Α-globin Gene Research Articles

Molecular characterization of alpha globin and beta globin genes in patients with hemoglobinopathies in Central Vietnam

Background: Hemoglobinopathy is the most common monogenic disease worldwide. The aims of the current study were: (1) to investigate some hematological characteristics of patients with hemoglobinopathies; and (2) to detect the mutation of α-globin and β-globin genes, as well as the association between genotype and degree of anemia. Materials and method: 251 patients with hemoglobinopathies were examined for the α-globin or β-globin gene mutations. Results: 51% were the carriers, and 49% were thalassemia intermedia or thalassemia major. Hematological characteristics were suitable for α-thalassemia or β-thalassemia. Elevenβ-globin gene mutations were observed. The β0/βA, βE/βA, βE/βE, βE/β+, β+/β+ genotypes were only found in β-thalassemia intermedia individuals; the β0/β0 genotype was limited to β-thalassemia major patients; the β+/β0 and βE/β0 genotypes were seen in both types. Four α-globin gene mutations were observed. All α-thalassemia patients were intermedia, the most common genotype was --SEA/-α3.7. Conclusion: There were differences in anemia degree between β-globin genotypes Key words: hemoglobinopathies, α-globin, β-globin.

Novel Decision Tool for More Severe α-Thalassemia Genotypes Screening with Functional Loss of Two or More α-Globin Genes: A Diagnostic Test Study.

After the exclusion of iron deficiency and β-thalassemia, molecular research for α-thalassemia is recommended to investigate microcytic anemia. Aiming to suggest more efficiently the molecular analysis for individuals with a greater chance of having a symptomatic form of the disease, we have developed and validated a new decision tool to predict the presence of two or more deletions of α-thalassemia, increasing considerably the pre-test probability. The model was created using the variables: the percentage of HbA2, serum ferritin and mean corpuscular volume standardized by age. The model was trained in 134 patients and validated in 160 randomly selected patients from the total sample. We used Youden's index applied to the ROC curve methodology to establish the optimal odds ratio (OR) cut-off for the presence of two or more α-globin gene deletions. Using the OR cut-off of 0.4, the model's negative predictive value (NPV) was 96.8%; the cut-off point accuracy was 85.4%; and the molecular analysis pre-test probability increased from 25.9% to 65.4% after the use of the proposed model. This tool aims to assist the physician in deciding when to perform molecular studies for the diagnosis of α-thalassemia. The model is useful in places with few financial health resources.

Alpha-Thalassemia: Diversity of Clinical Phenotypes and Update on the Treatment

One of the more common single-gene disorders worldwide is α-thalassemia, carriers of which are found at variable frequencies (>1%) across all tropical and subtropical countries. Two linked α-globin genes on each allele of chromosome 16 regulate α-globin chain production. Deletion of one or more α-globin genes is the most frequent molecular defect found in α-thalassemia, whereas non-deletional mutations also occur, leading to unstable α-globin chains. HbH is the most common clinically important α-thalassemia disease and occurs when three α-globin genes are deleted/mutated, leaving only one copy of the gene intact. HbH can be divided into deletional (--/-α) and non-deletional genotypes (--/αTα). Whereas clinical phenotypes of the former are usually homogenously mild to moderate, those of the latter can be diverse. As HbH disease is particularly prevalent in Southeast Asia and some parts of the Mediterranean region, where β-thalassemia is also prevalent, affected patients are sometimes left undertreated. Therefore, hematologists and general physicians need to be educated to provide optimal disease monitoring and early identification of those with more severe phenotypes. Some issues regarding transfusion and iron chelation management differ from those of β-thalassemia, and these need to be recognized. Hb Bart’s hydrops fetalis syndrome (BHFS) is the most severe form of α-thalassemia; affected patients lack production of α-globin chains. Recent advances in fetal medicine and neonatal intensive care have made it possible for BHFS to no longer constitute a universally fatal disorder. Transfusion and chelation strategies for rare survivors are distinct and require updating.

Lnp-Targeting Hematopoietic Stem Cells and Lentiviral Gene Transfer to Generate and Rescue a Novel Mouse Model of Lethal Alpha-Thalassemia

