Analysis of CircRNAs and CircRNA-Associated Competing Endogenous RNA Networks in β-Thalassemia

The involvement of circRNAs in β-thalassemia and their actions on fetal hemoglobin (HbF) is unclear. Here, the circRNAs in β-thalassemia carriers with high HbF levels were comprehensively analyzed in comparison with healthy individuals. Differential expression of 2183 circRNAs was observed and their correlations with hematological parameters were investigated. Down-regulated has-circRNA-100466 had a strong negative correlation with HbF and HbA2. Bioinformatics was employed to construct a has-circRNA-100466‑associated competing endogenous RNA (ceRNA) network with the determination of hub genes and associated miRNAs. In combination with previous reports, the has-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified. The ceRNA network was verified by qRT-PCR on β-thalassemia samples and RNA immunoprecipitation of K562 cell lysates. Has-circRNA-100466, miR-19b-3p, and SOX6 were present together in anti-argonaute 2 immunoprecipitates, indicating involvement with HbF induction. Furthermore, spearman correlation coefficients revealed their significant correlations with HbF. In conclusion, a novel has-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified, providing insight into HbF induction and suggesting targets β-thalassemia treatment.

Epigenomic analysis of KLF1 haploinsufficiency in primary human erythroblasts

Haploinsufficiency for the erythroid-specific transcription factor KLF1 is associated with hereditary persistence of fetal hemoglobin (HPFH). Increased HbF ameliorates the symptoms of β-hemoglobinopathies and downregulation of KLF1 activity has been proposed as a potential therapeutic strategy. However, the feasibility of this approach has been challenged by the observation that KLF1 haploinsufficient individuals with the same KLF1 variant, within the same family, display a wide range of HbF levels. This phenotypic variability is not readily explained by co-inheritance of known HbF-modulating variants in the HBB, HBS1L-MYB and/or BCL11A loci. We studied cultured erythroid progenitors obtained from Maltese individuals in which KLF1 p.K288X carriers display HbF levels ranging between 1.3 and 12.3% of total Hb. Using a combination of gene expression analysis, chromatin accessibility assays and promoter activity tests we find that variation in expression of the wildtype KLF1 allele may explain a significant part of the variability in HbF levels observed in KLF1 haploinsufficiency. Our results have general bearing on the variable penetrance of haploinsufficiency phenotypes and on conflicting interpretations of pathogenicity of variants in other transcriptional regulators such as EP300, GATA2 and RUNX1.

The Participation of the Organism Adaptation Mechanisms to the Lack of Oxygen According to the Assessment of the Fethemoglobin Content in the Peripheral Blood of Patients with Community-acquired Pneumonia

Introduction. Сommunity-acquired pneumonia (COP) is a global socio-medical problem. At emergence of pneumonia by any genesis, hypoxia develops. Oxygen homeostasis of the body is provided by the coordinated interaction of external respiration, circulatory system and oxygen-transport system of the blood. Hypoxia, due to the malfunction of the external respiratory system, causes the formation of compensatory changes, in the implementation of which involved components of the oxygen transport system. Molecular genetic mechanisms play an important role in the body's adaptation to oxygen deficiency. Fetal hemoglobin (FetHb), having an increased affinity for oxygen, makes a significant contribution to the body's adaptation to new conditions with altered gaseous environment in the presence of pathological processes occurring with hypoxia. In this regard, it is interest to determine FetHb in adults with COP to study its effect on the diagnosis, prognosis and outcome of the disease. The aim of the study. To determinate the participation of the organism adaptation mechanisms to the lack of oxygen according to the assessment of the content of fetal hemoglobin in the peripheral blood of patients with community-acquired pneumonia. Materials and methods. We examined 34 adult patients (18 women and 16 men) with COP, aged 18 to 80 years, who were in the therapeutic department of the City Clinical Hospital № 25 in Kharkiv. The control group was formed of 20 healthy individuals. Spirography was performed on the diagnostic complex "Valenta"; hematological examinations – on the analyzer "ADVIA 60"; measurement of pO2 and pCO2, oxygen saturation, content of fetal hemoglobin – on the device "RAPIDLAB865". Results. In patients with community-acquired pneumonia, there was a decrease of the ventilatory function of external respiration, which is confirmed by a marked decrease in partial oxygen pressure. Oxygen saturation of blood was reduced in the group of patients with COP, but the difference was not statistically significant 94.8 ± 1.0 %. This indicates the presence of compensatory mechanisms aimed at maintaining adequate blood oxygen saturation. Significant increase in pH (from 7.40 to 7.53) and decrease in standard bicarbonate (from 1.27 to 0.68 mmol/l) resulting from violation of the gas composition of the blood can be regarded as a manifestation of partially compensated respiratory alkalosis. In patients with COP, there was a reduction in the total time of hemolysis, a shift of the maximum erythrogram to the left and an increase in the maximum itself, indicating a sharp decline in erythrocyte resistance. The proportion of erythrocytes with reduced resistance was twice as large as similar forms in the control group and the number of highly resistant cells in patients with COP sharply decreased. Obviously, oxygen starvation-mediated stress erythropoiesis is accompanied by the entry into the circulation of functionally defective erythrocytes. They are subject to accelerated elimination from the vascular bed, which causes a decrease in the quantitative indicators of red blood (erythrocyte content, hemoglobin) while maintaining corpuscular parameters (Mean Corpuscular Volume, Mean Cell Hemoglobin Concentration). At the same time, the analysis of individual hemoglobin fractions revealed an increase in the proportion of fetal hemoglobin (from 2.90 ± 0.31 % in the group of healthy individuals to 5.43 ± 1.05 % in patients with COP) (p less than 0.05). Conclusions. Changes in the parameters of acid hemolysis, fetal hemoglobin in the peripheral blood of patients with community-acquired pneumonia with impaired pulmonary ventilation function indicate their participation in the mechanisms of adaptation to oxygen deficiency and they have informative potential. Elevated fetal hemoglobin in peripheral blood in these patients can be used as an indicator of hypoxia, accompanied by impaired oxygen delivery to tissues, which should be used as an additional criterion for diagnosing tissue hypoxia and justify the timely appointment of antihypoxia drugs. Keywords: hypoxia, community-acquired pneumonia, red blood cells, fetal hemoglobin.

