Β-globin Chain Research Articles

Estimated Level of Plasma Micro RNA-210 in Beta Thalassemia Patients and Its Relation to Hemoglobin F Level and Disease Severity

BACKGROUND: Beta-thalassemia is an autosomal recessive hereditary blood disorder occurs due to absent or diminished β-globin chains synthesis. The transformation from fetal to adult in globin synthesis is controlled by different mechanisms; one of those is BCL11A which is downregulated by microRNA-210 (miRNA-210). Therefore, this would lead to reactivation of fetal hemoglobin (HbF) production that improves anemia and reduces the needs for transfusion. OBJECTIVE: To study miRNA-210 in the plasma of beta thalassemia patients and its relation to HbF level and disease severity. SUBJECTS AND METHODS: The study was carried out on 50 individuals divided into three groups. Group A included 30 thalassemia major patients, Group B included 10 thalassemia intermedia patients, and Group C is 10 healthy volunteers as control. All individuals underwent to history taking, physical examination, and laboratory investigations, complete blood count, serum ferritin, hemoglobin electrophoresis, and measurement of miRNA-210 expression levels in plasma of peripheral blood samples by quantitative real-time polymerase chain reaction. RESULTS: Examination of miRNA-210 plasma level among different groups revealed no statistically significant association between control and thalassemia group or its subgroups (Major and intermedia) (P = 0.224, P = 0.116, and P = 0.734, respectively). Moreover, miRNA-210 was slightly higher in responders than nonresponders to hydroxyurea but did not reach significant level. CONCLUSION: miRNA-210 levels were not associated with increase in HbF levels in thalassemia patients.

Relationships Of Vitamin D And Vitamin B12 With Malonaldehyde In Patients With Beta Thalassemia Major

Background: Beta thalassemia major (Cooley's anemia) is a congenital genetic blood disorder resulting from a deficiency of β-globin chains due to severe anemia that depends on continuous blood transfusions for survival. Patients suffer from hemoglobinopathy, infection, diabetes mellitus, growth retardation, enlargement and damage to the liver and spleen, bone marrow aplasia and delayed sexual maturity. This complication arises due to the accumulation of iron, the impairment of antioxidants from vitamins, and an increase in lipid peroxides (MDA).Objectives: The study aimed to evaluate vitamin D and B12 levels and their relationship with MDA, liver enzymes (AST, ALT, ALP), iron and (Total Billirubin, Direct Billirubin, Indirect Billirubin).Materials and methods: The samples of BTM patients under study were (100 samples), (50 samples) for both males and females. Patients' ages ranged from (2-30 years). While the control sample was (40), with (20 samples) for each of the two sexes. Their ages ranged from (2-30 years). Their disease was confirmed by blood tests, hemoglobin electrophoresis, DNA analysis, and elevated ferritin levels. Vitamin D and B12 levels were assessed by immunoassay. While (MDA, Fe, AST, ALT, ALP) and (Total Billirubin, Direct Billirubin, Indirect Billirubin) were evaluated by colorimetric methods.Results: Vitamin D and B12 levels decreased significantly (p≤0.05). While the levels of (MDA, Fe, AST, ALT, ALP) and (Total Billirubin, Direct Billirubin, Indirect Billirubin) increased significantly at (p≤0.05) in BTM patients compared with the control group. Where the effect of the disease according to the results was clear in patients with age.
 conclusion: Our results indicate that the weakness of the antioxidant systems (vitamin D and B12) with the increase in oxidative factors resulting from the complications of the disease led to the exacerbation of the disease with iron overload, causing an increase in the levels of liver enzymes in the blood, damaging the liver and spleen.

Flurbiprofen inhibits heme induced NLRP3 inflammasome in Berkeley sickle cell disease mice.

Sickle cell disease (SCD) is accompanied by several complications, which emanate from the sickling of erythrocytes due to a point mutation in the β-globin chain of hemoglobin. Sickled erythrocytes are unable to move smoothly through small blood capillaries and therefore, cause vaso occlusion and severe pain. Apart from pain, continuous lysis of fragile sickled erythrocytes leads to the release of heme, which is a strong activator of the NLRP3 inflammasome, thus producing chronic inflammation in sickle cell disease. In this study, we identified flurbiprofen among other COX-2 inhibitors to be a potent inhibitor of heme-induced NLRP3 inflammasome. We found that apart from being a nociceptive agent, flurbiprofen exerts a strong anti-inflammatory effect by suppressing NF-κB signaling, which was evidenced by reduced levels of TNF-α and IL-6 in wild-type and sickle cell disease Berkeley mice models. Our data further demonstrated the protective effect of flurbiprofen on liver, lungs, and spleen in Berkeley mice. The current sickle cell disease pain management regime relies mainly on opiate drugs, which is accompanied by several side effects without modifying the sickle cell disease-related pathology. Considering the potent role of flurbiprofen in inhibiting NLRP3 inflammasome and other inflammatory cytokines in sickle cell disease, our data suggests that it can be explored further for better sickle cell disease pain management along with the possibility of disease modification.

