Hemoglobin Bart's hydrops fetalis: charting the past and envisioning the future.

ABSTRACT: Hemoglobin Bart's hydrops fetalis syndrome (BHFS) represents the most severe form of α-thalassemia, arising from deletion of the duplicated α-globin genes from both alleles. The absence of α-globin leads to the formation of nonfunctional hemoglobin (Hb) Bart's (γ4) or HbH (ß4) resulting in severe anemia, tissue hypoxia, and, in some cases, variable congenital or neurocognitive abnormalities. BHFS is the most common cause of hydrops fetalis in Southeast Asia; however, owing to global migration, the burden of this condition is increasing worldwide. With the availability of intensive perinatal care and intrauterine transfusions, an increasing number of patients survive with this condition. The current approach to long-term management of survivors involves regular blood transfusions and iron chelation, a task made challenging by the need for intensified transfusions to suppress the production of nonfunctional HbH-containing erythrocytes. Although our knowledge of outcomes of this condition is evolving, it seems, in comparison to individuals with transfusion-dependent ß-thalassemia, those with BHFS may face an elevated risk of complications arising from chronic anemia and hypoxia, ongoing hemolysis, iron overload, and from their respective treatments. Although stem cell transplantation remains a viable option for a select few, it is not without potential side effects. Looking ahead, potential advancements in the form of genetic engineering and innovative therapeutic approaches, such as the reactivation of embryonic α-like globin gene expression, hold promise for furthering the treatment of this condition. Prevention remains a crucial aspect of care, particularly in areas with high prevalence or limited resources.

Survival of transfused red blood cells from a donor with alpha-thalassemia trait in a recipient with sickle cell disease.

BACKGROUND: Post-transfusion survival of donor red blood cells (RBCs) is important for effective chronic transfusion therapy in conditions including sickle cell disease (SCD). Biotin labeling RBCs allows direct in vivo measurement of multiple donor RBC units simultaneously post-transfusion. STUDY DESIGN AND METHODS: In an observational trial of patients with SCD receiving monthly chronic transfusion therapy, aliquots of RBCs from one transfusion episode were biotin-labeled and infused along with the unlabeled RBC units. Serial blood samples were obtained to measure RBC survival. Donor units were tested for RBC indices, hemoglobin fractionation, and glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. For microcytic donor RBCs (MCV < 70 fL), HBA1 and HBA2 genetic testing was performed on whole blood. RESULTS: We present one recipient, a pediatric patient with SCD and splenectomy who received two RBC units with aliquots from each unit labeled at distinct biotin densities (2 and 18 µg/mL biotin). One donor unit was identified to have microcytosis (MCV 68.5 fL after biotinylation); whole blood sample obtained at a subsequent donation showed 2-gene deletion alpha-thalassemia trait (ɑ-3.7kb/ɑ-3.7kb) and normal serum ferritin. G6PD activity was >60% of normal mean for both. The RBCs with alpha-thalassemia RBC had accelerated clearance and increased surface phosphatidylserine post-transfusion, as compared with the normocytic RBC (half life 65 vs. 86 days, respectively). DISCUSSION: Post-transfusion RBC survival may be lower for units from donors with alpha-thalassemia trait, although the impact of thalassemia trait donors on transfusion efficacy requires further study.

Suppression of Hb Bart's to improve tissue oxygenation and fetal development in homozygous alpha-thalassemia.

Intra-uterine reduction of Hb Bart's only reached with exchange transfusions.

Essential genetic modifiers and their measurable impact in a community-recruited population analysis for non-severe hemoglobin E/ß-thalassemia prenatal genetic counseling.

