Hemoglobin Beta Gene Research Articles

Iron Restricted Erythropoiesis Under Hepcidin Mimetic Treatment (PN23114) Improved Disease Parameters in a Mouse Model for Sickle Cell Disease

Sickle Cell Disease (SCD) is an inherited hemolytic disorder that results from a single point mutation in the hemoglobin-beta (HBB) gene that encodes for the beta-globin subunit of hemoglobin, causing the production of abnormal hemoglobin S (HbS). HbS polymerizes under low oxygen conditions, causing sickling effects on red blood cells (RBCs). Sickling of RBCs initiates vaso-occlusion, downstream inflammatory responses, and hemolysis (Zhang D, et al., Blood. 2016;127:801-9). Previous studies have shown that polymerization under low oxygen conditions is extremely sensitive to HbS concentrations within the RBCs (Hofrichter J, et al, Proc Natl Acad Sci USA. 1974;71:4864-8; Sunshine HR, et al. Nature. 1978;275:238-40). Controlling iron delivery for erythropoiesis provides a mechanism to potentially down-modulate the HbS concentration, thereby reducing the potential for HbS polymerization and sickling of RBCs (Parrow NL, et al., Blood. 2021;137:1553-5). The potent hepcidin mimetic peptide PN23114 targets the iron exporter membrane protein ferroportin to trigger its degradation, thus preventing iron export from cells. We hypothesize that the resulting pharmacodynamic effect of limiting iron availability for erythropoiesis should reduce HbS concentrations within the RBCs that may prevent sickling and hemolysis. Townes mice (genetically altered to contain the human SCD HbSS homozygous mutation) are a well-established experimental model for human SCD (Li-Chen W, et al., Blood. 2006;108:1183-8). They exhibit severe hemolysis as indicated by elevated bilirubin and lactate dehydrogenase (LDH) levels, and a shortened RBC half-life compared to wild type (WT) mice. These mice also develop vaso-occlusive events and elevated inflammatory responses (for example, increases in peripheral blood white blood cell [WBC] and neutrophil counts). To test whether iron restriction mediated by a hepcidin mimetic may alter outcomes in this model, PN23114 was administered subcutaneously to Townes HbSS mice. Mice were treated over 4-weeks (3 times weekly) with vehicle or PN23114 at 1 or 2.5 mg/kg (vehicle treated WT mice served as controls). At the end of 4 weeks, total bilirubin was elevated in the HbSS-vehicle group compared to WT-vehicle (Figure 1A). Total bilirubin was dose-dependently reduced by PN23114 treatment compared to the HbSS-vehicle group (Figure 1A). LDH was also elevated in the HbSS-vehicle group and was dose-dependently reduced with PN23114 treatment (557±105 units/L in WT-vehicle, 809±145 units/L in HbSS-vehicle, 611±341 units/L in HbSS-PN23114-1mg/kg and 372±71 units/L in HbSS-PN23114-2.5mg/kg groups). Peripheral blood WBC counts were dose-dependently reduced by PN23114 treatment compared to HbSS-vehicle (Figure 1B), with concomitant decreases in peripheral blood neutrophil counts. Reduction in hemoglobin values was observed with PN23114 treatment in HbSS (5.9±0.9 g/dL in HbSS-1mg/kg group and 4.4±0.4 g/dL in HbSS-2.5 mg/kg group) compared to the HbSS-vehicle group (7.1±0.3 g/dL). These results suggest that pharmacologic iron restriction with PN23114, a hepcidin mimetic peptide, led to a reduction in hemolysis and inflammatory markers in a SCD model. Therefore, a hepcidin mimetic therapeutic could potentially be a novel approach to prevent hemolysis, inflammation and downstream clinical complications associated with human SCD.

Molecular characterization of HBB gene mutations in beta-thalassemia patients of Southern Iraq

Introduction and Aim: Beta-thalassemia is a serious inherited genetic disorder and an increasing health burden globally. Beta -thalassemia is caused by genetic globin abnormalities within the hemoglobin beta (HBB) gene. This study aimed to characterize the HBB gene mutations in beta -thalassemia among southern Iraqi patients.
 