α-Thalassemia (α-thal) is caused by insufficient production of the α-globin protein. Clinical presentation of α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to Bart's Hydrops Fetalis Syndrome/BHFS (complete knockout). In patients with severe α-thal, allogeneic bone marrow transplantation may be required for survival. Alternatively, patients could be treated through autologous BMT following ex vivo transduction with a human α-globin expressing lentiviral vector. However, a proper model system and vector must be established for the development of such a treatment. We crossed animals that had deletion of both Hba-a1 and Hba-a2 in heterozygosity (Hba-a1+/ko/Hba-a2+/ko) to generate knockout (KO) embryonic mice lacking all the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko). Although these embryos die perinatally, their fetal liver cells (FLC) at 13.5 dpc are still viable and represent a source of engraftable hematopoietic stem cells (HSC). KO-FLC were transplanted into congenic myeloablated WT mice to generate mice carrying Hba-a1ko/ko/Hba-a2ko/ko HSC. These animals developed a severe phenotype ~7 weeks post engraftment. As these mice lack α-globin expression, we observed β-tetramer formation (β4, HbH, Fig1A), resulting in aberrant red blood cell (RBC) morphology, β-globin precipitates, high RBC oxygen binding affinity, elevated erythropoietin, hematocrit and hemoglobin levels, splenomegaly and vaso-occlusive episodes, due to the large number of abnormal RBCs. Furthermore, we observed iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver. However, FLC engraftment is cumbersome and requires extensive breeding and many animals to generate enough FLC for few recipients. To facilitate the generation of α-thal mice, we created conditional knockout (cKO) mice that have been edited to remove Hba-a2, while inserting loxP sites flanking Hba-a1 (Hba-a1fl/Hba-a2ko). Homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) demonstrate a mild phenotype. To achieve complete deletion of the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko) in HSC, we used lipid nanoparticles (LNPs), which are one of the most promising delivery systems to package and make mRNA into a useful therapeutic. We developed a novel LNP preparation with an antibody to target CD117 and deliver nucleoside-modified mRNA to HSC, (see abstract Breda et al). We treated HSC from homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) ex vivo with CD117-LNP embedded with Cre mRNAs (Cre-CD117-LNP). Cells showed the expected deletion of the α-globin genes and were injected into myeloablated recipient mice. These chimeras recapitulated the phenotype observed following transplantation of constitutive α-globin-KO FLC, including elevated hematocrit, splenomegaly, and culminating in lethality ~7 weeks post-transplant. We screened multiple potential erythroid specific lentiviral vectors in both mouse and human cell lines and identified a promising candidate, ALS20αI, which at VCN=1 produced human α-globin protein equivalent to that of a single endogenous α-globin gene. Myeloablated recipient mice transplanted with Cre-CD117-LNP-treated cKO BM die roughly 7 weeks post-transplant with the expected pathological phenotype (Fig1A). In contrast, mice receiving BM treated with Cre-CD117-LNP and ALS20αI (VCN>1) all survived long term (>4-month) with normalization of erythropoiesis and iron metabolism. VCNs were proportional to chimeric hemoglobin (human α/mouse β) levels and concurrently decreased or absent HbH (Fig1B-C-D). Additionally, we tested ALS20αI in erythroid progenitors derived from CD34 cells isolated from patients with deletional and non-deletional HbH disease. Preliminary data demonstrates improvement in α-globin/β-globin mRNA ratio and reduction in formation of HbH by HPLC. In summary, we have developed new mouse models for α-thal with a lethal and reproducible phenotype. Remarkably, use of LNP technology accelerated the generation of α-thal mice and their characterization. Furthermore, our new vector ALS20αI rescued these animals with stable expression of the human α-globin gene. These data strongly support the use of ALS20αI for the cure of severe forms of α-thal, as 2 copies may be sufficient to rescue patients affected by BHFS, while 1 copy may be sufficient to cure patients affected by HbH disease. HP, SR corresponding Authors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Ancient Transcriptional Hub Couples Developmentally Regulated Gene Expression with Metabolism during Erythropoiesis

