Β-globin Chain Research Articles

Impaired physical ability in patients with transfusion-dependent β-thalassaemia: Can regular physical activity be a countermeasure?

Transfusion-dependent β-thalassaemia (TDβT) is a genetic disorder characterised by reduced or absent β-globin chain synthesis, resulting in chronic anaemia. Treatment consists of regular blood transfusions and chelation therapy to limit iron overload and its negative effects on organs (e.g. heart, lungs and liver). Despite improved life expectancy with transfusions and chelation therapy, TDβT patients experience poor quality of life and impaired exercise capacity (mainly due to muscle deconditioning and reduced cardiac inotropism). The aims of this review are (i) to discuss the role of organ dysfunction in the impairment of exercise capacity and reduced quality of life of TDβT patients; and (ii) to discuss physical activity as a potential therapeutic strategy to alleviate the multifactorial impact of the disease.

Deciphering the Impact of miR-92a-3p on Controlling BCL11A Gene Expression and Its Implication in Easing Thalassemia Symptoms

Deciphering the Impact of miR-92a-3p on Controlling BCL11A Gene Expression and Its Implication in Easing Thalassemia Symptoms

7568 Sickle Cell Variant Promotes Glycemic Dysregulation In A Mouse Model Of Human Sickle Cell Trait And Sickle Cell Disease

Abstract Disclosure: J.L. Zapater: None. B.T. Layden: None. Since cases of type 2 diabetes mellitus (T2DM) are rising due to environmental and societal factors, it is estimated that in the coming decades that T2DM will affect twenty percent of the United States population and that mortality from diabetic complications will continue to rise. It is therefore critical to accurately diagnose and treat T2DM early, however this proves difficult in certain populations. Sickle cell anemia is a common hemoglobinopathy in which changes in red blood cell turnover lead to inaccurate measurement of hemoglobin A1c and incongruencies between this and other glucose measures, making it difficult to establish a diagnosis of T2DM. The sickle cell variant has been linked to higher hemoglobin A1c, and with 1-in-10 African Americans having the sickle cell trait (SCT), an increasing prevalence of sickle cell disease (SCD), and a higher risk of diabetic complications in those with SCT or SCD, it is critical that we elucidate the precise mechanisms by which the sickle cell variant affects glycemic homeostasis. In this work, we utilized a mouse model of human sickle cell anemia to begin to determine the biochemical mechanisms governing glucose handling in the presence of the sickle cell variant by examining glycemic homeostasis under normal and obesogenic conditions. Townes sickle cell mice containing human hemoglobin with either two normal β-globin chains (littermate controls) or one normal and one sickle β-globin chain (SCT) were placed on either a normal chow diet or on a high fat diet (HFD) until 20 weeks of age to induce an obesogenic state. Although no overt glycemic phenotype was noted, 20-week-old HFD-fed SCT mice exhibited an increased glucose excursion following an oral glucose load compared to littermate controls. Though serum insulin levels were comparable, HFD-fed SCT mice also exhibited a reduced whole-body insulin sensitivity during insulin tolerance testing compared to controls, correlating with the noted increased glycemic excursion. With these findings, we have initiated work with Townes mice containing two normal α-chains and two sickle β-chains (SCD), which is found in human sickle cell anemia. Early results show that chow-fed SCD mice exhibit greater random and fasting glucose levels at 8 and 12 weeks of age, despite similar fasting insulin levels. These findings suggest that the presence of one sickle β-globin chain, as in SCT, promotes hyperglycemia in the obesogenic state through reduction of whole-body insulin sensitivity. Furthermore, the presence of two sickle β-globin chains, as in SCD, is sufficient to dysregulate glycemic control under normal conditions. In total, these findings demonstrate that the presence of the sickle cell variant promotes glucose dysregulation and hyperglycemia, meaning that it is essential to understand the glycemic regulation in patients with sickle cell trait and sickle cell anemia. Presentation: 6/1/2024

Exploring the bone marrow micro environment in thalassemia patients: potential therapeutic alternatives.

