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1.
Int J Mol Sci ; 22(7)2021 Mar 31.
Article in English | MEDLINE | ID: mdl-33807258

ABSTRACT

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.


Subject(s)
Erythroid Cells/classification , Erythroid Cells/metabolism , MicroRNAs/genetics , Adult , Age Factors , Cell Differentiation/genetics , Cell Line , Erythropoiesis/genetics , Fetal Blood/cytology , Fetal Hemoglobin/genetics , Fetus/metabolism , Humans , RNA, Messenger/genetics , Transcription Factors , gamma-Globins/genetics
2.
Int J Mol Sci ; 21(18)2020 Sep 11.
Article in English | MEDLINE | ID: mdl-32933098

ABSTRACT

The ß-thalassemias are an increasing challenge to health systems worldwide, caused by absent or reduced ß-globin (HBB) production. Of particular frequency in many Western countries is HBBIVSI-110(G > A) ß-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 ß-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) ß-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 ß-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.


Subject(s)
Alternative Splicing/genetics , Mutation/genetics , beta-Globins/genetics , beta-Thalassemia/genetics , Cells, Cultured , Humans , RNA, Messenger/genetics
5.
Hum Gene Ther Methods ; 29(1): 60-74, 2018 02.
Article in English | MEDLINE | ID: mdl-29325430

ABSTRACT

The ß-hemoglobinopathies sickle cell anemia and ß-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous ß-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human ß-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood-derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human ß-globin locus. At run times of 8 min for separation of murine and human ß-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for ß-hemoglobinopathies.


Subject(s)
Anemia, Sickle Cell , Cell- and Tissue-Based Therapy/methods , Genetic Therapy/methods , Genetic Vectors , beta-Globins , gamma-Globins , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/metabolism , Anemia, Sickle Cell/pathology , Anemia, Sickle Cell/therapy , Animals , Cell Line , Disease Models, Animal , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Mice , Mice, Transgenic , beta-Globins/biosynthesis , beta-Globins/genetics , gamma-Globins/genetics
6.
Cytotherapy ; 19(2): 311-326, 2017 02.
Article in English | MEDLINE | ID: mdl-28088294

ABSTRACT

BACKGROUND AIMS: Primary hematopoietic stem and progenitor cells (HSPCs) are key components of cell-based therapies for blood disorders and are thus the authentic substrate for related research. We propose that ubiquitous small-volume diagnostic samples represent a readily available and as yet untapped resource of primary patient-derived cells for cell- and gene-therapy studies. METHODS: In the present study we compare isolation and storage methods for HSPCs from normal and thalassemic small-volume blood samples, considering genotype, density-gradient versus lysis-based cell isolation and cryostorage media with different serum contents. Downstream analyses include viability, recovery, differentiation in semi-solid media and performance in liquid cultures and viral transductions. RESULTS: We demonstrate that HSPCs isolated either by ammonium-chloride potassium (ACK)-based lysis or by gradient isolation are suitable for functional analyses in clonogenic assays, high-level HSPC expansion and efficient lentiviral transduction. For cryostorage of cells, gradient isolation is superior to ACK lysis, and cryostorage in freezing media containing 50% fetal bovine serum demonstrated good results across all tested criteria. For assays on freshly isolated cells, ACK lysis performed similar to, and for thalassemic samples better than, gradient isolation, at a fraction of the cost and hands-on time. All isolation and storage methods show considerable variation within sample groups, but this is particularly acute for density gradient isolation of thalassemic samples. DISCUSSION: This study demonstrates the suitability of small-volume blood samples for storage and preclinical studies, opening up the research field of HSPC and gene therapy to any blood diagnostic laboratory with corresponding bioethics approval for experimental use of surplus material.