 Materials and Methods: The study included 30 beta -thalassemia patients referred to the Thi-Qar Center for Genetic Diseases, Iraq and 15 control samples from a random group of apparently healthy individuals. Genomic DNA was isolated from blood sample collected from each individual. The DNA was amplified for specific regions of the HBB gene and the amplified products sequenced. The sequences generated were analysed for mutations using sequence analysis tools.
 
 Results: Molecular analysis revealed several mutations in the HBB gene including translocation, deletion and substitution mutations in the population tested positive for the beta -thalassemia trait.
 
 Conclusion: Thalassemia major is a serious concern in southern Iraq and therefore this study emphasizes a need for complete mutation profiling of the beta -globin gene as a strategy for screening of carriers within the population. Such examinations could be useful in pre-marital genetic counseling and for undertaking prevention and treatment measures.

Intraosseous injection of SMNP vectors enables CRISPR/Cas9-mediated knock-in of HBB gene into hematopoietic stem and progenitor cells

β-Hemoglobinopathies are hereditary diseases originating from mutations in the hemoglobin beta (HBB) gene. Allogeneic transplantation of hematopoietic stem cells (HSCs) is the only approved treatment to date providing a cure, which is limited due to low histocompatibility from potential donors. Although several clinical trials are introducing the functional HBB gene into autologous HSCs, the ex vivo viral transfection method raises safety concerns for clinical translation. Here, we reported a novel strategy that features direct bone marrow delivery of a CRISPR/Cas9-mediated knock-in system that integrates a copy of the functional HBB gene to the genome by supramolecular nanoparticle (SMNP) nonviral vectors. We demonstrated the feasibility of SMNP vectors for the delivery of the knock-in system. First, the biodistribution of SMNPs was monitored after intraosseous (i.o.) injection in a mouse model. Second, the in vitro CRISPR/Cas9-mediated knock-in of the HBB/GFP gene via SMNP vectors was evaluated in mouse stem cells. Finally, in vivo CRISPR/Cas9-mediated knock-in of mouse hematopoietic stem and progenitor cells (HSPCs) was performed by i.o. injection of SMNP vectors into mouse bone marrow. We believe this strategy presents a unique and innovative approach to treating β-hemoglobinopathies.

Modelling the Applications of CRISPR‐Cas9 for Treating Sickle Cell Disease

Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR‐associated protein 9 (CRISPR‐Cas9) is a genome editing tool that can remove, modify, or add sections of DNA. It is made up of two main molecules: the Cas9 protein and the guide‐RNA (gRNA), which is used to match a target gene to the CRISPR‐Cas9 molecule. The desired sequence of DNA must be between 2 and 5 nucleotides and must be followed by the protospacer adjacent motif (PAM) at the 3’ end of the gRNA. This enables the target gene to be found and cut by the Cas9 endonuclease. Once cut, the DNA can be repaired using non‐homologous end joining (where a random insertion or deletion of DNA occurs) or homology directed repair (where a homologous piece of DNA is used as a template to repair the DNA). The latter is used for genome editing, as the target sequence can be changed with the CRISPR‐Cas9 system.Sickle Cell Disease (SCD) is an autosomal recessive hereditary disorder that affects erythrocytes and is caused by a single base substitution of adenine to thymine on the hemoglobin beta (HbB) gene. Currently two different groups are examining two different techniques to treat SCD. One group has shown that by deleting the BCL11A gene, fetal hemoglobin production can occur, which greatly diminishes the amount of Sickle hemoglobin (HbS). An example of this application of CRISPR‐Cas9 for treating SCD is the story of Victoria Gray, the first person to have received genome editing treatment for SCD.

Acute soft head syndrome in sickle anemia: The first case report in Kano

Sickle cell anemia is an autosomal recessive disorder resulting from insertion of the valine codon at position sixth of beta-hemoglobin gene instead of glutamate leading to the production of insoluble beta-hemoglobin chain. Although sickle anemia is mostly associated with bone pain and anemia, other manifestations also occur. Acute head syndrome is an uncommon complication resulting from skull bone infarction, cortical osteopenia, and rupture of tortuous periosteal blood vessels leading to extravasation of blood into the subgaleal space. This report therefore intends to create awareness that nontraumatic scalp swelling is an uncommon complication in sickle cell disease and no traditional practice or surgical excision should be employed in managing it.

