Therapeutic Gene Editing for Hemoglobinopathies.

In the last ten years, a consistent number of clinical studies have evaluated different gene approaches for the treatment of patients with sickle cell disease (SCD) and transfusion-dependent ß-thalassemia (TDT). Initial studies of gene therapy for hemoglobinopathies involved the use of lentiviral vectors to add functional copies of the gene encoding ß-globin in defective CD34 cells; more recently, gene editing techniques have been used involving either CRISPR-Cas9, transcription activation-like effector protein nuclease, zinc finger nuclease, and base editing to either induce fetal hemoglobin production at therapeutic levels or to genetically repair the underlying molecular defect causing the disease. Here, we review recent gene editing studies that have started the development of a new era in the treatment of hemoglobinopathies and, in general, monoallelic hereditary diseases.

Unmasking hemoglobin Köln: a rare cause of discrepancies between pulse oximetry and arterial oxygen saturation-a case report.

Discrepancies between pulse oximetry and arterial oxygen saturation can pose challenges in clinical assessment. Possible underlying causes include poor peripheral perfusion, skin pigmentation, motion artifacts, and conditions like unstable hemoglobin and methemoglobinemia. Unstable hemoglobin variants, such as hemoglobin Köln, are rare inherited mutations affecting globin genes, potentially disrupting the folding, assembly, or interactions among subunits in globin molecules and the essential interactions between heme and globin for oxygen-binding properties. In this case report, we present the case of a 44-year-old Arabic woman who underwent extensive investigations due to disparities in pulse oximetry and arterial oxygen saturation, ultimately leading to the diagnosis of the unstable hemoglobin variant, hemoglobin Köln.

Prevalence, Risk Factors, and Complications of Sickle Cell Disease in Saudi Arabia: A Systematic Review.

This study examined sickle cell disease (SCD) in Saudi Arabia. A systematic search of relevant databases was conducted to identify studies investigating SCD in the Saudi population. Studies were then screened based on predefined criteria and critically appraised for methodological quality. Data was extracted and synthesized to provide an overall picture of the SCD burden in Saudi Arabia. The most commonly reported complications were vaso-occlusive crises (VOC), acute chest syndrome (ACS), acute painful crisis, splenic sequestration, osteomyelitis, aplastic crisis, hemolytic crisis, serious bacterial infections, chronic vascular occlusion (CVO), depression, sickle cell nephropathy (SCN), obstructive sleep apnea (OSA), and renal complications. Reduced blood levels of antioxidant trace elements (Cu, Zn, and Se) may encourage oxidative stress, which in turn may contribute to the pathophysiology of SCD. Infections and ACS were common among young children (<7 years) while pain attacks were common in older children (>7 years). The high rate of hospitalizations among SCD patients highlights the need for better management strategies. Future research should focus on understanding the underlying causes of SCD complications and developing new ways to control them.

Case report-unveiling a case of hemoglobin D-Punjab variant with iron deficiency anemia in Sindh (Pakistan).

Hemoglobin D variations are a group of hemoglobinopathies caused by mutations in the genes that control the synthesis of new globin chains. Hemoglobin D-Punjab is the most prevalent but frequently asymptomatic, it can occasionally cause mild to moderate hemolytic anemia, making diagnosis difficult and raising the risk of misdiagnosis. This article discusses a rare instance of a seventeen-year-old male in Sindh, Pakistan with iron deficiency anemia who was later found to have the Punjab variation of the hemoglobin D. The patient had signs of weakness, exhaustion, and shortness of breath, which were initially alleviated by iron supplementation but eventually became refractory. Hemoglobin electrophoresis demonstrated the distinctive hypochromic, microcytic red blood cell shape, and laboratory tests verified the presence of the Hemoglobin D-Punjab feature. The instance emphasizes how crucial it is to distinguish Hemoglobin D-Punjab from other anemias in order to guarantee proper care. This case underscores the importance of recognizing hemoglobin D-Punjab trait, to provide appropriate genetic counseling and ensure the patient's well-being. Increased awareness among healthcare professionals regarding the diverse spectrum of hemoglobinopathies is essential for accurate diagnosis and management.

Identification of genetic variants associated with clinical features of sickle cell disease.

