High‐level induction of fetal haemoglobin by pomalidomide in β‐thalassaemia/HbE erythroid progenitor cells

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155942/1/bjh16670.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155942/2/bjh16670_am.pd

Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure

Background: Plasmodium vivax remains the malaria species posing a major threat to human health worldwide owing to its relapse mechanism. Currently, the only drugs of choice for radical cure are the 8-aminoquinolines (primaquine and tafenoquine), which are capable of killing hypnozoites and thus preventing P. vivax relapse. However, the therapeutic use of primaquine and tafenoquine is restricted because these drugs can cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. This study aimed to assess and understand the hemolytic risk of using 8-aminoquinolines for radical treatment in a malaria endemic area of Thailand. Methods: The prevalence of G6PD deficiency was determined using a quantitative test in 1,125 individuals. Multiplexed high-resolution meltinging (HRM) assays were developed and applied to detect 12 G6PD mutations. Furthermore, biochemical and structural characterization of G6PD variants was carried out to understand the molecular basis of enzyme deficiency. Results: The prevalence of G6PD deficiency was 6.76% (76/1,125), as assessed by a phenotypic test. Multiplexed HRM assays revealed G6PD Mahidol in 15.04% (77/512) of males and 28.38% (174/613) of females, as well as G6PD Aures in one female. G6PD activity above the 30% cut-off was detected in those carrying G6PD Mahidol, even in hemizygous male individuals. Two variants, G6PD Murcia Oristano and G6PD Songklanagarind + Viangchan, were identified for the first time in Thailand. Biochemical characterization revealed that structural instability is the primary cause of enzyme deficiency in G6PD Aures, G6PD Murcia Oristano, G6PD Songklanagarind + Viangchan, and G6PD Chinese 4 + Viangchan, with double G6PD mutations causing more severe enzyme deficiency. Conclusion: In western Thailand, up to 22% of people may be ineligible for radical cure. Routine qualitative tests may be insufficient for G6PD testing, so quantitative tests should be implemented. G6PD genotyping should also be used to confirm G6PD status, especially in female individuals suspected of having G6PD deficiency. People with double G6PD mutations are more likely to have hemolysis than are those with single G6PD mutations because the double mutations significantly reduce the catalytic activity as well as the structural stability of the protein

Reactivation of a developmentally silenced embryonic globin gene

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues

Glucose-6-phosphate dehydrogenase mutations in malaria endemic area of Thailand by multiplexed high‐resolution melting curve analysis

Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymopathy in humans, is prevalent in tropical and subtropical areas where malaria is endemic. Anti-malarial drugs, such as primaquine and tafenoquine, can cause haemolysis in G6PD-deficient individuals. Hence, G6PD testing is recommended before radical treatment against vivax malaria. Phenotypic assays have been widely used for screening G6PD deficiency, but in heterozygous females, the random lyonization causes difficulty in interpreting the results. Over 200 G6PD variants have been identified, which form genotypes associated with differences in the degree of G6PD deficiency and vulnerability to haemolysis. This study aimed to assess the frequency of G6PD mutations using a newly developed molecular genotyping test. Methods A multiplexed high-resolution melting (HRM) assay was developed to detect eight G6PD mutations, in which four mutations can be tested simultaneously. Validation of the method was performed using 70 G6PD-deficient samples. The test was then applied to screen 725 blood samples from people living along the Thai–Myanmar border. The enzyme activity of these samples was also determined using water-soluble tetrazolium salts (WST-8) assay. Then, the correlation between genotype and enzyme activity was analysed. Results The sensitivity of the multiplexed HRM assay for detecting G6PD mutations was 100 % [95 % confidence interval (CI): 94.87–100 %] with specificity of 100 % (95 % CI: 87.66–100 %). The overall prevalence of G6PD deficiency in the studied population as revealed by phenotypic WST-8 assay was 20.55 % (149/725). In contrast, by the multiplexed HRM assay, 27.17 % (197/725) of subjects were shown to have G6PD mutations. The mutations detected in this study included four single variants, G6PD Mahidol (187/197), G6PD Canton (4/197), G6PD Viangchan (3/197) and G6PD Chinese-5 (1/197), and two double mutations, G6PD Mahidol + Canton (1/197) and G6PD Chinese-4 + Viangchan (1/197). A broad range of G6PD enzyme activities were observed in individuals carrying G6PD Mahidol, especially in females. Conclusions The multiplexed HRM-based assay is sensitive and reliable for detecting G6PD mutations. This genotyping assay can facilitate the detection of heterozygotes, which could be useful as a supplementary approach for high-throughput screening of G6PD deficiency in malaria endemic areas before the administration of primaquine and tafenoquine

Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines

Background: It was hypothesized that glucose-6-phosphate dehydrogenase (G6PD) deficiency confers a protective effect against malaria infection, however, safety concerns have been raised regarding haemolytic toxicity caused by radical cure with 8-aminoquinolines in G6PD-deficient individuals. Malaria elimination and control are also complicated by the high prevalence of G6PD deficiency in malaria-endemic areas. Hence, accurate identification of G6PD deficiency is required to identify those who are eligible for malaria treatment using 8-aminoquinolines. Methods: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency. Results: Based on phenotypic testing, the prevalence of G6PD deficiency (T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals. Conclusions: With varying degrees of enzyme deficiency, G6PD genotyping can be used as a complement to phenotypic screening to identify those who are eligible for 8-aminoquinolines. The information gained from this study could be useful for management and treatment of malaria, as well as for the prevention of unanticipated reactions to certain medications and foods in the studied population

Understanding regulation of zeta-globin transcription as the first step towards embryonic globin induction in patients with severe alpha-thalassemia

It is estimated by the World Health Organization that 250,000 individuals with severe hemoglobinopathies are born each year. A significant proportion of these suffer from @alpha;-thalassemia, which is one of the most common human monogenic disorders known with a carrier rate of &amp;GT;1% among all tropical and subtropical populations that have been studied. A common cause of alpha-thalassemia is an intra chromosomal deletion, termed the Southeast Asian (SEA) deletion, which removes both adult alpha-globin genes leaving the embryonically expressed zeta-globin gene intact. The SEA deletion is very common in some areas of the world, including Northern Thailand, where it is present at an allele frequency of approximately 15%. Individuals homozygous for the SEA deletion die of severe anaemia and tissue hypoxia in the third trimester of pregnancy, a condition termed Hb Bart's Hydrops Fetalis Syndrome (BHFS). Although BHFS has hitherto been considered a universally fatal disorder, an increasing number of patients have survived because of prenatal and immediate postnatal blood transfusion. The first aim of this work is to fully document the natural history and clinical outcomes of long-term survivors with the BHFS to gain insight into whether this disease should now be considered manageable and to assess whether the burden of treatment is too great. To achieve this, I have initiated a BHFS survivor registry and recruited 60 cases. Analysis suggests that as many as 82% of the BHFS survivors have favorable long-term neurodevelopmental outcomes. However, 50% of the patients suffer from severe growth retardation and 14% have inoperable limb defects. In addition, the majority (83%) have the burden of life-long transfusion dependence. Previous work suggests that zeta-globin can functionally substitute for alpha globin in adulthood, therefore there is a need for improved understanding of the regulation of the embryonic zeta-globin gene to allow development of targeted therapeutic approaches for embryonic hemoglobin induction to ameliorate BHFS. The second aim of this work is to investigate the cis- and trans-regulation of the zeta-globin gene with the ultimate aim of preventing silencing of this gene or reactivating its expression in definitive hematopoiesis. I have investigated the zeta-globin cis-regulatory network during murine primitive erythropoiesis using a DNaseI hypersensitivity assay coupled with an approach (termed Capture-C) to determine the cis-acting regulatory landscape of the zeta-globin gene. Interestingly, the data show that all five previously characterized DNaseI Hypersensitive Sites (DHSs) 5' of the α-globin cluster are present in primitive erythroid cells and that no extra sites are present at this developmental stage. These DHSs interact with both the zeta-globin and alpha-globin gene promoters in primitive erythroid cells as determined by Capture-C. I have also identified differential contributions of the individual alpha-globin cis-acting elements on alpha- and zeta-globin expression during primitive erythropoiesis using mouse lines harboring specific cis-element deletions. To identify novel trans-acting regulators of zeta-globin I have compared the transcriptomes of primitive and definitive erythroblasts using high-throughput sequencing and gene expression arrays. I have prioritized differentially expressed genes, using ontology analysis, for future functional testing using a Cas9/CRISPR library screen. In the last part of this work, I have integrated information on the cis- and trans-regulation of zeta-globin in an attempt to gain insight into an unusual BHFS survivor, who remarkably survived the first year of life with minimal transfusion, most likely because of persistent expression of zeta-globin.</p

