Preclinical safety assessment of modified gamma globin lentiviral vector-mediated autologous hematopoietic stem cell gene therapy for hemoglobinopathies.

Previously, we reported the development of a human Aγ-globin gene lentivirus (LV), GbG, which expresses high levels of HbF to correct the sickle cell anemia (SCA) phenotype in the Berkeley SCA mouse model, and then modified the γ-globin gene by substituting glycine at codon 16 with aspartic acid in the Aγ-globin gene to generate GbGM LV. In the present study, we evaluated the long-term safety of human Aγ-globin gene carrying GbGM LV in wild-type mice after primary and secondary transplants of GbGM-modified hematopoietic stem cells (HSC) over 18 months. The safety of the GbGM bone marrow transplant was assessed by monitoring the effects on body weight, hematology, histopathology, malignancy formation, and survival. Mice transplanted with Mock-transduced and spleen focus forming virus (SFFV) γ-retroviral vector (RV)-transduced HSC served as negative and positive controls, respectively. The mean donor-cell engraftment was comparable across Mock, GbGM LV, and SFFV RV groups. There were no significant differences in body weight, clinical signs, immunophenotype, or histopathology in the GbGM-treated mice compared to controls. Four SFFV RV-treated mice, but none of the GbGM-treated mice, developed donor-derived, vector-positive lymphomas as demonstrated by flow cytometry analysis and in situ hybridization. These results highlight the safety of the administration of GbGM LV-modified HSC with long-term follow-up after primary and secondary transplants in mice. This data supported the initiation of phase 1/2 first-in-human SCA clinical trial in the United States.

The Application of Clinical and Molecular Diagnostic Techniques to Identify a Rare Haemoglobin Variant.

Haemoglobin disorders represent a heterogeneous group of inherited conditions that involve at least one genetic abnormality in one or more of the globin chains, resulting in changes in the structure, function, and/or amount of haemoglobin molecules, which are very important for their related clinical aspects. Detecting and characterizing these disorders depends primarily on laboratory methods that employ traditional approaches and, when necessary, newer methodologies essential for solving a number of diagnostic challenges. This review provides an overview of key laboratory techniques in the diagnosis of haemoglobinopathies, focusing on the challenges, advancements, and future directions in this field. Moreover, many haemoglobinopathies are benign and clinically silent, but it is not uncommon to find unexpected variants during routine laboratory tests. The present work reported a rare and clinically interesting case of identification of haemoglobin fractions in an adult man by the determination of glycated haemoglobin (HbA1c) during a routine laboratory assessment, highlighting how the correct use of laboratory data can modify and improve the patient's clinical management.

Non-invasive prenatal testing of beta-hemoglobinopathies using next generation sequencing, in-silico sequence size selection, and haplotyping.

AIM: To develop a non-invasive prenatal test for beta-hemoglobinopathies based on analyzing maternal plasma by using next generation sequencing. METHODS: We applied next generation sequencing (NGS) of maternal plasma to the non-invasive prenatal testing (NIPT) of autosomal recessive diseases, sickle cell disease and beta-thalassemia. Using the Illumina MiSeq, we sequenced plasma libraries obtained via a Twist Bioscience probe capture panel covering 4 Kb of chromosome 11, including the beta-globin (HBB) gene and >450 genomic single-nucleotide polymorphisms (SNPs) used to estimate the fetal fraction (FF). The FF is estimated by counting paternally transmitted allelic sequence reads present in the plasma but absent in the mother. We inferred fetal beta-globin genotypes by comparing the observed mutation (Mut) and reference (Ref) read ratios to those expected for the three possible fetal genotypes (Mut/Mut; Mut/Ref; Ref/Ref), based on the FF. RESULTS: We bioinformatically enriched the FF by excluding reads over a specified length via in-silico size selection (ISS), favoring the shorter fetal reads, which increased fetal genotype prediction accuracy. Finally, we determined the parental HBB haplotypes, which allowed us to use the read ratios observed at linked SNPs to help predict the fetal genotype at the mutation site(s). We determined HBB haplotypes via Oxford Nanopore MinION sequencing of a 2.2 kb amplicon and aligned these sequences using Soft Genetics' NextGENe LR software. CONCLUSION: The combined use of ISS and HBB haplotypes enabled us to correctly predict fetal genotypes in cases where the prediction based on variant read ratios alone was incorrect.

New Born Screening of Hemoglobinopathies in a Center Tunisian Population.