Effect of maternal hemoglobin and iron status on fetal hemoglobin, iron status, growth and maturity

Background: Iron deficiency anemia during pregnancy is a major health issue in India. However, the status of iron stores in infants born to iron depleted mothers remains controversial and inadequately investigated. The present study is therefore an attempt to understand whether maternal anemia and iron stores have any significant effect on iron status and growth of fetus.Methods: This is a prospective, cross-sectional, hospital based study conducted at Mata Chanan Devi Hosital, Janakpuri, New Delhi. Hemoglobin and iron profile of 100 newborns and their mothers were taken. Mothers were divided into anemic and non anemic group to see the effect of maternal anemia on fetus.Results: A total of 100 newborns and their mothers were analysed. Cord hemoglobin and iron profile was significantly reduced in anemic compared to non anemic group. Birth weight of newborn was significantly reduced only in moderately anemic group as compared to non anemic group but there was no difference seen in length and head circumference.Conclusions: Maternal serum ferritin levels should be measured for the diagnosis of occult iron deficiency in the fetus so that timely measures can be taken to prevent iron deficiency anemia in the newborn.

Expression of γ-globin genes in β-thalassemia patients treated with sirolimus: results from a pilot clinical trial (Sirthalaclin)

Introduction: The β-thalassemias are due to autosomal mutations of the β-globin gene, inducing absence or low-level synthesis of β-globin in erythroid cells. It is widely accepted that high production of fetal hemoglobin (HbF) is beneficial for β-thalassemia patients. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus found to be a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular and clinical results of the sirolimus-based NCT03877809 clinical trial (A Personalized Medicine Approach for β-thalassemia Transfusion Dependent Patients: Testing sirolimus in a First Pilot Clinical Trial: Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed by reverse-transcription-quantitative PCR and the hemoglobin pattern by HPLC. The immunophenotype was analyzed by FACS using antibodies against CD3, CD4, CD8, CD14, CD19, CD25. Results: The results were obtained in 8 patients with β+/β+ and β+/β0 genotypes, treated with a starting dosage of 1 mg/day sirolimus for 24-48 weeks. The first finding of the study was that expression of γ-globin mRNA was increased in blood and erythroid precursor cells isolated from β-thalassemia patients treated with low-dose sirolimus. A second important conclusion of our trial was that sirolimus influences erythropoiesis and reduces biochemical markers associated to ineffective erythropoiesis (I.E.) (excess of free α-globin chains, bilirubin, soluble transferrin receptor and ferritin). In most (7/8) of the patients a decrease of the transfusion index was observed. The drug was well tolerated with minor effects on immunophenotype, the only side effect being frequently occurring stomatitis. Conclusions: The data obtained indicate that sirolimus given at low doses modifies hematopoiesis and induces increased expression of γ-globin genes in a sub-set of β-thalassemia patients. Further clinical trials are warranted, considering the possibility to test the drug in patients with less severe forms of the disease and exploring combination therapies.