The study aimed to identify essential phenotype-modulating factors among the pre-existence of several important ones and clarify their measurable impact on the clinical severity of hemoglobin (Hb) E/ß-thalassemia in a community-recruited population analysis. This prospective study was designed to compare modifiers between community- (less or no symptoms) and hospital-recruited individuals with Hb E/ß-thalassemia. The formerly included couples previously assessed for prenatal thalassemia at-risk status at 42 community and 7 referral hospitals in Thailand through on-site investigations between June 2020 and December 2021. The control included Hb E/ß-thalassemia patients undergoing transfusions. The Mahidol score classified disease severity. Beta-globin, α0-thalassemia (-SEA, -THAI), α+-thalassemia (-α3.7, -α4.2), Hb Constant Spring (αCS) alleles, rs766432 in BCL11A, rs9399137 in HBS1L-MYB, and rs7482144-XmnI were evaluated. Modifiers were compared between 102 community- and 104 hospital-recruited cases. Alleles of ß+, -SEA, -α3.7, αCS, and a minor allele of rs9399137 were prevalent in the community and mild severity groups (p < 0.05). Multiple linear regression analysis associated modulating alleles with -4.299 (-SEA), -3.654 (ß+), -3.065 (rs9399137, C/C), -2.888 (αCS), -2.623 (-α3.7), -2.361 (rs7482144, A/A), -1.258 (rs9399137, C/T), and -1.174 (rs7482144, A/G) severity score reductions (p < 0.05). Certain modifiers must be considered in routine prenatal genetic counseling for Hb E/ß-thalassemia.

Optimizing transfusion therapy for survivors of Haemoglobin Bart's hydrops fetalis syndrome: Defining the targets for haemoglobin-H fraction and "functional" haemoglobin level.

Owing to the unique pathophysiology of anaemia in haemoglobin Bart's hydrops fetalis (HBHF), a transfusion strategy based on beta-thalassemia guidelines is suboptimal for chronically transfused HBHF patients. A more aggressive transfusion aimed at reducing the proportion of non-functional HbH and improving the "functional" haemoglobin (f-Hb) can lead to reduced haemolysis and improved tissue oxygenation. However, the optimal transfusion targets for these parameters are not yet defined. In this retrospective, longitudinal study on four chronically transfused patients with HBHF, we used receiver operating characteristic curves to find a pre-transfusion f-Hb of 106 g/l and a HbH of 16.1% to be the optimal thresholds to achieve a normal soluble transferrin receptor and lactate dehydrogenase, respectively.

The pleiotropic effects of α-thalassemia on HbSS and HbSC sickle cell disease: Reduced erythrocyte cation co-transport activity, serum erythropoietin, and transfusion burden, do not translate into increased survival.

α-Thalassemia is one of the most important genetic modulators of sickle cell disease (SCD). Both beneficial and detrimental effects have been described previously. We use a 12-year data set on a large cohort of patients with HbSS (n = 411) and HbSC (n = 146) to examine a wide range of these clinical and laboratory associations. Our novel findings are that α-thalassemia strongly reduces erythrocyte potassium chloride co-transporter (KCC) activity in both HbSS and HbSC (p = .035 and p = .00045 respectively), suggesting a novel mechanism through which α-thalassemia induces a milder phenotype by reducing red cell cation loss. This may be particularly important in HbSC where reduction in mean cell hemoglobin concentration is not seen and where KCC activity has previously been found to correlate with disease severity. Additionally, we show that α-thalassemia not only increases hemoglobin in patients with HbSS (p = .0009) but also reduces erythropoietin values (p = .0005), demonstrating a measurable response to improved tissue oxygenation. We confirm the reno-protective effect of α-thalassemia in patients with HbSS, with reduced proteinuria (p = .003) and demonstrate a novel association with increased serum sodium (p = .0004) and reduced serum potassium values (p = 5.74 × 10-10 ). We found patients with α-thalassemia had a reduced annualized transfusion burden in both HbSS and HbSC, but α-thalassemia had no impact on annualized admission rates in either group. Finally, in a larger cohort, we report a median survival of 62 years in patients with HbSS (n = 899) and 80 years in those with HbSC (n = 240). α-thalassemia did not influence survival in HbSS, but a nonsignificant trend was seen in those with HbSC.

20 years review of antenatal diagnosis of haemoglobin Bart's disease and treatment with intrauterine transfusion.

