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G6PD deficiency is the commonest enzyme deficiency found in humans. Current diagnostic methods lack sensitivity to detect all cases of G6PD deficiency. We evaluated the reverse dot blot flow-through hybridisation assay designed to detect simultaneously multiple known G6PD mutations in a group of Malaysian neonates. Archival DNA samples from 141 G6PD-deficient neonates were subjected to reverse dot blot flow-through hybridisation assay using the GenoArray Diagnostic Kit (Hybribio Limited, Hong Kong) and DNA sequencing. The method involved PCR amplification of 5 G6PD exons using biotinylated primers, hybridisation of amplicons to a membrane containing oligoprobes designed for G6PD mutations known to occur in the Malaysian population and colour detection by enzyme immunoassay. The assay detected 13 of the 14 G6PD mutations and genotyped 133 (94.3%) out of 141 (102 males, 39 females) cases. Among the 39 female G6PD-deficient neonates, there were 7 homozygous and 6 compound heterozygous cases. The commonest alleles were G6PD Viangchan 871G > A (21%) and G6PD Mahidol 487G > A(20%) followed by G6PD Mediterranean 563C > T, (14%), G6PD Vanua Lava 383T > C (12%), G6PD Canton 1376G > T (10%), G6PD Orissa 131C > G (6.3%) G6PD Coimbra 592C > T (5.6%) plus 6 other mutations. DNA sequencing of remaining cases revealed 6 cases of intron 11 nt 93C > T not previously reported in Malaysia and two novel mutations, one case each of nt 1361G > T and nt 1030G > A. We found the reverse dot blot assay easy to perform, rapid, accurate and reproducible, potentially becoming an improved diagnostic test for G6PD deficiency. 2019, The Author(s), under exclusive licence to The Japan Society of Human Genetics.

Alpha (α) thalassaemia is the most common inherited disorder in Malaysia. The clinical severity is dependant on the number of α genes involved. Full blood count (FBC) and haemoglobin (Hb) analysis using either gel electrophoresis, high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) are unable to detect definitively alpha thalassaemia carriers. Definitive diagnosis of α-thalassaemias requires molecular analysis and methods of detecting both common deletional and non-deletional molecular abnormailities are easily performed in any laboratory involved in molecular diagnostics. We carried out a retrospective analysis of 1623 cases referred to our laboratory in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) for the diagnosis of α-thalassaemia during the period October 2001 to December 2012. We examined the frequency of different types of alpha gene abnormalities and their haematologic features. Molecular diagnosis was made using a combination of multiplex polymerase reaction (PCR) and real time PCR to detect deletional and non-deletional alpha genes relevant to southeast Asian population. Genetic analysis confirmed the diagnosis of α-thalassaemias in 736 cases. Majority of the cases were Chinese (53.1%) followed by Malays (44.2%), and Indians (2.7%). The most common gene abnormality was αα/--SEA (64.0%) followed by αα /- α 3.7 (19.8%), - α 3.7/--SEA (6.9%), αα/αα CS (3.0%), --SEA/--SEA (1.2%), - α 3.7/- α 3.7 (1.1%), αα/-α4.2 (0.7%), -α 4.2/--SEA (0.7%), -α3.7/-α 4.2 (0.5%), αα CS/-- SEA (0.4%), αα CS/ααCd59 (0.4%), ααCS/αα CS (0.4%), -α3.7/αα Cd59 (0.3%), αα/ααCd59 (0.1%), αα Cd59/ ααIVS I-1 (0.1%), -α3.7/ααCS (0.1%) and --SEA /αα Cd59 (0.1%). This data indicates that the molecular abnormalities of α-thalassaemia in the Malaysian population is heterogenous. Although α-gene deletion is the most common cause, non-deletional α-gene abnormalities are not uncommon and at least 3 different mutations exist. Establishment of rapid and easy molecular techniques is important for definitive diagnosis of alpha thalassaemia, an important prerequisite for genetic counselling to prevent its deleterious complications.

Quantitative measurement of G6PD enzyme activity using Hb normalization OSMMR2000D G6PD assay method has been introduced as routine service in UKMMC since 2011. However, it requires expensive laboratory equipment and skilled personnel. A rapid, mobile and reliable quantitative tool would be ideal especially in centers that depend solely on FST to detect G6PD deficiency. In this study, we evaluated the performance of two rapid POCT G6PD assay kits; Carestart Biosensor (single kit) and Carestart Biosensor 1 (combo kit), and compared the findings to OSMMR2000D G6PD assay method. Cord blood samples in EDTA tube from 153 neonates (91 normal and 62 deficient) measured by OSMMR2000D G6PD assay method in Haematology Unit, UKMMC were used to evaluate both POCT kits. The G6PD enzyme activity was then calculated and expressed in U/gmHb. The enzyme activities measured by each method were then compared with OSMMR2000D G6PD assay method. All deficient samples were analysed for underlying G6PD genetic mutations by Hybribio Flow-through hybridization. The correlation study of both POCT with OSMMR2000D showed strong Spearman correlation coefficient; 0.783 for the single kit and 0.769 for the combo kit. Both POCT kits also showed strong agreement with OSMMR2000D G6PD assay method, as illustrated by kappa value of 0.805 and 0.795 for single and combo kit respectively. Ten G6PD gene mutations were identified; namely Viangchan, Mediterranean, Vanua Lava, Mahidol, Kaiping, Coimbra, Chatham, Orissa, Union and Canton. This study has shown that the G6PD enzyme activities measured by both POCT kits were comparable to the established method, OSMMR2000D G6PD assay method.

Background. Anaemia is a global health problem including Malaysia. In adults, anaemia may affect work productivity. Iron deficiency anaemia and thalassaemia are common causes of anaemia in Malaysia. However, there is scarcity of data on national prevalence of iron deficiency anaemia and thalassaemia, especially in young adults. This cross sectional study was performed to determine the prevalence of iron deficiency anaemia and thalassaemia among medical students of Universiti Kebangsaan Malaysia Medical Centre (UKMMC). Materials and Methods. Blood samples collected in EDTA tubes were analyzed for haemoglobin level and red cell parameters such as MCV, MCH and red cell counts. Samples with abnormal red cell indices were sent for analysis of RBC morphology, iron status, haemoglobin analysis and DNA analysis. Results. A total of 400 samples were available for this study. Fiftyeight (14.5%) students had hypochromic microcytic red cell indices in which 44 (11%) showed thalassaemia red cell indices while 14 (3.5%) had iron deficiency red cell indices which were finally confirmed by serum iron/TIBC analysis. Amongst those suspected to have thalassaemia, 12 (27.3%) were confirmed as alpha thalassaemia trait (??/--SEA), 11 (25%) as Haemoglobin-E trait, 8 (18.2%) as beta thalassaemia trait and 2 (4.5%) as Haemoglobin Constant Spring (??/?CS?). However, eleven students (25%) with thalassaemia red cell indices could not be confirmed with the common thalassaemia primers available, thus causes have yet to be established. Conclusion. Our prevalence of thalassaemia was high and thus we opine that better screening methods should be adopted. � Societ� Editrice Universo (SEU).