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Imbalanced globin chain output contributes to thalassemia pathophysiology. Hence, induction of fetal hemoglobin in β-thalassemia and other β-hemoglobinopathies are of continuing interest for therapeutic approaches. Genome-wide association studies have identified three common genetic loci: namely β-globin (HBB), an intergenic region between MYB and HBS1L, and BCL11A underlying quantitative fetal hemoglobin production. Here, we report that knockdown of HBS1L (all known variants) using shRNA in early erythroblast obtained from β0-thalassemia/HbE patients triggers an upregulation of γ-globin mRNA 1.69 folds. There is modest perturbation of red cell differentiation assessed by flow cytometry and morphology studies. The levels of α- and β-globin mRNAs are relatively unaltered. Knockdown of HBS1L also increases the percentage of fetal hemoglobin around 16.7 folds when compared to non-targeting shRNA. Targeting HBS1L is attractive because of the potent induction of fetal hemoglobin and the modest effect on cell differentiation. [ABSTRACT FROM AUTHOR]

Defective hemoglobin production and ineffective erythropoiesis contribute to the pathophysiology of thalassemia syndromes. Previous studies in the field of erythropoiesis mainly focused on the severe forms of thalassemia, such as β-thalassemia major, while mechanisms underlying the pathogenesis of other thalassemia syndromes remain largely unexplored. The current study aimed to investigate the intrinsic pathophysiological properties of erythroid cells derived from the most common forms of thalassemia diseases, including α-thalassemia (hemoglobin H and hemoglobin H-Constant Spring diseases) and β-thalassemia (homozygous β0-thalassemia and β0-thalassemia/hemoglobin E diseases), under an identical in vitro erythroid culture system. Cell proliferation capacity, differentiation velocity, cell death, as well as globin synthesis and the expression levels of erythropoiesis modifying factors were determined. Accelerated expansion was found in erythroblast cells derived from all types of thalassemia, with the highest degree in β0-thalassemia/hemoglobin E. Likewise, all types of thalassemia showed limited erythroid cell differentiation, but each of them manifested varying degrees of erythroid maturation arrest corresponding with the clinical severity. Robust induction of HSP70 transcripts, an erythroid maturation-related factor, was found in both α- and β-thalassemia erythroid cells. Increased cell death was distinctly present only in homozygous β0-thalassemia erythroblasts and associated with the up-regulation of pro-apoptotic (Caspase 9, BAD, and MTCH1) genes and down-regulation of the anti-apoptotic BCL-XL gene. [ABSTRACT FROM AUTHOR]

Reactivating of fetal hemoglobin (HbF; α2γ2) can ameliorate the severity of β-thalassemia disease by compensating for adult hemoglobin deficiency in patients. Previously, microarray analysis revealed that zinc finger protein (ZNF)802 (also known as Juxta-posed with another zinc finger gene-1 (JAZF1)) was upregulated in human erythroblasts derived from adult peripheral blood compared with fetal liver-derived cells, implying a potential role as a HbF repressor. However, deficiency in ZNF802 induced by lentiviral shRNA in β0-thalassemia/hemoglobinE erythroblasts had no effect on erythroblast proliferation and differentiation. Remarkably, the induction of HBG expression was observed at the transcriptional and translational levels resulting in an increase of HbF to 35.0 ± 3.5%. Interestingly, the embryonic globin transcripts were also upregulated but the translation of embryonic globin was not detected. These results suggest ZNF802 might be a transcriptional repressor of the γ-globin gene in adult erythroid cells. [ABSTRACT FROM AUTHOR]

