Genetic Modifiers Associated with Vaso-Occlusive Crises and Acute Pain Phenomena in Sickle Cell Disease: A Scoping Review.

Sickle cell disease (SCD) is a group of recessive diseases caused by the β sickling mutation of in homozygosity or in compound heterozygosity with other pathogenic mutations. Patients with severe SCD typically experience painful vaso-occlusive crises and other pain-related phenomena, including acute chest syndrome, priapism, dactylitis, avascular necrosis, and splenic sequestration and infarction. High variability of pain-related phenomena per SCD genotype indicates genetic disease modifiers (GDMs) as pathology determinants and, thus, as critical to prognosis, treatment choice, and therapy development.

Non-Invasive Determination of the Paternal Inheritance in Pregnancies at Risk for β-Thalassaemia by Analyzing Cell-Free Fetal DNA Using Targeted Next-Generation Sequencing.

Non-invasive prenatal testing (NIPT) has been widely adopted for the screening of chromosomal abnormalities; however, its adoption for monogenic disorders, such as β-thalassaemia, has proven challenging. Haemoglobinopathies are the most common monogenic disorders globally, with β-thalassaemia being particularly prevalent in Cyprus. This study introduces a non-invasive prenatal haplotyping (NIPH) assay for β-thalassaemia, utilizing cell-free DNA (cfDNA) from maternal plasma.

Case report: Rethinking NGS analysis in diagnosing Diamond-Blackfan anemia syndrome.

Diamond-Blackfan anemia syndrome (DBAS) is a rare inherited bone marrow failure (BMF) syndrome characterized by erythroid aplasia, congenital malformations, and cancer predisposition. With its genetic heterogeneity, variable penetrance and expressivity, DBAS poses significant diagnostic challenges, necessitating advancements in genetic testing for improved accuracy. Here, we present the case of an 18-year-old male with a long-standing macrocytic anemia that remained undiagnosed despite standard whole exome sequencing (WES).

Global Globin Network and adopting genomic variant database requirements for thalassemia.

Thalassemia is one of the most prevalent monogenic disorders in low- and middle-income countries (LMICs). There are an estimated 270 million carriers of hemoglobinopathies (abnormal hemoglobins and/or thalassemia) worldwide, necessitating global methods and solutions for effective and optimal therapy. LMICs are disproportionately impacted by thalassemia, and due to disparities in genomics awareness and diagnostic resources, certain LMICs lag behind high-income countries (HICs).

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

β-Thalassemia is brought about by defective β-globin (HBB [hemoglobin subunit β]) 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 splice mutation.

Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of β-Thalassemia by Gene Addition and Mutation-Specific RNA Interference.

Therapy via the gene addition of the anti-sickling β-globin transgene is potentially curative for all β-hemoglobinopathies and therefore of particular clinical and commercial interest. This study investigates GLOBE-based lentiviral vectors (LVs) for β-globin addition and evaluates strategies for an increased β-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-βAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior β-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs).

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on mRNA synthesis.

Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34 cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for manipulation.

The Optimized γ-Globin Lentiviral Vector GGHI-mB-3D Leads to Nearly Therapeutic HbF Levels In Vitro in CD34 Cells from Sickle Cell Disease Patients.

We have previously demonstrated that both the original γ-globin lentiviral vector (LV) GGHI and the optimized GGHI-mB-3D LV, carrying the novel regulatory elements of the 3D HPFH-1 enhancer and the 3' β-globin UTR, can significantly increase HbF production in thalassemic CD34 cells and ameliorate the disease phenotype in vitro. In the present study, we investigated whether the GGHI-mB-3D vector can also exhibit an equally therapeutic effect, following the transduction of sickle cell disease (SCD) CD34 cells at MOI 100, leading to HbF increase coupled with HbS decrease, and thus, to phenotype improvement in vitro. We show that GGHI-mB-3D LV can lead to high and potentially therapeutic HbF levels, reaching a mean 2-fold increase to a mean value of VCN/cell of 1.

Catching Them Early: Framework Parameters and Progress for Prenatal and Childhood Application of Advanced Therapies.

Advanced therapy medicinal products (ATMPs) are medicines for human use based on genes, cells or tissue engineering. After clear successes in adults, the nascent technology now sees increasing pediatric application. For many still untreatable disorders with pre- or perinatal onset, timely intervention is simply indispensable; thus, prenatal and pediatric applications of ATMPs hold great promise for curative treatments.

Global Globin Network Consensus Paper: Classification and Stratified Roadmaps for Improved Thalassaemia Care and Prevention in 32 Countries.

The Global Globin Network (GGN) is a project-wide initiative of the Human Variome/Global Variome Project (HVP) focusing on haemoglobinopathies to build the capacity for genomic diagnosis, clinical services, and research in low- and middle-income countries. At present, there is no framework to evaluate the improvement of care, treatment, and prevention of thalassaemia and other haemoglobinopathies globally, despite thalassaemia being one of the most common monogenic diseases worldwide. Here, we propose a universally applicable system for evaluating and grouping countries based on qualitative indicators according to the quality of care, treatment, and prevention of haemoglobinopathies.

CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation.

Molecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate γ-globin repressor in erythroid cells by tagging the 3' untranslated region (UTR) of with miRNA recognition sites (MRSs) for the abundant erythromiR miR-451a.

Regulation of ER Composition and Extent, and Putative Action in Protein Networks by ER/NE Protein TMEM147.

Nuclear envelope (NE) and endoplasmic reticulum (ER) collaborate to control a multitude of nuclear and cytoplasmic actions. In this context, the transmembrane protein TMEM147 localizes to both NE and ER, and through direct and indirect interactions regulates processes as varied as production and transport of multipass membrane proteins, neuronal signaling, nuclear-shape, lamina and chromatin dynamics and cholesterol synthesis. Aiming to delineate the emerging multifunctionality of TMEM147 more comprehensively, we set as objectives, first, to assess potentially more fundamental effects of TMEM147 on the ER and, second, to identify significantly TMEM147-associated cell-wide protein networks and pathways.

Adapting the ACMG/AMP variant classification framework: A perspective from the ClinGen Hemoglobinopathy Variant Curation Expert Panel.

Accurate and consistent interpretation of sequence variants is integral to the delivery of safe and reliable diagnostic genetic services. To standardize the interpretation process, in 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published a joint guideline based on a set of shared standards for the classification of variants in Mendelian diseases. The generality of these standards and their subjective interpretation between laboratories has prompted efforts to reduce discordance of variant classifications, with a focus on the expert specification of the ACMG/AMP guidelines for individual genes or diseases.

Therapy Development by Genome Editing of Hematopoietic Stem Cells.

Accessibility of hematopoietic stem cells (HSCs) for the manipulation and repopulation of the blood and immune systems has placed them at the forefront of cell and gene therapy development. Recent advances in genome-editing tools, in particular for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and CRISPR/Cas-derived editing systems, have transformed the gene therapy landscape. Their versatility and the ability to edit genomic sequences and facilitate gene disruption, correction or insertion, have broadened the spectrum of potential gene therapy targets and accelerated the development of potential curative therapies for many rare diseases treatable by transplantation or modification of HSCs.

Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells.

MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures.

Relative and Absolute Quantification of Aberrant and Normal Splice Variants in β-Thalassemia.

The β-thalassemias are an increasing challenge to health systems worldwide, caused by absent or reduced β-globin (HBB) production. Of particular frequency in many Western countries is β-thalassemia (HGVS name: HBB:c.93-21G > A).

TMEM147 interacts with lamin B receptor, regulates its localization and levels, and affects cholesterol homeostasis.

The structurally and functionally complex endoplasmic reticulum (ER) hosts critical processes including lipid synthesis. Here, we focus on the functional characterization of transmembrane protein TMEM147, and report that it localizes at the ER and nuclear envelope in HeLa cells. Silencing of drastically reduces the level of lamin B receptor (LBR) at the inner nuclear membrane and results in mistargeting of LBR to the ER.

Acid Ceramidase Depletion Impairs Neuronal Survival and Induces Morphological Defects in Neurites Associated with Altered Gene Transcription and Sphingolipid Content.

The gene encodes acid ceramidase (AC), an enzyme that is implicated in the metabolism of ceramide (Cer). Mutations in the gene cause two different disorders, Farber disease (FD), a rare lysosomal storage disorder, and a rare form of spinal muscular atrophy combined with progressive myoclonic epilepsy (SMA-PME). In the absence of human in vitro neuronal disease models and to gain mechanistic insights into pathological effects of deficiency, we established and characterized a stable knockdown () SH-SY5Y cell line.

The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements.

The common IVSI-110 (G>A) β-thalassemia mutation is a paradigm for intronic disease-causing mutations and their functional repair by non-homologous end joining-mediated disruption. Such mutation-specific repair by disruption of aberrant regulatory elements (DARE) is highly efficient, but to date, no systematic analysis has been performed to evaluate disease-causing mutations as therapeutic targets. Here, DARE was performed in highly characterized erythroid IVSI-110(G>A) transgenic cells and the disruption events were compared with published observations in primary CD34 cells.

Fe enrichment in mice for β-thalassaemia studies via Mössbauer spectroscopy of blood samples.

In this work, wild-type and heterozygous β-thalassaemic mice were enriched with Fe via gastrointestinal absorption to characterize in greater detail the iron complexes then identifiable via Mössbauer spectroscopy. The Fe enrichment method was validated and Mössbauer spectra were obtained at 80 K from blood samples from wild-type and β-thalassaemic mice at 1, 3, 6, and 9 months of age. As expected, the haemoglobin levels of the thalassaemic mice were lower than from normal mice, indicating anaemia.

Disruptive Technology: CRISPR/Cas-Based Tools and Approaches.

Designer nucleases are versatile tools for genome modification and therapy development and have gained widespread accessibility with the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology. Prokaryotic RNA-guided nucleases of CRISPR/Cas type, since first being adopted as editing tools in eukaryotic cells, have experienced rapid uptake and development. Diverse modes of delivery by viral and non-viral vectors and ongoing discovery and engineering of new CRISPR/Cas-type tools with alternative target site requirements, cleavage patterns and DNA- or RNA-specific action continue to expand the versatility of this family of nucleases.