The α-globin genes and their regulation by the α-globin superenhancer have been extensively characterised. An adjacent gene, Nprl3, contains 4 of the 5 α-globin enhancers in mice, and 3 of 4 in humans. The neighbouring genomic positions of Nprl3 and α-globin, which share a topologically associated domain, have been maintained for >500 million years. However, the potential functional requirement of this genomic linkage has not been investigated until now. Nprl3 is conserved across Animalia and Fungi, and has been characterised to provide negative regulation of mTORC1, a critical controller of cellular metabolism. Here we report phenotypes of Nprl3-deficient erythropoiesis in mouse fetal liver and bone marrow, and in human peripheral blood. We provide molecular mechanistic insight that Nprl3 is essential for optimal erythropoiesis and for responding to fluctuating nutrient and growth factor concentrations. Finally, we show that erythroid Nprl3 function depends on transcriptional support arising from the α-globin enhancers. Analysis of Nprl3-/- fetal livers revealed severe impairment of erythroid development. On day E13.5, erythroid cells fail to develop beyond the proerythroblast stage. Loss of Nprl3 is accompanied by elevated mTORC1 signalling, confirming the canonical signalling role of Nprl3 in erythroblasts. Active mTORC1 typically inhibits autophagy; in consistence with this, we observed suppressed autophagic activity in Nprl3-/- erythroblasts. We next investigated whether the erythroid defect results from haematopoietic-intrinsic Nprl3 requirements. This was tested using competitive bone marrow - fetal liver chimera experiments, in which Nprl3-/- fetal liver cells contributed poorly to erythroid lineage reconstitution of irradiated adult bone marrow. Thus, Nprl3 regulates baseline erythropoiesis in the mouse. To discover whether NPRL3 is also required for human erythropoiesis, NPRL3-knockout was induced in primary human CD34+ progenitors using a CRISPR-Cas9 RNP-editing system. These progenitors had reduced ability to produce enucleated erythroid cells compared to their negative control counterparts. NPRL3-knockout erythroblasts also demonstrated defective mTORC1 signalling responses to iron deficiency, amino acid withdrawal and erythropoietin stimulation. These results indicate that NPRL3 has a critical role in interpreting fluctuating nutritional environments, and in tuning the metabolic response of developing erythroid cells to the extracellular milieu. Importantly, Nprl3 expression is known to be elevated in erythroid cells, increasing ~30-fold during erythroid lineage commitment due to α-globin enhancer activity. We sought to discover whether this erythroid-specific increase in Nprl3 expression is required for the functionality outlined above. Using genetic engineering to delete the Nprl3 promoter on one allele, and to delete all α-globin enhancers on the other, we eliminated all interactions between Nprl3 and α-globin regulatory elements. Embryos heterozygous for both alleles (Nprl3+/-α-globin-enhancers+/-,'Nalph') retain non-erythroid 'un-enhanced' Nprl3 expression levels, and enhancer-regulated α-globin expression. Remarkably, at E13.5, the Nalph genotype presented impaired erythropoiesis reminiscent of that observed in Nprl3-/- embryos, with development inhibited at the same stage of differentiation. Thus, we conclude that the genomic contact hub Nprl3 shares with α-globin and its enhancers bestows the erythroid-specific transcriptional upregulation required for Nprl3 to perform its important erythropoietic role. This finding suggests that the deep evolutionary coupling of these two genes has enabled the α-globin enhancers to control metabolism, as well as α-globin production, in developing erythroid cells.

Pregnancy Surveillance in Sickle Cell Disease Patients: A Cohort Study in an African Country - Angola

Sickle Cell Disease (SCD) is a severe monogenic disease caused by the inherence of a recessive mutation in the β-globin gene, with an especially high prevalence in sub-Saharan Africa. In Angola, the prevalence of the disease is almost 2%, and the carriers reach 21% of the population. Although its presentation tends to be very heterogeneous, chronic hemolytic anemia, frequent painful crisis and extensive organ damage are common features of these patients. Pregnancy in SCD patients is associated with an increase in severe outcomes, namely, high risk of eclampsia and pre-eclampsia, stroke and even death. Therefore, it is crucial to maintain continuous medical surveillance during pregnancy, especially in women with previous strokes. Moreover, health services in low- and middle-income countries are generally not prepared to follow these patients. The present cohort study, conducted at the Lucrecia Paim Maternity Hospital (Luanda, Angola), aims to determine pregnancy complications in SCD women, especially those responsible for maternal death, and, by supporting the obstetric consultations in this hospital, contribute to the reduction of mortality and morbidity rates. Pregnancy monitoring includes analysis of clinical history and incidents (number of hospitalizations, blood transfusions, strokes and other clinical complications), hematological and biochemical analysis, transcranial doppler to assess cerebral hemodynamics and genetic analysis (confirmation of the diagnosis, genotyping of four SNPs in the β-cluster to assess the haplotype, and evaluation of the presence of the 3.7kb deletion of the α-globin gene). To date, 61 women are being followed in the obstetric consultations, with ages from 18 to 40 years old (mean 26.1±5.4). There are no records of previous strokes, although 83.9% of the patients have been previously transfused (47 out of 56), 98.2% have been hospitalized (55 out of 56) due to SCD complications and 19.6% (10 out of 54) had at least one miscarriage. At the first appointment, total hemoglobin values ranged from 4.70 to 10.40 g/dL (n=52, mean 7.18±1.30), erythrocytes from 1.46 to 5.42 x1012/L (n=52, mean 2.46±0,72), white blood cells count from 1.67 to 61.88 x109/L (n=51, mean 12.20±8.69), platelets from 24.2 to 710.0 x109/L (n=52, mean 272.2±155.9), and lactate dehydrogenase (LDH) from 263.3 to 2836.7 (n=50, mean 708.1±450.46). The CAR/CAR haplotype, which is usually associated with a more severe prognosis, is the most prevalent in this population (57.7%, 30 out of 52), followed by the CAR/SEN haplotype (25.0%, 13 out of 52). In this population, 17.3% (9 out of 52) are homozygous for the 3.7kb α-thalassemia deletion and 44.2% (23 out of 52) are carriers. This deletion influences hematological and clinical aspects of sickle cell disease patients, resulting in less severe phenotypes. TCD time-averaged mean of the maximum velocity (TAMMx) at the middle cerebral arteries ranged between 41 to 132 cm/s (n=61, mean 84cm/s) and peak systolic velocity (PSV) from 61 to 180 cm/s (mean 129 cm/s). At the basilar artery level, TAMMx obtained were between 29 to 102 cm/s (n=60, mean 52 cm/s) and PSV ranged from 43 to 141 cm/s (mean 78 cm/s). The main goal of this project is to study pregnancy-related complications and outcomes by giving support to an Angolan cohort of SCD pregnant women, seeking to reduce them and improve these women and neonates' future quality of life. By the end of the project, we also intend to obtain TCD reference values of cerebral blood flow velocities in pregnant women with SCD as a risk predictor of vascular events as there are no values in the literature for this specific population. This project is funded by Fundação Calouste Gulbenkian, Portugal, and by Instituto Camões - Instituto da Cooperação e da Língua, I.P.