Sickle cell diseases, ß-thalassemia, and other hemoglobinopathies are common in Africa. Their distribution differs from one region to another. There are higher frequencies in Western and Northern Africa. Their clinical complications presented a real public health problem in each country. For this, early treatment can improve the severity of these diseases. Hemoglobinopathies targeted by screening are associated with SCD, ß, and α thalassemia. Our study aim is to report our experience with newborn screening for hemoglobinopathy in Tunis. The 156 newborn's cord blood was collected at the time of childbirth in the center region (Farhat Hached Hôspital). We opted for hemoglobin exploration to achieve maximum efficiency and effectiveness in screening. After that, all patients suspected to have hemoglobinopathies are affected by molecular investigation. Our findings showed the presence of some hemoglobinopathies such as ß-thalassemia and α-thalassemia with the following frequencies: 12% and 0.33%. The molecular results show the presence of HBB: c.93-21G>A, IVS-I-110G>A, HBBc. -106G>A -56G>C, HBBc.404T>C, Hb Yaounde described for the first time in Tunisia and α 3,7 . In conclusion, newborn screening diagnoses neonates with different examples of hemoglobinopathies, which will be beneficial not only for the care of the child but also for genetic counseling of the potential risk's parents.

Molecular epidemiology and hematological profiles of hemoglobin variants in southern Thailand.

Data on hemoglobin (Hb) variants in southern Thailand are lacking. This study aimed to reassess the frequency of Hb variants and the clinical aspects of compound heterozygous Hb variant with other hemoglobinopathies. We enrolled 13,391 participants from ten provinces in southern Thailand during 2015-2022. Hb analysis was performed using capillary electrophoresis, and mutations in the HBA and HBB genes were identified using PCR or DNA sequencing. Hb variants were identified in 337 (2.5%) unrelated subjects. Nine ß-chain variants, namely Hb Malay (76.9%), Hb C (10.1%), Hb D-Punjab (2.9%), Hb G-Makassar (2.3%), Hb Dhonburi (2.3%), Hb Tak (1.4%), Hb J-Bangkok (1.4%), Hb New York (0.3%), and Hb Hope (0.3%), and four α-chain variants-Hb G-Georgia (HBA1) (0.9%), Hb G-Georgia (HBA2) (0.3%), Hb Q-Thailand (0.6%), and Hb St. Luke's-Thailand (0.3%)-were identified. The southern population exhibited a distinct spectrum of Hb variants compared to that observed in the populations from other areas. Several compound heterozygous genotypes were also identified. Combining Hb Malay with Hb E or high Hb F determinants did not require a blood transfusion. This study provides essential information for genetic counseling in thalassemia prevention and control programs in this region.

Premarital Screening is Pivotal in Reducing the Births of Babies Affected with Thalassemia Major in Iraq.

Thalassemia major is one of the health problems in Iraq, especially in Kurdistan. Pre-marriage mandatory preventive screening program was established in Kurdistan in 2008, which allowed us to study the prevalence of different hemoglobinopathies among newly married young adults in this region. A total of 1154 subjects (577 couples) attending the Koya district, premarital Health center, were screened using red cell indices. Those who had mean corpuscular volume (MCV)<80 fl and mean corpuscular hemoglobin (MCH)<27 pg had high-performance liquid chromatography and iron studies. Out of 1154 individuals that were evaluated, 183 (11.9%) had low MCV and MCH. Of the former 183 subjects, 69 (5.97%) had ß-thalassemia trait, 10 (0.86%) had δß-thalassemia trait, and no other hemoglobinopathies were recorded in our study. There was second-degree consanguinity in 4.7% of all 577 couples. In two couples, both partners had ß-thalassemia trait and both were consanguineous. Both couples decided to separate after counseling. Based on the current study, the role of the premarital screening program in decreasing the number of new thalassemia major cases among the Kurdish population is laudable. Therefore, mandatory premarital screening is advised in all parts of Iraq.

ß0-Thalassemia Caused by a Novel Nonsense Mutation [HBB:c.199A > T].

We report two hemoglobinopathy cases involving a novel ß-thalassemia (ß-thal) nonsense mutation, HBB:c.199A > T. One patient had Hb S/ß-thal, and a second unrelated patient had Hb D-Punjab/ß-thal. The HBB:c.199A > T mutation introduces a premature termination codon at amino acid codon 66 (AAA→TAA) in exon 2, resulting in typical high Hb A2 ß0-thal.

The First Iranian Case of Unstable Hemoglobin Santa Ana.

In this report, we describe a 6-year-old girl with a medical history of pallor, mild icterus, anemia, blood transfusion and abnormal hemoglobin variant analysis on capillary electrophoresis. She was referred for further analysis. DNA sequencing of the proband revealed a de novo mutation in Codon 88 (CTG > CCG) of the ß-globin gene (HBB: c.266T > C) in a heterozygous state compatible with hemoglobin Santa Ana, an unstable hemoglobin. This is the first case of Hb Santa Ana from Iran associated with moderate to severe anemia who underwent splenectomy with clinical improvement.

Autologous gene therapy for hemoglobinopathies: From bench to patient's bedside.

