Medicine
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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
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Item Hemoglobin E-beta-thalassemia: Progress report from the international study group(Blackwell Publishing, 2005) Premawardhena, A.; de Silver, S.; Arambepola, M.; Olivieri, N.F.; Vichinsky, E.P.; Merson, L.; Muraco, G.; Allen, A.; Fisher, C.; Peto, T.; Weatherall, D.J.A long-term observational study of Hb E-beta-thalassemia in Sri Lanka is beginning to define some of the genetic and environmental factors that are responsible for its remarkable phenotypic variability. In this population there is a very small difference between the steady-state hemoglobin levels between the mild and severe phenotypes, and it has been possible to stop transfusion in many of those who have been on long-term treatment of this kind. These preliminary observations, made over the last 7 years, provide directions for future research into this increasingly important disease.Item Base editing repairs the HBE mutation restoring the production of normal globin chains in severe HBE/β-thalassemia patient hematopoietic stem and erythroid cells(Elsevier, 2021) Badat, M.; Hua, P.; Mettananda, S.; Fisher, C.; Roy, N.; Rice, S.; Roy, A.; Higgs, D.; Davies, J.HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any severe β 0-thalassemia mutation on the other. These mutations lead to a reduction in β-globin production, resulting in a relative excess in α-globin chains that go on to cause ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies also rely on non-homologous end joining (NHEJ) targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Adenine base editors (ABEs) potentially circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C conversions through targeting with a Cas9-nickase and single-guide RNA (sgRNA). Conversion of the HbE codon to normal through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions (Figure 1A). ABEs are able to convert the HbE codon (AAG, lys) to wild-type (GAG, glu), but also to GGG (gly) or AGG (arg). GGG at codon 26 is found in a naturally occurring hemoglobin, Hb Aubenas. Heterozygotes have normal red cell indices and are phenotypically normal. We electroporated the latest generation of ABE8 editors (ABE8e, ABE8.13 and ABE8 V106W) as mRNA into WT CD34+ hematopoietic stem and progenitor cells (HSPCs) with sgRNAs targeting the middle A of the WT GAG codon. These had similar editing efficiencies although ABE8 V106W had marginally higher on-target efficiency. V106W has been evolved to have a favourable off-target profile. V106W mRNA/sgRNA was electroporated into 3 different severe HbE/β-thalassemia donor HSPCs. The HbE codon was converted to WT with a mean 28.7% efficiency, to Hb Aubenas 48.6% and to an undescribed AGG codon 2.1%. The mean conversion from HbE to a normal or normal variant was 78.7±8.7% (Figure 1B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure 1C), indicating a reduction in the relative excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Figure 1C and D). To prove that base editing using mRNA was possible in long-term HSCs, CD34+ cells from 4 WT cord blood donors were edited using ABEmax. Mice were culled after 16 weeks, and human cells were collected and transplanted into 7 secondary mice, which were also culled after 16 weeks. Each secondary mouse showed the presence of hCD45+ cells, indicating engraftment of LT-HSCs. All secondary replicates showed editing, with a mean editing efficiency of 34.5% (initial editing 46.3%). In both rounds of mice, there was robust lymphoid and myeloid engraftment and expected levels of erythroid engraftment for the NSG model in bone marrow and spleen. Potential off-target effects were assessed in-vitro by CIRCLE-seq in triplicate. These sites were assessed by targeted oligonucleotide capture of DNA from mRNA edited patient cells to detect in-vivo editing. Together these data provide robust evidence for base editing as an effective and safe therapeutic strategy for HbE/β-thalassemia.Item Base editing repairs the hbe mutation restoring the production of normal globin chains in severe hbe/β-Thalassemia patient hematopoietic stem and erythroid cells(European Hematology Association, 2022) Badat, M.; Hua, P.; Mettananda, S.; Fisher, C.; Roy, N.; Rice, S.; Roy, A.; Higgs, D.; Davies, J.Aims: HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide1. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any β0-thalassemia mutation on the other. There is a reduction in β-globin production, resulting in a relative excess in α-globin chains that leads to ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype shared by 1.5% of the world’s population2. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies rely on non-homologous end joining targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Methods: Adenine base editors (ABEs) circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C transitions. Conversion of the HbE codon to WT through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions. ABEs are able to convert the HbE codon (AAG) to wild-type (GAG), but also to GGG or AGG (Fig A). GGG at codon 26 is found in a naturally occurring haemoglobin, Hb Aubenas3. Heterozygotes have normal red cell indices and are phenotypically normal. We electroporated the latest generation of ABE8 editors4 as mRNA into 3 different severe HbE/β-thalassemia donor HSPCs with sgRNAs targeting the HbE codon. Results: The mean conversion from the HbE codon to a normal or normal variant in unselected cells was 86.2 (SD±8.1%, Fig B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure C), indicating a reduction in the excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Fig D & E). In serial NSG-murine xenotransplantation experiments, base edited cells were found to persist in secondary transplants, showing editing is possible in long-term HSCs (mean editing efficiency 34.5%, Fig F). Potential off-target effects were assessed in-vitro by CIRCLE-seq5; most candidate sites were in intergenic and intronic regions (Fig G & H). The top 250 sites were sequenced using deep targeted NGS. Only 5 sites showed OT deaminations at low levels in patient cells (mean 1.5%). We developed a machine learning model to assess potential OT-effects on chromatin accessibility, at all candidate sites in 49 different blood cell types6. Only 17 potential edits were predicted to result in a significant change in chromatin state (Fig I). 3 of these were in the top 250 sites sequenced previously, and none showed deamination in-vivo. Conclusion: Together these data provide robust evidence for base editing being used as an effective and safe therapeutic strategy for HbE/β-thalassemia.Item Survival and complications in patients with haemoglobin E thalassaemia in Sri Lanka: a prospective, longitudinal cohort study.(Elsevier Ltd, 2022) Premawardhena, A.P.; Ediriweera, D.S.; Sabouhanian, A.; Allen, A.; Rees, D.; de Silva, S.; Perera, W.; Katugaha, N.; Arambepola, M.; Yamashita, R.C.; Mettananda, S.; Jiffry, N.; Mehta, V.; Cader, R.; Bandara, D.; St Pierre, T.; Muraca, G.; Fisher, C.; Kirubarajan, A.; Khan, S.; Allen, S.; Lamabadusuriya, S.P.; Weatherall, D.J.; Olivieri, N.F.Background: Worldwide, haemoglobin E β-thalassaemia is the most common genotype of severe β-thalassaemia. The paucity of long-term data for this form of thalassaemia makes evidence-based management challenging. We did a long-term observational study to define factors associated with survival and complications in patients with haemoglobin E thalassaemia. Methods: In this prospective, longitudinal cohort study, we included all patients with haemoglobin E thalassaemia who attended the National Thalassaemia Centre in Kurunegala, Sri Lanka, between Jan 1, 1997, and Dec 31, 2001. Patients were assessed up to three times a year. Approaches to blood transfusions, splenectomy, and chelation therapy shifted during this period. Survival rates between groups were evaluated using Kaplan-Meier survival function estimate curves and Cox proportional hazards models were used to identify risk factors for mortality. Findings: 109 patients (54 [50%] male; 55 [50%] female) were recruited and followed up for a median of 18 years (IQR 14-20). Median age at recruitment was 13 years (range 8-21). 32 (29%) patients died during follow-up. Median survival in all patients was 49 years (95% CI 45-not reached). Median survival was worse among male patients (hazard ratio [HR] 2·51, 95% CI 1·16-5·43), patients with a history of serious infections (adjusted HR 8·49, 2·90-24·84), and those with higher estimated body iron burdens as estimated by serum ferritin concentration (adjusted HR 1·03, 1·01-1·06 per 100 units). Splenectomy, while not associated with statistically significant increases in the risks of death or serious infections, ultimately did not eliminate a requirement for scheduled transfusions in 42 (58%) of 73 patients. Haemoglobin concentration less than or equal to 4·5 g/dL (vs concentration >4·5 g/dL), serum ferritin concentration more than 1300 μg/L (vs concentration ≤1300 μg/L), and liver iron concentration more than 5 mg/g dry weight of liver (vs concentration ≤5 mg/g) were associated with poorer survival. Interpretation: Patients with haemoglobin E thalassaemia often had complications and shortened survival compared with that reported in high-resource countries for thalassaemia major and for thalassaemia intermedia not involving an allele for haemoglobin E. Approaches to management in this disorder remain uncertain and prospective studies should evaluate if altered transfusion regimens, with improved control of body iron, can improve survival.Item Oxidative stress and antioxidant deficiency in the β-thalassaemia in Sri Lanka(Sri Lanka Medical Association, 2021) Perera, S.; Allen, A.; Mettananda, S.; Rodrigo, R.; Perera, L.; Darshana, T.; Moggach, F.; Jackson-Crawford, A.; Heirene, L.; Fisher, C.; Olivieri, N.; Rees, D.; Allen, S.; Premawardhena, A.Introduction In the β thalassaemias oxidative stress is potentially high and this could be further exacerbated in the absence of robust antioxidant defences, such as nutritional vitamin C and E deficiencies. We