Anemia in β-thalassemia is associated with ineffective erythropoiesis and a shortened lifespan of erythroid cells. The limited differentiation of β-thalassemic erythroblasts has been documented, but the characteristic feature of terminal erythroid maturation and its physiological relevance are not clearly described in β-thalassemias. Here, the red blood cell and reticulocyte cellular characteristics were determined in patients with β0-thalassemia/HbE in comparison to patients with iron deficiency anemia and healthy normal subjects. Severely affected β0-thalassemia/HbE patients showed the highest increase in immature reticulocytes, but the number of total erythrocytes was the lowest. Despite similar ranges of hemoglobin levels, β0-thalassemia/HbE patients had a higher number of reticulocytes and a greater proportion of immature fraction than patients with iron deficiency anemia did. In vitro CD34+ hematopoietic progenitor cells' culture and flow cytometry analysis were conducted to investigate the erythroid maturation and mitochondrial clearance in β0-thalassemia/HbE erythroid cells as compared to normal cells. The delayed erythroid maturation and evidence of impaired mitochondria clearance were observed in β0-thalassemia/HbE cells at the terminal stage of differentiation. Additionally, increased transcript levels of genes related to erythroid mitophagy, BNIP3L and PINK1, were revealed in β0-thalassemia/HbE erythroblasts. The findings indicate that the erythroid maturation is physiologically relevant, and that the restoration of terminal maturation represents a potential therapeutic target for β-thalassemias. [ABSTRACT FROM AUTHOR]

β-Thalassemia (β-thal) is highly prevalent in Myanmar, but limited data are available on the molecular basis and the clinical manifestations in Myanmar patients. In this study, we investigated the clinical features and β-globin gene abnormalities in 15 homozygous β-thal and 60 Hb E (HBB: c.79G>A)/β-thal pediatric patients who attended Yangon Children Hospital, the biggest thalassemia day care unit center in Myanmar. Eight different β0-thal mutations were identified, with four accounting for 88.9% of alleles studied (excluding the Hb E variant). A genotype-phenotype correlation was found; all homozygous β0-thalassemias had severe clinical courses, whereas the highly variable disease severity was demonstrated among Hb E/β0-thal patients. Interactions of IVS-I-1 (G>T) (HBB: c0.92+1G>T) β0-thal with Hb E are associated with milder clinical symptoms. The number of mildly affected Hb E/β-thal patients was lower than expected, suggesting that there may be a considerable number of patients in the population who have either not been admitted to hospital or diagnosed with carrying the disease. Although the clinical severity in the Myanmar β-thal patients seems to be similar to that in other populations, the levels of hemoglobin (Hb) appears to be very low. These findings indicate the need for the improvement of patient management and the development of prevention and control programs for β-thal in Myanmar. [ABSTRACT FROM AUTHOR]

Polycythaemia vera (PV) is a haematological disorder caused by an overproduction of erythroid cells. To date, the molecular mechanisms involved in the disease pathogenesis are still ambiguous. This study aims to identify aberrantly expressed proteins in erythroblasts of PV patients by utilizing mass spectrometry-based proteomic analysis. Haematopoietic stem cells (HSCs) were isolated from newly-diagnosed PV patients, PV patients who have received cytoreductive therapy, and healthy subjects. In vitro erythroblast expansion confirmed that the isolated HSCs recapitulated the disease phenotype as the number of erythroblasts from newly-diagnosed PV patients was significantly higher than those from the other groups. Proteomic comparison revealed 17 proteins that were differentially expressed in the erythroblasts from the newly-diagnosed PV patients compared to those from healthy subjects, but which were restored to normal levels in the patients who had received cytoreductive therapy. One of these proteins was S-methyl-5′-thioadenosine phosphorylase (MTAP), which had reduced expression in PV patients' erythroblasts. Furthermore, MTAP knockdown in normal erythroblasts was shown to enhance their proliferative capacity. Together, this study identifies differentially expressed proteins in erythroblasts of healthy subjects and those of PV patients, indicating that an alteration of protein expression in erythroblasts may be crucial to the pathology of PV. [ABSTRACT FROM AUTHOR]

