Medicine
Permanent URI for this communityhttp://repository.kln.ac.lk/handle/123456789/12
This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
Browse
23 results
Search Results
Item Chracterisation of beta giobin mutations in Sri Lankan patients with betathalassaemia intermedia(Sri Lanka Medical Association, 2013) Perera, S.; Silva, D.P.S.I.; Hapugoda, M.; Wickramarathne, M.N.; Wijesirwardhena, I.; Efremove, D.G.; Fisher, C.A.; Weatherall, D.J.; Premawardhena, A.P.INTRODUCTION AND OBJECTIVES: Patients with beta thalassaemia intermedia account for a third of patients attending thalassaemia clinics in Sri Lanka. They show immense phenotypic diversity, the genetic basis for which has not been identified so far. Objective were to characterise beta globin gene mutations in Sri Lankan thalassaemia intermedia patients and to determine how it to influences disease severity. METHODS: We identified 64 thalassaemia intermedia patients from the five main thalassaemia centers; Anuradhapura (n= 6), Kuruncgala (n= 4), Ragama (n= 42), Badulla (n=7) and Chilaw (n=5). Their beta globin DNA sequences were analyzed using ABI PRISM 313lx genetic analyser. RESULTS: Of sixteen patients identified to be homozygous for beta mutations, eleven carried mild beta alleles, IVSI 5 G_C (n= 10) and a rare homozygous promoter mutation - 90 C_T (N=l). Other five were shown to have different types of severe iputations in homozygous state. Nearly half the sample (n=39) was heterozygous for beta mutations. Of them 33 showed mild to severe mutation in one of the alleles IVSI-5 G_C (n=12), IVSI-1 G_A (n= 11) were the commonest. Two patients who were hetcrozygones for beta mutation had a highly unstable Hb variant haemoglobin Mizuho causing severe haemolytic anacma. Hb variants Hb G-Szuhu and Hb G-Coushatta were identified in two patients. CONCLUSIONS: We identified types of beta mutations in some patients with thalassaemia intermedia, which account for the clinical severity.Item Direct correction of haemoglobin E β-thalassaemia using base editors(Nature Pub. Group, 2023) Badat, M.; Ejaz, A.; Hua, P.; Rice, S.; Zhang, W.; Hentges, L.D.; Fisher, C.A.; Denny, N.; Schwessinger, R.; Yasara, N.; Roy, N.B.A.; Issa, F.; Roy, A.; Telfer, P.; Hughes, J.; Mettananda, S.; Higgs, D.R.; Davies, J.O.J.Haemoglobin E (HbE) β-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE β-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamatic acid → AAG; lysine, E26K), and any mutation causing severe β-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (β-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 90% in primary human CD34 + cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.Item Genotype-phenotype association analysis identifies the role of α globin genes in modulating disease severity of β thalassaemia intermedia in Sri Lanka(Nature Publishing Group, 2019) Perera, S.; Allen, A.; Silva, I.; Hapugoda, M.; Wickramarathne, M.N.; Wijesiriwardena, I.; Allen, S.; Rees, D.; Efremov, D.G.; Fisher, C.A.; Weatherall, D.J.; Premawardhena, A.β thalassaemia intermedia (βTI) are a heterogeneous group of disorders known to be extremely phenotypically diverse. This group is more complex to manage as no definitive treatment guidelines exist unlike for β thalassaemia major (βTM). There are only a few studies looking at genotype phenotype associations of βTI outside the Mediterranean region. The reasons for the diverse clinical phenotype in βTI are unknown. We categorized fifty Sri Lankan patients diagnosed with βTI as mild, moderate or severe according to published criteria. DNA samples were genotyped for β thalassaemia mutations, α globin genotype and copy number and known genetic modifiers of haemoglobin F production. There were 26/50 (52.0%) in mild group and 12/50 (24.0%) each in moderate and sever categories. 18/26 (69.2%) classified as mild were β heterozygotes and 17/18 (94.4%) had excess α globin genes. 