OBJECTIVE: To review prenatal diagnosis and outcome of alpha thalassaemia major through universal antenatal screening. METHOD: This was a retrospective study on ultrasound features, antenatal diagnosis, in-utero intervention and long term outcome of pregnancies at risk of Haemoglobin Bart's hydrops foetalis syndrome attending prenatal diagnosis from 2000 to 2019 at Tsan Yuk Hospital in Hong Kong. RESULTS: Among 390 foetuses from 373 at-risk pregnancies, 122 (31%) prenatal invasive procedures were performed and 65 affected foetuses were diagnosed antenatally. For foetuses with ultrasound features of anaemia, the diagnostic yield of BHFS was 73%. Cardiomegaly carried a positive predictive value of 65.2% while its absence had the highest negative predictive value (96.0%). Three women having affected foetuses continued pregnancy and received intrauterine transfusion beginning 20 weeks of gestation. All babies were born alive and non-hydropic. They were managed with regular transfusion and cured by haematopoietic stem cell transplantation. CONCLUSIONS: Absence of ultrasound features of anaemia had high negative predictive value for alpha thalassaemia major. Couple at risk of having affected foetus could be offered serial ultrasound surveillance. Invasive testing for pregnancies with features of foetal anaemia provided high diagnostic yield. Intrauterine transfusion corrected foetal anaemia and allowed long term transfusion free survival without significant neurological sequelae following postnatal transplant therapy.

ATR-X syndrome: genetics, clinical spectrum, and management.

ATR-X, an acronym for alpha thalassemia and mental retardation X-linked, syndrome is a congenital condition predominantly affecting males, characterized by mild to severe intellectual disability, facial, skeletal, urogenital, and hematopoietic anomalies. Less common are heart defects, eye anomalies, renal abnormalities, and gastrointestinal dysfunction. ATR-X syndrome is caused by germline variants in the ATRX gene. Until recently, the diagnosis of the ATR-X syndrome had been guided by the classical clinical manifestations and confirmed by molecular techniques. However, our new systematic analysis shows that the only clinical sign shared by all affected individuals is intellectual disability, with the other manifestations varying even within the same family. More than 190 different germline ATRX mutations in some 200 patients have been analyzed. With improved and more frequent analysis by molecular technologies, more subtle deletions and insertions have been detected recently. Moreover, emerging technologies reveal non-classic phenotypes of ATR-X syndrome as well as the description of a new clinical feature, the development of osteosarcoma which suggests an increased cancer risk in ATR-X syndrome. This review will focus on the different types of inherited ATRX mutations and their relation to clinical features in the ATR-X syndrome. We will provide an update of the frequency of clinical manifestations, the affected organs, and the genotype-phenotype correlations. Finally, we propose a shift in the diagnosis of ATR-X patients, from a clinical diagnosis to a molecular-based approach. This may assist clinicians in patient management, risk assessment and genetic counseling.

Telomere shortening correlates with disease severity in hemoglobin H disease patients.

Hemoglobin H (Hb H) disease is the most significant health problem of the α-thalassemia syndromes. The Hb disease patients are categorized based on their genotype to deletional and nondeletional, with the latter genotype presents the more severe clinical symptoms. Since telomere length is an indicator of biological aging and health, we hypothesized that telomere length could reflect Hb H disease's severity. In this study, we recruited 48 deletional and 47 nondeletional Hb H disease patients, along with 109 normal controls, for telomere length assessment. The leukocyte telomere length was assessed by monochromatic multiplex real-time PCR and reported as the telomere to single-copy gene (T/S) ratio. When telomere length was adjusted for age, the analysis of covariance between the control and the two Hb H disease groups revealed no significant difference. However, the telomere shortening rate was more rapid in the nondeletional Hb H disease group than those of the control and deletional Hb H disease groups. Gender analysis found that male patients have a significantly lower T/S ratio than females in the nondeletional group but not in the control and deletional groups. In the two disease groups, the T/S ratio was not influenced by ferritin level or transfusion burden but was positively correlated with the absolute reticulocyte count.