Introduction: β-Thalassemia/hemoglobin E represents one-half of all the clinically severe β-thalassemias worldwide. Despite similar genetic backgrounds, patients show clinical heterogeneity ranging from nearly asymptomatic to transfusion-dependent thalassemia. The underlying disease modifying factors remain largely obscure. Methods: To elucidate the correlation between ineffective erythropoiesis and β0-thalassemia/hemoglobin E (HbE) disease severity, in vitro culture of erythroid cells derived from patients with different clinical symptoms was established. Cell proliferation, viability, and differentiation were investigated. To identify potential molecular mechanisms leading to the arrested erythroid maturation, the expression levels of erythropoiesis modifying factors were measured. Results: The β0-thalassemia/HbE cells exhibited enhanced proliferation, limited differentiation, and impaired erythroid terminal maturation but did not show accelerated erythroblast differentiation and increased cell death. Erythroblasts derived from mild patients showed the highest proliferation rate with a faster cell division time, while erythroblasts derived from severe patients displayed extremely delayed erythroid maturation. Downregulation of growth differentiation factor 11 and FOXO3a was observed in mild β0-thalassemia/HbE erythroblasts, while upregulation of heat shock protein 70 and activin receptor 2A was revealed in severe erythroblasts. Discussion/Conclusion: The degree of erythroid expansion and maturation arrest contributes to the severity of β0-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients. [ABSTRACT FROM AUTHOR]

Abstract Background Patients with Hb E/β0 thalassemia display remarkable variability in disease severity. To identify genetic modifiers influencing disease severity, we conducted a two-stage genome scan in groups of 207 mild and 305 severe unrelated patients from Thailand with Hb E/β0 thalassemia and normal α-globin genes. Methods First, we estimated and compared the allele frequencies of approximately 110,000 gene-based single nucleotide polymorphisms (SNPs) in pooled DNAs from different severity groups. The 756 SNPs that showed reproducible allelic differences at P < 0.02 by pooling were selected for individual genotyping. Results After adjustment for age, gender and geographic region, logistic regression models showed 50 SNPs significantly associated with disease severity (P < 0.05) after Bonferroni adjustment for multiple testing. Forty-one SNPs in a large LD block within the β-globin gene cluster had major alleles associated with severe disease. The most significant was bthal_bg200 (odds ratio (OR) = 5.56, P = 2.6 × 10-13). Seven SNPs in two distinct LD blocks within a region centromeric to the β-globin gene cluster that contains many olfactory receptor genes were also associated with disease severity; rs3886223 had the strongest association (OR = 3.03, P = 3.7 × 10-11). Several previously unreported SNPs were also significantly associated with disease severity. Conclusions These results suggest that there may be an additional regulatory region centromeric to the β-globin gene cluster that affects disease severity by modulating fetal hemoglobin expression.

Introduction: Several DNA‐based approaches including a reverse dot‐blot hybridization (RDB) have been established for detection of β‐thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe β‐thalassemia. However, one of major concerns of these techniques is a risk of misdiagnosis due to a lack of DNA controls. Here, we constructed positive DNA controls for β‐thalassemia genotyping in order to ensure that all steps in the analysis are performed properly. Methods: Four recombinant β‐globin plasmids, including a normal sequence and three different mutant panels covering 10 common β‐thalassemia mutations in Asia, were constructed by a conventional cloning method followed by sequential rounds of site‐directed mutagenesis. These positive DNA controls were further validated by RDB analysis. Results: We demonstrated the applicability of established positive DNA controls for β‐thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant β‐globin plasmids into a single positive control, which showed positive signals for both normal and mutant probes of all tested mutations. Therefore, only two positive DNA controls, normal and combined mutant β‐globin plasmids, are required for detecting 10 common β‐thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis. Conclusion: The β‐globin DNA controls established here provide efficient alternatives to a conventional DNA source from peripheral blood, which is more difficult to obtain. They also provide a platform for future development of β‐globin plasmid controls with other mutations, which can also be suitable for other DNA‐based approaches. [ABSTRACT FROM AUTHOR]