Genetic mutations in the β-globin gene lead to a decrease or removal of the β-globin chain, causing the build-up of unstable alpha-hemoglobin. This condition is referred to as beta-thalassemia (BT). The present treatment strategies primarily target the correction of defective erythropoiesis, with a particular emphasis on gene therapy and hematopoietic stem cell transplantation. However, the presence of inefficient erythropoiesis in BT bone marrow (BM) is likely to disturb the previously functioning BM microenvironment. This includes accumulation of various macromolecules, damage to hematopoietic function, destruction of bone cell production and damage to osteoblast(OBs), and so on. In addition, the changes of BT BM microenvironment may have a certain correlation with the occurrence of hematological malignancies. Correction of the microenvironment can be achieved through treatments such as iron chelation, antioxidants, hypoglycemia, and biologics. Hence, This review describes damage in the BT BM microenvironment and some potential remedies.

Potential Use of MicroRNA Technology in Thalassemia Therapy.

Thalassemia encompasses a group of inherited hemoglobin disorders characterized by reduced or absent production of the α- or β-globin chains, leading to anemia and other complications. Current management relies on lifelong blood transfusions and iron chelation, which is burdensome for patients. This review summarizes the emerging therapeutic potential of modulating microRNAs (miRNAs) to treat thalassemia. MiRNAs are small non-coding RNAs that regulate gene expression through sequence-specific binding to messenger RNAs (mRNAs). While they commonly repress gene expression by binding to the 3' untranslated regions (UTRs) of target mRNAs, miRNAs can also interact with 5'UTRs and gene promoters to activate gene expression. Many miRNAs are now recognized as critical regulators of erythropoiesis and are abnormally expressed in β-thalassemia. Therapeutically restoring levels of deficient miRNAs or inhibiting overexpression through miRNA mimics or inhibitors (antagomir), respectively, has shown preclinical efficacy in ameliorating thalassemic phenotypes. The miR-144/451 cluster is especially compelling for targeted upregulation to reactivate fetal hemoglobin synthesis. Advances in delivery systems are addressing previous challenges in stability and targeting of miRNA-based drugs. While still early, gene therapy studies suggest combinatorial approaches with miRNA modulation may provide synergistic benefits. Several key considerations remain including enhancing delivery, minimizing off-target effects, and demonstrating long-term safety and efficacy. While no miRNA therapies have yet progressed to clinical testing for thalassemia specifically, important lessons are being learned through clinical trials for other diseases and conditions, such as cancer, cardiovascular diseases, and viral. If limitations can be overcome through multi-disciplinary collaboration, miRNAs hold great promise to expand and transform treatment options for thalassemia in the future by precisely targeting pathogenic molecular networks. Ongoing innovations, such as advancements in miRNA delivery systems, improved targeting mechanisms, and enhanced understanding of miRNA biology, continue to drive progress in this emerging field towards realizing the clinical potential of miRNA-based medicines for thalassemia patients.

Revolutionary breakthrough: FDA approves CASGEVY, the first CRISPR/Cas9 gene therapy for sickle cell disease.

Sickle cell disease (SCD) is a hereditary hemoglobinopathy resulting from a β-globin chain mutation that causes abnormal hemoglobin (HbS) polymerization and leads to severe complications. Current treatment options primarily focus on symptom management, with limited curative potential. Recently, Casgevy, the first CRISPR/Cas9-based gene therapy for SCD, has received breakthrough FDA approval. Clinical trials have shown that Casgevy administered to patients aged older than or equal to 12 years enables precise modifications in hematopoietic stem cells, resulting in elevated fetal hemoglobin (HbF) levels and a significant reduction in vaso-occlusive events. Unlike conventional treatments, this therapy offers a curative approach and eliminates the need for recurrent transfusions and transplants, thereby improving the quality of life of patients with SCD. Casgevy has emerged as a beacon of hope for SCD patients and signifies a potential paradigm shift in SCD management due to its safety, curative potential, and transformative impact, positioning it as a groundbreaking intervention. Nevertheless, ethical considerations surrounding CRISPR technology and regulatory frameworks must be addressed to ensure responsible application and equitable access to this one-time gene editing therapy. As the authors celebrate this scientific advancement, sustained interdisciplinary collaboration and ethical scrutiny are essential to navigating the evolving landscape of CRISPR technology in medicine. This review aims to provide a detailed insight into the application of Casgevy, challenges associated with its application, future prospects of this therapy, and its comparison with existing treatment options for SCD.