Cardiorenal syndrome in thalassemia patients.

BACKGROUND: Cardiorenal syndrome (CRS), a serious condition with high morbidity and mortality, is characterized by the coexistence of cardiac abnormality and renal dysfunction. There is limited information about CRS in association thalassemia. This study aimed to investigate the prevalence of CRS in thalassemia patients and also associated risk factors. METHODS: Thalassemia patients who attended the out-patient clinic of a tertiary care university hospital from October 2016 to September 2017 were enrolled onto this cross-sectional study. Clinical and laboratory findings from 2 consecutive visits, 3 months apart, were assessed. The criteria for diagnosis of CRS was based on a system proposed by Ronco and McCullough. Cardiac abnormalities are assessed by clinical presentation, establishment of acute or chronic heart failure using definitions from 2016 ESC guidelines or from structural abnormalities shown in an echocardiogram. Renal dysfunction was defined as chronic kidney disease according to the 2012 KDIGO guidelines. RESULTS: Out of 90 thalassemia patients, 25 (27.8%) had CRS. The multivariable analysis showed a significant association between CRS and extramedullary hematopoiesis (EMH) (odds ratio (OR) 20.55, p = 0.016); thalassemia type [ß0/ßE vs ß0/ß0 thalassemia (OR 0.005, p = 0.002)]; pulmonary hypertension (OR 178.1, p = 0.001); elevated serum NT-proBNP (OR 1.028, p = 0.022), and elevated 24-h urine magnesium (OR 1.913, p = 0.016). There was no association found between CRS and frequency of blood transfusion, serum ferritin, liver iron concentration, cardiac T2*, type of iron chelating agents, or urine neutrophil gelatinase-associated lipocalin level. CONCLUSIONS: CRS is relatively common in thalassemia patients. Its occurrence is associated with laboratory parameters which are easily measured in clinical practice.

[Clinical practice guidelines for alpha-thalassemia].

Alpha-thalassemia is an autosomal recessive genetic disease as well as a relatively common hemoglobinopathy. Severe alpha-thalassemia (also known as Hb Bart's Hydrops fetalis syndrome) and intermediate alpha-thalassemia (also known as Hb H disease) are among the most common birth defects in southern China. To implement carrier screening and large population prevention program in high incidence areas can significantly reduce the incidence of alpha-thalassemia. This guideline was established by combining the discoveries of basic research, clinical research and guidelines from other countries and the actual data of Chinese population. It has summarized the medical genetics knowledge and key points in the clinical treatment for alpha-thalassemia, and provided suggestions for the clinical diagnosis and standard management of patients.

CRISPR/Cas9 gene correction of HbH-CS thalassemia-induced pluripotent stem cells.

Haemoglobin (Hb) H-constant spring (CS) alpha thalassaemia (- -/-αCS) is the most common type of nondeletional Hb H disease in southern China. The CRISPR/Cas9-based gene correction of patient-specific induced pluripotent stem cells (iPSCs) and cell transplantation now represent a therapeutic solution for this genetic disease. We designed primers for the target sites using CRISPR/Cas9 to specifically edit the HBA2 gene with an Hb-CS mutation. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm the mutation correction, we verified that the purified clones retained full pluripotency and exhibited a normal karyotype. This strategy may be promising in the future, although it is far from representing a solution for the treatment of HbH-CS thalassemia now.

Increased endothelial activation in α-thalassemia disease.

