Regulation of N6-methyladenosine modification in erythropoiesis and thalassemia.

In eukaryotic RNA, N6-methyladenosine (m6A) is a prevalent form of methylation modification. The m6A modification process is reversible and dynamic, written by m6A methyltransferase complex, erased by m6A demethylase, and recognized by m6A binding proteins. Through mediating RNA stability, decay, alternative splicing, and translation processes, m6A modification regulates gene expression at the post-transcriptional level. Erythropoiesis is the process of hematopoietic stem cells undergoing proliferation, a series of differentiation and maturation to form red blood cells (RBCs). Thalassemia is a common monogenic disease characterized by excessive production of ineffective RBCs in the peripheral circulation, resulting in hemolytic anemia. Increasing evidence suggests that m6A modification plays a crucial role in erythropoiesis. In this review, we comprehensively summarize the function of m6A modification in erythropoiesis and further generalize the mechanism of m6A modification regulating ineffective erythropoiesis and fetal hemoglobin expression. The purpose is to improve the understanding of the pathogenesis of erythroid dysplasia and offer new perspectives for the diagnosis and treatment of thalassemia.

A dataset of digital holograms of normal and thalassemic cells.

Digital holographic microscopy (DHM) is an intriguing medical diagnostic tool due to its label-free and quantitative nature, providing high-contrast images of phase samples. By capturing both intensity and phase information, DHM enables the numerical reconstruction of quantitative phase images. However, the lateral resolution is limited by the diffraction limit, which prompted the recent suggestion of microsphere-assisted DHM to enhance the DHM resolution straightforwardly. The use of such a technique as a medical diagnostic tool requires testing and validation of the proposed assays to prove their feasibility and viability. This paper publishes 760 and 609 microsphere-assisted DHM images of normal and thalassemic red blood cells obtained from a normal and thalassemic male individual, respectively.

CHMMOTv1 - cardiac and hepatic multi-echo (T2*) MRI images and clinical dataset for Iron overload on thalassemia patients.

INTRODUCTION: Regarding deep learning networks in medical sciences for improving diagnosis and treatment purposes and the existence of minimal resources for them, we decided to provide a set of magnetic resonance images of the cardiac and hepatic organs. DATABASE DESCRIPTION: The dataset included 124 patients (67 women and 57 men) with thalassemia (THM), the age range of (5-52) years. Patients were divided into two groups: with follow-up (1-5 times) at time intervals of about (5-6) months and without follow-up. T2* and, R2* values, the results of the Cardiac and Hepatic overload report (normal, mild, moderate, severe), and laboratory tests including Ferritin, Bilirubin (D, and T), AST, ALT, and ALP levels were provided as an Excel file. Also, the details of the patients' Echocardiogram data have been made available. This dataset CHMMOTv1) has been published in Mendeley Dataverse and also is accessible through the web at: http://databiox.com .

Magnetic resonance imaging assessment of the changes of cardiac and hepatic iron load in thalassemia patients before and after hematopoietic stem cell transplantation.

To investigate the value of T2* technique on 3.0 T magnetic resonance imaging (MRI) in evaluating the changes of cardiac and hepatic iron load before and after hematopoietic stem cell transplantation (HSCT) in patients with thalassemia (TM), the 141 TM patients were divided into 6 group for subgroup analysis: 6, 12, 18, 24 and > 24 months group, according to the postoperative interval. The T2* values of heart and liver (H-T2*, L-T2*) were quantified in TM patients before and after HSCT using 3.0 T MRI T2* technology, and the corresponding serum ferritin (SF) was collected at the same time, and the changes of the three before and after HSCT were compared. The overall H-T2* (P = 0.001) and L-T2* (P = 0.041) of patients after HSCT were higher than those before HSCT (mean relative changes = 19.63%, 7.19%). The H-T2* (P < 0.001) and L-T2* (P < 0.001) > 24 months after HSCT were significantly higher than those before HSCT (mean relative changes = 69.19%, 93.73%). The SF of 6 months (P < 0.001), 12 months (P = 0.008), 18 months (P = 0.002) and > 24 months (P = 0.001) were significantly higher than those before HSCT (mean relative changes = 57.93%, 73.84%, 128.51%, 85.47%). There was no significant improvement in cardiac and liver iron content in TM patients within 24 months after HSCT, while the reduction of cardiac and liver iron content in patients is obvious when > 24 months after HSCT.

