In Malaysia, Sabah population constitutes the most number of β-thalassaemia cases ranging from asymptomatic to transfusion dependent. Filipino β°-deletion has been reported as the predominant mutation in Sabah [1]. Despite having the same primary mutation, co-inheritance of genetic variants at HbF quantitative trait loci of HBS1L-MYB intergenic region may cause variability in clinical features by affecting the haemoglobin (Hb) subtypes level, especially HbF. Study suggested that MYB would activate γ-globin repressor gene directly and subsequently initiate the molecular HbF repression mechanisms. Polymorphisms within HBS1L-MYB intergenic region would inhibit binding of transcription factor on MYB and leading to elevation of HbF levels [2]. This can act as an ameliorating factor in the clinical presentation of β-thalassaemia patients [3]. This study aimed to elucidate the association of Hb subtypes levels with three HBS1L-MYB variants among 134 Filipino β°-deletion carriers. PCR-RFLP analysis was done for HBSIL-MYB rs4895441 (A→G) while tetra-primers ARMS PCR analysis was done for HBSIL-MYB rs9399137 (T→C) and rs11759553 (A→T) (Fig.1).
Introduction: Reduction or complete absence of α-globin chain production may result α-thalassemia. Alpha thalassemia carrier may have normal haemoglobin level and thus will be eligible as blood donor. Few complications may happen in which the carrier who donated the blood might be at risk of hypoxia and their blood components might not suitable for transfusion. Thus, it is important to screen for α-thalassemia to prevent any complications happen
after donation. The objective of this study is to investigate the interaction of red blood cell indices and α-globin genotypes among eligible blood donors in a private university, Universiti Tunku Abdul Rahman (UTAR), Malaysia. Methods: A total of 270 eligible blood donors were recruited for this study. Red cell indices were analysed using Horiba hematology analyser and α-globin genotyping was performed for seven alpha deletions, six alpha point mutations
and two alpha triplications. Results: Our study showed high prevalence of α-thalassemia carriers among the eligible blood donors (7.7%, 21/270), with all of them showed normal Hb level (>12 gm/dl). Five genotypes were detected consisting of 249 αα/αα (92.2%), 9 -α3.7/αα (3.3%), 9 --SEA/αα (3.3%), 2 -α4.2/αα (0.7%) and 1 ααCS/αα (0.4%). All α-globin genotypes showed normal Hb level with no significant difference between genotypes (p=0.167). Different
α-globin genotypes showed significant difference in RBC, MCV, MCH, MCHC, RDW and Hct/Hb ratio at the p
Globally, α-thalassaemia is a highly prevalent disease. In Malaysia, this disorder is a well-known public health problem [1]. The three most common deletional α-thalassaemia found in this region include --SEA deletion, -α3.7 and -α4.2 deletions [2]. The prevalence rate of triplication alpha cases such as αααanti3.7 and αααanti4.2 is unknown in Malaysia although it plays a pivotal role in exacerbating the clinical phenotypes in beta thalassaemia carriers [3]. Therefore, the purpose of this study was to design an assay for the detection of triplications and common deletional alpha thalassaemia using droplet digital PCR (ddPCR). Copy number changes were analysed using Quanta-SoftTM software version 1.6.6 after performing ddPCR. Sensitivity and validation analysis were also performed on the DNA samples. The changes in copy number changes (common deletions, duplications and triplications) in the alpha globin gene has been quantitatively detected using ddPCR. For the samples validation as determined by ddPCR, the mean copy number values for αα/αα are 2.0275±0.0177 (HS-40), 1.8175±0.0389 (HBA2), 2.0450±0.0848 (HB 3.7), 2.0050±0.0000 (HBA1). For -α3.7 /--SEA, the mean copy number values are 2.0225±0.2180 (HS-40), 0.9325±0.1213 (HBA2), 0 (HB 3.7), 0.9984±0.1333 (HBA1). As for –α4.2 /--SEA, the mean copy number values are 1.9350 (HS-40), 0 (HBA2), 0.7945 (HB 3.7), 0.8480 (HBA1). The mean copy number values for --SEA/αα samples are 1.9067±0.1327 (HS-40), 0.8164±0.0364 (HBA2), 0.8920±0.0434 (HB 3.7), 0.9148±0.0338 (HBA1) respectively. This study has found that the use of ddPCR is convenient as it allows direct quantification without the requirement of a calibration curve unlike qPCR [4]. Secondly, this study also showed that ddPCR is accurate and precise in the detection of alpha thalassaemia deletions and triplications based on the gene dosages using absolute quantification. In addition, the non-requirement of post-PCR work has minimised the risk of PCR carryover contamination. Thirdly, ddPCR saves time with less turnaround time and minimise the labour work required as compared to techniques such as MLPA which requires DNA denaturation and hybridisation reaction on day 1 while ligation and PCR reaction on day 2. Fourthly, this study found that the detection of α-thalassaemia using ddPCR is sensitive. DNA samples with low concentration as low as 1 ng were able to be detected for α-thalassaemia using ddPCR. The ability to detect minute amount of DNA concentration is crucial particularly in the diagnosing of the lethal HbH hydrops foetalis during the neonatal stage in α-thalassaemia. In conclusion, this is an alternative method (ddPCR) that can be employed for rapid detection of alpha thalassaemia variants in Malaysia.