Understanding regulation of zeta-globin transcription as the first step towards embryonic globin induction in patients with severe alpha-thalassemia: Regulation of zeta-globin transcription

It is estimated by the World Health Organization that 250,000 individuals with severe hemoglobinopathies are born each year. A significant proportion of these suffer from @alpha;-thalassemia, which is one of the most common human monogenic disorders known with a carrier rate of &GT;1% among all tropical and subtropical populations that have been studied. A common cause of alpha-thalassemia is an intra chromosomal deletion, termed the Southeast Asian (SEA) deletion, which removes both adult alpha-globin genes leaving the embryonically expressed zeta-globin gene intact. The SEA deletion is very common in some areas of the world, including Northern Thailand, where it is present at an allele frequency of approximately 15%. Individuals homozygous for the SEA deletion die of severe anaemia and tissue hypoxia in the third trimester of pregnancy, a condition termed Hb Bart's Hydrops Fetalis Syndrome (BHFS). Although BHFS has hitherto been considered a universally fatal disorder, an increasing number of patients have survived because of prenatal and immediate postnatal blood transfusion. The first aim of this work is to fully document the natural history and clinical outcomes of long-term survivors with the BHFS to gain insight into whether this disease should now be considered manageable and to assess whether the burden of treatment is too great. To achieve this, I have initiated a BHFS survivor registry and recruited 60 cases. Analysis suggests that as many as 82% of the BHFS survivors have favorable long-term neurodevelopmental outcomes. However, 50% of the patients suffer from severe growth retardation and 14% have inoperable limb defects. In addition, the majority (83%) have the burden of life-long transfusion dependence. Previous work suggests that zeta-globin can functionally substitute for alpha globin in adulthood, therefore there is a need for improved understanding of the regulation of the embryonic zeta-globin gene to allow development of targeted therapeutic approaches for embryonic hemoglobin induction to ameliorate BHFS. The second aim of this work is to investigate the cis- and trans-regulation of the zeta-globin gene with the ultimate aim of preventing silencing of this gene or reactivating its expression in definitive hematopoiesis. I have investigated the zeta-globin cis-regulatory network during murine primitive erythropoiesis using a DNaseI hypersensitivity assay coupled with an approach (termed Capture-C) to determine the cis-acting regulatory landscape of the zeta-globin gene. Interestingly, the data show that all five previously characterized DNaseI Hypersensitive Sites (DHSs) 5' of the α-globin cluster are present in primitive erythroid cells and that no extra sites are present at this developmental stage. These DHSs interact with both the zeta-globin and alpha-globin gene promoters in primitive erythroid cells as determined by Capture-C. I have also identified differential contributions of the individual alpha-globin cis-acting elements on alpha- and zeta-globin expression during primitive erythropoiesis using mouse lines harboring specific cis-element deletions. To identify novel trans-acting regulators of zeta-globin I have compared the transcriptomes of primitive and definitive erythroblasts using high-throughput sequencing and gene expression arrays. I have prioritized differentially expressed genes, using ontology analysis, for future functional testing using a Cas9/CRISPR library screen. In the last part of this work, I have integrated information on the cis- and trans-regulation of zeta-globin in an attempt to gain insight into an unusual BHFS survivor, who remarkably survived the first year of life with minimal transfusion, most likely because of persistent expression of zeta-globin

