay. Patient #1 showed mild in vitro induction of β-globin expression, which is lower than the control group, but no apparent cell proliferation. Patient #2 showed no induction of β-globin expression and hyperproliferation at a later stage of expansion (See Figure); however, the levels of BCL11A and γ-globin transcripts were indistinguishable from controls. Although we were unable to detect any abnormality of the BCL11A transcript as a cause of high fetal hemoglobin expression in these patients, we cannot rule out the possibility that the intronic mutations in patient #1 may interfere with BCL11A gene translation, perhaps by interference with non-coding RNA. The potential molecular mechanism of γ-to-β switch is being explored by gene expression profiling and microRNA analyses. Disclosures: No relevant conflicts of interest to declare.