Induction of fetal hemoglobin: Lentiviral shRNA knockdown of HBS1L in ß0-thalassemia/HbE erythroid cells.

Imbalanced globin chain output contributes to thalassemia pathophysiology. Hence, induction of fetal hemoglobin in ß-thalassemia and other ß-hemoglobinopathies are of continuing interest for therapeutic approaches. Genome-wide association studies have identified three common genetic loci: namely ß-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 ß0-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 ß-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.

In Vitro Study of Ineffective Erythropoiesis in Thalassemia: Diverse Intrinsic Pathophysiological Features of Erythroid Cells Derived from Various Thalassemia Syndromes.

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 ß-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 ß-thalassemia (homozygous ß0-thalassemia and ß0-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 ß0-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 ß-thalassemia erythroid cells. Increased cell death was distinctly present only in homozygous ß0-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.

Clinical Severity of ß-Thalassemia Pediatric Patients in Myanmar.

ß-Thalassemia (ß-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 ß-globin gene abnormalities in 15 homozygous ß-thal and 60 Hb E (HBB: c.79G>A)/ß-thal pediatric patients who attended Yangon Children Hospital, the biggest thalassemia day care unit center in Myanmar. Eight different ß0-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 ß0-thalassemias had severe clinical courses, whereas the highly variable disease severity was demonstrated among Hb E/ß0-thal patients. Interactions of IVS-I-1 (G>T) (HBB: c0.92+1G>T) ß0-thal with Hb E are associated with milder clinical symptoms. The number of mildly affected Hb E/ß-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 ß-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 ß-thal in Myanmar.

Impaired Terminal Erythroid Maturation in ß0-Thalassemia/HbE Patients with Different Clinical Severity.

Anemia in ß-thalassemia is associated with ineffective erythropoiesis and a shortened lifespan of erythroid cells. The limited differentiation of ß-thalassemic erythroblasts has been documented, but the characteristic feature of terminal erythroid maturation and its physiological relevance are not clearly described in ß-thalassemias. Here, the red blood cell and reticulocyte cellular characteristics were determined in patients with ß0-thalassemia/HbE in comparison to patients with iron deficiency anemia and healthy normal subjects. Severely affected ß0-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, ß0-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 ß0-thalassemia/HbE erythroid cells as compared to normal cells. The delayed erythroid maturation and evidence of impaired mitochondria clearance were observed in ß0-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 ß0-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 ß-thalassemias.

Down-regulation of the transcriptional repressor ZNF802 (JAZF1) reactivates fetal hemoglobin in ß0-thalassemia/HbE.

Reactivating of fetal hemoglobin (HbF; α2γ2) can ameliorate the severity of ß-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 ß0-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.

MTAP-related increased erythroblast proliferation as a mechanism of polycythaemia vera.

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.

Association of the Degree of Erythroid Expansion and Maturation Arrest with the Clinical Severity of ß0-Thalassemia/Hemoglobin E Patients.

INTRODUCTION: ß-Thalassemia/hemoglobin E represents one-half of all the clinically severe ß-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 ß0-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 ß0-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 ß0-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 ß0-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients.

Lysine-specific histone demethylase 1 inhibition enhances robust fetal hemoglobin induction in human ß0-thalassemia/hemoglobin E erythroid cells.

Induction of fetal hemoglobin (HbF) ameliorates the clinical severity of ß-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 ß0-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 ß0-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 ß0-thalassemia/HbE patients.

Prevalence and antimicrobial susceptibility of Campylobacter isolated from retail chickens in Thailand.

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.

Development of DNA controls for detection of ß-thalassemia mutations commonly found in Asian.

INTRODUCTION: Several DNA-based approaches including a reverse dot-blot hybridization (RDB) have been established for detection of ß-thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe ß-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 ß-thalassemia genotyping in order to ensure that all steps in the analysis are performed properly. METHODS: Four recombinant ß-globin plasmids, including a normal sequence and three different mutant panels covering 10 common ß-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 ß-thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant ß-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 ß-globin plasmids, are required for detecting 10 common ß-thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis. CONCLUSION: The ß-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 ß-globin plasmid controls with other mutations, which can also be suitable for other DNA-based approaches.

UNC0638 induces high levels of fetal hemoglobin expression in ß-thalassemia/HbE erythroid progenitor cells.