Two Is Better Than One: Fetal Hemoglobin Reactivation and Alpha-Globin Downregulation to Correct the β-Hemoglobinopathy Phenotype through Base Editing

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult hemoglobin β-chain. In β-thalassemia, the reduced (β+) or absent (β0) production of β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent RBC sickling, vaso-occlusive crises, organ damage and reduced life expectancy. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors or CRISPR/Cas9 nuclease are not equally effective in all the patients and/or raise safety concerns. Base editing (BE) allows the introduction of point mutations (C>T by cytidine base editors, CBEs; A>G by adenine base editors, ABEs) without generating dangerous double strand breaks, thus allowing the multiplexed editing of several targets. Mutations leading to elevated fetal Hb (HbF) or reduced α-globin expression are associated with a better clinical phenotype in both β-thalassemia and SCD patients. Fetal β-like γ-globin compensates for adult β-globin deficiency and reduced α-globin levels impairs its precipitation and inhibit Hb polymerization in SCD. In this study, we have developed BE strategies aiming to (i) reactivate HbF by down-regulating BCL11A, a master repressor of γ-globin expression though inactivation of its enhancers, and (ii) reduce the α-globin levels through targeting of the MCS-R2 enhancer of the α-globin genes (HBA1/2). First, we used CBEs and ABEs to target the GATA1 or ATF4 activator binding site (BS) within the erythroid-specific BCL11A enhancers (+58-kb and +55-kb regions) in SCD HSPCs through RNA delivery and achieved efficiencies of up to ~90%. A colony forming cell (CFC) assay showed no impact on progenitors' viability. RBCs derived from base-edited HSPCs exhibited high HbF expression levels that were sufficient to ameliorate the sickling phenotype. Samples carrying different mutations within the GATA1 or ATF4 BS were associated with different HbF levels, implying that some nucleotides are more essential for GATA1 and ATF4 binding and the consequent BCL11A gene activation. In parallel, we used CBEs and ABEs to target the GATA1 or NF-E2 activator BS within the MCS-R2 enhancer of HBA1/2. We screened single guide RNAs (sgRNAs) in an erythroid cell line (K562) that allowed editing of different nucleotides within the GATA1 and NF-E2 BS and we identified the most efficient sgRNAs diminishing α-globin expression. Next, selected sgRNAs were delivered to SCD HSPCs, achieving high editing efficiency (~85%). A CFC assay showed a good progenitor viability. RT-qPCR and Western Blot analyses in the erythroid progeny of edited SCD HSPCs confirmed α-globin downregulation at RNA and protein levels. Lastly, we combined both strategies using ABEs to target simultaneously BS within the BCL11A and MCS-R2 enhancers in SCD HSPCs. The concomitant usage of two sgRNAs did not affect editing efficiency at the different targeted sites, which was ~80% at both BCL11A and MCS-R2 enhancers. The erythroid progeny of edited SCD HSPCs harboring mutations in both BCL11A and MCS-R2 enhancers showed downregulation of α-globin and up-regulation of γ-globin at RNA and protein expression level. We expect that this combined strategy will efficiently rescue the pathological phenotype of RBCs derived from both SCD and β-thalassemic HSPCs. In conclusion, our study identified the core nucleotides of both the BCL11A and HBA1/2 MCS-R2 enhancers, which are crucial for regulating the expression of these genes and represent potential targets for scarless editing through BE to finely modulate globin expression. We also developed efficient BE strategies to simultaneously reactivate HbF and downregulate the α-globin expression in patient HSPCs. Validation of the above-described results in HSPCs in vivo will provide sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies.