Keywords: fetal haemoglobin induction; -thalassaemia/HbE; pomalidomide; hydroxyurea; decitabine EN fetal haemoglobin induction -thalassaemia/HbE pomalidomide hydroxyurea decitabine e240 e245 6 06/18/20 20200615 NES 200615 Studies have shown that increased expression of fetal haemoglobin (HbF; SB 2 sb SB 2 sb ) can ameliorate red blood cell deficiencies in patients with -thalassaemia and sickle cell disease (SCD).[[1], [3]] Pharmacological induction of HbF expression in -thalassaemia has been investigated using several classes of small molecules,[4] including 5-azacytidine,[5] decitabine,[6] hydroxyurea,[7] LSD1 inhibitors (tranylcypromine and RN-1),[[8]] and short chain fatty acid derivatives.[[10]] Among these molecules, hydroxyurea is the only U.S. Food and Drug Administration (FDA) currently approved drug for the treatment of SCD and/or -thalassaemia. SP 0 sp -thalassaemia/HbE precursors from patients of different SP 0 sp -thalassemic mutations (Table SI) showed similarly increased levels of HbF induction in response to pomalidomide treatment. GLO:1XW/15jun20:bjh16670-fig-0002.jpg PHOTO (COLOR): 2 Effect of pomalidomide and its combinations on erythroid differentiation and mRNA expression of HbF regulators in cultured erythroid cells from 0-thalassaemia/HbE patients. HbF-inducing effects of hydroxyurea (HU), decitabine (DAC), and RN-1 in erythroid cells from 0-thalassaemia/HbE patients. [Extracted from the article]

The article offers information on the characterization and identification of hemoglobin (Hb) Bart's hydrops fetalis caused by a compound heterozygous mutation. It discusses the Alpha thalassemia as one of the most common inherited hemoglobin (Hb) disorders, characterized by reduction or absence of the α-globin chain synthesis due to deletion or mutation of alpha-globin gene.

Background. A key event in human development is the establishment of erythropoietic progenitors in the bone marrow, which is accompanied by a fetal-to-adult switch in hemoglobin expression. Understanding of this event could lead to medical application, notably treatment of sickle cell disease and -thalassemia. The changes in gene expression of erythropoietic progenitor cells as they migrate from the fetal liver and colonize the bone marrow are still rather poorly understood, as primary fetal liver (FL) tissues are difficult to obtain. Methods. We obtained human FL tissue and adult peripheral blood (AB) samples from Thai subjects. Primary CD34+ cells were cultured in vitro in a fetal bovine serumbased culture medium. After 8 days of culture, erythroid cell populations were isolated by flow cytometry. Gene expression in the FL- and AB-derived cells was studied by Affymetrix microarray and reverse-transcription quantitative PCR. The microarray data were combined with that from a previous study of human FL and AB erythroid development, and meta-analysis was performed on the combined dataset. Results. FL erythroid cells showed enhanced proliferation and elevated fetal hemoglobin relative to AB cells. A total of 1,391 fetal up-regulated and 329 adult up-regulated genes were identified from microarray data generated in this study. Five hundred ninety-nine fetal up-regulated and 284 adult up-regulated genes with reproducible patterns between this and a previous study were identified by metaanalysis of the combined dataset, which constitute a core set of genes differentially expressed between FL and AB erythroid cells. In addition to these core genes, 826 and 48 novel genes were identified only from data generated in this study to be FL up- and AB up-regulated, respectively. The in vivo relevance for some of these novel genes was demonstrated by pathway analysis, which showed novel genes functioning in pathways known to be important in proliferation and erythropoiesis, including the mitogenactivated protein kinase (MAPK) and the phosphatidyl inositol 3 kinase (PI3K)-Akt pathways. Discussion. The genes with upregulated expression in FL cells, which include many novel genes identified from data generated in this study, suggest that cellular proliferation pathways are more active in the fetal stage. Erythroid progenitor cells may thus undergo a reprogramming during ontogenesis in which proliferation is modulated by changes in expression of key regulators, primarily MYC, and others including insulinlike growth factor 2 mRNA-binding protein 3 (IGF2BP3), neuropilin and tolloid-like 2 (NETO2), branched chain amino acid transaminase 1 (BCAT1), tenascin XB (TNXB) and proto-oncogene, AP-1 transcription factor subunit (JUND). This reprogramming may thus be necessary for acquisition of the adult identity and switching of hemoglobin expression. [ABSTRACT FROM AUTHOR]

