Initiation of regular transfusion in transfusion-dependent thalassemia (TDT) is based on the assessment of clinical phenotype. Pathogenic HBB variants causing β-thalassemia are important determinants ...of phenotype and could be used to aid decision making. We investigated the association of HBB genotype with survival in a cohort study in the four thalassemia centres in Cyprus. HBB genotype was classified as severe (β0/β0 or β+/β0), moderate (β+/β+), or mild (β0/β++ or β+/β++). Risk factors for mortality were evaluated using multivariate Cox proportional-hazards regression. 537 subjects were followed for a total of 20,963 person years. 80.4% (95% CI 76.4-84.7) of individuals survived to 50 years of age with increasing rates of liver, infection and malignancy-related deaths observed during recent follow-up. We evaluated non-modifiable risk factors and found worse outcomes associated with male sex (Hazard ratio 1.9, 95% CI 1.1-3.0, p=0.01) and milder genotype (Hazard ratio 1.6, 95% CI 1.1-2.3, p=0.02). The effect of genotype was confirmed in a second model, which included treatment effects. Patients with a milder genotype initiated transfusion significantly later and had reduced blood requirements compared to those with moderate or severe genotypes, although pre-transfusion hemoglobin levels did not differ between genotypes. Our results suggest that early treatment decisions to delay transfusion and different long-term treatment strategies in milder genotypes have led to adverse long-term effects of under-treated thalassemia and worse survival. We propose that HBB genotype determination and use of this information to aid in decision making can improve long-term outcomes of thalassaemia patients.
Several types of haemoglobinopathies are caused by copy number variants (CNVs). While diagnosis is often based on haematological and biochemical parameters, a definitive diagnosis requires molecular ...DNA analysis. In some cases, the molecular characterisation of large deletions/duplications is challenging and inconclusive and often requires the use of specific diagnostic procedures, such as multiplex ligation-dependent probe amplification (MLPA). Herein, we collected and comprehensively analysed all known CNVs associated with haemoglobinopathies. The dataset of 291 CNVs was retrieved from the IthaGenes database and was further manually annotated to specify genomic locations, breakpoints and MLPA probes relevant for each CNV. We developed IthaCNVs, a publicly available and easy-to-use online tool that can facilitate the diagnosis of rare and diagnostically challenging haemoglobinopathy cases attributed to CNVs. Importantly, it facilitates the filtering of available entries based on the type of breakpoint information, on specific chromosomal and locus positions, on MLPA probes, and on affected gene(s). IthaCNVs brings together manually curated information about CNV genomic locations, functional effects, and information that can facilitate CNV characterisation through MLPA. It can help laboratory staff and clinicians confirm suspected diagnosis of CNVs based on molecular DNA screening and analysis.
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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.
cells displayed reduced proliferation due to elevated apoptosis and G1/S cell cycle arrest. Distribution of LAMP1-positive lysosomes towards the cell periphery and significantly shortened and less branched neurites upon differentiation, implicate AC for lysosome positioning and neuronal development, respectively. Lipidomic analysis revealed changes in the intracellular levels of distinct sphingolipid species, importantly without Cer accumulation, in line with altered gene transcription within the sphingolipid pathway. Additionally, the transcript levels for Rho GTPases (RhoA, Rac1, and Cdc42), which are key regulators of axonal orientation, neurite branching and lysosome positioning were found to be dysregulated. This study shows the critical role of AC in neurons and suggests how AC depletion leads to defects seen in neuropathology of SMA-PME and FD.
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Haemoglobinopathies are the commonest monogenic diseases worldwide and are caused by variants in the globin gene clusters. With over 2400 variants detected to date, their interpretation using the ...American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines is challenging and computational evidence can provide valuable input about their functional annotation. While many in silico predictors have already been developed, their performance varies for different genes and diseases. In this study, we evaluate 31 in silico predictors using a dataset of 1627 variants in
,
and
. By varying the decision threshold for each tool, we analyse their performance (a) as binary classifiers of pathogenicity and (b) by using different non-overlapping pathogenic and benign thresholds for their optimal use in the ACMG/AMP framework. Our results show that CADD, Eigen-PC, and REVEL are the overall top performers, with the former reaching moderate strength level for pathogenic prediction. Eigen-PC and REVEL achieve the highest accuracies for missense variants, while CADD is also a reliable predictor of non-missense variants. Moreover, SpliceAI is the top performing splicing predictor, reaching strong level of evidence, while GERP++ and phyloP are the most accurate conservation tools. This study provides evidence about the optimal use of computational tools in globin gene clusters under the ACMG/AMP framework.