Evaluation of conventional renal function tests in β-thalassemia major patients in Nineveh province

β-thalassemia is an inherited anemia, caused by impaired β-globin chain synthesis. The disease leads to serious health problems unless treated by regular blood transfusion and iron chelating therapy. However, complications from continuous therapy may arise due to iron overload and toxicity of iron chelating therapy, it may affects many organs in the body including the renal system. The present study was directed to study the changes in conventional renal function tests that may occur in β-thalassemia patients. The present study consisted of two groups of β-thalassemia patients; group A, 40 transfusion dependent β-thalassemia patients receiving only regular blood transfusion, age ranged 1-4 years; Group B, 40 transfusion dependent β- thalassemia patients receiving regular blood transfusion and deferroxamine chelation therapy, age ranged 4-20 years old. In addition to a control group C, 40 subjects, age ranged 1-20 years. Blood samples were collected and serum was separated for measurement of creatinine, urea, and ferritin. Urine samples were also collected for measurement of microalbuminuria. There was a significant decrease in serum creatinine, and a significant increase in serum urea in group B when compared with their control (P≤0.01). In addition, there was a significant increase in serum ferritin in groups A and B when compared with their controls (P≤0.01). On the other hand, there were no significant changes in microalbuminuria between groups A and B when compared with their controls. The results of the present study showed no deterioration in renal functions in β-thalassemia patients in both groups A and B regarding serum creatinine and urea in addition to microalbuminuria, although, subclinical alteration in renal functions could be expected in those patients, so that measurement of other early markers of renal dysfunction is occasionally recommended.

Gene Therapy for β-Hemoglobinopathies: From Discovery to Clinical Trials.

Investigations to understand the function and control of the globin genes have led to some of the most exciting molecular discoveries and biomedical breakthroughs of the 20th and 21st centuries. Extensive characterization of the globin gene locus, accompanied by pioneering work on the utilization of viruses as human gene delivery tools in human hematopoietic stem and progenitor cells (HPSCs), has led to transformative and successful therapies via autologous hematopoietic stem-cell transplant with gene therapy (HSCT-GT). Due to the advanced understanding of the β-globin gene cluster, the first diseases considered for autologous HSCT-GT were two prevalent β-hemoglobinopathies: sickle cell disease and β-thalassemia, both affecting functional β-globin chains and leading to substantial morbidity. Both conditions are suitable for allogeneic HSCT; however, this therapy comes with serious risks and is most effective using an HLA-matched family donor (which is not available for most patients) to obtain optimal therapeutic and safe benefits. Transplants from unrelated or haplo-identical donors carry higher risks, although they are progressively improving. Conversely, HSCT-GT utilizes the patient's own HSPCs, broadening access to more patients. Several gene therapy clinical trials have been reported to have achieved significant disease improvement, and more are underway. Based on the safety and the therapeutic success of autologous HSCT-GT, the U.S. Food and Drug Administration (FDA) in 2022 approved an HSCT-GT for β-thalassemia (Zynteglo™). This review illuminates the β-globin gene research journey, adversities faced, and achievements reached; it highlights important molecular and genetic findings of the β-globin locus, describes the predominant globin vectors, and concludes by describing promising results from clinical trials for both sickle cell disease and β-thalassemia.

Foetal haemoglobin inducers for reducing blood transfusion in non-transfusion-dependent beta-thalassaemias.

Foetal haemoglobin inducers for reducing blood transfusion in non-transfusion-dependent beta-thalassaemias.

Clinical genome editing to treat sickle cell disease-A brief update.