Investigating the prolongation of blood transfusion frequency in thalassemic children by omega 3 fatty acid and protein supplementations through milk

<p class="abstract"><strong>Background:</strong> Thalassemia is an inherited genetic hemoglobin disorder wherein, afflicted child is born when both parents are carriers for defective alpha or beta hemoglobin gene. The thalassemias are the most common genetic disorder on a worldwide basis. The requirement of frequent blood transfusions in these patients pose a substantial burden on the health care system.</p><p class="abstract"><strong>Methods:</strong> A prospective observational study was conducted across 6 months (July 2018-December 2018) in a tertiary care hospital, Pune. The present study included 30 registered patient & their past 6 months record of blood transfusion (esp frequency), previous hemoglobin levels, height & weight. All these parameters were compared 6 months after supplementation with Omega 3 fatty Acids & proteins.</p><p class="abstract"><strong>Results:</strong> Total 16 of 28 patient showed that the average durations between two blood transfusions was increased by minimum 01 day to a maximum of 5 days. The average number of blood bag required was less than required blood bags in the period of pre supplementation. 20 of 28 patients showed a rise in hemoglobin level from a range of 0.5 to 1.2 gm/dl. </p><p class="abstract"><strong>Conclusions: </strong>In a country like India, with the high frequency of hemoglobinopathies, causing increased burden on the society, it is necessary to control the incidence by effective steps. Low cost and easily administered supplementation by omega 3 fatty acid and proteins may reduce the requirement of repeated blood transfusion along with increase in hemoglobin level. Implementation of carrier screening program offering genetic counseling and prenatal diagnosis followed by selective termination of affected cases would help in preventing the disease.</p>

Delaying Blood Transfusion in Thalassemic Children with Supplementation of Omega 3 Fatty Acids and Protein: A Novel Strategy

Background: Thalassemia is an inherited genetic hemoglobin disorder wherein, afflicted child is born when both parents are carriers for defective alpha or beta hemoglobin gene. The thalassemias are the most common genetic disorder on a worldwide basis. The requirement of frequent blood transfusions in these patients pose a substantial burden on the health care system. Methods: A prospective observational study was conducted across 6 months (July 2018-December 2018) in a tertiary care hospital, Pune. The present study included 30 registered patient and their past 6 months record of blood transfusion (esp frequency), previous hemoglobin levels, height and weight. All these parameters were compared 6 months after supplementation with omega 3 fatty acids and proteins. Results: 16 of 28 patient showed that the average durations between two blood transfusions was increased by minimum 01 day to a maximum of 5 days. The average number of blood bag required was less than required blood bags in the period of pre supplementation. 20 of 28 patients showed a rise in hemoglobin level from a range of 0.5 to 1.2 gm/dl. Conclusion: In a country like India, with the high frequency of hemoglobinopathies, causing increased burden on the society, it is necessary to control the incidence by effective steps. Low cost and easily administered supplementation by omega 3 fatty acid and proteins may reduce the requirement of repeated blood transfusion along with increase in hemoglobin level.

Increased Adhesiveness of Red Blood Cells from Sickle Cell Trait Carriers after Exposure to Daunirubicin Breast Cancer Chemotherapeutic Agent