Sickle cell disease (SCD) is an inherited blood disorder marked by homozygosity of hemoglobin S, which is a defective hemoglobin caused by a missense mutation in the ß-globin gene. However, clinical phenotypes of SCD vary among patients. To investigate genetic variants associated with various clinical phenotypes of SCD, we genotyped DNA samples from 520 SCD subjects and used a genome-wide association study (GWAS) approach to identify genetic variants associated with phenotypic features of SCD. For HbF levels, the previously reported 2p16.1 locus (BCL11A) reached genome significance (rs1427407, P = 8.58 × 10-10) in our GWAS as expected. In addition, we found a new genome-wide significance locus at 15q14 (rs8182015, P = 2.07 × 10-8) near gene EMC7. GWAS of acute chest syndrome (ACS) detected a locus (rs79915189, P = 3.70 × 10-8) near gene IDH2 at 15q26.1. The SNP, rs79915189, is also an expression quantitative trait locus (eQTL) of IDH2 in multiple tissues. For vasoocclusive episode (VOE), GWAS detected multiple significant signals at 2p25.1 (rs62118798, P = 4.27 × 10-8), 15q26.1 (rs62020555, P = 2.04 × 10-9) and 15q26.3 (rs117797325, P = 4.63 × 10-8). Our findings provide novel insights into the genetic mechanisms of SCD suggesting that common genetic variants play an important role in the presentation of the clinical phenotypes of patients with SCD.

Chronic automated red cell exchange therapy for sickle cell disease.

BACKGROUND: The data to support chronic automated red cell exchange (RCE) in sickle cell disease (SCD) outside of stroke prevention, is limited, especially in adults. STUDY DESIGN AND METHODS: A retrospective analysis was conducted of patients with SCD who were referred for chronic RCE at our institution over a 10-year period. Data that were evaluated included patient demographics, referral indications, and procedural details (e.g., vascular access, adverse events, etc.). In a subanalysis, the number of annual acute care encounters during 3 years of chronic RCE was compared with that in the year preceding the first RCE. RESULTS: A total of 164 patients were referred for chronic RCE: median age was 28 years (interquartile range [IQR] = 22-36) at referral and 60% were female. Seventy (42.6%) were naïve to chronic transfusion (simple or RCE) prior to referral. The leading indications for referral were refractory pain (73/164, 44.5%) and iron overload (57/164, 34.7%). A total of 5090 procedures occurred during the study period (median = 19, IQR = 5-45). Of the 138 patients who had central vascular access, 8 (6%) and 16 (12%) had ≥1 central-line-related thrombosis and/or infection, respectively. Of those who were not RBC alloimmunized at initiation of RCE, 12/105 (11.4%) developed new antibodies during chronic RCE. In those 30 patients who were adherent to therapy for 3 years, there was no significant difference in acute care encounters following initiation of RCE. CONCLUSION: Prospective clinical trials are needed to determine which patients are most likely to benefit from chronic RCE and refine selection accordingly.

DAHEAN: A Danish nationwide study ensuring quality assurance through real-world data for suspected hereditary anemia patients.

BACKGROUND: Hereditary anemias are a group of genetic diseases prevalent worldwide and pose a significant health burden on patients and societies. The clinical phenotype of hereditary anemias varies from compensated hemolysis to life-threatening anemia. They can be roughly categorized into three broad categories: hemoglobinopathies, membranopathies, and enzymopathies. Traditional therapeutic approaches like blood transfusions, iron chelation, and splenectomy are witnessing a paradigm shift with the advent of targeted treatments. However, access to these treatments remains limited due to lacking or imprecise diagnoses. The primary objective of the study is to establish accurate diagnoses for patients with hereditary anemias, enabling optimal management. As a secondary objective, the study aims to enhance our diagnostic capabilities. RESULTS: The DAHEAN study is a nationwide cohort study that collects advanced phenotypic and genotypic data from patients suspected of having hereditary anemias from all pediatric and hematological departments in Denmark. The study deliberates monthly by a multidisciplinary anemia board involving experts from across Denmark. So far, fifty-seven patients have been thoroughly evaluated, and several have been given diagnoses not before seen in Denmark. CONCLUSIONS: The DAHEAN study and infrastructure harness recent advancements in diagnostic tools to offer precise diagnoses and improved management strategies for patients with hereditary anemias.