Treatment of Erythroid Precursor Cells from β-Thalassemia Patients with Cinchona Alkaloids: Induction of Fetal Hemoglobin Production

β-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding β-globin, causing an absence or low-level production of adult hemoglobin (HbA). Induction of fetal hemoglobin (HbF) is considered to be of key importance for the development of therapeutic protocols for β-thalassemia and novel HbF inducers need to be proposed for pre-clinical development. The main purpose on this study was to analyze Cinchona alkaloids (cinchonidine, quinidine and cinchonine) as natural HbF-inducing agents in human erythroid cells. The analytical methods employed were Reverse Transcription quantitative real-time PCR (RT-qPCR) (for quantification of γ-globin mRNA) and High Performance Liquid Chromatography (HPLC) (for analysis of the hemoglobin pattern). After an initial analysis using the K562 cell line as an experimental model system, showing induction of hemoglobin and γ-globin mRNA, we verified whether the two more active compounds, cinchonidine and quinidine, were able to induce HbF in erythroid progenitor cells isolated from β-thalassemia patients. The data obtained demonstrate that cinchonidine and quinidine are potent inducers of γ-globin mRNA and HbF in erythroid progenitor cells isolated from nine β-thalassemia patients. In addition, both compounds were found to synergize with the HbF inducer sirolimus for maximal production of HbF. The data obtained strongly indicate that these compounds deserve consideration in the development of pre-clinical approaches for therapeutic protocols of β-thalassemia.

Investigational curative gene therapy approaches to sickle cell disease

Sickle cell disease (SCD) is an inherited blood condition resulting from abnormal hemoglobin production. It is one of the most common genetic diseases in the world. The clinical manifestations are variable and range from recurrent acute and debilitating painful crises to life-threatening pulmonary, cardiovascular, renal, and neurologic complications. The only curative treatment of SCD at this time is bone marrow transplantation (also called hematopoietic stem cell transplantation) using healthy blood stem cells from an unaffected brother or sister or from an unrelated donor if one can be identified who is a match in tissue typing. Unfortunately, only a minority of patients with sickle cell has such a donor available. The use of autologous hematopoietic stem cells and alternative types of genetic modifications is currently under study in clinical research trials for this disease. The approaches include the use of viral vectors to express globin genes that are modified to prevent sickle hemoglobin polymerization or to express interfering RNAs to “flip the switch” in adult red cells from adult β-sickle hemoglobin to fetal hemoglobin using a physiologic switch, and several gene editing approaches with the goal of inducing fetal hemoglobin or correcting/modifying the actual sickle mutation. In this audio review, we will discuss these different approaches and review the current progress of curative therapy for SCD using gene therapy.

Salubrinal induces fetal hemoglobin expression via the stress-signaling pathway in human sickle erythroid progenitors and sickle cell disease mice

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24µM SAL increased F-cells levels 1.4-fold (p=0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p=0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p=0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.

LSD1 inhibition enhances robust fetal hemoglobin induction in human β0-thalassemia/HbE erythroid cells

Induction of fetal hemoglobin (HbF) ameliorates the clinical severity of β-thalassemias. Histone methyltransferase LSD1 enzyme removes methyl groups from the activating chromatin mark histone 3 lysine 4 at silenced genes, including the γ-globin genes. LSD1 inhibitor RN-1 induces HbF levels in cultured human erythroid cells. Here, the HbF-inducing activity of RN-1 was investigated in erythroid progenitor cells derived from β0-thalassemia/HbE patients. The significant and reproducible increases in γ-globin transcript and HbF expression upon RN-1 treatment was demonstrated in erythroid cells with divergent HbF baseline levels, the average of HbF induction was 17.7 + 0.8%. RN-1 at low concentration did not affect viability and proliferation of erythroid cells, but decreases in cell number was observed in cells treated with RN-1 at high concentration. Delayed terminal erythroid differentiation was revealed in β0-thalassemia/HbE erythroid cells treated with RN-1 as similar to other compounds that target LSD1 activity. Downregulation of repressors of γ-globin expression; NCOR1 and SOX6, was observed in RN-1 treatment. These findings provide a proof of concept that a LSD1 epigenetic enzymes is a potential therapeutic target for β0-thalassemia/HbE patients.