Increased expression of fetal hemoglobin (HbF) improves the clinical severity of ß-thalassemia patients. EHMT1/2 histone methyltransferases are epigenetic modifying enzymes that are responsible for catalyzing addition of the repressive histone mark H3K9me2 at silenced genes, including the γ-globin genes. UNC0638, a chemical inhibitor of EHMT1/2, has been shown to induce HbF expression in human erythroid progenitor cell cultures. Here, we report the HbF-inducing activity of UNC0638 in erythroid progenitor cells from ß-thalassemia/HbE patients. UNC0638 treatment led to significant increases in γ-globin mRNA, HbF expression, and HbF-containing cells in the absence of significant cytotoxicity. Moreover, UNC0638 showed additive effects on HbF induction in combination with the immunomodulatory drug pomalidomide and the DNMT1 inhibitor decitabine. These studies provide a scientific proof of concept that a small molecule targeting EHMT1/2 epigenetic enzymes, used alone or in combination with pomalidomide or decitabine, is a potential therapeutic approach for HbF induction. Further development of structural analogs of UNC0638 with similar biological effects but improved pharmacokinetic properties may lead to promising therapies and possible clinical application for the treatment of ß-thalassemia.

Visual genotyping of thalassemia by using pyrrolidinyl peptide nucleic acid probes immobilized on carboxymethylcellulose-modified paper and enzyme-induced pigmentation.

A simple probe pair was designed for the detection of hemoglobin E (HbE) genotype, a single-point mutation that leads to abnormal red blood cells commonly found in South East Asia. The key to differentiation is the use of a conformationally constrained peptide nucleic acid (PNA) that was immobilized on carboxymethylcellulose-modified paper. This was then used for target DNA binding and visualization by an enzyme-catalyzed pigmentation. The biotinylated target DNA bound to the immobilized probe was visually detected via alkaline phosphatase-linked streptavidin. This enzyme conjugate catalyzed the dephosphorylation of the substrate 5-bromo-4-chloro-3-indolyl phosphate, leading to a series of reactions that generate an intense, dark blue pigment. The test was validated with 100 DNA samples, which shows good discrimination among different genotypes (normal, HbE, and heterozygous) with 100% accuracy when optimal conditions of analysis were applied. The method does not require temperature control and can be performed at ambient temperature. This is an attractive feature for diagnosis in primary care, which accounts for a large part of affected population. Graphical abstract Schematic representation of a paper-based sensor for the detection of the gene Hemoglobin E. The interaction between an immobilized peptide nucleic acid and a DNA target leads to enzymatic pigmentation, allowing simple visual readout with up to 100% accuracy.

Comparison of gene expression profiles between human erythroid cells derived from fetal liver and adult peripheral blood.

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 ß-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 serum-based 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 meta-analysis 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 mitogen-activated 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 insulin-like 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.

Genetic variation of Krüppel-like factor 1 (KLF1) and fetal hemoglobin (HbF) levels in ß0-thalassemia/HbE disease.

Heterogeneity of HbF levels in ß0-thalassemia/HbE disease has been reported to be associated with variations in clinical manifestations of the disease, and several genetic-modifying factors beyond the ß-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 ß0-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 ß0-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 ß0-thalassemia/HbE erythroblasts can increase HbF levels, it is not sufficient to alleviate the clinical phenotype.

Fetal hemoglobin regulation in ß-thalassemia: heterogeneity, modifiers and therapeutic approaches.

INTRODUCTION: Stress erythropoiesis induces fetal hemoglobin (HbF) expression in ß-thalassemias, however the level of expression is highly variable. The last decade has seen dramatic advances in our understanding of the molecular regulators of HbF production and the genetic factors associated with HbF levels, leading to the promise of new methods of the clinical induction of HbF. Areas covered: This article will review the heterogeneity and genetic modifiers of HbF and HbF induction therapy in ß-thalassemia. Expert commentary: One promising curative ß-thalassemia therapy is to induce HbF synthesis in ß-thalassemic erythrocytes to therapeutic levels before clinical symptom occurs. Further understanding of HbF level variation and regulation is needed in order to predict the response from HbF-inducing approaches.

Insight into the peopling of Mainland Southeast Asia from Thai population genetic structure.

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.

Genetic modifiers of Hb E/beta0 thalassemia identified by a two-stage genome-wide association study.

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_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.