Subject(s)
Blood Specimen Collection/methods , Blood Specimen Collection/standards , Cell Separation/methods , Cell Separation/standards , Cell- and Tissue-Based Therapy/methods , Leukocytes/pathology , Thalassemia/blood , Blood Preservation/methods , Blood Preservation/standards , Cell Proliferation , Cell Survival , Cells, Cultured , Cryopreservation , Feasibility Studies , Freezing , Hematopoietic Stem Cells/pathology , Hematopoietic Stem Cells/physiology , Humans , Leukocyte Count , Leukocytes/physiology , Serologic Tests , Thalassemia/pathology
7.
Hematology ; 22(5): 304-309, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28007020

ABSTRACT

OBJECTIVES: Thalassaemia is a potentially lethal inherited anaemia, caused by reduced or absent synthesis of globin chains. Measurement of the minor adult haemoglobin Hb A2, combining α- with δ-globin, is critical for the routine diagnosis of carrier status for α- or ß-thalassaemia. Here, we aim to characterize a novel δ-globin variant, Hb A2 Episkopi, in a single family of mixed Lebanese and Cypriot ancestry with mild hypochromic anaemia and otherwise normal globin genotype, which also presents with a coincidental 0.78-Mb sequence duplication on chromosome 1 (1q44) and developmental abnormalities. METHODS: Analyses included comprehensive haematological analyses, cation-exchange high-performance liquid chromatography (CE-HPLC), cellulose acetate electrophoresis (CAE), Sanger sequencing and structure-based stability predictions for Hb A2 Episkopi. RESULTS: The GCT > GTT missense mutation, underlying Hb A2 Episkopi, HBD:c.428C > T, introduces a cd142 codon change in the mature protein, resulting in reduced normal Hb A2 amounts and a novel, less abundant Hb A2 variant (HGVS: HBD:p.A143V), detectable as a delayed peak by CE-HPLC. The latter was in line with structure-based stability predictions, which indicated that the substitution of a marginal, non-helical and non-interface residue, five amino acids from the δ-globin chain carboxy-terminus, was moderately destabilizing. DISCUSSION: Detection of the new variant depends on the diagnostic set-up and had failed by CAE and on an independent CE-HPLC system, which, in unfavourable circumstances, may lead to misdiagnoses of ß-thalassaemia as α-thalassaemia. Given the mixed background of the affected family, the ethnic origin of the mutation is unclear, and this study thus suggests awareness for possible detection of Hb A2 Episkopi in both the Cypriot and the Lebanese populations.


Subject(s)
Anemia, Hypochromic/genetics , Chromosomes, Human, Pair 1/genetics , Hemoglobins, Abnormal/genetics , Mutation, Missense , delta-Globins/genetics , Adult , Amino Acid Substitution , Cyprus , Family , Female , Humans , Lebanon , Male
9.
Pediatr Endocrinol Rev ; 8 Suppl 2: 290-4, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21705980

ABSTRACT

In patients with b-thalassemia major (TM), the anterior pituitary gland is particularly sensitive to free radical stresses. It has been reported that the GH deficiency (GHD) may be secondary to either pituitary or hypothalamic dysfunction. The duration of the disease, the patient's age and the severity of iron overload are the most important factors responsible for the defect of growth hormone (GH) secretion. Recent reports have documented a frequency of severe growth hormone deficiency in 13%-32% of patients with b-thalassemia major. All of these patients underwent GH-releasing hormone (GH-RH) plus arginine (ARG) testing. We undertook the present study to evaluate the GH and adrenal response during glucagon stimulation test (GST) in patients with TM because the GH-RH plus ARG test in patients with hypothalamic GHD may be misleading. Thirty-three adult TM patients were recruited (mean age 36.6 years). Fifty four percent were included in the severe GHD group (GH peak below 3mg/l). The IGF-1 level in TM patients was consistently low (60.3 ± 35.3 mg/l) and 86.6% of patients with a normal GH response to GST had a low IGF-1 level. These findings are also indicative of a relative resistance to GH. In eight out of 18 TM patients (44.4%), the GHD was associated with hypogonadotropic hypogonadism. A positive correlation was found between GH peak after GST and IGF-1 level (r = 0.8, p: 0.003) and a negative correlation between the age of female TM patients and GH peak (r = 0.711, p: 0.007). All patients but one had no evidence of cardiac iron overload (mean T2* 30.4 ± 8.2 ms; range 14-44 ms). The mean LVEF (%) in TM patients was no different when compared to healthy controls. However, three patients with severe GHD and normal T2*were found to have reduced LVEF.One patient (4%) had a peak cortisol response to GST compatible to adrenal insufficiency. Nausea, headache and\or hypoglycemia occurred in 3 patients (12%) during GST. In conclusion, our study demonstrates that the presence of GHD is frequent in adult TM patients. According to the international guidelines for medical practice, we believe that before considering hormone replacement therapy, a second test to confirm the diagnosis of GHD and adrenal insufficiency is required.