Transient Elastography of Liver and Serum Hyaluronic Acid Levels In Adult Transfusion Dependent Thalassemia Patients.

Iron overload occurs as a result of multiple blood transfusions and increased iron absorption in thalassemia patients. Iron deposition in liver results in liver stiffness and fibrosis. Non invasive methods including imaging and serum biomarkers have been introduced for assessment of liver fibrosis. We aimed to study liver stiffness using transient elastography and serum hyaluronic acid levels and correlate them with serum ferritin levels in adult transfusion dependent beta thalassemia patients. MATERIAL: 70 transfusion dependent thalassemia patients of age ≥18 years, registered at Thalassemia Day Care Centre were subjected to investigations like CBC, Liver function tests, viral markers, serum ferritin, serum hyaluronic acid levels and transient elastography. Fibrosis indices like FIB-4, AAR and APRI were also calculated. 45 patients had T2*MRI reports with them; which were also included and analysed. Spearman coefficient r was used to test correlations between TE values and serum HA levels with other variables. OBSERVATION: 70 patients (41 male and 29 female) with mean age of 24.09±5.38 years and BMI 20.51 ±3.47 kg/m², were enrolled. Median values of hemoglobin, AST, ALT, TE, serum HA and serum ferritin were, 9.15 g/dl, 42 IU/L, 47.50 IU/L, 9.1 kPa, 284 ng/dl and 1841 ng/ml, respectively . TE values had significant positive correlation with serum ferritin (r=0.5, p < 0.001), ALT (r=0.59, p < 0.001), AST (r=0.58, p< 0.001), APRI (r=0.5, p<0.001) and FIB-4 (p=0.02), respectively and significant negative correlation with T2* MRI (ms) (r= -0.5, p<0.001). No significant correlation of HA was found with any variable. CONCLUSION: Transient elastography can be used as a non expensive, easily accessible and non invasive marker of liver iron overload. Further detailed studies are required to establish the role of serum Hyaluronic acid in thalassemia patients.

National networking in rare diseases and reduction of cardiac burden in thalassemia major.

AIMS: A tailored chelation therapy guided by magnetic resonance imaging (MRI) is a strategy to improve the prognosis in iron-loaded patients, in many cases still hampered by limited MRI availability. In order to address this issue, the Myocardial Iron Overload in Thalassemia (MIOT) network was established in Italy and we aimed to describe the impact of 10-year activity of this network on cardiac burden in thalassemia major (TM). METHODS AND RESULTS: Within the MIOT network, 1746 TM patients (911 females; mean age 31.2 ± 9.1 years) were consecutively enrolled and prospectively followed by 70 thalassemia and 10 MRI centres. Patients were scanned using a multiparametric approach for assessing myocardial iron overload (MIO), biventricular function, and myocardial fibrosis. At the last MRI scan, a significant increase in global heart T2* values and a significantly higher frequency of patients with no MIO (all segmental T2* ≥20 ms) were detected, with a concordant improvement in biventricular function, particularly in patients with baseline global heart T2* <20 ms. Forty-seven percentage of patients changed the chelation regimen based on MRI. The frequency of heart failure (HF) significantly decreased after baseline MRI from 3.5 to 0.8% (P < 0.0001). Forty-six patients died during the study, and HF accounted for 34.8% of deaths. CONCLUSION: Over 10 years, continuous monitoring of cardiac iron and a tailored chelation therapy allowed MIO reduction, with consequent improvement of cardiac function and reduction of cardiac complications and mortality from MIO-related HF. A national networking for rare diseases therefore proved effective in improving the care and reducing cardiac outcomes of TM patients.