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. Ongoing developments seek to address efficiency and precision of HSC modification, tolerability of treatment and the distribution and affordability of corresponding therapies. Here, we give an overview of recent progress in the field of HSC genome editing as treatment for inherited disorders and summarize the most significant findings from corresponding preclinical and clinical studies. With emphasis on HSC-based therapies, we also discuss technical hurdles that need to be overcome en route to clinical translation of genome editing and indicate advances that may facilitate routine application beyond the most common disorders.
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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. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.
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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. To this end, we employed nucleofection of CRISPR/Cas9 ribonucleoprotein (RNP) particles alongside double- or single-stranded oligodeoxynucleotides for, respectively, non-homologous-end-joining (NHEJ)- or homology-directed-repair (HDR)-mediated MRS insertion. NHEJ-based tagging was imprecise and inefficient (≤6%) and uniformly produced knock-in- and indel-containing MRS tags, whereas HDR-based tagging was more efficient (≤18%), but toxic for longer donors encoding concatenated and thus potentially more efficient MRS tags. Isolation of clones for robust HEK293T cells tagged with a homozygous quadruple MRS resulted in 25% spontaneous reduction in BCL11A and up to 36% reduction after transfection with an miR-451a mimic. Isolation of clones for human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells tagged with single or double MRS allowed detection of albeit weak γ-globin induction. Our study demonstrates suitability of TAMED for physiologically relevant modulation of gene expression and its unsuitability for therapeutic application in its current form.
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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 HBBIVSI−110(G > ...A) β-thalassemia (HGVS name: HBB:c.93-21G > A). Its underlying mutation creates an abnormal splice acceptor site in the HBB gene, and while partially retaining normal splicing of HBB, it severely reduces HBB protein expression from the mutant locus and HBB loci in trans. For the assessment of the underlying mechanisms and of therapies targeting β-thalassemia, accurate quantification of aberrant and normal HBB mRNA is essential, but to date, has only been performed by approximate methods. To address this shortcoming, we have developed an accurate, duplex reverse-transcription quantitative PCR assay for the assessment of the ratio and absolute quantities of normal and aberrant mRNA species as a tool for basic and translational research of HBBIVSI−110(G > A) β-thalassemia. The method was employed here to determine mRNA ratios and quantities in blood and primary cell culture samples and correlate them with HBB protein levels. Moreover, with its immediate utility for β-thalassemia and the mutation in hand, the approach can readily be adopted for analysis of alternative splicing or for quantitative assays of any disease-causing mutation that interferes with normal splicing.
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To develop a sensitive, specific, simple, cost-effective and reproducible platform for the non-invasive prenatal detection of paternally inherited alleles for β-thalassaemia. The development of such ...an assay is of major significance in order to replace currently-applied invasive methods containing inherent fetal loss risks.
We present a fast Temperature-Gradient Co-amplification at Lower Denaturation Temperature Polymerase Chain Reaction (fast TG COLD PCR) methodology for the detection of the paternally-inherited fetal alleles in maternal plasma. Two single-nucleotide polymorphisms (SNPs), rs7480526 (G/T) and rs968857 (G/A) that are located on the β-globin gene cluster and exhibit a high degree of heterozygosity in the Cypriot population were selected for evaluation. Seventeen maternal plasma samples from pregnancies at risk for β-thalassemia were analysed for the selected SNPs using the novel fast TG COLD PCR assay.
Using fast TG COLD PCR, the paternally inherited allele in cell free fetal DNA was correctly determined for all the 17 maternal plasma samples tested, showing full agreement with the Chorionic Villus Sampling (CVS) analysis.
Our findings are encouraging and demonstrate the efficiency and sensitivity of fast TG COLD PCR in detecting the minor paternally-inherited fetal alleles in maternal plasma for the development of a NIPD assay for β-thalassaemia.
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