Sickle cell disease (SCD) is one of the most common hemoglobinopathies. Due to its high prevalence, with about 20 million affected individuals worldwide, the development of novel effective treatments is highly warranted. While transplantation of allogeneic hematopoietic stem cells (HSC) is the standard curative treatment approach, a variety of gene transfer and genome editing strategies have demonstrated their potential to provide a prospective cure for SCD patients. Several stratagems employing CRISPR-Cas nucleases or base editors aim at reactivation of γ-globin expression to replace the faulty β-globin chain. The fetal hemoglobin (HbF), consisting of two α-globin and two γ-globin chains, can compensate for defective adult hemoglobin (HbA) and reverse the sickling of hemoglobin-S (HbS). Both disruption of cis-regulatory elements that are involved in inhibiting γ-globin expression, such as BCL11A or LRF binding sites in the γ-globin gene promoters (HBG1/2), or the lineage-specific disruption of BCL11A to reduce its expression in human erythroblasts, have been demonstrated to reestablish HbF expression. Alternatively, the point mutation in the HBB gene has been corrected using homology-directed repair (HDR)-based methodologies. In general, genome editing has shown promising results not only in preclinical animal models but also in clinical trials, both in terms of efficacy and safety. This review provides a brief update on the recent clinical advances in the genome editing space to offer cure for SCD patients, discusses open questions with regard to off-target effects induced by the employed genome editors, and gives an outlook of forthcoming developments.

β-Thalassemia in childhood: Current state of health in a high-income country.

β-thalassemia is an haemoglobinopathy characterized by a defective synthesis of the β-globin chain. To assess the current state of health of paediatric patients with β-thalassemia, data from the French national registry regarding children born between 2005 and 2020 with β-thalassemia intermedia (TI) or major (TM) were collected. A total of 237 patients (median age 7.1 years at last visit) were analysed, of whom 156 (65.8%) were born in France and 162 (68.4%) had a TM phenotype. The probability of survival for children with TM born in France was 98.3% at 15 years. Fifty-four (22.8%) children received a haematopoietic stem cell transplant with a success rate of 88.8%. Hepatic and cardiac iron overload monitoring in non-transplanted patients showed moderate overload in 15.7% (18/115) and 7.1% (7/99) of cases, respectively, while clinical complications were found in only 4 patients with TM (hepatic in 3 cases). At last visit, mean ferritinemia was 1293 ng/ml (±759). Overall, less than 10% of children underwent splenectomy. No significant impact of the disease on growth or academic achievement was observed. Deferasirox was the main first-line chelator, prescribed in 78.2% of cases, with side effects reported in 11.7% of instances.

Haematopoietic Stem Cell Transplantation in Thalassaemia Major: A Narrative Review

Thalassaemia constitutes an especially prevalent human monogenic illness caused by a lack of synthesis of the α- or β-globin chains. The clinical impact of β-thalassaemia is worse since it consists of the same pair gene configuration, thalassaemia major, causing significant health discouragement and loss of life due to life threateningly insufficient haemoglobin (Hb) levels. Only a few nations have successfully reduced the prevalence of β-thalassaemia major, even though comprehensive screening, group counselling, pre-natal detection and public education can all be used. Since over ½ century ago, the fundamental elements of treatment for thalassaemia major have been iron chelation and hypertransfusion. The globin chain that makes up the adult Hb molecule is missing or synthesised at a reduced rate in β-thalassemia. The aberrant buildup of the α-globin chain and faulty formation of red blood cells (RBCs) leading to RBC haemolysis are the outcomes of this genetic abnormality. Since allogeneic haematopoietic stem cell transplantation (Allo-HSCT) has been a well-established gene replacement therapy for individuals with thalassaemia major for several years, it has had very successful outcomes for patients with access to it. Over the past 20 years, the consequences for more susceptible patients have also steadily improved, leading to 80%–90% longer-term life expectancy amongst this group of patients. However, providing Allo-HSCT as a treatment for these patients globally presents numerous difficulties. Replacing genes in autologous HSCs employing viral vectors has been possible in recent years.

Fetal Hemoglobin Inducer Activity of Moringa oleifera, Curcuma aueruginosa Roxb., and Artocarpus altilis Based on the Gamma Globin Expression

BACKGROUND: Beta (β)-thalassemia is a common single-gene blood disorder resulting in a decrease or absence of β-globin chain synthesis. In β-thalassemia major, an imbalance of α/β globin chains results in severe oxidative stress that leads to pathological conditions. Reactivating the gamma (γ)-globin gene will overcome the excess alpha (α)-globin chains and relieve β-thalassemia patients' clinical course. AIMS AND OBJECTIVES: This study aimed to determine the most competent herbal extract with high efficiency in inducing γ-globin gene expression to facilitate β-thalassemia patients. MATERIALS AND METHODS: This study used K562 cell line culture to assess the fetal hemoglobin (HbF) induction efficacy of Moringa oleifera (MO), Curcuma aueruginosa Roxb. (CA), and Artocarpus altilis (AA). We carried out the benzidine test to count hemoglobin-containing cells and RT-qPCR to measure γ-globin gene expression. RESULTS: The benzidine test showed that MO extract was the highest value (3%) in inducing fetal hemoglobin. However, based on RT-PCR analysis, CA extract had the most significant (2.39 fold change) ability to reactivate the γ-globin gene compared to Hydroxyurea as a positive control. CONCLUSION: The different properties and levels of MO and CA testing in antioxidant and reactivation of gene expression mechanism most probably influenced the discrepancy between the benzidine test and RT-qPCR results.