Pediatric thalassemic patients have higher incidence of asthma: A nationwide population-based retrospective cohort study.

INTRODUCTION: Patients with hemoglobinopathies have been reported to have higher rates of pulmonary complications. Few studies have investigated the association between thalassemia and asthma in children. METHODS: We used the data of one million individuals randomly selected from the Registry for Beneficiaries of the National Health Insurance Research Database. One thalassemic child was matched with four control children without thalassemia according to sex, birth year, birth season, prematurity, and previous enteroviral infection. RESULTS: A total of 800 hundred thalassemic children and 3200 controls were included. Children with thalassemia had higher rates of developing asthma (41.81 vs 25.70 per 1000 person-years, P < 0.001) than the non-thalassemia controls with an adjusted hazard ratio of 1.37 (95% confidence interval [CI] = 1.19-1.58). Boys in the thalassemia cohort had a significantly higher adjusted incidence hazard ratio (IRR) of asthma than those in the non-thalassemia cohort (adjusted IRR = 1.45, 95% CI = 1.02-1.73). The risk of atopic and nonatopic asthma was higher in the thalassemia cohort than in the non-thalassemia cohort (IRR = 1.3, 1.61, respectively). CONCLUSIONS: Children with thalassemia were more likely to develop asthma. More attention should be paid to the early diagnosis of asthma and prevention of asthma attacks.

Compounded with hemoglobin Port Phillip and -α4.2 or --SEA deletions were identified in Chinese population.

INTRODUCTION: Although over 1000 hemoglobin (Hb) variants were identified so far, Hb Port Phillip compound with α-thalassemia deletion had no reported before. METHODS: Two patients and the associated families from Guangdong province in China were recruited. Hematological parameters were determined by blood routine examination and hemoglobin electrophoresis. Genotyping was performed by Gap-PCR and Sanger sequencing. RESULTS: One patient was diagnosed as Hb Port Phillip, while her daughter was compounded with -α4.2 deletion, with normal Hb level (150 g/L), mean corpuscular volume (MCV) 108.4 fl and mean corpuscular hemoglobin (MCH) (30.5 pg). Another patient was diagnosed as compound Hb Port Phillip and --SEA deletion. This proband presented with more severe α-thalassemia trait than the patient compounded with -α4.2 deletion, with hemoglobin 80 g/L, MCV 61.7 fl, and MCH 18.7 pg. CONCLUSION: Here we first time identified two patients compound with Hb Port Phillip and -α4.2 and --SEA deletions, respectively, which had never been reported. Our study widens the genotypes of hemoglobinopathy and provides reference for genetic counselling and prenatal diagnosis in this population.

Pharmacological and clinical evaluation of deferasirox formulations for treatment tailoring.

Deferasirox (DFX) is the newest among three different chelators available to treat iron overload in iron-loading anaemias, firstly released as Dispersible Tablets (DT) and more recently replaced by Film-Coated Tablets (FCT). In this retrospective observational study, pharmacokinetics, pharmacodynamics, and safety features of DFX treatment were analyzed in 74 patients that took both formulations subsequently under clinical practice conditions. Bioavailability of DFX FCT compared to DT resulted higher than expected [Cmax: 99.5 (FCT) and 69.7 (DT) µMol/L; AUC: 1278 (FCT) and 846 (DT), P < 0.0001]. DFX FCT was also superior in scalability among doses. After one year of treatment for each formulation, no differences were observed between the treatments in the overall iron overload levels; however, DFX FCT but not DT showed a significant dose-response correlation [Spearman r (dose-serum ferritin variation): - 0.54, P < 0.0001]. Despite being administered at different dosages, the long-term safety profile was not different between formulations: a significant increase in renal impairment risk was observed for both treatments and it was reversible under strict monitoring (P < 0.002). Altogether, these data constitute a comprehensive comparison of DFX formulations in thalassaemia and other iron-loading anaemias, confirming the effectiveness and safety characteristics of DFX and its applicability for treatment tailoring.