Regulatory Assessment of Casgevy for the Treatment of Transfusion-Dependent β-Thalassemia and Sickle Cell Disease with Recurrent Vaso-Occlusive Crises.

Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are hereditary haemoglobinopathies characterized by a reduction in functional β-globin chains. Both conditions cause tiredness and increase susceptibility to infection, which can lead organ failure, significantly reducing life expectancy and typically requiring those affected to undergo regular erythrocyte transfusion. Recently, a novel therapeutic treatment for SCD and TDT was approved by the UK regulatory body (Medicines and Healthcare products Regulatory Agency; MHRA). Exagamglogene autotemcel (Casgevy) is the first licensed therapy globally to utilize CRIPSR/Cas9 technology and induces an increase in expression of γ-globin chains to compensate for the reduction in functional β-globin. Casgevy represents a first-in-class therapeutic, and numerous considerations were made by the MHRA throughout its assessment of the medicine. These include, but are not limited to, the risk of tumorigenicity and off-target editing, a limited cohort size, the validity of proposed dosing and the conduction of only single-arm studies. The MHRA's analyses of the data to support the proposed indications are presented and discussed throughout this manuscript. Overall, the sponsors claims were considered well supported by their data, and Casgevy was licensed for the treatment of TDT or SCD in patients 12 years of age and older for whom hematopoietic stem cell (HSC) transplantation is appropriate, but a human leukocyte antigen-matched related HSC donor is not available.

Sickle Cell Disease: Current Drug Treatments and Functional Foods with Therapeutic Potential.

Sickle cell anemia (SCA), the most common form of sickle cell disease (SCD), is a genetic blood disorder. Red blood cells break down prematurely, causing anemia and often blocking blood vessels, leading to chronic pain, organ damage, and increased infection risk. SCD arises from a single-nucleotide mutation in the β-globin gene, substituting glutamic acid with valine in the β-globin chain. This review examines treatments evaluated through randomized controlled trials for managing SCD, analyzes the potential of functional foods (dietary components with health benefits) as a complementary strategy, and explores the use of bioactive compounds as functional food ingredients. While randomized trials show promise for certain drugs, functional foods enriched with bioactive compounds also hold therapeutic potential. Further research is needed to confirm clinical efficacy, optimal dosages, and specific effects of these compounds on SCD, potentially offering a cost-effective and accessible approach to managing the disease.

The Discovery of Selective Protein Arginine Methyltransferase 5 Inhibitors in the Management of β-Thalassemia through Computational Methods.

β-Thalassemia is an inherited genetic disorder associated with β-globin chain synthesis, which ultimately becomes anemia. Adenosine-2,3-dialdehyde, by inhibiting arginine methyl transferase 5 (PRMT5), can induce fetal hemoglobin (HbF) levels. Hence, the materialization of PRMT5 inhibitors is considered a promising therapy in the management of β-thalassemia. This study conducted a virtual screening of certain compounds similar to 5'-deoxy-5'methyladenosine (3XV) using the PubChem database. The top 10 compounds were chosen based on the best docking scores, while their interactions with the PRMT5 active site were analyzed. Further, the top two compounds demonstrating the lowest binding energy were subjected to drug-likeness analysis and pharmacokinetic property predictions, followed by molecular dynamics simulation studies. Based on the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) score and molecular interactions, (3R,4S)-2-(6-aminopurin-9-yl)-5-[(4-ethylcyclohexyl)sulfanylmethyl]oxolane-3,4-diol (TOP1) and 2-(6-Aminopurin-9-yl)-5-[(6-aminopurin-9-yl)methylsulfanylmethyl]oxolane-3,4-diol (TOP2) were identified as potential hit compounds, while TOP1 exhibited higher binding affinity and stabler binding capabilities than TOP2 during molecular dynamics simulation (MDS) analysis. Taken together, the outcomes of our study could aid researchers in identifying promising PRMT5 inhibitors. Moreover, further investigations through in vivo and in vitro experiments would unquestionably confirm that this compound could be employed as a therapeutic drug in the management of β-thalassemia.

