Peripheral blood samples were collected from 252 unrelated healthy Lao Mon-Khmer people, including 174 females and 78 males. Participants’ ages ranged from 18-50 years old. The participants were living in the Feuang District, Vientiane Province, and Lao PDR. This study was a community-based survey which was approved by the Ethics Committee of the university of Health Sciences, Lao PDR. The sample collection was performed in December 2016. Written informed consent was obtained before blood sampling from all individual participants included in the study.
G6PD enzyme activity assay
The blood samples were stored at 4 ºC and analysed for G6PD enzyme activity within 7 days after collection. All samples were measured for G6PD enzyme activity in duplicate using a quantitative G6PD kit (Trinity G-6-PDH Kit, Trinity Biotech, Bray, Ireland). The assay was carried out at 30 ºC, and the method was performed following the manufacturer’s instructions. The haemoglobin (Hb) concentration was determined using a HemoCue Haemoglobin System (HemoCue Hb 201 Analyzer, Fisher Scientific, Inc, Waltham, MA, USA). The G6PD enzyme activity values were calculated in units per gram of Hb (U/g Hb).
Identification of 8 common G6PD Asian types
All 252 subjects were genotyped for the 8 common G6PD mutations detected in Asia using a multiplex allele-specific PCR-based assay (DiaplexCTM G6PD Genotyping Kit (Asian type), SolGent, Daejeon, Korea). The genomic DNA of all blood samples was extracted by using a QIAamp® DNA Blood Kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s recommended protocol and kept at -80°C until use. The commercial kit enabled the detection of 8 common G6PD mutations, including Vanua Lava c.383T>C, Mahidol c.487 G>A, Mediterranean c.563 C>T, Coimbra c.592 C>T, Viangchan c.871 G>A, Union c.1360 C>T, Canton c.1376 G>T and Kaiping c.1388 G>A. The PCR mixture contained 12 µl of 2X Multiplex PCR Smart Mix, 2 µl of primer mixture, 20-50 ng DNA template and nuclease- free water to obtain a total volume of 25 µl. The PCR cycling conditions used were as follows: initial denaturation at 95°C for 15 min, followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 40 sec; this was followed by a final extension at 72°C for 5 min. After PCR amplification, 5 µl of the PCR product and 5 µl of a standard marker were separated by 3% agarose gel electrophoresis, stained with SYBR® Green (Thermo Fisher Scientific, USA) and visualized under UV light. The positive results were validated by PCR sequencing.
Identification of G6PD gene mutations by PCR sequencing
PCR sequencing was performed to identify the G6PD gene mutation in the G6PD-deficient samples that could not be identified by DiaplexCTM G6PD Genotyping Kit. The entire coding sequence (exons 2-13) of the G6PD gene was amplified with specific primers following a protocol previously described . The PCR products were purified using an AccuPre® PCR Purification Kit (Bioneer, Daejeon, Korea) and subsequently sequenced by using the Sanger sequencing method (Bioneer Sequencing Service, Daejeon, Korea). The sequence of G6PD was analysed by BLAST and compared to the G6PD gene mutation with database accession no. NC_000023.11.
Detection of G6PD Aures by the amplification refractory mutation system (ARMS)-PCR method
The ARMS-PCR method for detecting the G6PD Aures mutation was first developed in this study. The G6PD Aures allele-specific PCR primer pair consisted of a forward primer (5'-ACCTGGCCAAGAAGAAGAT-3') and a reverse primer (5'-CTCACTCTGTTTGCGGATG -3'), which produce a 226 bp fragment. Two additional primers consisting of a forward primer (5'-TGGTTCTGCCCTCTCTAC-3') and a reverse primer (5'-GAGACACGGACAGA CAGA-3') were added to produce a 519 bp fragment of the 3'-UTR region to serve as an internal amplification control (Fig. 1). The multiplex ARMS-PCR mixture contained 2x HotStar Taq Master Mix (Qiagen Multiplex PCR Kit, Qiagen, Düsseldorf, Germany), 200 μM of each dNTP, 10 pmol of each primer, 5x Q-solution and 150 ng genomic DNA. PCR cycling was performed with an initial denaturation at 95°C for 15 minutes, followed by 35 cycles of amplification at 95°C for 30 sec, 55°C for 1 min and 72°C for 1 min. The final extension was performed at 72°C for 5 min. The PCR product was separated on a 1.5% agarose gel and visualized under UV light. The multiplex ARMS-PCR method was validated by direct DNA sequencing. After validation, multiplex ARMS-PCR was used to detect G6PD Aures mutations in all 252 Lao Theung DNA samples. Heterozygosity of the G6PD Aures mutation was identified by direct DNA sequencing.
Data were analysed for the mean, SD, median, and interquartile range (IQR) by using IBM SPSS statistics software version 22 (IBM Corp., Armonk, NY, USA). The 95% confidential intervals were calculated by Java Stat Binomial Confidence Intervals. The adjusted male median G6PD activity was calculated to identify the cut-off values for G6PD deficiency following a calculation method described previously . In detail, the adjusted male median (AMM) values were defined as 100% activity of males after excluding the data of all participants with G6PD activity equal to or less than 10% of the overall median G6PD activity. The cut-off points for G6PD deficiency were median values < 30 %of the AMM for G6PD deficient, 30 % to 70% of the AMM for G6PD intermediate. Subjects with G6PD activity over 70 % of the AMM were defined as having normal activity.