Heterogeneity of HbF levels in β0-thalassemia/HbE disease has been reported to be associated with variations in clinical manifestations of the disease, and several genetic-modifying factors beyond the β-globin gene cluster have been identified as HbF regulators. Down-regulation or heterozygous mutations of Krüppel-like factor 1 (KLF1) is associated with elevated HbF levels in non-thalassemia subjects. This study confirms that experimental down-regulation of KLF1 in β0-thalassemia/HbE-derived erythroblasts significantly increases HbF production (up to 52.3 ± 2.4%), albeit with slightly delayed erythroid terminal differentiation. KLF1 exome sequencing of 130 Thai β0-thalassemia/HbE patients without co-inheritance of α-thalassemia found six patients with KLF1 heterozygous mutations including rs2072596 (p.F182L; n = 5) and rs745347362 (p.P284L; n = 1) missense mutations. However, while these patients had high HbF levels (38.1 ± 7.5%), they were all associated with a severe clinical phenotype. These results suggest that while reduction of KLF1 expression in β0-thalassemia/HbE erythroblasts can increase HbF levels, it is not sufficient to alleviate the clinical phenotype. [ABSTRACT FROM AUTHOR]

β-Thalassemia/HbE disease is clinically variable. In searching for genetic factors modifying the disease severity, patients were selected based on their disease severities, and a genome-wide association study (GWAS) was performed. Genotyping was conducted with the Illumina Human 610-Quad BeadChips array using DNAs from 618 Thai β0-thalassemia/HbE patients who were classified as 383 severe and 235 mild phenotypes by a validated scoring system. Twenty-three SNPs in three independent genes/regions were identified as being significantly associated with the disease severity. The highest association was observed with SNPs in the β-globin gene cluster (chr.11p15), and rs2071348 of the HBBP1 gene revealed the most significant association [ P = 2.96 × 10−13, odds ratio (OR) = 4.33 (95% confidence interval (CI), 2.74–6.84)]. The second was identified in the intergenic region between the HBS1L and MYB genes (chr.6q23), among which rs9376092 was the most significant [ P = 2.36 × 10−10, OR = 3.07 (95% CI, 2.16–4.38)]. The third region was located in the BCL11A gene (chr.2p16.1), and rs766432 showed the most significant association [ P = 5.87 × 10−10, OR = 3.06 (95% CI, 2.15–4.37)]. All three loci were replicated in an independent cohort of 174 Indonesian patients. The associations to fetal hemoglobin levels were also observed with SNPs on these three regions. Our data indicate that several genetic loci act in concert to influence HbF levels of β0-thalassemia/HbE patients. This study revealed that all the three reported loci and the α-globin gene locus are the best and common predictors of the disease severity in β-thalassemia. [ABSTRACT FROM AUTHOR]

Background: Patients with Hb E/b0 thalassemia display remarkable variability in disease severity. To identify genetic modifiers influencing disease severity, we conducted a two-stage genome scan in groups of 207 mild and 305 severe unrelated patients from Thailand with Hb E/β0 thalassemia and normal α-globin genes. Methods: First, we estimated and compared the allele frequencies of approximately 110,000 gene-based single nucleotide polymorphisms (SNPs) in pooled DNAs from different severity groups. The 756 SNPs that showed reproducible allelic differences at P < 0.02 by pooling were selected for individual genotyping. Results: After adjustment for age, gender and geographic region, logistic regression models showed 50 SNPs significantly associated with disease severity (P < 0.05) after Bonferroni adjustment for multiple testing. Forty-one SNPs in a large LD block within the b-globin gene cluster had major alleles associated with severe disease. The most significant was bthal•bg200 (odds ratio (OR) = 5.56, P = 2.6 × 10-13). Seven SNPs in two distinct LD blocks within a region centromeric to the b-globin gene cluster that contains many olfactory receptor genes were also associated with disease severity; rs3886223 had the strongest association (OR = 3.03, P = 3.7 × 10-11). Several previously unreported SNPs were also significantly associated with disease severity. Conclusions: These results suggest that there may be an additional regulatory region centromeric to the β-globin gene cluster that affects disease severity by modulating fetal hemoglobin expression. [ABSTRACT FROM AUTHOR]