A Novel Frameshift Mutation of HBB Causing Dominant β-Thalassemia in a Chinese Individual

We reported a rare β-thalassemia patient, a 41-year-old Chinese male with small cell hypopigmentation anemia, jaundice and splenomegaly as the main clinical symptoms. By using Next-Generation Sequencing (NGS), we identified a novel de novo HBB mutation(c.358_365dup, p.Phe123Alafs*39) which resulted in an abnormally prolonged β-globin chain comprising 159 amino acid residues. The secondary and three-dimensional structures of the β-globin predicted that the novel prolonged β-globin chain has a considerable risk of instability in the hemoglobin, and leads to clinical phenotype. This study contributes to the enrichment of the genetic pathogenic mutation database for thalassemia and underscores the significance of NGS in the screening of mutations for thalassemia families.

New Insights in the Treatment of Sickle Cell Disease in Childhood

Sickle cell disease encompasses diseases that are pathophysiologically caused by hemoglobin S. The HbS component of total hemoglobin in SCD is normally over 50%. HbS is based on an amino acid substitution at position 6 of the β-globin chain, where glutamic acid is replaced by valine. This substitution replaces a hydrophilic amino acid with a hydrophobic amino acid at an outward-facing position in the h molecule, explaining the reduced water solubility and altered molecular organization of HbS compared to normal hemoglobin. Diseases caused by HbS include homozygous SCD, where both alleles are affected by the sickle cell mutation (SCD-S/S), HbSC disease, where one allele is affected by the sickle cell mutation and the other by the HbC mutation (SCD-S/C), and sickle cell β-thalassemia with mixed heterozygosity for the sickle cell mutation and a β-thalassemia mutation (SCD-S/β-thalassemia). In SCD-S/β-thalassemia, forms are distinguished where the β-thalassemia mutation completely inactivates the affected gene (SCD-S/β0-thalassemia) and forms where the allele with the thalassemia mutation still has residual activity (SCD-S/β+-thalassemia). Rarely, the sickle cell mutation can also be combined with other hemoglobin variants (SCD-S/D, SCD-S/OArab, SCD-S/Lepore). The carrier status for the sickle cell mutation generally has no clinical significance except in extreme situations (e.g., eye trauma or surgeries involving cardiopulmonary bypass). Carriers have a normal life expectancy. However, there are individual reports of complications in heterozygous carriers of the sickle cell mutation under common circumstances such as pregnancy, mountain sports, intense physical activity, or air travel. It is also unclear whether the carrier status is associated with an increased rate of kidney complications. Nevertheless, it is not appropriate to indicate specific medical care needs based on these individual case reports given the frequency of carrier status. However, the familial risk of developing SCD should be considered in adult carriers. This manuscript focus on new targets to treat sickle cell anemia in children.

Hospitalization Events Among Adolescents and Adults With Sickle Cell Disease in a Tertiary Care Center in Central India.

Sickle cell disease (SCD) is an inherited red blood cell disorder, wherein mutation causes the substitution of glutamic acid to valine at the sixth position of the β-globin chain. These include sickle cell anemia (homozygous sickle mutation), sickle-beta thalassemia, and hemoglobin SCD. The clinical manifestations of SCD are protean. Individuals with SCD suffer from both acute and chronic complications, which include recurring episodes of pain commonly called vaso-occlusive crisis (VOC) - acute chest syndrome (ACS); aseptic necrosis of the bone; micro-infarction of the spleen, brain, and kidney; infections; stroke; and organ damage affecting every part of the body. SCD necessitates frequent hospitalizations because of severe complications, which pose a significant burden on caregivers and economic strain on healthcare systems. The pattern of hospital admission with SCD varies in different parts of the world. This study aimed to determine the causes of hospitalization among adolescent and adult patients with SCD and to determine factors associated with their hospital stay. The study was a hospital-based prospective observational study comprising adolescent and adult patients diagnosed with SCD, aged 15-45 years, who were hospitalized in the Department of General Medicine at All India Institute of Medical Sciences in Raipur from August 2021 to August 2022. According to our study, the primary reason for hospitalization was a painful crisis, accounting for 63% of cases, followed by infection (17%), ACS (11%), and acute hemolytic crisis (9%). Notably, we did not observe any significant differences between genders and causes of admission (p > 0.05). Joint pain (p = 0.005), back pain (p = 0.001), and chest pain (p = 0.001) were more frequently reported by adults over the age of 19. In addition, our analysis of the duration of hospital stays and various factors revealed that patients admitted for infections had a significantly longer mean hospital stay duration (p = 0.040). Acute painful crises were the primary cause of hospital admission among individuals with SCD; many patients also encountered infections and ACS. Furthermore, patients who experienced infections and VOC had a lengthier duration of hospital stay. Therefore, it is essential to provide them with comprehensive instructions on various preventive measures against infections and the factors that trigger painful crises.