Subject(s)
Glucagon , Human Growth Hormone/blood , Insulin-Like Growth Factor I/metabolism , Laron Syndrome , Stroke Volume/physiology , beta-Thalassemia , Adolescent , Adrenal Glands/drug effects , Adrenal Glands/metabolism , Adult , Comorbidity , Diagnostic Techniques, Endocrine , Female , Gastrointestinal Agents/administration & dosage , Glucagon/administration & dosage , Human Growth Hormone/deficiency , Humans , Insulin-Like Growth Factor I/biosynthesis , Laron Syndrome/diagnosis , Laron Syndrome/epidemiology , Laron Syndrome/metabolism , Male , Middle Aged , Young Adult , beta-Thalassemia/diagnosis , beta-Thalassemia/epidemiology , beta-Thalassemia/metabolism
10.
Blood Cells Mol Dis ; 44(2): 100-6, 2010.
Article in English | MEDLINE | ID: mdl-19914848

ABSTRACT

We examined the effect of the anthracyclines aclarubicin, bleomycin, daunorubicin, doxorubicin and idarubicin on human gamma- and beta-globin promoter activity in an in vitro luciferase assay, ex vivo in erythroid cultures and in vivo in transgenic mice carrying the human gamma-globin gene. Effects in erythroid liquid cultures derived from healthy donors were assayed by evaluating HbF production with high performance liquid chromatography and by measuring mRNA levels of the globin genes and the proportion of erythroblasts containing HbF. Compounds testing positive in the in vitro and ex vivo assays were applied to erythroid cultures derived from thalassaemic patients. Doxorubicin, idarubicin and daunorubicin increased HbF production in cultures of both, healthy and thalassaemic donors. Daunorubicin induced HbF in thalassaemic cells ex vivo with the highest statistical significance and, importantly and in contrast to the clinical HbF inducer hydroxyurea, showed specific induction of gamma-globin without associated induction of alpha-globin. Daunorubicin was screened in transgenic mice carrying the human (A)gamma-globin gene, and it resulted in increased (A)gamma-globin mRNA levels. Our results indicate that anthracyclines are a promising group of compounds with the potential to provide lead substances for the synthesis of new agents with clinical applications as gamma-globin gene inducers. In parallel, future studies of the epigenetic effects of the five anthracyclines on the beta-globin locus will generate possible mechanistic leads on the regulation of the globin genes.


Subject(s)
Anthracyclines/pharmacology , Anti-Bacterial Agents/pharmacology , Gene Expression Regulation/drug effects , gamma-Globins/genetics , Animals , Anthracyclines/administration & dosage , Anti-Bacterial Agents/administration & dosage , Cell Line , Cells, Cultured , Erythroid Cells/drug effects , Fetal Hemoglobin/genetics , Fetal Hemoglobin/metabolism , Humans , Mice , Mice, Transgenic , Promoter Regions, Genetic/drug effects , RNA, Messenger/genetics , Thalassemia/drug therapy , Thalassemia/genetics , beta-Globins/genetics
11.
Pediatr Endocrinol Rev ; 6 Suppl 1: 116-22, 2008 Oct.
Article in English | MEDLINE | ID: mdl-19337164