Effect of Luspatercept on Red Blood Cell (RBC) Transfusion Burden, Iron Chelation Therapy (ICT), and Iron Overload in Adults with Transfusion-Dependent β-Thalassemia (TDT) from the BELIEVE Trial: A Long-Term Analysis

Effect of Luspatercept on Red Blood Cell (RBC) Transfusion Burden, Iron Chelation Therapy (ICT), and Iron Overload in Adults with Transfusion-Dependent β-Thalassemia (TDT) from the BELIEVE Trial: A Long-Term Analysis

Effect of Luspatercept in β-Thalassemia Patients with β0/β0 Genotype: A Subgroup Analysis of the BELIEVE Study

Introduction: β-thalassemia is an inherited hemoglobinopathy caused by mutations in the HBB gene that encodes β-globin. Patients (pts) homozygous for mutations that severely reduce or silence expression of HBB (β0/β0 genotype) do not produce any functional β-globin, resulting in an imbalance of globin chains, ineffective erythropoiesis, and severe anemia. Pts with a β0/β0 genotype require frequent, lifelong red blood cell (RBC) transfusions to address their anemia. However, regular RBC transfusions are associated with increased risk of morbidities and mortality. As such, interventions that can reduce dependence on RBC transfusions are important for improving outcomes in this pt population. Results from the phase 3 BELIEVE study showed that a higher percentage of pts with transfusion-dependent β-thalassemia who received luspatercept experienced reductions in RBC transfusion burden than pts receiving placebo in the first 48 weeks (wk) of the study (Cappellini MD, et al. N Engl J Med 2020;382:1219-1231) and the efficacy of luspatercept has been shown to persist with longer-term treatment (Cappellini MD, et al. HemaSphere 2022;6 [Suppl 3]. Abstract 270). The aim of this post-hoc sub-analysis was to investigate the long-term efficacy of luspatercept in pts with β0/β0 genotypes from the BELIEVE trial. Methods: Pts ≥ 18 years of age with β-thalassemia or hemoglobin E/β-thalassemia (compound β-thalassemia mutation and/or multiplication of α-globin genes allowed) and requiring regular RBC transfusions (6-20 RBC units in the 24 wk prior to randomization, no transfusion-free period > 35 days) were randomized 2:1 to receive luspatercept (1.0-1.25 mg/kg) or placebo subcutaneously every 3 wk. Response was defined as ≥ 33% or ≥ 50% reduction from baseline in RBC transfusion burden during any 12- or 24-wk interval over the entire study period. Genotyping was performed if this information was not included in the pt's medical history. Results: Of the 336 pts enrolled in the BELIEVE study, 103 had a β0/β0 genotype. HBB mutations represented in the β0/β0 subgroup were: c.17_18delCT; c.20delA; c.25_26delAA; c.27dupG; c.52A>T; c.84_85insC; c.92G>A; c.92+1G>A (IVS1-1); c.92+1G>T (IVS1-1); c.92+2T>G; c.93-15T>G (IVS1-116); c.112delT; c.114G>A; c.118C>T; c.126_129delCTTT; c.135delC; c.217dupA; c.315+1G>A (IVS2-1); c.315+1G>T (IVS2-1); c.316-2A>C (IVS2-849); and deletion of HBB. In the intent-to-treat (ITT) population, 224 pts received luspatercept, 68 of whom had a β0/β0 genotype. Median (range) duration of treatment for pts receiving luspatercept was 153.57 (1.7-215.0) wk. In the ITT luspatercept population, 173 (77.2%) pts achieved ≥ 33% reduction in RBC transfusion burden and 112 (50.0%) pts achieved ≥ 50% reduction in RBC transfusion burden during any 12-wk interval over the entire study period (Figure 1A). For pts with a β0/β0 genotype, 52 (76.5%) achieved ≥ 33% reduction in RBC transfusion burden and 36 (52.9%) achieved ≥ 50% reduction during any 12-wk interval. Transfusion burden reductions of ≥ 33% and ≥ 50% over any 24-wk interval were reported in 116 (51.8%) and 53 (23.7%) pts in the ITT population, respectively. Of the pts with a β0/β0 genotype, 36 (52.9%) achieved ≥ 33% reduction and 11 (16.2%) achieved ≥ 50% reduction in RBC transfusion burden during any 24-wk interval. Responses in the β0/β0 subgroup were also evaluated by splenectomy status (n = 40 splenectomized and n = 28 non-splenectomized pts). Of the splenectomized pts, 34 (85.0%) achieved ≥ 33% reduction in RBC transfusion burden and 27 (67.5%) ≥ 50% reduction during any 12-wk interval; 18 (64.3%) and 9 (32.1%) non-splenectomized pts, respectively, achieved these responses (Figure 1B). Over any 24-wk interval, 24 (60.0%) and 8 (20.0%) splenectomized pts achieved ≥ 33% reduction and ≥ 50% reduction in RBC transfusion burden, respectively. For non-splenectomized pts, reductions in RBC transfusion burden of ≥ 33% and ≥ 50% were reported in 12 (42.9%) and 3 (10.7%) pts, respectively. Conclusion: Pts with a β0/β0 genotype in the BELIEVE trial experienced similar reductions in RBC transfusion burden with luspatercept as the overall ITT luspatercept population. Furthermore, greater proportions of splenectomized pts with a β0/β0 genotype achieved reductions in RBC transfusion burden than β0/β0 pts who were not splenectomized. These longer-term data demonstrate the efficacy of luspatercept in a pt population with more severe disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Co-inherited α-Globin Gene Cluster Duplication Compromises RBC Indices-Based Thalassemia Screening.