Introduction: African-Americans (AA) with breast cancer continue to experience worse morbidity and mortality than white patients. In addition to barriers to care, an active area of investigation has been the etiology of the increased risk adverse events (AEs) related to chemotherapy. An emerging question is if sickle cell trait (i.e., heterozygotes for hemoglobin beta gene), present in an estimated 1 in 12 of the AA population, confers increased susceptibility to chemotherapy-related complications. We posit that conditions of high physiological stress might be manifest during systemic treatment with toxic agents resulting in alterations of red blood cells (RBCs). Using an experimental biomechanical model system, we hypothesized that RBCs from SCT carriers would be prone to increased adhesiveness after exposure to a common systemic anti-cancer agent used in breast cancer. Increased adhesiveness of RBCs can be a precipitating factor of blocked vasculature and subsequent sickling crises, potentially leading to AEs during treatment. Methods: Our study testedex vivoRBCs from two groups of healthy female participants: 20 African-American sickle cell trait carriers (AA-SCT); and 15 white subjects with wild-type hemoglobin (W-WT). Isolated RBCs were treated with scaled Daunorubicin (DNR) doses. The unbinding forces between αvβ3 ligands and intercellular adhesion molecule-4 (ICAM-4) receptors are reported using the frequency distribution, which states the percentage of events whose unbinding forces are within each width of the bin. We then compared the median values of the forces measured in experiments without and with treatment for each drug. The collective frequency (CF%) is related to the population of active ICAM-4 receptors and is defined as the percentage of all unbinding events divided by the total number of measurements, which is 32 × 32 =1024 for each cell multiplied by the number of tested cells for each blood sample. Results: For AA-SCT RBCs, pre-treatment baseline CF of active ICAM-4 receptors was 4.88 ± 0.87%, which was significantly increased after administration of DNR (13.13 ± 2.30%, p<0.0001). In contrast, treatment of W-WT RBCs held the CF of active ICAM-4 receptors at a comparable level as untreated RBCs (9.54 ± 1.48%; 7.47 ± 1.07%, p=0.50, respectively). Conclusion: Our findings could support hypotheses for adhesion-related RBC clumping among AAs with SCT during systemic treatment with anti-neoplastic agents, and, putatively, resultant AEs. Given past studies showing African-American women with breast cancer have higher rates of self-withdrawal from treatment, further exploration with additional systemic agents are warranted. Our novel study is limited by a small sample size as well lack of potential confounding factors in analyses. Future studies are planned with additional agent(s) and AAs without SCT. Should it be confirmed that SCT carrier status predicts RBC alterations during systemic treatment for breast cancer, and are linked to subsequent adverse events, it could lead to precision treatment planning. Disclosures Andemariam: Novartis:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;Hemanext:Membership on an entity's Board of Directors or advisory committees;Global Blood Therapeutics:Consultancy, Membership on an entity's Board of Directors or advisory committees;Vertex:Honoraria;Imara:Research Funding;Emmaus:Membership on an entity's Board of Directors or advisory committees;bluebird bio:Consultancy, Membership on an entity's Board of Directors or advisory committees;NovoNordisk:Consultancy, Membership on an entity's Board of Directors or advisory committees;CRISPR/Vertex:Consultancy, Membership on an entity's Board of Directors or advisory committees;CHNCT:Consultancy;Accordant:Membership on an entity's Board of Directors or advisory committees;Guidepoint:Honoraria;Sanofi Genzyme:Consultancy, Membership on an entity's Board of Directors or advisory committees;Terumo BCT:Consultancy, Membership on an entity's Board of Directors or advisory committees;Roche:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;Cyclerion:Consultancy, Membership on an entity's Board of Directors or advisory committees;Pfizer:Consultancy, Membership on an entity's Board of Directors or advisory committees.

Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies.

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate-mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)-encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

CRISPR-mediated gene modification of hematopoietic stem cells with beta-thalassemia IVS-1-110 mutation

Backgroundβ-Thalassemias represent a group of genetic disorders caused by human hemoglobin beta (HBB) gene mutations. The radical curative approach is to correct the mutations causing the disease. CRISPR-CAS9 is a novel gene-editing technology that can be used auspiciously for the treatment of these disorders. The study aimed to investigate the utility of CRISPR-CAS9 for gene modification of hematopoietic stem cells in β-thalassemia with IVS-1-110 mutation.Methods and resultsWe successfully isolated CD34+ cells from peripheral blood of β-thalassemia patients with IVS-1-110 mutation. The cells were transfected with Cas9 endonuclease together with guide RNA to create double-strand breaks and knock out the mutation. The mutation-corrected CD34+ cells were subjected to erythroid differentiation by culturing in complete media containing erythropoietin.ConclusionCRISPR/Cas-9 is an effective tool for gene therapy that will broaden the spectrum of therapy and potentially improve the outcomes of β-thalassemia.