A Comparative Study to Assess Parathyroid Hormone Status in Different Age Groups of b-Thalassaemia Major Individuals from Eastern India

Introduction: β-thalassaemia major, due to imbalance, missing or deficiency of β-globin chain synthesis pathway, is marked as a hereditary disorder. Homozygous state presents with severe anaemia. Regular blood transfusions and chelation therapy increase the life expectancy in thalassaemia patients. Due to recent advances in the treatment paradigm of β-thalassaemia major patients, there has been a significant increase in their lifespan but, due to the treatment related iron overload, endocrine defects like hypogonadism, diabetes mellitus, hypothyroidism and Hypoparathyroidism (HPT) have been seen to progress. Diverse studies state the occurrence of HPT to be from none upto almost 22.5% in patients. It has also been recognised that asymptomatic hypocalcaemia is much more common and can be overlooked unless precisely viewed. Aim: To estimate and compare the biochemical parameters related to bone turnover in regularly transfused thalassaemia patients in different age groups and to find out significant correlation among the biochemical markers. Materials and Methods: In this cross-sectional study conducted at Calcutta National Medical College, Kolkata, West Bengal, India, from January 2017 till January 2018, 100 β-thalassaemia major patients were enrolled. Serum Parathyroid Hormone (PTH), serum ferritin, ionic calcium (Ca2+), Alkaline Phosphatase (ALP), phosphorus (P) and 25-Hydroxy vitamin D (25-OHD) levels were estimated in these patients. Statistical evaluation was done by the Statistical Package for the Social Sciences (SPSS), version 12.0. Result was reported as mean±SD. Pearson correlation coefficient was used to determine the association of serum ferritin levels and parathormone levels. The p-value of <0.05 was considered as statistically significant. Results: Out of 100 patients, 19 patients had HPT. The mean age was found to be 12.6 years on diagnosis, mean ionised calcium was 0.95 mmol/L, mean serum ferritin was 3045 μg/L (range 1209-10,000 µg/L) and mean serum phosphate was 1.88 mmol/L (range 1.50-2.73 mmol/L). Serum PTH values of 7.04 pg/mL was found to be low. Negative correlation serum PTH and ferritin levels were found. Significant higher values of mean serum ferritin (2789 μg/L) and lower values of parathormone (17.13 pg/mL) was found in the age group of 11-18 years as compared to the other age group of ≤10 years with serum ferritin and parathormones values being 1648 μg/L and 23.44 pg/mL, respectively. Conclusion: Regularly transfused β-thalassaemia major patients, inspite of receiving chelation therapy tend to develop altered calcium and vitamin D homeostasis once they enter second decade of life. Thereby, biochemical parameters related to bone profile which includes ionic calcium, vitamin D, phosphate and parathyroid hormones levels should stringently monitored once the patient crosses first decade of life to prevent any development of hypocalcaemia or overt HPT.

Knowledge and Attitudes towards Thalassemia among Public University Students in Bangladesh

Background: Thalassemia is the most common congenital single-gene condition globally, characterized by a lack of or reduced synthesis of either the α- or β-globin chains and passed down from parents to offspring. This study aimed to determine the level of knowledge of thalassemia among Bangladeshi students from universities and their opinion on prevention. Methods: A cross-sectional descriptive online survey was conducted on public university students in Bangladesh using a structured questionnaire between June and November 2020, completed by students online. Demographic characteristics, knowledge, and attitude of the participants were assayed with 10 questions on each topic. The data were analyzed using Python. Descriptive statistics methods such as frequencies and percentages were used to present data. Result: A total of 681 students participated in the online survey. The average age of the respondents in this study was 21.97 years, with a standard deviation of 2.9. Most of the students, 611(89.72%), had heard about Thalassemia, yet only 248 (36.42%) students had good knowledge of Thalassemia. The knowledge level of students had no significant relationship with sex (p-value = 0.0819), marital status (p-value = 0.2281), and year of study (p-value = 0.4619), but had a considerable difference with the field of study (0.0042). However, 478 (78.36%) participants showed a positive attitude toward "Premarital Screening" to prevent Thalassemia. Conclusions: Long-term and target-based preventive initiatives are recommended for university students and the general population of Bangladesh, where the rate of consanguineous marriage is high. These initiatives would provide crucial information and increase awareness of premarital screening and genetic counseling to prevent thalassemia.