Post-GWAS Validation of Target Genes Associated with HbF and HbA2 Levels.

Genome-Wide Association Studies (GWASs) have identified a huge number of variants associated with different traits. However, their validation through in vitro and in vivo studies often lags well behind their identification. For variants associated with traits or diseases of biomedical interest, this gap delays the development of possible therapies. This issue also impacts beta-hemoglobinopathies, such as beta-thalassemia and sickle cell disease (SCD). The definitive cures for these diseases are currently bone marrow transplantation and gene therapy. However, limitations regarding their effective use restrict their worldwide application. Great efforts have been made to identify whether modulators of fetal hemoglobin (HbF) and, to a lesser extent, hemoglobin A2 (HbA2) are possible therapeutic targets. Herein, we performed the post-GWAS in vivo validation of two genes, cyclin D3 (CCND3) and nuclear factor I X (NFIX), previously associated with HbF and HbA2 levels. The absence of Ccnd3 expression in vivo significantly increased g (HbF) and d (HbA2) globin gene expression. Our data suggest that CCND3 is a possible therapeutic target in sickle cell disease. We also confirmed the association of Nfix with γ-globin gene expression and present data suggesting a possible role for Nfix in regulating Kruppel-like transcription factor 1 (Klf1), a master regulator of hemoglobin switching. This study contributes to filling the gap between GWAS variant identification and target validation for beta-hemoglobinopathies.

Irregular red blood cell antibodies, abnormal hemoglobin and dangerous universal blood donor insights from a public blood center in a Brazilian metropolitan area.

BACKGROUND: Immunohematology tests are crucial in transfusion safety. This study aimed to assess irregular red blood cell (RBC) antibodies, abnormal hemoglobin and dangerous universal blood donors at a public blood center in a Brazilian metropolitan area. METHODS: A cross-sectional study included all consecutive blood donors from January 2018 to December 2021 at the Brasília Blood Center Foundation, Federal District (FD), Brazil. RESULTS: Among 205,965 blood donations, irregular RBC antibodies were found in 743 (0.4 %). Abnormal hemoglobin was observed in 5396 (2.6 %): 3959 (1.9 %) with Hb AS, 1344 (0.7 %) with Hb AC, and 93 (< 0,1 %) with other hemoglobin variants. Of O group donors, 12.5 % (9646) had hemolysins: 12.5 % (2410) both anti-A and anti-B, 8.7 % (9646) only anti-A, and 1.6 % (1763) only anti-B hemolysins. Female sex (p < 0.001) and increasing age (p < 0.001) were associated with irregular RBC antibodies. O and/or Rh(D)-positive blood groups had a lower prevalence of irregular RBC antibodies compared to other ABO and/or Rh(D)-negative groups. Age (p < 0.001) and female sex (p < 0.001) were associated with anti-A/anti-B hemolysins, while FD residency was associated with reduced incidence (p < 0.001). CONCLUSION: Anti-A/anti-B hemolysins in O group donors, abnormal hemoglobin and irregular RBC antibodies pose risks to transfusion practice and should not be overlooked. Advancing age, female sex, ABO blood group other than O, or Rh(D)- negative are independently associated with the presence of irregular RBC antibodies. Dangerous universal blood donors were associated with advanced age, female gender, Rh(D)-positive blood type, and individuals residing in a Brazilian state other than where the blood center was located.

Safety and efficacy studies of CRISPR-Cas9 treatment of sickle cell disease highlights disease-specific responses.

Fetal hemoglobin (HbF) reactivation expression through CRISPR-Cas9 is a promising strategy for the treatment of sickle cell disease (SCD). Here, we describe a genome editing strategy leading to reactivation of HbF expression by targeting the binding sites (BSs) for the lymphoma-related factor (LRF) repressor in the γ-globin promoters. CRISPR-Cas9 treatment in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny. LRF BS disruption did not impair HSPC engraftment and differentiation but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared with HD cells. We detected off-target activity and chromosomal rearrangements, particularly in SCD samples (likely because of the higher overall editing efficiency) but did not impact the target gene expression and HSPC engraftment and differentiation. Transcriptomic analyses showed that the editing procedure results in the up-regulation of genes involved in DNA damage and inflammatory responses, which was more evident in SCD HSPCs. This study provides evidence of efficacy and safety for an editing strategy based on HbF reactivation and highlights the need of performing safety studies in clinically relevant conditions, i.e., in patient-derived HSPCs.