The Safety of Chelators for Iron Overload in Sickle Cell Disease: A Brief Systematic Review

Sickle cell disease (SCD) is a group of disorders that affects hemoglobin due to a mutation of the hemoglobin beta gene (HBB) on chromosome 11. Patients with SCD have atypical hemoglobin molecules called hemoglobinS (HbS), which distort erythrocytes into a “sickle-shape”. Typical symptoms of SCD include periodic episodes of pain, repeated infections, and anemia. This disorder is abundant in sub-Saharan African countries, the Mediterranean region, and also appears in some southern provinces in Turkey. Because of the high concentration of HbS in patients, a high risk of chronic anemia and vaso-occlusive events, such as stroke may deteriorate suddenly. In these conditions, transfusion of blood, especially erythrocytes, can be life-saving. However, chronic blood transfusions may lead to iron overload in SCD patients. Erythrocyte transfusion is associated with a higher risk in most patients with SCD than in the general population. Therefore, chelation therapy has become an important component of the transfusion program to prevent complications of iron accumulation in organs such as liver and heart. In this study, we sought to conduct a systematic review to assess the safety of iron chelating agents used by SCD patients with iron overload mainly due to necessary blood transfusion regime. Our evaluation revealed that in general iron chelation therapy, either deferasirox, deferoxamine or deferiprone, remains the most effective and safest available method to treat iron overload in SCD. Furthermore, current reports do not reflect any significant safety concerns against the use of available chelators.

Fine-Scale Characterization of Genomic Structural Variation in the Human Genome Reveals Adaptive and Biomedically Relevant Hotspots.

Genomic structural variants (SVs) are distributed nonrandomly across the human genome. The “hotspots” of SVs have been implicated in evolutionary innovations, as well as medical conditions. However, the evolutionary and biomedical features of these hotspots remain incompletely understood. Here, we analyzed data from 2,504 genomes to construct a refined map of 1,148 SV hotspots in human genomes. We confirmed that segmental duplication–related nonallelic homologous recombination is an important mechanistic driver of SV hotspot formation. However, to our surprise, we also found that a majority of SVs in hotspots do not form through such recombination-based mechanisms, suggesting diverse mechanistic and selective forces shaping hotspots. Indeed, our evolutionary analyses showed that the majority of SV hotspots are within gene-poor regions and evolve under relaxed negative selection or neutrality. However, we still found a small subset of SV hotspots harboring genes that are enriched for anthropologically crucial functions and evolve under geography-specific and balancing adaptive forces. These include two independent hotspots on different chromosomes affecting alpha and beta hemoglobin gene clusters. Biomedically, we found that the SV hotspots coincide with breakpoints of clinically relevant, large de novo SVs, significantly more often than genome-wide expectations. For example, we showed that the breakpoints of multiple large SVs, which lead to idiopathic short stature, coincide with SV hotspots. Therefore, the mutational instability in SV hotpots likely enables chromosomal breaks that lead to pathogenic structural variation formations. Overall, our study contributes to a better understanding of the mutational and adaptive landscape of the genome.

Dilated Cardiopathy Associated with Sickle Cell Disease in a 68 Years Old Female: An Emerging Complication in Sub-sahara Africa

Sickle Cell Anemia (SCA) is an autosomal recessive disease caused by a point mutation in the hemoglobin beta gene found on chromosome 11p15.5 [1]. Specifically, it occurs when a single base from A to T in the codon for glutamic acid at position 6 is changed to valine of the beta globin and thus disrupts the tertiary structure and stability of the hemoglobin molecule [2]. Sickle hemoglobin is responsible for wide spectrum of disorders which vary with respect to severity of anemia, frequency of crises and duration of survival [3]. We present the case of a dilated cardiopathy in an elderly female sickle cell patient. A 68 years old female sickle cell patient with no known major cardiovascular risk factor presented with progressive onset of dyspnea. Clinical examination showed signs of left ventricular failure; an electrocardiogram showed a sinus regular rhythm, left ventricular hypertrophy with systolic overload, Q waves in the anteroseptal leads. Cardiac ultrasound showed a dilated cardiopathy with a preserved left ventricular systolic function at 57%, normal regional wall motion and normal pulmonary pressure. We concluded of heart failure due to probable chronic anemia or anischemic cardiopathy with a conserved systolic function in an elderly sickle cell patient but we were not able to confirm the main etiology without CT Coronary Angiogram or coronarography. Sickle cell anemia is a common genetic condition in sub-Saharan Africa associated with early death. This case is special because we have an elderly female presenting with heart failure on a dilated cardiopathy. This enhances the necessity of strict cardiovascular follow up of Sickle cell patient.