One complication of thalassemia is thromboembolism (TE), which is caused by an abnormal red blood cell surface, as well as endothelial and platelet activation. These findings are commonly observed in severe ß-thalassemia. However, limited information on α-thalassemia exists. This study enrolled subjects with deletional and non-deletional α-thalassemia and normal controls (NC). Plasma and serum of subjects were tested for endothelial activation markers including thrombomodulin (TM), vascular cell adhesion molecule-1 (VCAM-1), and von Willebrand factor antigen as well as platelet activation markers including thromboxane B2 and platelet factor 4. A total of 179 subjects were enrolled: 29 in the deletional group (mean age 13.3 ± 4.4 years), 31 in the non-deletional group (mean age 12.9 ± 4.8 years), and 119 in the NC group (mean age 13.6 ± 3.0 years). Twenty nine percent of subjects in the non-deletional group received regular red blood cell transfusion and iron chelator administration. Serum ferritin level was higher in the non-deletional group than that in the deletional group. Multivariate analysis demonstrated that VCAM-1 and TM levels were increased significantly in α-thalassemia compared with NC group (816.8 ± 131.0 vs 593.9 ± 49.0 ng/ml, and 4.9 ± 0.7 vs 4.0 ± 0.4 ng/ml, P < 0.001 respectively). VCAM-1 and TM levels in the non-deletional group were significantly higher than that in the deletional group. The present study demonstrated endothelial activation in children with α-thalassemia disease, especially those in the non-deletional group, which might be one risk factor for TE in α-thalassemia disease.

Frequency of Alpha Thalassaemia in homozygous Beta Thalassaemia paediatric patients and its clinical impact at a blood disease centre in Karachi, Pakistan.

OBJECTIVE: To find frequency ofalpha Thalsaemia nhomozygous beta Thalsaemia patients, and to se any difernce infrequency and age ofirst ransfusion and mean haemoglobin concentration. METHODS: The single-centred, escriptive cros-sectional study was conducted athe National Instiute of Blod Disease and Bone Marow Transplantaion, Karchi, from June 1,2012, to May 31, 2013. Patients of homozygous beta halsaemia, diagnosed by polymerase chain reaction, wer tested for coinheritance of alpha Thalsaemia nd foetal haemoglobin XMN1 polymorphism using polymerase chain reaction. SPS 17 was used for dat anlysi. RESULTS: Of the 286 patients, 19(41.6%) wer males, and 9(34.6%) showed coinheritance ofalpha thalsaemia. In the coinheritance group, 50(50%) and 1(1%) patients recived 1-20 and 21-40 times transfusions per year espectively, while inthe non-coinheritance group, the coresponding numbers wer 125(67%) and 27(14.%). Overal, 73(25.%) patients had nevr ben transfused, including 38(13.%) patients inthe alpha Thalsaemia group. XMN1 polymorphism was found in 86(41%) ofthe 208 patients who wer tested and anlysed on this count. CONCLUSIONS: Alpha thalsemia was presnt inmore than one-third homozygous beta halsemia patients.

Molecular basis of α-thalassemia.

α-Thalassemia is an inherited, autosomal recessive, disorder characterized by a microcytic hypochromic anemia. It is one of the most common monogenic gene disorders in the world population. The clinical severity varies from almost asymptomatic, to mild microcytic hypochromic, and to a lethal hemolytic condition, called Hb Bart's Hydrops Foetalis Syndrome. The molecular basis are usually deletions and less frequently, point mutations affecting the expression of one or more of the duplicated α-genes. The clinical variation and increase in disease severity is directly related to the decreased expression of one, two, three or four copies of the α-globin genes. Deletions and point mutations in the α-globin genes and their regulatory elements have been studied extensively in carriers and patients and these studies have given insight into the α-globin genes are regulated. By looking at naturally occurring deletions and point mutations, our knowledge of globin-gene regulation and expression will continue to increase and will lead to new targets of therapy.

Variability in State-Based Recommendations for Management of Alpha Thalassemia Trait and Silent Carrier Detected on the Newborn Screen.

We conducted an inventory of state-based recommendations for follow-up of alpha thalassemia silent carrier and trait identified on newborn screen. We found wide variability in the nature and timing of these recommendations. We recommend a standardized recommendation to guide pediatricians in evidenced-based care for this population.

Transfusion practices and complications in thalassemia.