ABSTRACT

BACKGROUND: The pathogenesis of bone disease in thalassaemia major (TM) is multifactorial and remains unclear, although gonadal dysfunction probably has the most dominant role. OBJECTIVE: The aim of the study is to investigate the impact of several factors on the development of reduced bone mass in patients of both sexes with TM, treated in our Center. SUBJECTS AND METHODS: 76 thalassaemic patients (26 males, 50 females) of Greek Cypriot origin with a mean age of 31.4 (17-53) years were included in the study. All patients were on the standard treatment protocol for thalassemia at our Center. Bone mineral density (BMD) of lumbar spine and femoral neck was measured by dual-energy X-ray absorptiometry. Factors known to be associated with low bone mass (gender, endocrine disorders, iron overload) were included in the analysis to ascertain possible associations. Iron overload calculation was based on the mean ferritin level over a 6-year period and T2* MRI of the liver and the heart. Statistical analysis was performed with the SPSS program. RESULTS: Bone disease was present in the spine of 89.5% of the patients and in 84.2% at the femoral site. Male patients were more frequently affected than females (92.3% vs. 88.0% in the spine and 88.5% vs. 82% at the femoral neck). Hypogonadal patients were found to be more frequently affected compared to eugonadal patients (94.1% in spine and 88.2% cyat the femoral neck compared to 89.5% and 81.6% respectively). Males with normal gonadal function were more severely affected in the lumbar spine than eugonadal women (male mean BMD z-score was -3.0 vs. -2.037 in women, p=0.004). Low BMD values were found to be more common in the presence of endocrinopathies. No correlation was found between ferritin status and severity of bone disease. Patients with severe liver iron overload seemed to be more affected in the spine. Heart T2* MRI measurements showed that patients with severe and moderate iron overload in the heart were more affected with bone disease in both the spine and femoral neck. CONCLUSIONS: This study demonstrates a gender difference not only in the prevalence of osteoporosis/osteopenia in patients with TM, but also in the severity of the disorder, as males are more frequently and severely affected than females. Moreover, hypogonadism may have a greater impact on spine BMD in females than in males. The underlying pathogenic mechanisms contributing to the development of bone disease in thalassaemia are multiple and complicated, indicating the necessity of further investigation in order to understand the pathophysiology of this highly prevalent complication. Further research in this field will allow the design of preventive and therapeutic measures.


Subject(s)
Osteoporosis/pathology , beta-Thalassemia/pathology , Adolescent , Adult , Bone Density , Cyprus/epidemiology , Female , Humans , Iron Overload/metabolism , Male , Middle Aged , Osteoporosis/epidemiology , Osteoporosis/metabolism , Prevalence , Retrospective Studies , Sex Factors , Young Adult , beta-Thalassemia/epidemiology , beta-Thalassemia/metabolism
12.
Pediatr Endocrinol Rev ; 6 Suppl 1: 144-8, 2008 Oct.
Article in English | MEDLINE | ID: mdl-19337169

ABSTRACT

BACKGROUND: Bisphosphonates are potent inhibitors of osteoclastic bone resorption and have been recently used in thalassaemia major (TM) osteoporosis with encouraging results. OBJECTIVE: The aim of the study is to investigate the effect of two Bisphosphonate drugs, Alendronate and Pamidronate on bone mass in patients of both genders with TM, treated in our Center. SUBJECTS AND METHODS: 53 (22 males, 31 females) Thalassaemic patients of Greek Cypriot origin were randomly divided into two groups. 29 patients in group A with a mean age of 33, 32 years were treated with alendronate and 24 patients in group B with a mean age of 34, 36 years received pamidronate for a period of 2 years. The effectiveness of both drugs was estimated based on the change of Bone mineral density (BMD) values of lumbar spine and femoral neck. Bone mineral density (BMD) of lumbar spine and femoral neck was measured by dual-energy X-ray absiorptiometry. All patients were on the standard treatment protocol of Thalassaemia. Statistical analysis was performed with the SPSS program. RESULTS: After completion of treatment with pamidronate the mean lumbar spine BMD has improved from -2.813 to -2.174 (p<0.001) and the mean hip BMD from -2.138 to -2.078 (p=0.018). The change of spine BMD in patients who received alendronate was from -2.720 to -2.602 (p=0.059) and the changes in BMD at the femoral neck from -2.035 to -2.007 (p=0.829). CONCLUSIONS: This study demonstrates the efficacy of two bisphosphonate drugs in improving BMD values in patients with TM and osteoporosis. Since the origin of bone disease in TM is multifactorial and some of the underlying pathogenic mechanisms are still unclear, further research in this field is needed, which will allow the design of optimal therapeutic measures.