Increase in fetal hemoglobin (Hb F) reduces globin chain imbalance in beta-thalassemia, consequently improving symptoms. QTL mapping together with previous genome-wide association study involving approximately 110,000 gene-based SNPs in mild and severe beta(0)-thalassemia/Hb E patients revealed SNPs in HBS1L significantly associated with severity and Hb F levels. Given its potential as binding site for transcription factor activator protein 4, HBS1L exon 1 C32T polymorphism was genotyped in 455 cases, providing for the first time evidence that C allele is associated with elevated Hb F level among beta(0)-thalassemia/Hb E patients with XmnI-(G)gamma-/-and XmnI-(G)gamma+/-polymorphisms. [ABSTRACT FROM AUTHOR]

Hemoglobin E (Hb E)-β-thalassemia patients display a range of clinical severities, from nearly asymptomatic to transfusion-dependent thalassemia major. Given this clinical heterogeneity, additional genetic factors modifying disease severity remain to be discovered. Association studies are being conducted to elucidate the role of genetic polymorphisms as disease severity modifiers in Hb E-β-thalassemia patients. Using strict scoring criteria, 1060 Hb E-β-thalassemia patients were categorized into mild, moderate, and severe groups. Taking a candidate gene approach, we found no statistically significant differences between the mild and severe patients groups in allelic or genotypic frequencies for single nucleotide polymorphisms within five genes known to influence globin gene expression and erythropoiesis. [ABSTRACT FROM AUTHOR]

There is considerable ethno-linguistic and genetic variation among human populations in Asia, although tracing the origins of this diversity is complicated by migration events. Thailand is at the center of Mainland Southeast Asia (MSEA), a region within Asia that has not been extensively studied. Genetic substructure may exist in the Thai population, since waves of migration from southern China throughout its recent history may have contributed to substantial gene flow. Autosomal SNP data were collated for 438,503 markers from 992 Thai individuals. Using the available self-reported regional origin, four Thai subpopulations genetically distinct from each other and from other Asian populations were resolved by Neighbor-Joining analysis using a 41,569 marker subset. Using an independent Principal Components-based unsupervised clustering approach, four major MSEA subpopulations were resolved in which regional bias was apparent. A major ancestry component was common to these MSEA subpopulations and distinguishes them from other Asian subpopulations. On the other hand, these MSEA subpopulations were admixed with other ancestries, in particular one shared with Chinese. Subpopulation clustering using only Thai individuals and the complete marker set resolved four subpopulations, which are distributed differently across Thailand. A Sino-Thai subpopulation was concentrated in the Central region of Thailand, although this constituted a minority in an otherwise diverse region. Among the most highly differentiated markers which distinguish the Thai subpopulations, several map to regions known to affect phenotypic traits such as skin pigmentation and susceptibility to common diseases. The subpopulation patterns elucidated have important implications for evolutionary and medical genetics. The subpopulation structure within Thailand may reflect the contributions of different migrants throughout the history of MSEA. The information will also be important for genetic association studies to account for population-structure confounding effects. [ABSTRACT FROM AUTHOR]