Co-inherited α-Globin Gene Cluster Duplication Compromises RBC Indices-Based Thalassemia Screening.

Genotype-Phenotype Study of β-Thalassemia Patients in Sabah

β-thalassemia is a serious public health problem in Sabah due to its high prevalence. This study aimed to investigate the effects of different types of β-globin gene mutations, coinheritance with α-globin gene mutations, XmnI-Gγ, and rs368698783 polymorphisms on the β-thalassemia phenotypes in Sabahan patients. A total of 111 patients were included in this study. The sociodemographic profile of the patients was collected using a semi-structured questionnaire, while clinical data were obtained from their medical records. Gap-PCR, ARMS-PCR, RFLP-PCR, and multiplex PCR were performed to detect β- and α-globin gene mutations, as well as XmnI-Gγ and rs368698783 polymorphisms. Our data show that the high prevalence of β-thalassemia in Sabah is not due to consanguineous marriages (5.4%). A total of six different β-globin gene mutations were detected, with Filipino β°-deletion being the most dominant (87.4%). There were 77.5% homozygous β-thalassemia patients, 16.2% compound heterozygous β-thalassemia patients, and 6.3% β-thalassemia/Hb E patients. Further evaluation on compound heterozygous β-thalassemia and β-thalassemia/Hb E patients found no concomitant α-globin gene mutations and the rs368698783 polymorphism. Furthermore, the XmnI-Gγ (−/+) genotype did not demonstrate a strong impact on the disease phenotype, as only two of five patients in the compound heterozygous β-thalassemia group and two of three patients in the β-thalassemia/Hb E group had a moderate phenotype. Our findings indicate that the severity of the β-thalassemia phenotypes is closely related to the type of β-globin gene mutations but not to the XmnI-Gγ and rs368698783 polymorphisms.

The prevalence and genetic disorders spectrum of thalassemia among breast cancer patients in Jiangxi province, China.

Background: Thalassemia is a common inherited hematological disease with genetic disorders characterized by imbalanced synthesis of the globin chains. Due to the improvement of treatment methods, patients with thalassemia can survive for a long time. Therefore, it is not uncommon for patients with thalassemia suffering from malignant tumors. However, there are quite few reports on thalassemia patients complicated with breast cancer. Herein, we try to investigate the prevalence and genetic disorders spectrum of thalassemia in patients with breast cancer. Methods: Blood routing tests and serum ferritin analysis were conducted in 1887 breast cancer patients treated in the department of radiation oncology during 1 April 2020 and 30 March 2022. The suspected thalassemia carriers with small mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH) or mean corpuscular hemoglobin concentration (MCHC) but the concentration of serum ferritin within normal limits were further investigated by polymerase chain reaction (PCR) and flow through hybridization gene chip to detect common mutations of α-globin and β-globin genes using Thalassemia Geno Array Diagnostic Kit. The prevalence and genetic mutation spectrum of thalassemia among breast cancer patients were analyzed. Results: Four hundred and eighty-nine suspected thalassemia carriers were detected by complete blood cell counts and serum ferritin analysis among 1887 breast cancer patients. One hundred and seven cases (5.7%) were identified as carriers of thalassemia, of which 55 cases (51.4%) were α-thalassemia, 50 cases (46.7%) were β-thalassemia, and 2 cases (1.9%) were co-inheritance of α-thalassemia and β-thalassemia simultaneously. In α-thalassemia, the most prevalent genotype is -SEA/αα; as for β-thalassemia, βIVS-II-654/β is the most common genotype. The degree of anemia is more severe in β-thalassemia than in α-thalassemia. Conclusion: This is the first comprehensive molecular epidemiological investigation on thalassemia among breast cancer patients. Our data indicated that thalassemia was not uncommon in breast cancer patients. The physicians should have the knowledge to avoid misdiagnosis as iron deficiency anemia.

Accurate genotype diagnosis of Hong Kongαα thalassemia based on third-generation sequencing.