Increased Expression of α-Hemoglobin Stabilizing Protein (AHSP) mRNA in Erythroid Precursor Cells Isolated from β-Thalassemia Patients Treated with Sirolimus (Rapamycin).

Background/Objectives: in β-thalassemia, important clinical complications are caused by the presence of free α-globin chains in the erythroid cells of β-thalassemia patients. These free α-globin chains are present in excess as a result of the lack of β-globin chains to bind with; they tend to aggregate and precipitate, causing deleterious effects and overall cytotoxicity, maturation arrest of the erythroid cells and, ultimately, ineffective erythropoiesis. The chaperone protein α-hemoglobin-stabilizing protein (AHSP) reversibly binds with free α-globin; the resulting AHSP-αHb complex prevents aggregation and precipitation. Sirolimus (rapamycin) has been previously demonstrated to induce expression of fetal hemoglobin and decrease the excess of free α-globin chain in the erythroid cells of β-thalassemia patients. The objective of this study was to verify whether sirolimus is also able to upregulate AHSP expression in erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. Methods: the expression of AHSP genes was analyzed by measuring the AHSP mRNA content by real-time quantitative PCR (RT-qPCR) and the AHSP protein production by Western blotting. Results: AHSP gene expression was found to be higher in ErPCs of β-thalassemia patients in comparison to ErPCs isolated from healthy subjects. In addition, AHSP expression was further induced by treatment of β-thalassemia ErPCs with sirolimus. Finally, AHSP mRNA was expressed at an increased level in ErPCs of sirolimus-treated β-thalassemia patients participating in the NCT03877809 Sirthalaclin clinical trial. Conclusions: this exploratory study suggests that AHSP expression should be considered as an endpoint in clinical trials based on sirolimus.

Unraveling DEHP influence on hemoglobin S polymerization in sickle cell disease: Ex vivo, in vitro and in silico analysis

Sickle cell disease (SCD) is a hereditary hemoglobinopathy, caused by a mutation at position 6 of the β-globin chain and patients are frequently exposed to several blood transfusions in order to maintain physiological function. Transfusion blood bags are composed of PVC and phthalates (as DEHP) are often introduced to the material in order to confer malleability. In this sense, DEHP can easily elute to the blood and cause harmful effects. This study aimed to unravel DEHP effect on SCD patient's hemoglobin function. We found that HbS polymerization using whole erythrocytes is decreased by DEHP in ex vivo experiments and this effect might be mediated by the DEHP-VAL6 interaction, evaluated in silico. Isolated HbS exhibited less polymerization at low DEHP concentrations and increased polymerization rate at higher concentration. When analyzing the propensity to aggregate, HbS is more inclined to aggregate when compared to HbA due to the residue 6 mutation. Circular dichroism showed characteristic hemoglobin peaks for oxygenated HbS that are lost when oxygen is sequestered, and DEHP at higher concentration mildly recovers a peak close to the second hemoglobin one. Finally, by transmission electron microscopy we demonstrated that high DEHP concentration increased polymer formation with a more organized structure. These findings show for the first-time the beneficial effect of low-dose DEHP on HbS polymerization.

Natural Anticoagulant Protein Levels in Patients With Beta-Thalassemia Major: A Case-Control Study.