Estimating the Incidence Frequency of Helicobacter pylori Co-infection with Hepatitis C Virus among Thalassemia Individual in Wasit Governorate of Iraq

Objective(s): Helicobacter pylori (H. pylori) is a gram-negative bacterium that plays a significant role in developing gastrointestinal diseases such as peptic ulcers. H. pylori infection affects more than half of the world’s population. This pathogen incidence becomes more effective among thalassemia patients, a common genetic disorder characterized by a point mutation that leads to impaired production of the β-globin chain. Chronic hepatitis C is a chronic disease caused by the hepatitis C virus (HCV) that has significantly increased morbidity and mortality rates in these patients due to liver failure or hepatocellular carcinoma. Methods: Current study included 90 β-thalassemia patients admitted to the thalassemia hematology center in Al-Kut women and children Hospital, Wasit province, Iraq from September 2020 to November 2021. The patients are divided into two groups (β-thalassemia with HCV group and β-thalassemia without HCV). First and second groups contain 32 and 58 specimens of blood and stool samples, respectively. The patient diagnosed with thalassemia syndrome regularly attended the thalassemia hematology center for transfusion and chelation and follow up on Hb level and iron status. This study aimed at serological and molecular diagnosis of co-infection H. pylori with Hepatitis C virus (HCV) among thalassemia patients and diagnosis infection rate. Diagnosis was used Serological enzyme-linked immunoassay (ELISA) techniques for HP-IgG, Level of ferritin, liver function test will be determined and Nested PCR method targeting the molecular diagnosis will be used to confirm H. pylori 23S rRNA using DNA extracted from stool samples. Results: 90 β-thalassemia patients sample were diagnosed by Serological ELISA techniques for HP-IgG and the result showed 30 samples were positive (18 from the β-thalassemia with HCV group and 12 from the β-thalassemia without HCV)and 60 samples negative for HP-IgG and Nested polymerase chain reaction (PCR) method targeting the molecular diagnosis will be used to confirm H. pylori 23S rRNA using DNA extracted from stool for 30 stool samples revealed 13 samples positive (9 from the β-thalassemia with HCV and 4 from β-thalassemia without HCV) while 17 samples were negative. Conclusions: The predominance of H. pylori among patients in Wasit was 43.3%. Levels of ferritin have significant correlation with H. pylori in HCV patients. There was a positive correlation between hepatitis C status and high H. pylori rate when compared with β-thalassemia without HCV. Splenoctomy was a positive correlation with H. pylori in β-thalassemia with HCV. Moreover, these results recommend study about the virulence factor and genes. Other molecular studies are needed for detecting more virulence genes in H. pylori bacteria such as real-time PCR and sequencing of H. pylori in patients with thalassemia, especially those with high serum ferritin.

Neuronal haemoglobin induces loss of dopaminergic neurons in mouse Substantia nigra, cognitive deficits and cleavage of endogenous α-synuclein

Parkinson’s disease (PD) presents the selective loss of A9 dopaminergic (DA) neurons of Substantia Nigra pars compacta (SNpc) and the presence of intracellular aggregates called Lewy bodies. α-synuclein (α-syn) species truncated at the carboxy-terminal (C-terminal) accumulate in pathological inclusions and promote α-syn aggregation and toxicity. Haemoglobin (Hb) is the major oxygen carrier protein in erythrocytes. In addition, Hb is expressed in A9 DA neurons where it influences mitochondrial activity. Hb overexpression increases cells’ vulnerability in a neurochemical model of PD in vitro and forms cytoplasmic and nucleolar aggregates upon short-term overexpression in mouse SNpc. In this study, α and β-globin chains were co-expressed in DA cells of SNpc in vivo upon stereotaxic injections of an Adeno-Associated Virus isotype 9 (AAV9) and in DA iMN9D cells in vitro. Long-term Hb over-expression in SNpc induced the loss of about 50% of DA neurons, mild motor impairments, and deficits in recognition and spatial working memory. Hb triggered the formation of endogenous α-syn C-terminal truncated species. Similar α-syn fragments were found in vitro in DA iMN9D cells over-expressing α and β- globins when treated with pre-formed α-syn fibrils. Our study positions Hb as a relevant player in PD pathogenesis for its ability to trigger DA cells’ loss in vivo and the formation of C-terminal α-syn fragments.

