Rapid detection of α-thalassaemia variants using droplet digital PCR.

INTRODUCTION: 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, -α(3.7) rightward and -α(4.2) leftward deletions. The prevalence rate of triplication alpha cases such as ααα(anti3.7) and ααα(anti4.2) 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. METHODS: The purpose of this study was to design an assay for the detection of triplications, common and rare deletional alpha thalassaemia using droplet digital PCR (ddPCR). RESULTS: This is a quantitative detection method to measure the changes of copy number which can detect deletions, duplications and triplications of the alpha globin gene simultaneously. CONCLUSION: In conclusion, ddPCR is an alternative method for rapid detection of alpha thalassaemia variants in Malaysia.

Analysis of α1 and α2 globin genes among patients with hemoglobin Adana in Malaysia.

Hemoglobin (Hb) Adana [HBA2: c179G>A (or HBA1); p.Gly60Asp] is a non-deletional α-thalassemia variant found in Malaysia. An improvement in the molecular techniques in recent years has made identification of Hb Adana much easier. For this study, a total of 26 Hb Adana α-thalassemia intermedia and 10 Hb Adana trait blood samples were collected from patients. Common deletional and non-deletional α-thalassemia genotypes were determined using multiplex gap polymerase chain reaction (PCR) and multiplex ARMS PCR techniques. Identification of the Hb Adana location on the α-globin gene was carried out using genomic sequencing and the location of the mutation was confirmed via restriction fragment length polymorphism-PCR. Among the 36 samples, 24 (66.7%) had the -α(3.7)/α(Cd59)α mutation, while the -α(3.7)/α(Cd59)α mutation accounted for 2 samples (5.6%) and the remaining 10 (27.8%) samples were α/α(Cd59)α. All 36 samples were found to have the Hb Adana mutation on the α2-globin gene. The position of the α-globin gene mutation found in our cases was similar to that reported in Indonesia (16%) but not to that in Turkey (0.6%). Our results showed that the Hb Adana mutation was preferentially present in the α2-globin genes in Malays compared to the other ethnicities in Malaysia. Thus, the Malays might have similar ancestry based on the similarities in the Hb Adana position.

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

INTRODUCTION: In Malaysia, ß-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 ß-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 ß-thalassaemia mutations can be identified in samples with low DNA concentrations. METHODS: Genotype identification of common ß-thalassaemia mutations in Malays was carried out using Taqman genotyping assays. RESULTS: Results show that the Taqman assays allow mutation detection with DNA template concentrations as low as 2-100 ng. In addition, consistent reproducibility was observed in the Taqman assays when repeated eight times and at different time intervals. CONCLUSION: The developed sensitive Taqman assays allow molecular characterization of ß-thalassaemia mutations in samples with low DNA concentrations. The Taqman genotyping assays have potential as a diagnostic tool for foetal blood, chorionic villi or pre-implantation genetic diagnosis where DNA is limited and precious.

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. METHODS: The methods assessed in this study were Tris-EDTA (TE) buffer-based method by Bereczky et al. (American Journal of Tropical Medicine and Hygiene 72, 2005, 249), NaCL/NaOH/Sodium dodecyl sulphate (SDS) method by Huang et al. (Human Genetics 84, 1990, 129) and NaOH method by Zhou et al. (Analytical Biochemistry 354, 2006, 159). Extracted DNA was amplified for three common beta-thalassaemia mutations in Malaysia. RESULTS: Amplicons derived from TE buffer-based method were very faint and almost nonexistent while the NaCl/NaOH/SDS method did not produce any visible amplicons. The extraction using NaOH method produced visible bands that were comparable to the standard method using extraction kit. CONCLUSION: The NaOH method is a simple method that uses minimal equipment and reagents that make it labour- and cost-effective. This method could be adopted by poorer countries to extract DNA for beta-thalassaemia mutation characterization.

Induced pluripotent stem cells: history, properties and potential applications.

The development of induced pluripotent stem cells (iPSCs) has been met with much enthusiasm and hailed as a breakthrough discovery by the scientific and research communities amidst the divisive and ongoing debates surrounding human embryonic stem cells (hESC) research. The discovery reveals the fact that embryonic pluripotency can be generated from adult somatic cells by the induction of appropriate transcriptional factor genes essential for maintaining the pluripotency. They provide an alternative source for pluripotent stem cells, thus representing a powerful new research tool besides their potential application in the field of regenerative medicine. In this review, the historical background of iPSCs generation will be discussed together with their properties and characteristics as well as their potential therapeutic applications.