β-thalassemia is a group of inherited blood disorders that affect the production of β-globin chains, leading to the reduction or absence of these chains. One of the complications observed in patients with β-thalassemia major (β-TM) is thrombosis, especially in those who receive frequent blood transfusions. This may be due to a decrease in the levels of the natural anticoagulants: protein C (PC), total protein S (PS), and antithrombin (AT). In this case-control study, patients with β-TM, who had received at least 20 packed cell transfusions during their lifetime, were included. Patients with other underlying diseases like bleeding or thrombotic disorders were excluded. Totally, 118 patients with β-TM and 120 healthy individuals were included. The mean level of PC and AT was significantly lower in patients with β-TM (48.2 ± 65.4 and 57.42 ± 13.6, respectively) compared to the control group (97.1 ± 21.46 and 81.79 ± 14.3, respectively), with P value of 0.001 and 0.01, respectively. Although the difference was not statistically significant (P = 0.1), a similar trend was observed for total PS (61.12 ± 21.12 for patients versus 72.2 ± 35.2 for the control group). Of note, the decrease in PC, AT, and total PS levels compared to the control group was 50.36%, 27.5%, and 15.34%, respectively. It seems that β-TM patients who receive prolonged blood transfusions frequently are at an increased risk of decreased in natural anticoagulants levels and therefore potentially are at risk of thrombosis.

Wide spectrum of novel and rare hemoglobin variants in the multi-ethnic Indian population: A review.

The hemoglobin (Hb) variants are qualitative abnormalities due to production of structurally abnormal globin proteins. They are categorized based on the type of mutation present in the α1, α2, β, Gγ, Aγ and δ globin genes. So far, more than 1550 Hb variants are reported in the database. They could lead to Hb polymerization, Hb instability, altered oxygen affinity and decreased oxygen-carrying capacity of Hb or have no clinical manifestations. In India, ethnic diversity, consanguinity, regional variations and migration result in the presence of different Hb variants. We have compiled all the variants of α, β and δ globin chains in heterozygous, homozygous and in compound heterozygous forms reported from India in the last 52 years. Of the 63 rare and novel hemoglobin variants reported from India, 22 were α-globin chain variants, 37 were β-globin chain variants and 4 were δ-globin chain variants. Twelve novel Hb variants (Hb J Rajappan, Hb Koya Dora, Hb Rampa, Hb Godavari, Hb Chandigarh, Hb D Agri, Hb Lucknow, Hb Vellore, Hb Midnapore, Hb Bijnor, Hb A2Tianhe and Hb A2Saurashtra) were identified among persons of Indian origin. Majority of them were picked up on HPLC. Some of the variants like Hb Titusville, Hb Shimonoseki, Hb Chandigarh, Hb D Agri, Hb Yaizu and Hb Vellore eluted in the HbS window whereas variants like HbD Iran, Hb St. Louis, Hb G Coushata, HbM Saskatoon, Hb Lucknow, Hb Grange-Blanche and Hb Tianshui showed falsely elevated HbA2. Hence, careful and systematic investigations are required to identify them.

Bone Health Impairment in Patients with Hemoglobinopathies: From Biological Bases to New Possible Therapeutic Strategies.

Hemoglobinopathies are monogenic disorders affecting hemoglobin synthesis. Thalassemia and sickle cell disease (SCD) are considered the two major hemoglobinopathies. Thalassemia is a genetic disorder and one of the major hemoglobinopathies determined by an impairment of globin chain production, which causes an alteration of erythropoiesis, an improvement in hemolysis, and an alteration of iron homoeostasis. In SCD, the mutations are on the β-globin chain of hemoglobin which results in a substitution of glutamic acid by valine with consequent formation of Hemoglobin S (HbS). Several factors are involved in bone metabolism alteration in patients with hemoglobinopathies, among them hormonal deficiency, bone marrow hyperplasia, iron overload, inflammation, and increased bone turnover. Bone metabolism is the result of balance maintenance between bone deposition and bone resorption, by osteoblasts (OBs) and osteoclasts (OCs). An impairment of this balance is responsible for the onset of bone diseases, such as osteoporosis (OP). Therefore, here we will discuss the alteration of bone metabolism in patients with hemoglobinopathies and the possible therapeutic strategies to contain and/or counteract bone health impairment in these patients, taking into consideration not only the pharmacological treatments already used in the clinical armamentarium, but also the new possible therapeutic strategies.

Identification of a rare [Gγ(Aγδβ)0] -thalassemia using tandem mass spectrometry.