Prevalence and Regional Distribution of Beta-Hemoglobin Variants in Saudi Arabia: Insights from the National Premarital Screening Program".

BACKGROUND: Hemoglobinopathies are among the most prevalent inherited disorders globally, with carrier prevalence varying significantly across regions. In Saudi Arabia, high rates of consanguineous marriages amplify the risk of these disorders. AIM: This study aims to assess the burden of hemoglobinopathies by evaluating the prevalence and regional distribution of beta-hemoglobin variants, including rare variants, among couples participating in the national premarital screening program. METHODS: Data were collected from the premarital genetic screening program and entered into the SEHA platform, covering the 13 administrative regions of Saudi Arabia. Blood samples underwent various screening tests for infectious and genetic diseases. Hemoglobin electrophoresis samples were analyzed using capillary electrophoresis, High-Performance Liquid Chromatography (HPLC), or a combination of both methods. RESULTS: From 2011 to 2018, 1,871,184 individuals were included in the study, with 49.8% male and 50.2% female. The average age was 30.2 years. Hemoglobin S (HbS) was identified in 88,431 individuals (4.7% of the tested population and 78.5% of abnormal screening results), primarily as a sickle cell trait. ß-thalassemia was the second most common disorder, identified in 22,420 individuals (1.2% of the population and 19.9% of hemoglobin disorders). HbC and HbD were each detected in 0.04% of cases, while HbO-Arab was identified in 0.007% and HbG in 0.006%. Hemoglobin E and hemoglobin Lepore were found to be extremely rare. CONCLUSION: The study demonstrates regional variation in the prevalence of hemoglobin genetic variants in Saudi Arabia. To effectively mitigate this risk, it is imperative to strengthen public education and awareness, particularly focusing on genetic screening and counseling.

Gene therapy and gene editing strategies in inherited blood disorders.

Gene therapy has shown significant potential in treating various diseases, particularly inherited blood disorders such as hemophilia, sickle cell disease, and thalassemia. Advances in understanding the regulatory network of disease-associated genes have led to the identification of additional therapeutic targets for treatment, especially for ß-hemoglobinopathies. Erythroid regulatory factor BCL11A offers the most promising therapeutic target for ß-hemoglobinopathies and reduction of its expression using the commercialized gene therapy product Casgevy was approved for use in the UK and USA in 2023. Notably, the emergence of innovative gene editing technologies has further broadened the gene therapy landscape, presenting new possibilities for treatment. Intensive studies indicate that base editing and prime editing, built upon CRISPR technology, enable precise single-base modification in hematopoietic stem cells for addressing inherited blood disorders ex vivo and in vivo. In this review, we present an overview of the current landscape of gene therapies, focusing on clinical research and gene therapy products for inherited blood disorders, evaluation of potential gene targets, and the gene editing tools employed in current gene therapy practices, which provides an insight for the establishment of safer and more effective gene therapy methods for a wider range of diseases in the future.

Exploring the Interplay between Asthma and Hemoglobinopathies: A Comprehensive Review.

Asthma, a prevalent chronic respiratory condition characterized by inflammation of the airways and bronchoconstriction, has demonstrated a potential association with hemoglobinopathies such as thalassemia and sickle cell disease (SCD). Numerous studies have highlighted a higher prevalence of asthma among thalassemia patients compared to the general population, with rates ranging around 30%. Similarly, asthma frequently coexists with SCD, affecting approximately 20-48% of patients. Children with SCD often experience heightened lower airway obstruction and airway hyper-reactivity. Notably, the presence of asthma in SCD exacerbates respiratory symptoms and increases the risk of severe complications like acute chest syndrome, stroke, vaso-occlusive episodes, and early mortality. Several studies have noted a decrease in various cytokines such as IFN-γ and IL-10, along with higher levels of both IL-6 and IL-8, suggesting an overactivation of pro-inflammatory mechanisms in patients with hemoglobinopathies, which could trigger inflammatory conditions such as asthma. The exact mechanisms driving this association are better elucidated but may involve factors such as chronic inflammation, oxidative stress, and immune dysregulation associated with thalassemia-related complications like chronic hemolytic anemia and iron overload. This review aims to comprehensively analyze the relationship between asthma and hemoglobinopathies, with a focus on thalassemia and SCD. It emphasizes the importance of interdisciplinary collaboration among pulmonologists, hematologists, and other healthcare professionals to effectively manage this complex interplay. Understanding this link is crucial for improving care and outcomes in affected individuals.