Alpha-Thalassemia: Diversity of Clinical Phenotypes and Update on the Treatment

One of the more common single-gene disorders worldwide is α-thalassemia, carriers of which are found at variable frequencies (>1%) across all tropical and subtropical countries. Two linked α-globin genes on each allele of chromosome 16 regulate α-globin chain production. Deletion of one or more α-globin genes is the most frequent molecular defect found in α-thalassemia, whereas non-deletional mutations also occur, leading to unstable α-globin chains. HbH is the most common clinically important α-thalassemia disease and occurs when three α-globin genes are deleted/mutated, leaving only one copy of the gene intact. HbH can be divided into deletional (--/-α) and non-deletional genotypes (--/αTα). Whereas clinical phenotypes of the former are usually homogenously mild to moderate, those of the latter can be diverse. As HbH disease is particularly prevalent in Southeast Asia and some parts of the Mediterranean region, where β-thalassemia is also prevalent, affected patients are sometimes left undertreated. Therefore, hematologists and general physicians need to be educated to provide optimal disease monitoring and early identification of those with more severe phenotypes. Some issues regarding transfusion and iron chelation management differ from those of β-thalassemia, and these need to be recognized. Hb Bart’s hydrops fetalis syndrome (BHFS) is the most severe form of α-thalassemia; affected patients lack production of α-globin chains. Recent advances in fetal medicine and neonatal intensive care have made it possible for BHFS to no longer constitute a universally fatal disorder. Transfusion and chelation strategies for rare survivors are distinct and require updating

Alpha-Thalassemia: Diversity of Clinical Phenotypes and Update on the Treatment

One of the more common single-gene disorders worldwide is &alpha;-thalassemia, carriers of which are found at variable frequencies (&gt;1%) across all tropical and subtropical countries. Two linked &alpha;-globin genes on each allele of chromosome 16 regulate &alpha;-globin chain production. Deletion of one or more &alpha;-globin genes is the most frequent molecular defect found in &alpha;-thalassemia, whereas non-deletional mutations also occur, leading to unstable &alpha;-globin chains. HbH is the most common clinically important &alpha;-thalassemia disease and occurs when three &alpha;-globin genes are deleted/mutated, leaving only one copy of the gene intact. HbH can be divided into deletional (--/-&alpha;) and non-deletional genotypes (--/&alpha;T&alpha;). Whereas clinical phenotypes of the former are usually homogenously mild to moderate, those of the latter can be diverse. As HbH disease is particularly prevalent in Southeast Asia and some parts of the Mediterranean region, where &beta;-thalassemia is also prevalent, affected patients are sometimes left undertreated. Therefore, hematologists and general physicians need to be educated to provide optimal disease monitoring and early identification of those with more severe phenotypes. Some issues regarding transfusion and iron chelation management differ from those of &beta;-thalassemia, and these need to be recognized. Hb Bart&rsquo;s hydrops fetalis syndrome (BHFS) is the most severe form of &alpha;-thalassemia; affected patients lack production of &alpha;-globin chains. Recent advances in fetal medicine and neonatal intensive care have made it possible for BHFS to no longer constitute a universally fatal disorder. Transfusion and chelation strategies for rare survivors are distinct and require updating

Inherited platelet dysfunction and hematopoietic transcription factor mutations

Transcription factors (TFs) are proteins that bind to specific DNA sequences and regulate expression of genes. The molecular and genetic mechanisms in most patients with inherited platelet dysfunction are unknown. There is now increasing evidence that mutations in hematopoietic TFs are an important underlying cause for the defects in platelet production, morphology, and function. The hematopoietic TFs implicated in the patients with impaired platelet function include Runt related TF 1 (RUNX1), Fli-1 proto-oncogene, ETS TF (FLI1), GATA-binding protein 1 (GATA1), and growth factor independent 1B transcriptional repressor (GFI1B). These TFs act in a combinatorial manner to bind sequence-specific DNA within a promoter region to regulate lineage-specific gene expression, either as activators or as repressors. TF mutations induce rippling downstream effects by simultaneously altering the expression of multiple genes. Mutations involving these TFs affect diverse aspects of megakaryocyte biology and platelet production and function, culminating in thrombocytopenia, platelet dysfunction, and associated clinical features. Mutations in TFs may occur more frequently in the patients with inherited platelet dysfunction than generally appreciated. This review focuses on the alterations in hematopoietic TFs in the pathobiology of inherited platelet dysfunction