Thalassemias are a group of inherited monogenic disorders characterized by defects in the synthesis of one or more of the globin chain subunits of the hemoglobin tetramer. Delta-beta (δβ-) thalassemia has large deletions in the β globin gene cluster involving δ- and β-globin genes, leading to absent or reduced synthesis of both δ- and β-globin chains. Here, we used direct globin-chain analysis using tandem mass spectrometry for the diagnosis of δβ-thalassemia. Two cases from unrelated families were recruited for the study based on clinical and hematological evaluation. Peptides obtained after trypsin digestion of proteins extracted from red blood cell pellets from two affected individuals and their parents were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mass spectrometric analysis revealed a severe reduction in δ, β, and Aγ globin proteins with increased Gγ globin protein in the affected individuals. The diagnosis of Gγ(Aγδβ)0 -thalassemia in the homozygous state in the affected individuals and in the heterozygous state in the parents was made from our results. The diagnosis was confirmed at the genetic level using multiplex ligation-dependent probe amplification (MLPA). Our findings demonstrate the utility of direct globin protein quantitation using LC-MS/MS to quantify individual globin proteins reflecting changes in globin production. This approach can be utilized for accurate and timely diagnosis of hemoglobinopathies, including rare variants, where existing diagnostic methods provide inconclusive results.

Towards detection of structurally-diverse glycated epitopes in native proteins: Single-chain antibody directed to non-A1c epitope in human haemoglobin

Over 500 million people worldwide are affected by diabetes mellitus, a chronic disease that leads to high blood glucose levels and causes severe side effects. The predominant biological marker for diagnosis of diabetes is glycated haemoglobin (GHb). In human blood the predominant reducing sugar, glucose, irreversibly conjugates onto accessible amine groups within Hb. Most methods for diagnosis and monitoring of diabetes selectively detect N-terminal glycation at Val-1 on the β-globin chain, but not glycation at other sites. Detection of other glycated epitopes of GHb has the potential to provide new information on the extent, duration and timing of elevated glucose, facilitating personalised diagnosis and intelligent diabetic control. In this work, a new anti-GHb Fab antibody (Fab-1) specific for haemoglobin A1c (HbA1c) with nanomolar affinity was discovered via epitope-directed immunisation and phage display. A single chain variable fragment (scFv) antibody derived from Fab-1 retained affinity and specificity for HbA1c, and affinity was enhanced tenfold upon addition of an enhanced green fluorescent protein tag. Both the scFv and Fab-1 recognised an epitope within HbA1c that was distinct from β-Val-1, and our data suggest that this epitope may include glycation at Lys-66 in the β-globin chain. To our knowledge, this is the first report of an scFv/Fab anti-glycated epitope antibody that recognises a non-A1c epitope in GHb, and confirms that fructosamine attached to different, discrete glycation sites within the same protein can be resolved from one another by immunoassay.

Identification of a Novel Variant c.163delG in HBB Gene Resulting in a Beta Null Phenotype in a Proband with Thalassemia Intermedia

A 21-year-old patient presented with a previous medical history of pallor, mild icterus, increased fatigue, low hemoglobin, and abnormal hemoglobin variant analysis with more than 70 transfusions. He was referred for genetic analysis to identify the pathogenic variations in the β-globin gene. Sanger’s sequencing of the proband and his family revealed the presence of a novel frame shift variant HBB:c.163delG in a compound heterozygous state with hemoglobin E (HbE) (HBB:c.79G > A) variant. The father and the sibling of the patient were found to be normal for the HBB gene. Mother was found to be heterozygous for HbE (HBB:c.79G > A) variant. In silico analysis by Mutalyzer predicted that c.163delG variant generated a premature stop codon after seven codons, leading to a truncated protein. FoldX protein stability analysis showed a positive ΔΔG value of 45.27 kcal/mol suggesting a decrease in protein stability. HBB:c.79G > A is a known variant coding for HbE variant, which results in the reduced synthesis of β-globin chain and shows mild thalassemia. Combined effect of HBB:c.163delG and HBB:c.79G > A variants in the proband might have led to the reduced synthesis of β-globin chains resulting in a thalassemia intermedia type of clinical manifestation.