undertook a comprehensive assessment of oxidant, antioxidant and inflammatory status in patients with subtypes of β-thalassaemia to study these factors in detail. Methods Consenting patients attending the thalassaemia centre in Ragama, (between November 2017 and June 2018) were assessed for the following: methaemoglobin, plasma haemoglobin, heme and ferritin as sources of oxidants, plasma haptoglobin, hemopexin and vitamins C and E as markers of antioxidants, plasma interleukin-6 and C-reactive protein for inflammation. Fruit and vegetable intake was determined by dietary recall. Results 162 patients were recruited. (59 HbE β-thalassaemia, 50 β-thalassaemia major, 40 β-thalassaemia intermedia, 13 HbS β-thalassaemia. Median age was 26.0 years (IQR 15.3-38.8), 101 (62.3%) were female. Oxidants were frequently increased and antioxidants depleted with high levels of oxidant damage, hypoxia and inflammation. Abnormalities were most severe in HbE-β thalassaemia and least severe in β thalassaemia intermedia. Oxidative stress was also more severe in splenectomised patients. Plasma vitamin C concentration was below the lower level of quantitation in 86/160 (53.8%) patients and vitamin E in 130/160 (81.3%) patients. Less than 15% of patients ate fresh fruits or raw vegetables frequently. Conclusion Markedly increased oxidative stress and antioxidant deficiency were observed in this study group, especially in those with HbE β-thalassaemia. Vitamin C & E supplementation may have a role in the long-term management of thalassaemia syndromes.Item Alpha thalassaemia and extended alpha globin genes in Sri Lanka(Elsevier-Academic Press, 2013) Suresh, S.; Fisher, C.; Ayyub, H.; Premawardhena, A.; Allen, A.; Perera, A.; Bandara, D.; Olivieri, N.; Weatherall, D.The α-globin genes were studied in nine families with unexplained hypochromic anaemia and in 167 patients with HbE β thalassaemia in Sri Lanka. As well as the common deletion forms of α(+) thalassaemia three families from an ethnic minority were found to carry a novel form of α(0) thalassaemia, one family carried a previously reported form of α(0) thalassaemia, --(THAI), and five families had different forms of non-deletional thalassaemia. The patients with HbE β thalassaemia who had co-inherited α thalassaemia all showed an extremely mild phenotype and reduced levels of HbF and there was a highly significant paucity of α(+) thalassaemia in these patients compared with the normal population. Extended α gene arrangements, including ααα, αααα and ααααα, occurred at a low frequency and were commoner in the more severe phenotypes of HbE β thalassaemia. As well as emphasising the ameliorating effect of α thalassaemia on HbE β thalassaemia the finding of a novel form of α(0) thalassaemia in an ethnic minority, together with an unexpected diversity of forms of non-deletion α thalassaemia in Sri Lanka, further emphasises the critical importance of micro-mapping populations for determining the frequency of clinically important forms of the disease.Item Oxidative status in the β-thalassemia syndromes in Sri Lanka; a cross-sectional survey(Elsevier Science-Pergamon Press, 2021) Allen, A.; Perera, S.; Mettananda, S.; Rodrigo, R.; Perera, L.; Darshana, T.; Moggach, F.; Crawford, A.J.; Heirene, L.; Fisher, C.; Olivieri, N.; Rees, D.; Premawardhena, A.; Allen, S.ABSTRACT: In the β-thalassemias, oxidative stress, resulting from chronic hemolysis, globin chain imbalance, iron overload and depleted antioxidant defences, likely contributes to cell death, organ damage, anemia, hypoxia and inflammation. We assessed variations in these parameters in β-thalassemia syndromes in Sri Lanka. Between November 2017 and June 2018, we assessed children and adults attending two thalassemia centres in Sri Lanka: 59 patients with HbE β-thalassemia, 50 β-thalassemia major, 40 β-thalassemia intermedia and 13 HbS β-thalassemia. Median age was 26.0 years (IQR 15.3-38.8), 101 (62.3%) were female and 152 (93.8%) of Sinhalese ethnicity. Methemoglobin, plasma hemoglobin, heme and ferritin were measured as sources of oxidants; plasma total antioxidant capacity, haptoglobin, hemopexin and vitamins C and E assessed antioxidant status; plasma thiobarbituric acid reactive substances and 8-hydroxy-2'-deoxyguanosine assessed oxidative damage; hemoglobin, plasma erythropoietin and transferrin receptor assessed anemia and hypoxia and plasma interleukin-6 and C-reactive protein assessed inflammation. Fruit and vegetable intake was determined by dietary recall. Physical fitness was investigated using the six-minute walk test and measurement of handgrip strength. Oxidant sources were frequently increased and antioxidants depleted, with consequent oxidative damage, anemia, hypoxia and inflammation. Biomarkers were generally most abnormal in HbE β-thalassemia and least abnormal in β-thalassemia intermedia but also varied markedly between individuals with the same thalassemia syndrome. Oxidative stress and damage were also more severe in splenectomized patients and/or those receiving iron chelation therapy. Less than 15% of patients ate fresh fruits or raw vegetables frequently, and plasma vitamins C and E were deficient in 132/160 (82.5%) and 140/160 (87.5%) patients respectively. Overall, physical fitness was poor in all syndromes and was likely due to anemic hypoxia. Studies of antioxidant supplements to improve outcomes in patients with thalassemia should consider individual patient variation in oxidative status both between and within the thalassemia syndromes. KEYWORDS: antioxidants; oxidative damage; oxidative stress; thalassemia; vitamins C and E.Item Methemoglobinemia and ascorbate deficiency in hemoglobin E β thalassemia: metabolic and clinical implications.