In recent years, a growing number of clinical trials have been initiated to evaluate gene therapy approaches for the treatment of patients with transfusion-dependent ß-thalassemia and sickle cell disease (SCD). Therapeutic modalities being assessed in these trials utilize different molecular techniques, including lentiviral vectors to add functional copies of the gene encoding the hemoglobin ß subunit in defective cells and CRISPR-Cas9, transcription activator-like effector protein nuclease, and zinc finger nuclease gene editing strategies to either directly address the underlying genetic cause of disease or induce fetal hemoglobin production by gene disruption. Here, we review the mechanisms of action of these various gene addition and gene editing approaches and describe the status of clinical trials designed to evaluate the potentially for these approaches to provide one-time functional cures to patients with transfusion-dependent ß-thalassemia and SCD.

Impact of α-Globin Gene Expression and α-Globin Modifiers on the Phenotype of ß-Thalassemia and Other Hemoglobinopathies: Implications for Patient Management.

In this short review, we presented and discussed studies on the expression of globin genes in ß-thalassemia, focusing on the impact of α-globin gene expression and α-globin modifiers on the phenotype and clinical severity of ß-thalassemia. We first discussed the impact of the excess of free α-globin on the phenotype of ß-thalassemia. We then reviewed studies focusing on the expression of α-globin-stabilizing protein (AHSP), as a potential strategy of counteracting the effects of the excess of free α-globin on erythroid cells. Alternative processes controlling α-globin excess were also considered, including the activation of autophagy by ß-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with ß-thalassemia and other hemoglobinopathies for which reduction in α-globin excess is clinically beneficial.

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

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

First Family Case of Hemoglobinopathy Titusville in China with Falsely Low SpO2 and Unmeasurable SaO2.

BACKGROUND: Normal hemoglobin is a tetrameric structure, consisting of two alpha-globin chains and two nonalpha (beta, gamma, delta) chains. Hemoglobinopathies occur when the presence of gene mutations affect the molecular structure or expression of the globin chains. METHODS: We reported the case of a 9-year-old Chinese girl who presented with abnormal low oxygen saturation values on pulse oximetry and no oximetry results were obtained during blood gas analysis (BGA). RESULTS: High-performance liquid chromatography (HPLC) and capillary electrophoresis demonstrated that the presence of a low oxygen affinity hemoglobin variant, characterized as hemoglobin Titusville, was proven by gene sequencing. The patient's mother and aunt also carry the hemoglobin variant, representing the first Chinese family case reported. CONCLUSIONS: Hemoglobin Titusville is a rare genetic hemoglobin structural defect. early diagnosis can help patients and clinicians avoid unnecessary anxiety and costly or excessive clinical investigations.

Revolutionising healing: Gene Editing's breakthrough against sickle cell disease.

Recent advancements in gene editing illuminate new potential therapeutic approaches for Sickle Cell Disease (SCD), a debilitating monogenic disorder caused by a point mutation in the ß-globin gene. Despite the availability of several FDA-approved medications for symptomatic relief, allogeneic hematopoietic stem cell transplantation (HSCT) remains the sole curative option, underscoring a persistent need for novel treatments. This review delves into the growing field of gene editing, particularly the extensive research focused on curing haemoglobinopathies like SCD. We examine the use of techniques such as CRISPR-Cas9 and homology-directed repair, base editing, and prime editing to either correct the pathogenic variant into a non-pathogenic or wild-type one or augment fetal haemoglobin (HbF) production. The article elucidates ways to optimize these tools for efficacious gene editing with minimal off-target effects and offers insights into their effective delivery into cells. Furthermore, we explore clinical trials involving alternative SCD treatment strategies, such as LentiGlobin therapy and autologous HSCT, distilling the current findings. This review consolidates vital information for the clinical translation of gene editing for SCD, providing strategic insights for investigators eager to further the development of gene editing for SCD.

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

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

Hb Mizuho Case Report; Early Genomic Testing Facilitates a Life Changing Diagnosis.

Unstable variant hemoglobinopathies are an uncommon cause of hemolysis in the pediatric patient and may cause a delay in diagnosis if there is not a high index of suspicion. Hemoglobin (Hb) Mizuho is a rare unstable hemoglobinopathy caused by a pathogenic variant of the HBB gene with a severe phenotype. Here we report on the first known case of Hb Mizuho in Australia, presenting with features of acute and chronic hemolysis. The morphological features on blood film review, in conjunction with biochemical findings and other clinical features, did not immediately suggest an alternative diagnosis and a Next Generation Sequencing gene analysis approach was taken to investigate genes associated with red blood cell disorders and atypical uremic syndrome. The HBB Mizuho variant was detected and established the diagnosis. This report highlights the challenge of diagnosing Hb Mizuho on conventional testing and the need for early genomic testing to clarify a diagnosis.