Differences in Protein S, Protein C, and Antithrombin Levels and Risk of Venous Thromboembolism Among American Black and White Enrollees in the Gate Study

Introduction: Black Americans have been reported to have a higher incidence of venous thromboembolism (VTE) compared to white Americans. The underlying cause of this disparity is not well characterized. Protein S, protein C, and antithrombin deficiency are known risk factors for VTE. There is inconsistent data regarding racial variation on the effect of protein S, protein C, and antithrombin levels and/or deficiency and risk of VTE. Most prior studies have not included a large black study population. The aim of this investigation is to compare levels of protein C, protein S, and antithrombin between VTE cases and controls by race among enrollees in the Genetic Attributes and Thrombosis Epidemiology (GATE) study, accounting for genetic differences such as hemoglobin genotype that may contribute to differences in protein levels by race.Methods: Testing for protein S, protein C, and antithrombin deficiency was performed on a subset of participants enrolled in the GATE study, a case-control study comprised of VTE cases and controls frequency-matched on age, sex, and race (self-identified). Controls had no history of VTE and did not receive anticoagulant therapy. Antigen and activity levels in cases were measured at least one month after completion of anticoagulation therapy. A priori cutoff levels for determining deficiency status were standardized per Centers for Disease Control Hemostasis Laboratory reference ranges. Beta hemoglobin genotype was determined by either a multiplex genotyping assay or sequencing of the beta hemoglobin gene. Differences in protein levels between cases and controls were compared using the Wilcoxon rank sum test. Conditional logistic regression analysis was performed to evaluate the association between protein S, protein C, or antithrombin deficiency and VTE. Crude models were conditioned on the matching factors, and adjusted models were conditioned on the matching factors and controlled for patient characteristics found to be associated with VTE and protein S, protein C, or antithrombin deficiency. Potential effect modification was assessed using likelihood ratio tests.Results: Among 2,454 subjects enrolled in the GATE study, 1,436 subjects (63.6%) had samples available for analysis. Protein S activity and total protein S antigen levels were significantly lower among VTE cases compared to controls in both white and black participants; however, antithrombin levels were higher among VTE cases compared to controls in white participants only. (Table 1) The prevalence of protein S and protein C deficiency was higher among VTE cases compared to controls in both white and black participants in the GATE study (Table 2). The prevalence of antithrombin deficiency was similar between VTE cases compared to controls. Adjusting for the effect of family history of VTE, history of cancer, and hemoglobin genotype, the effect of protein S and protein C deficiency on odds of VTE was stronger among black participants compared to white participants (Table 2).Conclusions: The effects of protein S and protein C deficiency on the odds of being a VTE case were higher among black participants in the GATE study, controlling for confounding variables including hemoglobin genotype. While these results indicate protein S and protein C deficiency may be important contributors to disparities in VTE occurrence, additional investigations using a larger study populations and different study designs are warranted. [Display omitted] DisclosuresPatel:Daiichi Sankyo: Other: DSMB member. Payne:Bioverativ: Other: treatment product donation; Novo Nordisk: Other: treatment product donation; Shire: Other: treatment product donation; Genentech: Membership on an entity's Board of Directors or advisory committees; Bayer: Other: treatment product donation.

A comprehensive review of the prevalence of beta globin gene variations and the co-inheritance of related gene variants in Saudi Arabians with beta-thalassemia.