(American Society of Hematology., 2012) Allen, A.; Fisher, C.; Premawardhena, A.; Bandara, D.; Perera, A.; Allen, S.; St Pierre, T.; Olivieri, N.; Weatherall, D.ABSTRACT: During investigations of the phenotypic diversity of hemoglobin (Hb) E β thalassemia, a patient was encountered with persistently high levels of methemoglobin associated with a left-shift in the oxygen dissociation curve, profound ascorbate deficiency, and clinical features of scurvy; these abnormalities were corrected by treatment with vitamin C. Studies of erythropoietin production before and after treatment suggested that, as in an ascorbate-deficient murine model, the human hypoxia induction factor pathway is not totally dependent on ascorbate levels. A follow-up study of 45 patients with HbE β thalassemia showed that methemoglobin levels were significantly increased and that there was also a significant reduction in plasma ascorbate levels. Haptoglobin levels were significantly reduced, and the high frequency of the 2.2 haptoglobin genotype may place an additional pressure on ascorbate as a free-radical scavenger in this population. There was, in addition, a highly significant correlation between methemoglobin levels, splenectomy, and factors that modify the degree of globin-chain imbalance. Because methemoglobin levels are modified by several mechanisms and may play a role in both adaptation to anemia and vascular damage, there is a strong case for its further study in other forms of thalassemia and sickle-cell anemia, particularly when splenic function is defective.Item The p.H63D allele of the HFE gene protects against low iron stores in Sri Lanka.(Academic Press, 2019) Allen, A.; Premawardhena, A.; Allen, S.; Rodrigo, R.; Manamperi, A.; Perera, L.; Wray, K.; Armitage, A.; Fisher, C.; Drakesmith, A.; Robson, K.; Weatherall, D.In hereditary hemochromatosis, iron overload is associated with homozygosity for the p.C282Y mutation. A second mutation, p.H63D, occurs at significant frequencies in Europe, North Africa, the Middle East and Asia. Early studies in Sri Lanka indicated that the variant had arisen independently, suggesting that it had been the subject of selective pressure. However, its role in iron absorption is unclear. In a survey of 7526 Sri Lankan secondary school students, we determined hemoglobin genotype and measured red cell indices, serum ferritin, transferrin receptor, iron zinc protoporphyrin and hepcidin. These variables were compared according to the presence or absence of the p.H63D variant in a subset of 1313 students for whom DNA samples were available. Students were classified as having low red cell indices if they had an MCV <80 fl and/or MCH <27 pg. Hetero and/or homozygosity for the p.H63D variant was more common in students with normal than low red cell indices (16.4% and 11.9% respectively; p = 0.019). Iron biomarkers and red cell indices were greater in children with the p.H63D variant than in normal and this was statistically significant for MCV (p = 0.046). Our findings suggest that selective pressure by mild iron deficiency contributes to the high frequencies of the p.H63D variant.Item The Evolutionary and clinical implications of the uneven distribution of the frequency of the inherited haemoglobin variants over short geographical distances(Wiley-Blackwell, 2017) Premawardhena, A.; Allen, A.; Piel, F.; Fisher, C.; Perera, L.; Rodrigo, R.; Goonathilaka, G.; Ramees, L.; Peto, T.; Olivieri, N.; Weatherall, D.Studies of the frequency of heterozygous carriers for common inherited diseases of haemoglobin in over 7500 adolescent children in 25 districts in Sri Lanka have disclosed a highly significant variation over very short geographical distances. A further analysis of these findings, including their relationship to the past frequency and distribution of malaria, climatic variation, altitude, ethnic origin and consanguinity rates, have provided evidence regarding the evolutionary basis for the variable distribution of these conditions over short distances. It is likely that the complex interplay between malaria and the environment, together with related ethnic and social issues, exists in many countries across the tropical belt. Hence, these observations emphasise the importance of micromapping heterozygote distributions in high-frequency countries in order to define their true burden and the facilities required for the prevention and management of the homozygous and compound heterozygous disorders that result from their interaction.