The Hong Kongαα (HKαα) allele is a complex structural rearrangement of the α-globin gene containing -α3.7 and αααanti 4.2 crossover junctions. Clinically, individuals carrying the HKαα allele are often misdiagnosed or missed using conventional thalassemia gene detection technology. This study aims to identify and validate different HKαα thalassemia subtypes using third-generation sequencing (TGS) technology. Between January 2015 and June 2021, 32 patients suspected of having HKαα thalassemia were included in this study. Genomic DNA was extracted, and gap-polymerase chain reaction (PCR), two-round nested PCR, multiplex ligation-dependent probe amplification (MLPA), and TGS were used for thalassemia gene detection. The results of HKαα/αα and HKαα/-α3.7 were similar to -α3.7/αα using the gap-PCR method. Two-round nested PCR could be used to verify the HKαα gene, but could not distinguish the subtypes of HKαα thalassemia. The MLPA assay was used to detect the change in the copy number of the α-globin gene, but it could not determine whether -α3.7 and αααanti 4.2 were in cis or in trans. Long-read TGS technology could accurately detect the HKαα allele and distinguish the genotypes of HKαα/αα, HKαα/-α3.7, HKαα/-α4.2, and HKαα/--SEA without pedigree analysis. The contiguous sequence of the HKαα allele was detected using the TGS approach. This study also demonstrated that individuals with HKαα/αα and βN/βN genotypes tended to have normal hematological phenotypes. Long-read TGS is a reliable and efficient approach for accurate detection of HKαα thalassemia, which can be widely used in clinical practice. Accurate molecular diagnosis of HKαα thalassemia will benefit clinical genetic counseling and prenatal diagnosis.

RARE UNSTABLE ALPHA GLOBIN VARIANT HB TAYBE (HBA1:C.118_120DELACC) WITH HBA2 POLY A MUTATIONS, CAUSES TO HEMOLYTIC ANEMIA IN TWO CASES FROM AZERBAIJAN

Sequence variants are usually silent and rarer in α-globin, some may lead to an unstable protein with hemolytic or thalassemic phenotype. The most common HBA variant is ConstantSpring which is leading to an unstable elongated protein chain. Others may be due to ins/del in the α-globin, one of them Hb-Taybe caused deletion of Thr residue at codon 40 of the HBA1. We report, for the first time, 2 cases with hemolytic anemia due to the presence of Hb-Taybe in trans with HBA2 poly-A mutations. Patients were managed in the Thalassemia Unit of the National Hematology Center. Detailed pedigrees are drawn, medical recordings are reviewed and peripheral blood samples are collected. Sanger sequencing was perfomed with in house designed primers ( HBA1, NM_000558.5 and HBA2 , NM_000517.6) on genome analyser (ABI3500). Deletion and duplication analysis is performed by MLPA. P-1: A 4-yr-old male who was diagnosed at the age of 1 yr with congenital hemolytic anemia. He presented with jaundice, the blood film showed moderate hypochromia and anisopoikilocytosis. He was transfusion dependent. P-2: A 45-yr-old female who was diagnosed at the age of 5 yr with congenital hemolytic anemia. She underwent splenectomy at the age of 32 yr due to moderate anemia. After splenectomy she was not transfusion dependent. Relevant clinical and laboratory data is presented at table. The clinical presentation is variable from mild hemolytic anemia to regular transfusion requirement. One patient had to have splenectomy in order to ameliorate the transfusion requirement. This study supports the requirement of α-globin gene analyses, and a careful evaluation of cases with hemolytic anemia, particularly in populations where thalassemias are endemic, in order to avoid missing any of the rare globin variants and to offer accurate genetic counseling.

Clinical and Haematological Parameters of Commonly Reported Non-deletional α-thalassaemia Mutations in Southeast Asia: A Review

Alpha (α)-thalassaemia is a common genetic disorder worldwide caused by the deletion and rarely non-deletional mutations of the α-globin gene. Nearly 70 types of non-deletional mutations have been reported worldwide, and this review focuses on the common ones affecting α-thalassaemia patients. The common mutations are initiation codon mutation, codon 30, haemoglobin (Hb) Constant Spring, Hb Quang Sze, Hb Adana and Hb Evora. The haematological parameters of non-deletional mutations usually show mild changes. However, a severe reduction in haemoglobin level, mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular haemoglobin count (MCHC) has been observed among compound heterozygous HbH disease, involving both deletional and non-deletional mutations. Although non-deletional mutations are rarely reported, it requires the study of more cases to understand the clinical phenotypes that lead to severe clinical manifestations.

Molecular characterisation of Hb Akron [β52 (D3) Asp→Val] combined with thalassaemia in a Chinese family

AimsHb Akron (HBB:c.158A>T) is a rare β-chain variant and many characteristics about its clinical features still remain unclear. In this study, we aimed to explore the molecular and haematological characterisations...

Detection of four rare thalassemia variants using Single-molecule realtime sequencing.