Campylobacter is an important foodborne pathogen causing bacterial gastroenteritis worldwide; however, there has been a lack of information over the past decade on its occurrence, antibiotic susceptibility and genetic diversity in Thailand. Poultry meat is considered as a reservoir for transmission of Campylobacter to humans. This study determines the prevalence and antimicrobial resistance patterns of Campylobacter spp. on chicken samples purchased from 50 local wet markets and supermarkets in central Thailand. Of the 296 samples, 99 (33.5%) were contaminated with C. jejuni , 54 (18.2%) were C. coli and 15 (5.1%) were contaminated with both species. Antibiotic resistance rate is higher among C. coli isolates; 100%, 76.8%, 37.7%, 36.2% and 13.0% were resistant to quinolones, cyclines, macrolides, clindamycin and gentamicin, respectively. Most of the C. jejuni isolates were resistant to quinolones (79.8%) and cyclines (38.6%) whereas resistance to macrolides, clindamycin and gentamicin was found to be 1.8%. Multi-drug resistance (i.e. to three or more unrelated antimicrobials) was detected in 37.7% of C. coli and 1.8% of C. jejuni isolates. This study has revealed high contamination rates and alarming levels of antimicrobial resistance in Campylobacter spp. isolated from retail chicken samples in Thailand, suggesting the necessity of implementing interventions to reduce its prevalence from farm to table in the country. • The prevalence rate of Campylobacter spp. in retail chicken samples was 57%. • C. jejuni was more predominant than C. coli among isolates from retail chicken. • Variable resistance rates towards the antimicrobials used in the study were observed. • The majority of Campylobacter isolates were resistant to quinolones and cyclines. • Multi-drug resistance was detected in 38% of C. coli and 2% of C. jejuni isolates. [ABSTRACT FROM AUTHOR]

Imbalanced globin chain output contributes to thalassemia pathophysiology. Hence, induction of fetal hemoglobin in β-thalassemia and other β-hemoglobinopathies are of continuing interest for therapeutic approaches. Genome-wide association studies have identified three common genetic loci: namely β-globin (HBB), an intergenic region between MYB and HBS1L, and BCL11A underlying quantitative fetal hemoglobin production. Here, we report that knockdown of HBS1L (all known variants) using shRNA in early erythroblast obtained from β0-thalassemia/HbE patients triggers an upregulation of γ-globin mRNA 1.69 folds. There is modest perturbation of red cell differentiation assessed by flow cytometry and morphology studies. The levels of α- and β-globin mRNAs are relatively unaltered. Knockdown of HBS1L also increases the percentage of fetal hemoglobin around 16.7 folds when compared to non-targeting shRNA. Targeting HBS1L is attractive because of the potent induction of fetal hemoglobin and the modest effect on cell differentiation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chumchuen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 β 0 -thalassemia/ hemoglobin E (HbE) patients. The significant and reproducible increases in γ-globin transcript and HbF expression upon RN-1 treatment were 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 were observed in cells treated with RN-1 at high concentration. Delayed terminal erythroid differentiation was revealed in β 0 -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 proof of the concept that LSD1 epigenetic enzyme is a potential therapeutic target for β 0 -thalassemia/HbE patients., Competing Interests: Conflict of interest: The authors declare no potential conflict of interest., (©Copyright: the Author(s).)

Introduction: β-Thalassemia/hemoglobin E represents one-half of all the clinically severe β-thalassemias worldwide. Despite similar genetic backgrounds, patients show clinical heterogeneity ranging from nearly asymptomatic to transfusion-dependent thalassemia. The underlying disease modifying factors remain largely obscure., Methods: To elucidate the correlation between ineffective erythropoiesis and β0-thalassemia/hemoglobin E (HbE) disease severity, in vitro culture of erythroid cells derived from patients with different clinical symptoms was established. Cell proliferation, viability, and differentiation were investigated. To identify potential molecular mechanisms leading to the arrested erythroid maturation, the expression levels of erythropoiesis modifying factors were measured., Results: The β0-thalassemia/HbE cells exhibited enhanced proliferation, limited differentiation, and impaired erythroid terminal maturation but did not show accelerated erythroblast differentiation and increased cell death. Erythroblasts derived from mild patients showed the highest proliferation rate with a faster cell division time, while erythroblasts derived from severe patients displayed extremely delayed erythroid maturation. Downregulation of growth differentiation factor 11 and FOXO3a was observed in mild β0-thalassemia/HbE erythroblasts, while upregulation of heat shock protein 70 and activin receptor 2A was revealed in severe erythroblasts., Discussion/conclusion: The degree of erythroid expansion and maturation arrest contributes to the severity of β0-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients., (© 2021 S. Karger AG, Basel.)