Feasibility of combining short tandem repeats (STRs) haplotyping with preimplantation genetic diagnosis (PGD) in screening for beta thalassemia.

β-thalassemia is an autosomal recessive disease with the reduction or absence in the production of β-globin chain in the hemoglobin, which is caused by mutations in the Hemoglobin subunit beta (HBB) gene. In Vietnam, the number of β-thalassemia carriers range from 1.5 to 25.0%, depending on ethnic and geographical areas, which is much higher than WHO's data worldwide (1.5%). Hence, preimplantation genetic diagnosis (PGD) plays a crucial role in reducing the rate of β-thalassemia affected patients/carriers. In this research, we report the feasibility and reliability of conducting PGD in combination with the use of short tandem repeat (STR) markers in facilitating the birth of healthy children. Six STRs, which were reported to closely linked with the HBB gene, were used on 15 couples of β-thalassemia carriers. With 231 embryos, 168 blastocysts were formed (formation rate of 72.73%), and 88 were biopsied and examined with STRs haplotyping and pedigree analysis. Thus, the results were verified by Sanger sequencing, as a definitive diagnosis. Consequently, 11 over 15 couples have achieved pregnancy of healthy or at least asymptomatic offspring. Only three couples failed to detect any signs of pregnancy such as increased Human Chorionic Gonadotropin (HCG) level, foetal sac, or heart; and one couple has not reached embryo transfer as they were proposed to continue with HLA-matching to screen for a potential umbilical cord blood donor sibling. Thus, these results have indicated that the combination of PGD with STRs analysis confirmed by Sanger sequencing has demonstrated to be a well-grounded and practical clinical strategy to improve the detection of β-thalassemia in the pregnancies of couples at-risk before embryo transfer, thus reducing β-thalassemia rate in the population.

Characterization of Hemoglobin Variants Due to in-Frame Deletions in the β-Globin Gene from CRISPR/Cas9 Gene Correction for Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene (HBB). CRISPR/Cas9 based gene correction approaches rely on inducing a double-strand break (DSB) at or near the sickle mutation, which subsequently results in gene correction via the homology-directed repair (HDR) or insertions/deletions (Indels) via non-homologous end joining (NHEJ). Frameshift Indels induce a premature stop codon and disrupt HBB production. Indels that preserve the reading frame may yield structurally abnormal β-globin chain with altered amino acid sequences and protein structures that can affect the globin chain stability, leading to thalassemia of uncertain pathogenic significance. The high rate and diversity of in-frame Indels as an unintended by-product of gene correction therapy confers a challenge for predicting therapeutic outcomes when it is not possible to frame the clinical behavior of hemoglobin (Hb) variants. Here we provided the molecular and structural characterization of the most commonly identified Hb variants due to CRISPR-induced in-frame deletions and proposed a putative pathogenic mechanism for the observed phenotype using human umbilical cord-derived erythroid progenitor cells (HUDEP2). Methods: HUDEP2 clonal cell lines carrying mutated Hb variants from in-frame deletions on HBB exon1 introduced by R-66S and R-02 CRISPR sgRNAs currently in use in preclinical and clinical studies for correcting sickle mutation, respectively, were established, and clonal genotypes validated by next-generation sequencing and droplet digital PCR based copy number assay. The Hb variants derived from R-66S include p.Val7_Ser10del (-12bp), p.Val7del (-3bp), p.Glut8del (-3bp), p.Glut8_Lys9del (-6bp), pVal7Thr13del (-21bp), p.His3_Lys9delinsGln (-18bp), and p.Thr5_Lys18del (-42bp), in the order of allelic abundance observed in the bulk population of edited cells. The Hb variants derived from R-02 include p.Val12_Ala14del (-9bp), p.Val12_Thr13del (-6bp), p.Ala14del (-3bp), p.Thr13_Gly17del (-15bp), and p.Thr13_Val19delinsMet (-18bp). Selected ten clonal cell lines carrying a unique combination of genotypes and the parent HUDEP2 were cultured for expansion in serum-free media for one week. All clones were subsequently subjected to erythroid differentiation media for ten days. Samples were collected on days 4, 7, and 10 of differentiation for analysis via high-performance liquid chromatography (HPLC) using an ionic column to access the Hb profile and spectrophotometry via a Hb assay kit to assess the total Hb quantity. Results: HPLC analysis demonstrated a significant qualitative variation in Hb profile in HUDEP2 clones carrying different Hb variants. p.Val7del is Hb Leiden, and the rest of Hb variants are not listed in the HbVar database. In clones carrying Hb Leiden in homozygous or heterozygous states, Hb Leiden eluted in the position of sickle Hb (HbS). A low percentage of multiple abnormal Hb X fractions was observed in all clones carrying Hb variants compared to parent HUDEP2, which hinted at the presence of unstable Hb variants or alpha precipitates. Levels of fetal Hb (HbF) increased in mutant clones, suggesting globin chain imbalance and hematopoietic stress. Clones missing multiple amino acids showed ineffective erythropoiesis and cell death by day 4 of differentiation, most likely exacerbated by the relative excess of the α-chains. Discussion: We found that unintended gene modifications can significantly impact Hb quality and quantity, reinforcing concerns about the safety of gene-editing therapy for SCD. In patients, cells carrying in-frame deletions in the β-globin chain could produce an unstable Hb and may contribute to a β-thalassemia-like phenotype. More work is required to further assess the consequences of the diverse Hb variants in combination with other unintended modifications that disrupt HBB in a bulk population of gene-edited cells. Detecting, profiling, and reducing these unintended modifications remains a priority to increase the efficacy and safety of gene-editing therapy for treating SCD or β-thalassemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigating Gene Expression Signatures and Cell-Type Composition Changes in Sickle Cell Disease Using Whole Blood Transcriptomics