11/12 (91.6%) classified as moderate were β heterozygotes and 8/11 (72.2%) had excess α globin genes. In contrast, 8/12 (66.7%) classified as severe were β homozygotes and 7/8(87.5%) had α globin gene deletions. In Sri Lanka, co-inheritance of either excess α globin genes in β thalassaemia heterozygotes or α globin gene deletions in β thalassaemia homozygotes is a significant factor in modulating disease severityItem Haemoglobin variants, iron status and anaemia in Sri Lankan adolescents with low red cell indices: A cross sectional survey(Academic Press, 2018) Rodrigo, R.; Allen, A.; Manamperi, A.; Perera, L.; Fisher, C.A.; Allen, S.; Weatherall, D.J.; Premawardhena, A.Iron deficiency complicates the use of red cell indices to screen for carriers of haemoglobin variants in many populations. In a cross sectional survey of 7526 secondary school students from 25 districts of Sri Lanka, 1963 (26.0%) students had low red cell indices. Iron deficiency, identified by low serum ferritin, was the major identifiable cause occurring in 550/1806 (30.5%) students. Low red cell indices occurred in iron-replete students with alpha-thalassaemia including those with single alpha-globin gene deletions. Anaemia and low red cell indices were also common in beta-thalassaemia trait. An unexpected finding was that low red cell indices occurred in 713 iron-replete students with a normal haemoglobin genotype. It is common practice to prescribe iron supplements to individuals with low red cell indices. Since low red cell indices were a feature of all forms of α thalassaemia and also of iron deficiency, in areas where both conditions are common, such as Sri Lanka, it is imperative to differentiate between the two, to allow targeted administration of iron supplements and avoid the possible deleterious effects of increased iron availability in iron replete individuals with low red cell indices due to other causes such as α thalassaemia.Item Phenotypic and molecular characterization of a serum-free miniature erythroid differentiation system suitable for high-throughput screening and single-cell assays(Elsevier Science Inc., 2018) Mettananda, S.; Clark, K.; Fisher, C.A.; Sloane-Stanley, J.A.; Gibbons, R.J.; Higgs, D.R.In vitro erythroid differentiation systems are used to study the mechanisms underlying normal and abnormal erythropoiesis and to test the effects of various extracellular factors on erythropoiesis. The use of serum or conditioned medium in liquid cultures and the seeding of cultures with heterogeneous peripheral blood mononuclear cells confound the reproducibility of these systems. Newer erythroid differentiation culture systems have overcome some of these limitations by using a fully defined, serum-free medium and initiating cultures using purified CD34+ cells. Although widely used in bulk cultures, these protocols have not been rigorously tested in high-throughput or single-cell assays. Here, we describe a serum-free erythroid differentiation system suitable for small-scale and single-cell experiments. This system generates large numbers of terminally differentiated erythroid cells of very high purity. Here we have adapted this culture system to a 96-well format and have developed a protocol to grow erythroid colonies from single erythroid progenitors in minute culture volumes.Item Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia(Nature Pub. Group, 2017) Mettananda, S.; Fisher, C.A.; Hay, D.; Badat, M.; Quek, L.; Clark, K.; Hublitz, P.; Downes, D.; Kerry, J.; Gosden, M.; Telenius, J.; Sloane-Stanley, J.A.; Faustino, P.; Coelho, A.; Doondeea, J.; Usukhbayar, B.; Sopp, P.; Sharpe, J.A.; Hughes, J.R.; Vyas, P.; Gibbons, R.J.; Higgs, D.R.β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with β-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for β-thalassemia.β-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.Item Selective silencing of α-globin by the histone demethylase inhibitor IOX1: A potentially new pathway for treatment of β-thalassemia(Pavia, Italy : Ferrata Storti Foundation, 2017) Mettananda, S.; Fisher, C.A.; Sloane-Stanley, J.A.; Taylor, S.; Oppermann, U.; Gibbons, R.J.; Higgs, D.R.Item Hepcidin is suppressed by erythropoiesis in hemoglobin E β-thalassemia and β-thalassemia trait(American Society of Hematology, 2015) Jones, E.; Pasricha, S.R.; Allen, A.; Evans, P.; Fisher, C.A.; Wray, K.; Premawardhena, A.; Bandara, D.; Perera, A.; Webster, C.; Sturges, P.; Olivieri, N.F.; St Pierre, T.; Armitage, A.E.; Porter, J.B.; Weatherall, D.J.; Drakesmith, H.Hemoglobin E (HbE) β-thalassemia is the most common severe thalassemia syndrome across Asia, and millions of people are carriers. Clinical heterogeneity in HbE β-thalassemia is incompletely explained by genotype, and the interaction of phenotypic variation with hepcidin is unknown. The effect of thalassemia carriage on hepcidin is also unknown, but it could be relevant for iron supplementation programs aimed at combating anemia. In 62 of 69 Sri Lankan patients with HbE β-thalassemia with moderate or severe phenotype, hepcidin was suppressed, and overallhepcidin inversely correlated with iron accumulation. On segregating