Background: Patients with Hb E/beta0 thalassemia display remarkable variability in disease severity. To identify genetic modifiers influencing disease severity, we conducted a two-stage genome scan in groups of 207 mild and 305 severe unrelated patients from Thailand with Hb E/beta0 thalassemia and normal alpha-globin genes., Methods: First, we estimated and compared the allele frequencies of approximately 110,000 gene-based single nucleotide polymorphisms (SNPs) in pooled DNAs from different severity groups. The 756 SNPs that showed reproducible allelic differences at P < 0.02 by pooling were selected for individual genotyping., Results: After adjustment for age, gender and geographic region, logistic regression models showed 50 SNPs significantly associated with disease severity (P < 0.05) after Bonferroni adjustment for multiple testing. Forty-one SNPs in a large LD block within the beta-globin gene cluster had major alleles associated with severe disease. The most significant was bthal&#95;bg200 (odds ratio (OR) = 5.56, P = 2.6 x 10(-13)). Seven SNPs in two distinct LD blocks within a region centromeric to the beta-globin gene cluster that contains many olfactory receptor genes were also associated with disease severity; rs3886223 had the strongest association (OR = 3.03, P = 3.7 x 10(-11)). Several previously unreported SNPs were also significantly associated with disease severity., Conclusions: These results suggest that there may be an additional regulatory region centromeric to the beta-globin gene cluster that affects disease severity by modulating fetal hemoglobin expression.

Beta-thalassemia intermediate patients show a remarkable clinical heterogeneity. We examined the phenotypic diversity of 950 beta-thalassemia/Hb E patients in an attempt to construct a system for classifying disease severity. A novel scoring system based on six independent parameters, hemoglobin level, age at disease presentation, age at receiving first blood transfusion, requirement for transfusion, spleen size, and growth and development, was able to separate patients into three distinctive severity categories: mild, moderate, and severe courses. This system, therefore, can increase the accuracy of studies of genotype-phenotype interactions and facilitate decisions for appropriate patient management., (Copyright 2008 Wiley-Liss, Inc.)

After the sequencing of the human genome is done, enormous genomic information and high-throughput profiling technologies are used. Increased attention has been paid to applying this knowledge to get better understanding of inherited diseases and complex disorders. Single nucleotide polymorphisms (SNPs) are DNA sequence variations that occur when a single nucleotide in the genome sequence is altered SNPs are an important tool for the study of the human genome. Application of SNPs analysis to human disease permits exploration of the influence of genetic polymorphisms on disease susceptibility, drug sensitivity/resistance, and ultimately health care. Databases of SNPs provide a powerful resource for association studies that try to establish a relationship between a phenotype and regions of the genome. Genomic approaches have garnered so much attention and investment because they offer the potential to provide better understanding of genetic factors in human health and disease, as well as more-precise definitions of the non-genetic factors involved.

Hemoglobin E (Hb E)-beta-thalassemia patients display a range of clinical severities, from nearly asymptomatic to transfusion-dependent thalassemia major. Given this clinical heterogeneity, additional genetic factors modifying disease severity remain to be discovered. Association studies are being conducted to elucidate the role of genetic polymorphisms as disease severity modifiers in Hb E-beta-thalassemia patients. Using strict scoring criteria, 1060 Hb E-beta-thalassemia patients were categorized into mild, moderate, and severe groups. Taking a candidate gene approach, we found no statistically significant differences between the mild and severe patients groups in allelic or genotypic frequencies for single nucleotide polymorphisms within five genes known to influence globin gene expression and erythropoiesis.