Extracellular vesicles from thalassemia patients carry iron-containing ferritin and hemichrome that promote cardiac cell proliferation.

Extracellular vesicles (EVs) are bioactive, submicron-sized membrane vesicles released from all cell types upon activation or apoptosis. EVs including microparticles (MPs) and exosomes have emerged as important mediators of cell-to-cell communication in both normal and pathological states including thalassemia (thal). However, the role of EVs derived from ß-thal patients with iron overload (+ IO) and without iron overload (-IO) on cardiac cells is unclear. We hypothesized plasma EVs in thal patients containing ferritin (iron storage protein) and a denaturated hemoglobin-hemichrome that induce cardiac cell proliferation. The origins and numbers of EVs isolated from plasma of normal, thal (+ IO), and (- IO) patients were compared and determined for their iron and iron-containing proteins along with their effects on cardiac and endothelial cells. Data shows that MPs were originated from many cell sources with marked numbers of platelet origin. Only the number of RBC-derived MPs in thal (+ IO) patients was significantly high when compared to normal controls. Although MPs derived from both normal and thal patients promoted cardiac cell proliferation in a dose-dependent manner, only exosomes from thal patients promoted cardiac cell proliferation compared to the untreated. Moreover, the exosomes from thal (+ IO) potentially induce higher cardiac cell proliferation and angiogenesis in terms of tube number than thal (- IO) and normal controls. Interestingly, ferritin content in the exosomes isolated from thal (+ IO) was higher than that found in the MPs isolated from the same patient. The exosomes of thal patients with higher serum ferritin level also contained greater level of ferritin inside the exosomes. Apart from ferritin, there were trends of increasing hemichrome and iron presented in the plasma EVs and EV-treated H9C2 cells. Findings from this study support the hypothesis that EVs from ß-thal patients carry iron-load proteins that leads to the induction of cardiac cell proliferation.

Forager and farmer evolutionary adaptations to malaria evidenced by 7000 years of thalassemia in Southeast Asia.

Thalassemias are inherited blood disorders that are found in high prevalences in the Mediterranean, Southeast Asia and the Pacific. These diseases provide varying levels of resistance to malaria and are proposed to have emerged as an adaptive response to malaria in these regions. The transition to agriculture in the Holocene has been suggested to have influenced the selection for thalassemia in the Mediterranean as land clearance for farming encouraged interaction between Anopheles mosquitos, the vectors for malaria, and human groups. Here we document macroscopic and microscopic skeletal evidence for the presence of thalassemia in both hunter-gatherer (Con Co Ngua) and early agricultural (Man Bac) populations in northern Vietnam. Firstly, our findings demonstrate that thalassemia emerged prior to the transition to agriculture in Mainland Southeast Asia, from at least the early seventh millennium BP, contradicting a long-held assumption that agriculture was the main driver for an increase in malaria in Southeast Asia. Secondly, we describe evidence for significant malarial burden in the region during early agriculture. We argue that the introduction of farming into the region was not the initial driver of the selection for thalassemia, as it may have been in other regions of the world.

Clinically relevant updates of the HbVar database of human hemoglobin variants and thalassemia mutations.

HbVar (http://globin.bx.psu.edu/hbvar) is a widely-used locus-specific database (LSDB) launched 20 years ago by a multi-center academic effort to provide timely information on the numerous genomic variants leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Here, we report several advances for the database. We made clinically relevant updates of HbVar, implemented as additional querying options in the HbVar query page, allowing the user to explore the clinical phenotype of compound heterozygous patients. We also made significant improvements to the HbVar front page, making comparative data querying, analysis and output more user-friendly. We continued to expand and enrich the regular data content, involving 1820 variants, 230 of which are new entries. We also increased the querying potential and expanded the usefulness of HbVar database in the clinical setting. These several additions, expansions and updates should improve the utility of HbVar both for the globin research community and in a clinical setting.