Subject(s)
Alendronate/therapeutic use , Bone Density Conservation Agents/therapeutic use , Bone Density/drug effects , Diphosphonates/therapeutic use , Osteoporosis/drug therapy , beta-Thalassemia/complications , beta-Thalassemia/drug therapy , Absorptiometry, Photon , Adult , Female , Humans , Male , Middle Aged , Osteoporosis/etiology , Pamidronate , Retrospective Studies , Young Adult
13.
Eur J Haematol ; 77(2): 150-6, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16800840

ABSTRACT

BACKGROUND: The clinical severity in thalassaemia major (TM) depends on the underlying mutations of the beta-globin gene and the degree of iron overload. OBJECTIVE: The aim of the study was to investigate the impact of genotype on the development of endocrine complications in TM in our center. SUBJECTS AND METHODS: 126 (62 males, 64 females) thalassaemic patients of Greek Cypriot origin with a mean age of 31.2 (17-68) yr were included in the study. All patients, who were on the standard treatment protocol, were subsequently divided into two groups according to their genotype, group A (92): TM with no mitigating factor and group B (34): TM carrying one or more mitigating factors in the beta- and/or alpha-globin genes. Iron overload calculation was based on the amount of red cell consumption and the mean ferritin level over a 12-year period. Statistical analysis was performed with the SPSS program. RESULTS: Patients in group A, who were consuming larger amounts of blood on transfusions, were more likely to develop hypogonadism (P = 0.001) compared with patients in group B, despite their similar mean ferritin levels. The incidence of other endocrinopathies (short stature, hypothyroidism, and diabetes mellitus) was similar in the two groups. The prevalence of hypothyroidism in splenectomized patients was significantly higher (P = 0.005), whereas the presence of hypogonadism, impaired glucose homeostasis and insulin resistance, although more frequent, was not statistically significant. The clinical severity of TM had no impact on bone mineral density (BMD) in both men and women. BMD was only influenced by gonadal function. CONCLUSIONS: This study demonstrates that the underlying genetic defect in TM is a contributing factor for gonadal dysfunction, because the patients with the more severe defects have a greater rate of iron loading through higher red cell consumption.


Subject(s)
Bone Diseases, Metabolic/etiology , Diabetes Mellitus/etiology , Dwarfism/etiology , Genotype , Globins/genetics , Hypogonadism/etiology , Hypothyroidism/etiology , beta-Thalassemia/genetics , Adolescent , Adult , Aged , Blood Glucose/analysis , Blood Transfusion/statistics & numerical data , Bone Density , Bone Diseases, Metabolic/epidemiology , Chelation Therapy/adverse effects , Combined Modality Therapy , Cyprus/epidemiology , DNA Mutational Analysis , Diabetes Mellitus/epidemiology , Dwarfism/epidemiology , Ethnicity/genetics , Female , Ferritins/blood , Genetic Predisposition to Disease , Humans , Hypogonadism/epidemiology , Hypothyroidism/epidemiology , Incidence , Insulin Resistance/genetics , Iron/metabolism , Iron Chelating Agents/adverse effects , Iron Chelating Agents/therapeutic use , Iron Overload/etiology , Male , Menstrual Cycle , Middle Aged , Retrospective Studies , Risk Factors , Splenectomy , Transfusion Reaction , beta-Thalassemia/complications , beta-Thalassemia/drug therapy , beta-Thalassemia/surgery , beta-Thalassemia/therapy
14.
Pediatr Endocrinol Rev ; 2 Suppl 2: 296-302, 2004 Dec.
Article in English | MEDLINE | ID: mdl-16462715

ABSTRACT

Therapeutic advances in thalassaemia major have significantly increased the average lifespan and improved the quality of life in thalassaemic patients. Therefore attainment of reproductive capacity and creation of a family has become a great task. Endocrine complications due to haemosiderosis and especially hypogonadotrophic hypogonadism are still present in a significant number of patients worldwide and often becomes a barrier in their desire for parenthood. The report of 358 successful pregnancies so far has provided strong evidence not only for the absence of any deleterious effect on the course of thalassaemia but also for the safety of the pregnancy in the thalassaemic woman. Ovarian function is well preserved in women suffering primary or secondary amenorrhea as they become able to conceive following a closely monitored stimulation therapy. The desire of the thalassaemic woman to become a mother is always viewed with special caution and sensitivity. Ambitions of this sort pose numerous medico legal and ethical issues that need to be addressed prudently if the patients' quality of life is to be optimized.


Subject(s)
Fertility , beta-Thalassemia , Adult , Chelation Therapy , Female , Humans , Hypogonadism , Male , Pregnancy , Pregnancy Complications, Hematologic , Pregnancy Outcome
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