by phenotype, there were no differences in hepcidin, erythropoiesis, orhemoglobin between severe or moderate disease, but multiple linear regression showed that erythropoiesis inversely correlated with hepcidin only in severe phenotypes. In moderate disease, no independent predictors of hepcidin were identifiable; nevertheless, the low hepcidin levels indicate a significant risk for iron overload. In a population survey of Sri Lankan schoolchildren, β-thalassemia (but not HbE) trait was associated with increased erythropoiesis and mildly suppressed hepcidin, suggesting an enhanced propensity to accumulate iron. In summary, the influence oferythropoiesis on hepcidin suppression associates with phenotypic disease variation and pathogenesis in HbE β-thalassemia and indicates that the epidemiology of β-thalassemia trait requires consideration when planning public health iron interventions.Item The clinical effects of excessive a globin genes : two family studies(Sri Lanka Medical Association, 2003) Premawardhena, A.P.; Fisher, C.A.; Rugless, M.; de Silva, S.; Perera, A.W.V.S.; Olivien, N.F.; Weatherall, D.J.INTRODUCTION: Globin chain imbalance is the central pathogenic abnormality in the thalassaemias, a condition where globin gene expression is reduced. Conversely, the inheritance of excess globin genes too may affect the phenotype. However such examples are rarely found. OBJECTIVES: To describe two families in whom the co-existence of excess a genes was noted together with p - thalassaemia trait. METHODS: During the routine P - globin gene analysis in patients attending the Thalassaemia Unit of the Kurunegala Hospital, two patients were identified to have thalassaemia intermedia phenotype, but with just one (3 - thalassaemia mutation. The clinical details of these patients and their families were studied in detail as was their h'aematological and genetic data. RESULTS: We describe two families in which the propositus had inherited six and eight a - genes respectively together with a single p - thalassaemia mutation. Both patients had the thalassaemia intermedia phenotype. The family members who did not inherit any thalassaemic mutations too had varying, but often marked hypochromic microcytosis. DISCUSSION: We describe the first ever family study of a patient with the combination of 8 a - genes and p - thalassaemia trait. We also describe another family where a member had 6 a - genes together with p thalassaemia trait. This highlights yet another mechanism for the intermedia phenotype in patients with a solitary (3 - globin gene mutation. It also highlights the need for the study of a globin genes in patients with unexplained hypochromic microcytic anaemia.Item The worldwide prevalence of the UGT-1A1 promoter polymorphism as a contributor for phenotypic variability in thalassaemia(Sri Lanka Medical Association, 2003) Premawardhena, A.P.; Lilt, Y.T.; Fisher, C.A.; Clegg, J.B.; Weatherall, D.J.INTRODUCTION: The number of TA repeats in the promoter region of the UGTIA1 gene is involved in fine-tuning of the serum bilirubin level. People who inherit more TA repeats than in the wild type (6), tend to have higher than normal Serum bilirubin levels. When homozygosity for 7 repeats is co-inherited with hereditary anaemias the patients have an elevated serum bilirubin level and an increased incidence of gallstone formation. OBJECTIVES: To investigate the pattern of UGTIAI promoter genotype in several diverse populations across the world. METHODS: A fluorescent labeled PCR method was designed which would amplify the region of interest of the UGTIAI gene. The PCR products were separated using (PAGE) polyacrelymide gel electrophoresis. DNA samples for the study were collected from people from 15 countries, from 5 continents. RESULTS: We found remarkable diversity of the UGTIAI polymorphism amongst people of African origin. The haplotype 7/7 was found in very high frequencies in India. Sri Lanka and Bangladesh, and was extremely rare amongst people of Southeast Asian origin. The Europeans and the Africans had intermediate frequencies. DISCUSSION: These results suggest that in people of Southeast Asian origin, a group that has a high prevalence of thalassaemia and other heamoglobinopathies, the UGTIAI polymorphisms are unlikely to be important genetic modifiers of the phenotype. However, Sri Lankans and other South Asians are very likely to be influenced by this polymorphism. These results also support the theory of African origins of Homo sapiens.
- «
- 1 (current)
- 2
- 3
- »