Conventional methods for the diagnosis of thalassemia include gap polymerase chain reaction (Gap-PCR), reverse membrane hybridization (RDB), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. In this study, we used single molecule real-time technology (SMRT) sequencing and discovered four rare variants that have not been identified by conventional diagnostic methods for thalassemia. We also performed genotype and phenotype analyses on family members of thalassemia patients. The SMRT technology detected five cases in which the proband had abnormal results by conventional diagnostic methods or inconsistencies between the genotype and phenotype. The variants included two cases of an α-globin gene cluster 27,311 bp deletion, --27.3/αα (hg38 chr16:158664-185974), one case of an HS-40 region 16,079 bp deletion (hg38 chr16:100600-116678), one case of a rearrangement of -α3.7α1α2 on one allele and one case of a ß-globin gene cluster HBG1-HBG2 4,924 bp deletion (hg38 chr11:5249345-5254268). This study clarified the hematological phenotypes of four rare variants and indicated the application value of SMRT in the diagnosis of rare α-globin and ß-globin gene cluster deletions, gene recombination and deletion breakpoints. The SMRT method is a comprehensive one-step technology for the genetic diagnosis of thalassemia and is particularly suitable for the diagnosis of thalassemia with rare deletions or genetic recombination.

The hemoglobinopathies, molecular disease mechanisms and diagnostics.

Hemoglobinopathies are the most common monogenic disorders in the world with an ever increasing global disease burden each year. As most hemoglobinopathies show recessive inheritance carriers are usually clinically silent. Programmes for preconception and antenatal carrier screening, with the option of prenatal diagnosis are considered beneficial in many endemic countries. With the development of genetic tools such as Array analysis and Next Generation Sequencing in addition to state of the art screening at the hematologic, biochemic and genetic level, have contributed to the discovery of an increasing number of rare rearrangements and novel factors influencing the disease severity over the recent years. This review summarizes the basic requirements for adequate carrier screening analysis, the importance of genotype–phenotype correlation and how this may lead to the unrevealing exceptional interactions causing a clinically more severe phenotype in otherwise asymptomatic carriers. A special group of patients are β‐thalassemia carriers presenting with features of β‐thalassemia intermedia of various clinical severity. The disease mechanisms may involve duplicated α‐globin genes, mosaic partial Uniparental Isodisomy of chromosome 11p15.4 where the HBB gene is located or haplo‐insufficiency of a non‐linked gene SUPT5H on chromosome 19q, first described in two Dutch families with β‐thalassemia trait without variants in the HBB gene.

Co-Inheritance of Heterozygous β0-Thalassemia with Single Functional α-Globin Gene: Challenges of Carrier Detection in Pre-Marital Screening Program for Thalassemia

This is a report of a couple with abnormal hematological indices who were investigated for α & β-thalassemia mutations. Based on CBC and capillary hemoglobin electrophoresis results, the male and female subjects were β & α-thalassemia carriers, respectively. Multiplex-Gap-PCR and Sanger sequencing techniques were used for the identification of mutations on α and β-globin genes. The DNA test showed the presence of c.315 + 1 G > A mutation on β-globin gene of male subject while the female case had – MED double gene deletion and c.427T > C mutation on α-globin and, interestingly, she was also a carrier for c.315 + 1 G > A mutation on β-globin gene. Cases with the coinheritance of heterozygous β0-thalassemia with one functional α-globin gene have normal HbA2 levels that may lead to their being misdiagnosed as β-thalassemia carriers, especially in premarital screening programs for thalassemia. Therefore, β-globin gene sequencing is recommended in cases with normal Hb electrophoresis and reduced hematological indices in premarital screening programs for thalassemia, especially in regions with a high frequency of β-globin mutations, in order to identify all the β-thalassemia carriers.

Detection of maternal carriers of common α-thalassemia deletions from cell-free DNA

α-Thalassemia is a common inherited blood disorder manifested mainly by the deletions of α-globin genes. In geographical areas with high carrier frequencies, screening of α-thalassemia carrier state is therefore of vital importance. This study presents a novel method for identifying female carriers of common α-thalassemia deletions using samples routinely taken for non-invasive prenatal tests for screening of fetal chromosomal aneuploidies. A total of 68,885 Vietnamese pregnant women were recruited and α-thalassemia statuses were determined by gap-PCR, revealing 5344 women (7.76%) carried deletions including αα/−−SEA (4.066%), αα/−α3.7 (2.934%), αα/−α4.2 (0.656%), and rare genotypes (0.102%). A two-stage model was built to predict these α-thalassemia deletions from targeted sequencing of the HBA gene cluster on maternal cfDNA. Our method achieved F1-scores of 97.14–99.55% for detecting the three common genotypes and 94.74% for detecting rare genotypes (−α3.7/−α4.2, αα/−−THAI, −α3.7/−−SEA, −α4.2/−−SEA). Additionally, the positive predictive values were 100.00% for αα/αα, 99.29% for αα/−−SEA, 94.87% for αα/−α3.7, and 96.51% for αα/−α4.2; and the negative predictive values were 97.63%, 99.99%, 99.99%, and 100.00%, respectively. As NIPT is increasingly adopted for pregnant women, utilizing cfDNA from NIPT to detect maternal carriers of common α-thalassemia deletions will be cost-effective and expand the benefits of NIPT.