An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

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.