Microcytic to hypochromic ratio as a discriminant index of thalassaemia trait in subjects with hypochromic anaemia.

INTRODUCTION: Differentiating between thalassaemia and iron deficiency anaemia (IDA) in hypochromic anaemia is a challenge to pathologists as it influences the choice of subsequent specialized confirmatory tests. In this study, we aimed to evaluate the performance of microcytic to hypochromic ratio (MicroR/ Hypo-He, M/H ratio) as a discriminant index in hypochromic anaemia. MATERIALS AND METHODS: A retrospective study was carried out on 318 subjects with hypochromic anaemia, which comprised 162 IDA and 156 thalassaemia trait subjects with α-thalassemia, ß-thalassemia and HbE trait. Optimal cut-off value, sensitivity and specificity of M/H ratio for thalassaemia trait discrimination was determined using Receiver Operating Characteristic (ROC) analysis. RESULTS: Subjects with thalassaemia trait showed higher MicroR compared to IDA ( p< 0.001) while subjects with IDA demonstrated higher Hypo-He than thalassaemia trait (p < 0.001). M/H ratio was significantly higher in thalassaemia trait compared to IDA, with medians of 3.77 (interquartile range: 2.57 - 6.52) and 1.73 (interquartile range: 1.27 - 2.38), respectively (p < 0.001). M/H ratio ≥ 2.25 was the optimal cut-off value for discriminating thalassaemia trait from IDA in hypochromic anaemia, with the area under ROC curve (AUC) of 0.83, sensitivity of 80.8% and specificity of 71.6%. CONCLUSIONS: M/H ratio is a useful discriminant index to distinguish thalassaemia trait from IDA in hypochromic anaemia prior to diagnostic analysis for thalassaemia confirmation. High M/H ratio is suggestive of thalassaemia trait than of IDA. However, more studies are required to establish the role of M/H ratio as a screening tool for thalassaemia discrimination in hypochromic anaemia.

Effect of Mutations on mRNA and Globin Stability: The Cases of Hb Bernalda/Groene Hart and Hb Southern Italy.

We identified two unstable variants in the third exon of α-globin genes: Hb Bernalda/Groene Hart (HBA1:c.358C>T), and Hb Caserta (HBA2:c.79G>A) in cis to Hb Sun Prairie (HBA2:c.391G>C), also named Hb Southern Italy. These mutations occurred in the H helix of the α-globin that is involved in heme contacting, specific recognition of α-hemoglobin-stabilizing protein (AHSP), and α1ß1 interactions. The carriers showed α-thalassemia phenotype, but one also jaundice and cholelithiasis. Molecular identification of clusters of families in Southern Italy encouraged molecular characterization of mRNA, globin chain analyses, molecular modeling studies, and comparison with globin variants to understand the mechanisms causing the α-thalassemia phenotype. A normal amount of Hb Bernalda/Groene Hart mRNA were found, and molecular modeling highlighted additional H bonds with AHSP. For Hb Southern Italy, showing an unexpected α/ß biosynthetic ratio typical of the ß-thalassemia type, two different molecular mechanisms were shown: Reduction of the variant mRNA, likely due to the No-Go Decay for the presence of unused triplet ACG at cod 26, and protein instability due to the impairment of AHSP interaction. The UDP glucuronosyltransferase 1A (UGT1A1) genotyping was conclusive in the case of jaundice and cholelithiasis. Multiple approaches are needed to properly identify the mechanisms leading to unstable variants and the effect of a mutation.

Extensive Extramedullary Hematopoiesis in Thalassemia: Soft Tissue Uptake on 99mTc-MDP SPECT/CT.

We report the case of a 45-year-old man with thalassemia major referred for a bone scan as a workup for generalized bone pain. Tc-MDP SPECT CT showed multiple elongated soft tissue masses along the inner aspects of ribs and along both sides of dorsal spine with calcification and mild tracer uptake. There was also a large lobulated presacral soft tissue mass displacing the bladder and involving the adjacent pelvic bone. Biopsy of mildly avid tracer uptake in pelvic soft tissue mass was extramedullary hematopoiesis confirmed by biopsy.