Background: Sickle cell disease (SCD) is a devastating Mendelian disease affecting millions of people worldwide. Although SCD is caused by a single-point mutation (Glu6Val) in the β-globin chain of hemoglobin, the clinical manifestations are heterogeneous and systemic, impacting the functions of multiple organs and leading to early mortality. Predicting disease severity and identifying the molecular mechanisms underlying disease pathology and progression remain a challenge. Objective: The goal of this work was to characterize the clinical heterogeneity associated with SCD using transcriptomics. Methods: We integrated publicly available bulk and single-cell RNA sequencing (scRNA-seq) data sets that included samples from pediatric to young adult patients and contained clinical phenotypes to identify molecular signatures of SCD severity and heterogeneity (Table). Our integrative transcriptomics analyses included the following: Identify cell-type clusters and derive novel progenitor signatures from an integrative analysis of the 2 independent scRNA-seq data sets capturing CD34+-enriched or unselected bone marrow (BM) cells from SCD and control samples.Calculate the relative proportions of cell types in whole blood by deconvolution using both the scRNA-seq-derived novel cell-type signatures (dtangle) and publicly available immune cell-type signatures (xCell, EPIC, quanTIseq).Identify signatures (genes, pathways, cell types) associated with disease severity and hemoglobin genotypes using various bioinformatics approaches, including differential gene expression and single-sample Gene Set Enrichment Analysis, among others, and examine relationships of enriched cell types and pathways (queried from MSigDB) in discovery followed by confirmation in validation data sets. Results: Cell-type clusters and gene signatures were generated by combining and integrating scRNA-seq data sets from BM SCD and control samples (Table). Using our fine-mapped cell-type clusters, we found an erythroid progenitor subclass to be depleted, whereas a lymphoid progenitor subclass expanded in SCD BM samples, increasing granularity of the cell types reported by Hua et al. Cell-type deconvolution of bulk transcriptome data using the combined scRNA-seq-derived signatures revealed enriched monocyte relative proportions in acute crisis compared with steady-state SCD samples (β=0.01, P=1.13×10−7) and enriched erythroblast relative proportions in SCD compared with control samples (β=0.02, P=1.10×10−12). Deconvolution using publicly available immune cell signatures showed significant enrichment of neutrophil and macrophage signatures in acute SCD samples (Padj<0.1). Analysis of bulk RNA-seq data indicated a set of highly correlated genes differentially expressed in steady-state SCD and associated with varied pathways, including red blood cell (RBC) morphology, hematopoiesis, anemia, and cholelithiasis. Higher pathway scores were seen in severe (HbSS) genotype samples than in milder (HbSC) genotype samples, consistent with known disease pathology and severity (Figure). Building on results from integrative analyses, we found that interferon and inflammatory response genes were enriched in acute SCD in both discovery and validation data sets. We found significant correlations between monocyte relative proportions and interferon signaling pathway scores, and between erythroblast relative proportions and the pathway scores affecting RBC production, morphology, and pathology and potential SCD comorbidities such as cholelithiasis (Figure). Conclusions: Our integrative scRNA-seq analysis led to robust cell cluster identification to improve deconvolution analysis of bulk transcriptomics data. We found elevated interferon and inflammatory pathway activity in acute SCD samples, consistent with findings in the literature (PMIDs: 34166491, 32764656). Furthermore, we found enriched neutrophils and monocyte signatures in acute SCD, suggesting their role in mediating the innate immune response. These findings provide motivation to characterize the role of neutrophils and monocytes in acute SCD crises. Our integrative transcriptomics analyses showed defective RBC production and maturation in SCD. Using the signatures from this work, we aim to elucidate the heterogeneity within SCD pathophysiology. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increased autophagy leads to decreased apoptosis during β-thalassaemic mouse and patient erythropoiesis