Carrier frequency estimation of pathogenic variants of autosomal recessive and X-linked recessive mendelian disorders using exome sequencing data in 1,642 Thais.

BACKGROUND: People with autosomal recessive disorders often were born without awareness of the carrier status of their parents. The American College of Medical Genetics and Genomics (ACMG) recommends screening 113 genes known to cause autosomal recessive and X-linked conditions in couples seeking to learn about their risk of having children with these disorders to have an appropriate reproductive plan. METHODS: We analyzed the exome sequencing data of 1,642 unrelated Thai individuals to identify the pathogenic variant (PV) frequencies in genes recommended by ACMG. RESULTS: In the 113 ACMG-recommended genes, 165 PV and likely PVs in 60 genes of 559 exomes (34%, 559/1642) were identified. The carrier rate was increased to 39% when glucose-6-phosphate dehydrogenase (G6PD) was added. The carrier rate was still as high as 14.7% when thalassemia and hemoglobinopathies were excluded. In addition to thalassemia, hemoglobinopathies, and G6PD deficiency, carrier frequencies of > 1% were found for Gaucher disease, primary hyperoxaluria, Pendred syndrome, and Wilson disease. Nearly 2% of the couples were at risk of having offsprings with the tested autosomal recessive conditions. CONCLUSIONS: Based on the study samples, the expanded carrier screening, which specifically targeted common autosomal recessive conditions in Thai individuals, will benefit clinical outcomes, regarding preconception/prenatal genetic carrier screening.

C2H2 Zinc Finger Transcription Factors Associated with Hemoglobinopathies.

In humans, specific aberrations in ß-globin results in sickle cell disease and ß-thalassemia, symptoms of which can be ameliorated by increased expression of fetal globin (HbF). Two recent CRISPR-Cas9 screens, centered on ∼1500 annotated sequence-specific DNA binding proteins and performed in a human erythroid cell line that expresses adult hemoglobin, uncovered four groups of candidate regulators of HbF gene expression. They are (1) members of the nucleosome remodeling and deacetylase (NuRD) complex proteins that are already known for HbF control; (2) seven C2H2 zinc finger (ZF) proteins, including some (ZBTB7A and BCL11A) already known for directly silencing the fetal γ-globin genes in adult human erythroid cells; (3) a few other transcription factors of different structural classes that might indirectly influence HbF gene expression; and (4) DNA methyltransferase 1 (DNMT1) that maintains the DNA methylation marks that attract the MBD2-associated NuRD complex to DNA as well as associated histone H3 lysine 9 methylation. Here we briefly discuss the effects of these regulators, particularly C2H2 ZFs, in inducing HbF expression for treating ß-hemoglobin disorders, together with recent advances in developing safe and effective small-molecule therapeutics for the regulation of this well-conserved hemoglobin switch.

Diagnosis and screening of abnormal hemoglobins.

Hemoglobin (Hb) abnormalities, such as thalassemia and structural Hb variants, are among the most prevalent inherited diseases and are associated with significant mortality and morbidity worldwide. However, there were not comprehensive reviews focusing on different clinical analytical techniques, research methods and artificial intelligence (AI) used in clinical screening and research on hemoglobinopathies. Hence the review offers a comprehensive summary of recent advancements and breakthroughs in the detection of aberrant Hbs, research methods and AI uses as well as the present restrictions anddifficulties in hemoglobinopathies. Recent advances in cation exchange high performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), isoelectric focusing (IEF), flow cytometry, mass spectrometry (MS) and polymerase chain reaction (PCR) etc have allowed for the definitive detection by using advanced AIand portable point of care tests (POCT) integrating with smartphone microscopic classification, machine learning (ML) model, complete blood counts (CBC), imaging-based method, speedy immunoassay, and electrochemical-, microfluidic- and sensing-related platforms. In addition, to confirm and validate unidentified and novel Hbs, highly specialized genetic based techniques like PCR, reverse transcribed (RT)-PCR, DNA microarray, sequencing of genomic DNA, and sequencing of RT-PCR amplified globin cDNA of the gene of interest have been used. Hence, adequate utilization and improvement of available diagnostic and screening technologies are important for the control and management of hemoglobinopathies.

Lessons learnt in the screening and diagnosis of haemoglobinopathies.

For this paper, cases reported formally and anecdotally to the authors in their screening and diagnostic roles have been selected to demonstrate areas where errors have occurred, and caution should be exercised. The cases demonstrate that it is vital that the performance and limitations of the techniques used, along with the phenotypic presentation of cases where haemoglobin variants and/or thalassaemias are coinherited are understood by those performing result interpretation. Those who deliver the service as well as those who receive reports and give results and counselling should be aware of the complexity of the topic.