MUTATIONAL ANALYSIS OF β-GLOBIN GENE OF THALASSEMIA IN LOCAL POPULATION OF PAKISTAN

Abstract

Thalassemia is a hereditary quantitative deficiency of functional α and β like globin chains synthesis that leads to ineffective erythropoiesis and reduced hemoglobin synthesis. Increased gastrointestinal iron absorption, following frequent blood transfusions in the absence of proper chelation therapy, builds iron overload leading to increased morbidity and mortality and shorten life expectancy. It is manifested by chronic anemia, hepatosplenomegaly, bone deformities especially for facial bones and improper growth rate. Regular blood transfusion and iron chelation therapy becomes essential for lifelong time. The geographical distribution of thalassemia can be divided broadly into four regions: Saharan Africa, Southeast Asia, Indian Asia and Mediterranean device. Migration from these affected areas into non-malarial areas results in heterogeneous diversity of thalassemia at global level. A multicentre study (including four cities in Pakistan) was aimed to investigate the frequency and spectrum of alpha and beta thalassemia genetic mutations and XmnI polymorphism of the Gamma Globin gene. Beta thalassemia individuals identified on the ground of haematological parameters were screened for mutations of the Alpha (HBA2 and HBA1) and Beta (HBB) Globin genes as well as Gamma (HBG2) Globin gene, -158 Gg XmnI polymorphism, using a combination of multiplex GAP polymerase chain reaction (PCR), Sanger sequencing and restriction fragment length polymerase (RFLP) based PCR respectively. Among 16 identified mutations in the beta gene by Sanger sequencing, NM_000158.4:c.27_28insG (p. Ser10ValfsTer14) was the most prevalent. In alpha thalassemia mutation, multiplex GAP-PCR run for seven most common deletion mutation showed that α-3.7 and α-4.2 deletions were coinherited with beta thalassemia mutations. Additionally, presence of rare mutations and novel variants has portrait the diversity of thalassemia mutations in Pakistani population. Moreover, NM_000184.2:c.-211C>T base-pair substitution (historically described as -158 GγXmnI polymorphism) was present in 36% of the patients. The hemolysis markers (red blood cell incidences), oxidative stress (lipid peroxidation, hemoglobin oxidation, carbonyl protein oxidation, and catalase activity), antioxidative level (glutathione reduced and oxidised, scavenging assays) and biochemical parameters were also analysed. Low copper, potassium, magnesium and phosphate levels and high AST, ALK and ALT levels which were observed importantly represent the iron overloaded peroxidative state in thalassemia. Not only these parameters but high values of lipid peroxidation, carbonyl contents and catalase activity and low values of glutathione reduce and scavenging assay also marked the oxidative damage caused mainly due to iron overload and its associated x complications. Additionally, three plants namely Curcuma longa (locally known as Haldi), Nigella sativa seeds (locally known as Kalonji), and Camellia sinensis (green tea) were studied to identify natural alternatives to treat disorders associated with hemolysis by reducing oxidative stress and to determine the effect of extraction systems on the functionality of phytochemicals. Based on Camellia sinensis and Curcuma longa well known bioactivities, it was assumed that they would significantly ameliorate the activities of oxidative markers and hence help in reducing the oxidative damage in beta thalassemia. However, treatment with both plant extract did not sufficiently improve the condition. This could be due to insufficient ameliorating effect as well as use of whole extract instead of isolated compound from plants was used. The psychological stress and its associated experiences were studied in index and their caregivers that duplicated the previous findings of low quality of life scores. The chronic nature of disease with enduring treatment has drastically affected their wellbeing. However, these changes vary between and within countries due to different health infrastructures, socioeconomic status and cultural and social norms. With advancement in reducing the thalassemia associated mortality and morbidity, some attention has now move into designing of improved and healthy lifestyles. Heterogeneity in clinical and haematological parameters in thalassemia major, show that monogenic disorders can present with a wide spectrum of disease severity. Present study will be useful in the prevention of highly prevalent disease of thalassemia in Pakistan following nationwide awareness campaign.