BACKGROUND: The severe forms of thalassemia are the most common inherited anemias managed with regular blood transfusion therapy. Transfusion policies and complications are critical to quality of life and survival, but there is a lack of standardized care. STUDY DESIGN AND METHODS: A survey of 58 items was completed in 2016 by 11 centers in California, Washington, Oregon, Nevada, and Arizona providing long-term care for thalassemia. The questionnaire addressed demographic information, transfusion practices and complications, and educational needs. RESULTS: The centers followed 717 patients with ß-thalassemia (314, 43.8%) or α-thalassemia (394, 55%). One-third (34.7%) of patients were transfusion-dependent. Indications and goals of transfusion therapy differed between centers. Prestorage leukoreduction was universal, while routine irradiation of units was limited to one site. Red blood cell antigen phenotype was determined before the first transfusion and patients received Rh/Kell-matched units. However, more than half of the transfused patients had received blood at multiple hospitals within or outside the United States. Alloantibodies were seen in 16.9% of transfused group, but management of such patients was variable. Unusual or emerging transfusion-transmitted pathogens were not observed. Multiple educational needs were recognized, with iron overload as the biggest challenge; the approach to iron chelation varied within the group. CONCLUSION: This study identified many patients not included in earlier surveys limited to major national centers, suggesting that the thalassemia population in the United States is vastly underestimated. Lack of evidence-based guidelines is a barrier to optimal care, which should be addressed through regional consortia of thalassemia centers.

Elevations of Thrombotic Biomarkers in Hemoglobin H Disease.

BACKGROUND: Thalassemia is a group of hereditary hemoglobinopathies caused by decreased or absent synthesis of α and/or ß globin chains. Studies have shown that hypercoagulability and thrombosis are common clinical symptoms in ß-thalassemia, especially ß-thalassemia intermedia, but little is known about in α-thalassemia. This study aims to examine phosphatidylserine (PS) levels, platelet activation, and coagulation markers in splenectomized (S) and nonsplenectomy (NS) patients with hemoglobin (Hb) H disease. METHODS: The NS group comprised 20 patients (median age 15.0 years, range, 14-16.5 years), and the S group consisted of 11 patients (median age 16.4 years, range, 14-19.9 years) with Hb H disease; the control group consisted of 20 normal subjects. Hematological parameters were collected. Flow cytometry was used to measure PS exposure on red blood cells. The levels of intercellular adhesive molecule (ICAM)-1, tumor necrosis factor α (TNFα), ß-thromboglobulin (TG) and prothrombin fragment 1 + 2 (F1.2) were determined using ELISA test kits. RESULTS: Significant increases in the levels of PS, ICAM-1, TNFα, ß-TG, and F1.2 were observed in both patient groups compared to normal controls (p < 0.01). CONCLUSION: This observation indicates blood coagulation, endothelial injury, chronic low-grade inflammation, platelet activation, and thrombin generation are present in Hb H disease; these findings merit further assessment in a larger prospective cohort to establish possible links with thrombotic manifestations.

Identification of a Novel Nonsense Mutation in a Patient with Transfusion-Dependent Hb H Disease.

BACKGROUND: Hb H disease is a form of α-thalassemia. The high clinical variability is influenced by the exact combination of mutations. Here we report on a 29-year-old female patient from Afghanistan who received regular blood transfusions since her childhood. METHODS: For diagnosis we employed Sanger sequencing, multiplex ligation-dependent probe amplification, hemoglobin-electrophoresis, and hematological analysis. RESULTS: Molecular genetic analysis revealed a non-deletional Hb H genotype with two in cis point mutations in HBA1 (c.183G>T;p.Lys61Asn and c.184A>T;p.Lys62*) in addition to the common deletions α4.2 and α3.7 in HBA2. The nonsense-mutation p.Lys62* has not been described before. Hematological data were in accordance with the genetic findings. CONCLUSIONS: We describe here a novel mutation in the HBA1 gene and support evidence for non-deletional type of Hb H leading to transfusion-dependent anemia.

The α-thalassemias.

More than 100 varieties of α-thalassemia have been identified. Their geographic distribution and the challenges associated with screening, diagnosis, and management suggest that α-thalassemias should have a higher priority on global public health agendas.