Beta-thalassemia is a genetic disorder that is caused by variations in the beta-hemoglobin (HBB) gene. Saudi Arabia is among the countries most affected by beta-thalassemia, and this is particularly problematic in the Eastern regions. This review article is an attempt to compile all the reported mutations to facilitate further national-level studies to prepare a Saudi repository of HBB gene variations. In Saudi Arabians, IVSI-5 (G>C) and Cd 39 (C>T) are the most prevalent HBB gene variations out of 42 variations. The coinheritance of HBB gene variations with ATRX, HBA1, HBA2, HBA12, AHSP, and KLF1 gene variations were observed to be common in the Saudi population. National surveys on the molecular nature of hemoglobinopathies should be set up through collaborations between research centers from various regions to create a well-documented molecular data bank. This data bank can be used to develop a premarital screening program and lead to the best treatment and prevention strategies for beta-thalassemia.

One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system

BackgroundThalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia.MethodsWe used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression.ResultsThe hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein.ConclusionsOur study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future.

Analysis of a mistake occurring during prenatal diagnosis of two couples respectively carrying CD41-42 (-TCTT) and CD43(G>T) mutations of the beta hemoglobin gene

To provide prenatal diagnosis for two couples who respectively carried heterozygous CD41-42 (-TCTT) and CD43 (G>T) mutations of the beta hemoglobin gene. The mutations were simultaneously detected with reverse dot blot (two diagnostic kits), multi-color melting curve analysis and sequencing analysis. The fetus of family 1 was shown to be heterozygous for CD43 (G>T) by the three methods, while the fetus of family 2 was shown to be double heterozygous for CD41-42 (-TCTT) and CD43 (G>T) by multi-color melting curve analysis and sequencing analysis. The two diagnostic kits yielded different results by reverse dot blot, one as double heterozygous for CD41-42 (-TCTT) and CD43 (G>T), and another as homozygous for CD41-42 (-TCTT). For prenatal diagnosis of couples carrying mutations of beta hemoglobin gene such as CD41-42 (-TCTT) and CD43 (G>T), other methods such as Sanger sequencing should be used in order to avoid misdiagnosis.

Traceability and distribution of Neisseria meningitidis DNA in archived post mortem tissue samples from patients with systemic meningococcal disease

BackgroundThe pathophysiology and outcome of meningococcal septic shock is closely associated with the plasma level of N. meningitidis lipopolysaccharides (LPS, endotoxin) and the circulating level of meningococcal DNA. The aim of the present study was to quantify the number of N. meningitidis in different formalin-fixed, paraffin-embedded (FFPE) tissue samples and fresh frozen (FF) tissue samples from patients with systemic meningococcal disease (SMD), to explore the distribution of N. meningitidis in the body.MethodsDNA in FFPE and FF tissue samples from heart, lungs, liver, kidneys, spleen and brain from patients with meningococcal shock and controls (lethal pneumococcal infection) stored at variable times, were isolated. The bacterial load of N. meningitidis DNA was analyzed using quantitative real-time PCR (qPCR) and primers for the capsule transport A (ctrA) gene (1 copy per N. meningitidis DNA). The human beta-hemoglobin (HBB) gene was quantified to evaluate effect of the storage times (2-28 years) and storage method in archived tissue.ResultsN. meningitidis DNA was detected in FFPE and FF tissue samples from heart, lung, liver, kidney, and spleen in all patients with severe shock. In FFPE brain, N. meningitidis DNA was only detected in the patient with the highest concentration of LPS in the blood at admission to hospital. The highest levels of N. meningitidis DNA were found in heart tissue (median value 3.6 × 107 copies N. meningitidis DNA/μg human DNA) and lung tissue (median value 3.1 × 107 copies N. meningitidis DNA/μg human DNA) in all five patients. N. meningitidis DNA was not detectable in any of the tissue samples from two patients with clinical meningitis and the controls (pneumococcal infection). The quantity of HBB declined over time in FFPE tissue stored at room temperature, suggesting degradation of DNA.ConclusionsHigh levels of N. meningitidis DNA were detected in the different tissue samples from meningococcal shock patients, particularly in the heart and lungs suggesting seeding and major proliferation of meningococci in these organs during the development of shock, probably contributing to the multiple organ failure. The age of archived tissue samples appear to have an impact on the amount of quantifiable N. meningitidis DNA.