Screening for sickle cell disease: focus on newborn investigations.

Drepanocytosis is a genetic disease relevant for its epidemiological, clinical and socio-economic aspects. In our country the prevalence is highly uneven with peaks in former malaria areas, but migration flows in recent years have led to significant changes. In this document we review the screening programs currently existing in Italy with particular emphasis on newborn screening, which in other countries around the world, including within Europe, is at most universal and mandatory. The essential laboratory issues are reviewed, from sampling aspects (cord blood or peripheral), to the analytical (analytical methods dedicated to neonatal screening and adult carrier detection) and post analytical (reporting, informative) ones. An economic analysis based on data collected in the province of Modena is also proposed, clearly showing that neonatal screening is also beneficial from an economic point of view.

Positive impacts of universal newborn screening on the outcome of children with sickle cell disease in the province of Quebec: A retrospective cohort study.

A universal newborn screening program for sickle cell disease (uNS-SCD) was implemented in the province of Québec (Qc) in November 2013, close in time to the recommendation of early initiation of hydroxyurea (HU) therapy for children. This retrospective cohort study evaluated the impact of such a program on children first seen between January 2000 and December 2019. Cohorts pre-SCD-uNS in Qc (pre-QcNS) (n = 253) and post-QcNS (n = 157) for patients seen prior to or after Nov 2013 were compared. Kaplan-Meier curves, Poisson regression, and logistic regressions were used for statistical analysis, using Software R version 4.2.1. Median age at first visit decreased significantly from 14.4 [interquartile range: 2.4-72.0] to 1.2 months [1.2-57.6] (p < 0.001). The percentage of children born in Qc undiagnosed at birth and referred after a first SCD-related complication dropped from 42.6% to 0.0% (p < 0.0001). The median age of HU introduction for patients with SS/Sß°-thalassemia decreased from 56.4 [31.2-96.0] to 9.0 months post-QcNS [8.0-12.1] (p < 0.001). Event-free survival improved significantly for any type of hospitalization as well as for vaso-occlusive crisis (VOC) (140-257 days (p < 0.001) and 1320 vs. 573 days (p < 0.002), respectively), resulting in a reduction from 2 [interquartile range: 1.0-3.0] to 1.0 hospitalizations/patient-year [0.6-1.4] (p < 0.001). Children with SS/Sß°-thalassemia referred post-QcNS also had fewer emergency department visits for VOC (RR: 0.69, 95% confidence interval: 0.54-0.88). The Universal NS program allows early detection and referral of children with SCD to comprehensive care centers. Earlier access ensures that children benefit from essential preventive interventions, reducing disease burden. This cohort study highlights that uNS-SCD is an essential public health measure.

Iron overload: The achilles heel of ß-thalassemia.

Systematic transfusions coupled with iron chelation therapy have substantially improved the life expectancy of thalassemia patients in developed nations. As the human organism does not have a protective mechanism to remove excess iron, iron overload is a significant concern in thalassemia, leading to organ damage, especially in the heart and liver. Thus, iron chelation therapy is crucial to prevent or reverse organ iron overload. There are three widely used iron chelators, either as monotherapy or in combination. The choice of iron chelator depends on several factors, including local guidelines, drug availability, and the individual clinical scenario. Despite treatment advancements, challenges persist, especially in resource-limited settings, highlighting the need for improved global healthcare access. This review discusses clinical management, current treatments, and future directions for thalassemia, focusing on iron overload and its complications. Furthermore, it underscores the progress in transforming thalassemia into a manageable chronic condition and the potential of novel therapies to further enhance patient outcomes.

Context base editing for splice correction of IVSI-110 ß-thalassemia.