β-Thalassaemia results from defects in β-globin chain production, leading to ineffective erythropoiesis and subsequently to severe anaemia and other complications. Apoptosis and autophagy are the main pathways that regulate the balance between cell survival and cell death in response to diverse cellular stresses. Herein, the death of erythroid lineage cells in the bone marrow from both βIVS2-654-thalassaemic mice and β-thalassaemia/HbE patients was investigated. Phosphatidylserine (PS)-bearing basophilic erythroblasts and polychromatophilic erythroblasts were significantly increased in β-thalassaemia as compared to controls. However, the activation of caspase 8, caspase 9 and caspase 3 was minimal and not different from control in both murine and human thalassaemic erythroblasts. Interestingly, bone marrow erythroblasts from both β-thalassaemic mice and β-thalassaemia/HbE patients had significantly increased autophagy as shown by increased autophagosomes and increased co-localization between LC3 and LAMP-1. Inhibition of autophagy by chloroquine caused significantly increased erythroblast apoptosis. We have demonstrated increased autophagy which led to minimal apoptosis in β-thalassaemic erythroblasts. However, increased PS exposure occurring through other mechanisms in thalassaemic erythroblasts might cause rapid phagocytic removal by macrophages and consequently ineffective erythropoiesis in β-thalassaemia.

Evaluation of the Function of a Rare Variant in the 3'-Untranslated Region of the β-Globin Gene

β-Thalassemia (β-thal) is an inherited genetic disease that occurs because of the absence or reduction of β-globin chain synthesis. Genetic changes occur in different regions of the β-globin gene, but these mutations are less reported in the 3' untranslated region (3'-UTR). The objective of the present investigation was to evaluate the functional effect of a rare variant in the 3'-UTR of the β-globin gene. A variant at the first nucleotide of the 3'-UTR of the β-globin gene (HBB: c.*1G > A) was identified by DNA sequencing in an individual with low hematological indices and a normal hemoglobin (Hb) electrophoresis pattern. To evaluate the functional effect of this variant, the normal and mutated 3'-UTR of the β-globin gene was synthesized separately and sub cloned in the psiCHEK2 vector. Next, using the calcium phosphate method, the psiCHEK2 vectors containing normal and mutated 3'-UTR were transfected separately into the HEK293T cell line. Finally, the transfected cell line was analyzed by dual luciferase assay. The ratio of Renilla to firefly for the mutant sample was 1.26 ± 0.06, while for normal samples it was 1.12 ± 0.04. The results of the luciferase assay showed that there was no significant difference in the functional effect between the mutant and wild type construct. Therefore, it was concluded that this variant might not reduce the expression of the β-globin gene. Future studies by globin chain synthesis or to evaluate the expression of the gene in erythroid cells, might be necessary to understand the regulatory function of this mutation.

Direct sequencing of β-globin gene reveals a rare combination of two exonic and two intronic variants in a β-thalassemia major patient: a case report

BackgroundDue to indels in the β-globin gene, patients with β-thalassemia major exhibit a range of severity, with genotype β0β0 > β0β+ > β+β+, according to the production level of the β-globin chain. More than 300 mutations have been identified in the β-globin gene.Case presentationIn this case study, we report a compound heterozygous condition with a rare concoction of four different variants (CD 3(T > C), CD41/42 (-CTTT), IVS II-16 (G > C), and IVS II-666 (C > T) in a single β-globin gene. A regular transfusion-dependent 4-year-old male patient from India was included in the study. Augmented direct sequencing of the β-globin gene helped reveal the presence of an unusual combination of different variants in a single gene. This patient clinically presented as β-thalassemia major and was genotypically considered as β0β+, although CD41/42(-CTTT) was the only causative/pathogenic mutation in the disease severity.ConclusionAlthough CD41/42-(CTTT) is the only pathogenic variant among the four variants, the clinical complications of such a combination of variants (pathogenic and benign) is not well understood. Intronic mutations may have the ability to modify clinical characteristics. The variants must therefore be reclassified using additional mRNA splicing and expression-based studies. Additionally, these types of combinations may have significance in studying population migration around the world.