Quantitative assessment of timing, efficiency, specificity and genetic mosaicism of CRISPR/Cas9-mediated gene editing of hemoglobin beta gene in rhesus monkey embryos.

Gene editing technologies offer new options for developing novel biomedical research models and for gene and stem cell based therapies. However, applications in many species demand high efficiencies, specificity, and a thorough understanding of likely editing outcomes. To date, overall efficiencies, rates of off-targeting and degree of genetic mosaicism have not been well-characterized for most species, limiting our ability to optimize methods. As a model gene for measuring these parameters of the CRISPR/Cas9 application in a primate species (rhesus monkey), we selected the β-hemoglobin gene (HBB), which also has high relevance to the potential application of gene editing and stem-cell technologies for treating human disease. Our data demonstrate an ability to achieve a high efficiency of gene editing in rhesus monkey zygotes, with no detected off-target effects at selected off-target loci. Considerable genetic mosaicism and variation in the fraction of embryonic cells bearing targeted alleles are observed, and the timing of editing events is revealed using a new model. The uses of Cas9-WT protein combined with optimized concentrations of sgRNAs are two likely areas for further refinement to enhance efficiency while limiting unfavorable outcomes that can be exceedingly costly for application of gene editing in primate species.

Upregulation of Renal Iron Metabolism in Sickle Cell Disease Mice

BACKGROUND: Hemolysis and frequent blood transfusions lead to the iron overload and organ iron accumulation in patients with red blood cells disorders. The pattern of iron accumulation within different organs is disease specific. Abnormalities of renal iron metabolism and cortical iron deposition is characteristic for sickle cell disease (SCD) but not for β-thalassemia. Renal iron deposition does not correlate with iron overload and blood transfusion. Iron is reabsorbed from primary urine in the renal proximal epithelial cells and released into the renal intersitium by ferroportin. Iron-regulating hormone, hepcidin controls ferroportin expression. Binding of hepcidin to the ferroportin induces ferroportin degradation and intracellular iron accumulation. Low concentrations of circulating hepcidin are common in SCD patients and do not explain paradoxical renal iron accumulation. SCD mice accumulate iron in the epithelial cells of proximal tubules and may be a suitable model to study iron metabolism in SCD.OBJECTIVES: To characterize proteins of the renal iron metabolism in SCD mouse model.METHODS: The SCD (Townes) mice do not express mouse α- or β-globin alleles, but carry two copies of a human α1-globin gene and two copies of a human Aγ-globin and βS-globin genes. These animals synthesize approximately 94% human sickle (HbS) and 6% human fetal hemoglobin (HbF), and no murine hemoglobin. Control animals carry two copies of the human α1-globin gene and two copies of the human hemoglobin gamma (Aγ) gene and the human wildtype hemoglobin beta (βA) gene. Kidneys were collected from 5 months old SCD and control mice. Renal cortex was used for RNA and protein isolation. Levels of renal hepcidin, ferroportin, transferrin receptor (TFR1), divalent cation receptor (DMT1), ferritin and hepheastin were determined by q-RT-PCR, WB and ELISA. Paraffin-embedded sections were used for immunostaining. Perl’s Prussian blue staining was used for detection of renal iron accumulation.RESULTS:We detected significant accumulation of iron in the epithelial cells of proximal tubules in SCD mice. Expression of renal hepcidin was increased in SCD mice compared to controls. Surprisingly mRNA levels of all other proteins involved in renal iron metabolism (ferroportin, TFR1, DMT1, ferritin and hephaestin) were decreased in SCD mice kidney. In contrast, we found increased protein levels of transferrin receptor (iron importer), ferritin (iron storage protein) and slightly increased level of ferroportin (iron exporter). We also observed significant renal macrophages infiltration in SCD mice.CONCLUSIONS: Increased levels of renal hepcidin expression in SCD mice may be associated with renal inflammation. Higher levels of locally expressed hepcidin may lead to the partial degradation of the iron exporter (ferroportin). Increased levels of iron importers (TFR1 and DMT1) and no significant change in ferroportin expression can cumulatively saturate iron storage in ferritin and lead to the accumulation of intracellular iron.ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. DisclosuresQuezado:IONIS Pharmaceuticals: Research Funding.