ß-Thalassemia is brought about by defective ß-globin (HBB [hemoglobin subunit ß]) formation and, in severe cases, requires regular blood transfusion and iron chelation for survival. Genome editing of hematopoietic stem cells allows correction of underlying mutations as curative therapy. As potentially safer alternatives to double-strand-break-based editors, base editors (BEs) catalyze base transitions for precision editing of DNA target sites, prompting us to reclone and evaluate two recently published adenine BEs (ABEs; SpRY and SpG) with relaxed protospacer adjacent motif requirements for their ability to correct the common HBBIVSI-110(G>A) splice mutation. Nucleofection of ABE components as RNA into patient-derived CD34+ cells achieved up to 90% editing of upstream sequence elements critical for aberrant splicing, allowing full characterization of the on-target base-editing profile of each ABE and the detection of differences in on-target insertions and deletions. In addition, this study identifies opposing effects on splice correction for two neighboring context bases, establishes the frequency distribution of multiple BE editing events in the editing window, and shows high-efficiency functional correction of HBBIVSI-110(G>A) for our ABEs, including at the levels of RNA, protein, and erythroid differentiation.

Haemoglobinopathies and other rare anemias in Spain: ten years of a nationwide registry (REHem-AR).

REHem-AR was created in 2013. The progressive implementation of neonatal screening for haemoglobinopathies in Spanish autonomous communities where the registry had not been implemented, as well as the addition of new centres during this period, has considerably increased the sample of patients covered. In this study, we update our previous publication in this area, after a follow-up of more than 5 years. An observational, descriptive, multicentre and ambispective study of adult and paediatric patients with haemoglobinopathies and rare anaemias registered in REHem was performed. The data are from a cross-sectional analysis performed on 1 June, 2023. The study population comprised 1,756 patients, of whom 1,317 had SCD, 214 had thalassaemia and 224 were diagnosed with another condition. Slightly more than one third of SCD patients (37%) were diagnosed based on neonatal bloodspot screening, and the mean age at diagnosis was 2.5 years; 71% of thalassaemia patients were diagnosed based on the presence of anaemia. Vaso-occlusive crisis and acute chest syndrome continue to be the most frequent complications in SCD. HSCT was performed in 83 patients with SCD and in 50 patients with thalassaemia. Since the previous publication, REHem-AR has grown in size by more than 500 cases. SCD and TM are less frequent in Spain than in other European countries, although the data show that rare anaemias are frequent within rare diseases. REHem-AR constitutes an important structure for following the natural history of rare anaemias and enables us to calculate investment needs for current and future treatments.

Prevalence of hemoglobinopathies among Malayali tribes of Jawadhu hills, Tiruvannamalai district, Tamil Nadu, India: a community-based cross-sectional study.

BACKGROUND: Hemoglobin (Hb), a red pigment of red blood cells (RBCs), carries oxygen from the lungs to different organs of the body and transports carbon dioxide back to the lungs. Any fault present in the Hb structure leads to undesirable functional effects of the RBCs, such as sickle cell anemia (SCA), thalassemia, etc. Hemoglobinopathies affect around 7% of people in both developed and developing countries globally. The aim of the present study was to determine the prevalence and carrier frequencies of hemoglobinopathies including SCA, thalassemia, and other abnormal Hb variants among Malayali tribes in the Jawadhu hills of Tiruvannamalai district, Tamil Nadu, India. METHODS: A community-based cross-sectional study was carried out among 443 Malayali tribes inhabiting the Jawadhu hills of Tiruvannamalai district from July 2022 to September 2022. The RBC indices were analyzed using an automated 5-part hematology analyzer (Mindray, BC-5150) and hemoglobin fractions were done using the HPLC system (Bio-Rad, D-10) following standard protocols. FINDINGS: A total of 443 participants were screened, out of whom 14.67% had an abnormal Hb fraction, 83.30% were identified as normal, and 2.03% were borderline. Notably, the study revealed a prevalence of 0.68% for the α-thalassemia trait and 13.99% for the ß-thalassemia trait. INTERPRETATION: Haemoglobinopathies, specifically the ß-thalassemia trait, were most prevalent among the Malayali tribal population of Tamil Nadu residing in the Jawadhu hills of Tiruvannamalai district. Hence, we need special attention for creating awareness, increasing hemoglobinopathies screening programs, and improving the importance of tribal health conditions by the government and non-governmental organizations (NGOs) for the betterment of the ethnic tribes.