Leukocyte telomere length in patients with transfusion-dependent thalassemia.

BACKGROUND: Thalassemia is a hereditary hemolytic anemia with a severity ranging from mild, non-transfusion dependent to severe chronic anemia requiring lifelong transfusion. Transfusional iron overload is a major complication in patients with transfusion-dependent thalassemia (TDT). Telomeres are sequences of nucleotides forming the end caps of chromosomes that act as a DNA repair system. Iron overload in thalassemia can cause increased oxidative stress which leads to cellular damage and senescence. This may result in telomere length shortening. The degree of telomere length shortening may reflect the severity of thalassemia. METHODS: This research aimed to study the leukocyte telomere length in patients with TDT in comparison to non-thalassemic individuals and to identify the clinical and laboratory parameters that are associated with telomere length. We conducted a cross-sectional study in patients with TDT aged ≥18 years. Leukocyte telomere length was measured by real-time quantitative PCR. RESULTS: Sixty-five patients with TDT were enrolled onto the study. There were 37 female patients (54.4%). The median age was 27 (18-57) years, and mean pre-transfusion hemoglobin level was 7.1 (± 1.07) g/dL. The mean telomere to single copy gene (T/S) ratios of patients with TDT and the controls were 0.72 ± 0.18 and 0.99 ± 0.25, respectively (p < 0.0001). There was a significant correlation between the T/S ratio and age (p = 0.0002), and hemoglobin level (p = 0.044). There was no correlation between telomere length and other factors. CONCLUSIONS: Our study showed that TDT patients had shorter leukocyte telomere length compared with controls. Leukocyte telomere shortening in TDT was an aging-dependent process and associated with lower hemoglobin level.

Myeloid diseases in the lung and pleura.

Myeloid diseases detected as primary or secondary lesions in the lung and pleura are rare. Clinical presentations and radiographic results may vary significantly depending on the nature of the diseases. The most common diseases associated with lung and pleura involvement are myeloid sarcoma/acute myeloid leukemia (AML) and extramedullary hematopoiesis (EMH). AML typically represents localized involvement by systemic acute leukemia, while EMH is frequently secondary to underlying benign hematolymphoid disorders or myeloproliferative neoplasms. This review provides an overview of the pathogenesis, clinical presentations, radiologic/imaging studies, pathologic and genetic findings, and treatment/outcomes associated with myeloid diseases in the lung and pleura.

Erythrocyte Indices as Differential Diagnostic Biomarkers of Iron Deficiency Anemia and Thalassemia.

INTRODUCTION: The most common causes of microcytic anemia are iron deficiency anemia (IDA) and thalassemia trait (TT). This study investigated the reliability of erythrocyte indices and formulas as screening tests in the differential diagnosis of IDA and TT before performing detailed tests for definitive diagnosis. MATERIALS AND METHODS: In total, 50 children with ß-TT, 31 with α-TT, 50 with IDA were included. For the 8 erythrocyte indices and formulas (red blood cells [RBC], red blood cell distribution width [RDW], red blood cell distribution width index [RDWI], Mentzer index [MI], Shine and Lal index [S&L], England and Fraser [E&F], Green and King index [G&K], Srivastava index) the sensitivity, specificity, positive and negative predictive values (PPVs and NPVs, respectively) were calculated according to the cutoff values in the literature and recalculated revised cutoff values. RESULTS: According to the cutoff values in the literature for the differential diagnosis of IDA and TT, the ranking of sensitivity, specificity, PPVs, and NPVs from the highest to the lowest was RDWI, RBC, E&F, G&K, MI, Srivastava, RDW, S&L. The sensitivity, specificity, PPVs, and NPVs of all the indices according to the revised cutoff values were higher than those according to the cutoff values in the literature. CONCLUSIONS: According to both the cutoff values in the literature and revised cutoff values, the most reliable indices were RBC and RDWI.