Mei I Lai

α-Haemoglobin stabilising protein expression is influenced by mean cell haemoglobin and HbF levels in HbE/β-thalassaemia individuals

The alpha haemoglobin stabilising protein (AHSP) acts as a molecular chaperone for α-globin by stabilising nascent α-globin before transferring it to waiting free β-globin chains. Binding of AHSP to α-globin renders α-globin chemically inert whereby preventing it from precipitating and forming reactive oxygen species byproducts. The AHSP has been actively studied in the recent years, particularly in its relation to β-thalassaemia. Studies have shown that AHSP is a modifier in β-thalassaemia mice models. However, this relationship is less established in humans. Studies by some groups showed no correlation between the AHSP haplotypes and the severity of β-thalassaemia, whereas others have shown that certain AHSP haplotype could modify the phenotype of β-thalassaemia intermedia patients. We investigated the expression of AHSP in relation to selected demographic data, full blood count, HPLC results, HbE/β-thalassaemia genotype, Xmn-1 Gγ polymorphism, α-globin, β-globin and γ-globin expression. We found that AHSP expression was significantly correlated to mean cell haemoglobin level, HbF %, α-globin, β-globin and excess α-globin expression. We concluded that AHSP could be a secondary compensatory mechanism in red blood cells to counterbalance the excess α-globin chains in HbE/β-thalassaemia individuals.

An assessment of three noncommercial DNA extraction methods from dried blood spots for beta-thalassaemia mutation identification

Introduction:  Dried blood spots (DBS) are currently the recommended sample collection method for newborn screening programmes in America. Early diagnosis of beta‐thalassaemia screening is essential as it provides an added advantage especially in sickle cell disease. Beta‐thalassaemia frequency is high in many poor countries, and the cost of using commercial DNA extraction kits can be prohibitive. Our study assessed three methods that use minimal reagents and materials to extract DNA from DBS for beta‐thalassaemia identification.

Rapid detection of α‐thalassaemia variants using droplet digital PCR

Alpha thalassaemia is a highly prevalent disease globally and is a well‐known public health problem in Malaysia. The deletional forms of the mutation are the most common forms found in alpha thalassaemia. The three most common deletional alpha thalassaemia found in this region include ‐‐SEA deletion, ‐α3.7 rightward and ‐α4.2 leftward deletions. The prevalence rate of triplication alpha cases such as αααanti3.7 and αααanti4.2 is not known in Malaysia although it plays a role in exacerbating the clinical phenotypes in beta thalassaemia carriers. Recently, there have been more reported cases of rare alpha thalassaemia mutations due to the advancement of molecular techniques involved in thalassaemia detections. Therefore, it is essential to develop a new method which allows the detection of different alpha thalassaemia mutations including the rare ones simultaneously and accurately.

The use of Taqman genotyping assays for rapid confirmation of β-thalassaemia mutations in the Malays: accurate diagnosis with low DNA concentrations

In Malaysia, β‐thalassaemia is a common inherited blood disorder in haemoglobin synthesis with a carrier rate of 4.5%. Currently, PCR‐incorporating techniques such as amplification refractory mutation system (ARMS) or reverse dot blot hybridization (RDBH) are used in β‐thalassaemia mutation detection. ARMS allows single‐mutation identification using two reactions, one for wild type and another for mutant alleles. RDBH requires probe immobilization and optimization of hybridization and washing temperatures which is time consuming. The aim of our study was to investigate whether β‐thalassaemia mutations can be identified in samples with low DNA concentrations.

Novel assay for efficient iron release from ferritin

Ferritin plays an important role in iron detoxification and iron storage by converting soluble iron(II) to insoluble iron(III) inside the protein cage. Mechanism of in vitro ferritin iron release occurs more readily under reducing conditions, whereby reducing agents may enter into the ferritin cage through the protein pores to convert insoluble iron(III) into soluble iron(II), after accepting an electron from reducing agents. The soluble iron(II) then liberates outside the protein cage and forms coloured and stable complexes in the presence of chromophore, an iron(II) chelating agent, to be quantified by spectrophotometry. However, iron(II) is readily converted back to iron(III) in the presence of oxygen. Thus, oxygen scavenger compounds were used to remove any dissolved oxygen. Studies suggested that low concentration of chaotropic agents regulate the protein pore opening and result in protein unfolding that is therefore accessible by reducing agents more effectively. According to in vivo studies, iron exit from ferritin for recycling or meeting the cells need via lysosomal and/or proteasomal pathways. We designed an assay to determine the total iron available inside the ferritin by first using the commercially available iron(II) or iron(III) compounds as a substrate to optimize the concentration of reactants involved. Apart from chromophore and reducing agents, oxygen scavenger compounds, chaotropic agents and enzymes were added in order to induce ferritin iron release effectively. Linear calibration curve of the iron(II)-chromophore complex were obtained and was used as a reference to correlate absorbance readings to concentration of iron level. Reducing agents was able to reduce the iron(III) to iron(II) and oxygen scavenger A seems to be more effective in removing any dissolved oxygen to avoid oxidation of both the reducing agents and iron(III). Taken altogether, this experiment setup will be beneficial for the improvement of efficiency in quantifying the total iron level inside the ferritin.

Iron deficiency anaemia: with the conclusion of a need for iron reader

In our bloodstream, there are plenty of red blood cells (RBC), which function as an important oxygen carrier in our bodies. Each RBC consists of millions of haemoglobin (Hb), which is made up from globin and iron. If any deficiency/malfunction of any globin, it will lead to anaemia as indicated in low Hb level while iron deficiency anaemia (IDA) is anaemic due to the lacking of iron as indicated in low Hb and ferritin levels. IDA affects almost two billion people globally while anaemia without iron deficiency, such as thalassaemia, affects almost 4.5% in Malaysian population. These anaemic conditions have similar clinical symptoms like fatigue, dizziness, in which disturb their cognitive development and productivity in workplace. In areas without proper medical access, many anaemic individuals were misdiagnosed and treated with iron tablets because they were thought to have iron deficiency anaemia due to low Hb content. But, excess iron is toxic to the body. Misdiagnosis can be avoided by iron status assessment. We hereby review the currently available iron status parameters in laboratory and field study with the conclusion of demonstrating the importance of a need for iron reader, in the effort to reduce the prevalence of IDA globally.

Ethnic specificity of beta-globin gene molecular defects among beta-thalassaemia major patients in the indigenous population of sabah

Beta-thalassaemia is the most common inherited disease in Malaysia with a carrier rate of 4.5%. The spectrum of β-globin mutations has been well documented in the major ethnic groups in West Malaysia. Sabah in East Malaysia has over 1000 cases of transfusion dependent β-thalassaemia major patients. The complete spectrum of β-thalassaemia mutations in Sabah is still unknown. This study was done to characterise these β-thalassaemia mutations incorporating PCR techniques. A total of 253 β-thalassaemia major patients were recruited in this study following institutional clearance and informed consent. Homozygosity of the Filipino β-thalassaemia deletion was found in 217 (85.8%) patients, comprising of 136 Kadazandusuns, 19 Rungus, 10 Murut, 9 Sungai, 4 Bajau, 2 Bisaya and 1 Kedayu. Compound heterozygosity of Filipino β-thalassaemia deletion was found in 24 (9.5%) patients. Mutations common in West Malaysia were found in 11 (4.34%) patients (6 Chinese, 2 Brunei, 1 Banjar, 1 Bajau and 1 Malay). This study indicates ethnicity is informative in assisting β-thalassaemia diagnosis in Sabah. The indigenous population in Sabah had a unique mutation, the Filipino deletion. This study provides a platform in developing a simple and accurate molecular diagnosis protocol for β-thalassaemia mutations identification in the indigenous population of Sabah.