Molecular Characterization of G6PD Mutations Reveals the High Frequency of G6PD Aures in the Mon-Khmer Population

doi:10.21203/rs.3.rs-45493/v1

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Molecular Characterization of G6PD Mutations Reveals the High Frequency of G6PD Aures in the Mon-Khmer Population

https://doi.org/10.21203/rs.3.rs-45493/v1

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published 07 Jan, 2021

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Background

The prevalence and genotypes of G6PD deficiency vary worldwide, with higher prevalence in malaria endemic areas. The first time assessment of G6PD deficiency prevalence and molecular characterization of G6PD mutations in the Mon-Khmer population were performed in this study.

Methods

A total of 252 unrelated Mon-Khmer participants residing in the Lao People's Democratic Republic (PDR) were recruited. All participant samples were tested for G6PD enzyme activity and G6PD gene mutations. The amplification refractory mutation system (ARMS)-PCR for detecting G6PD Aures was developed in this study.

Results

The G6PD mutations were detected in 11.51% (29/252) of the participants. The mutation pattern was homogenous, predominantly involving the G6PD Aures mutation (6.75%), followed by 1.19% G6PD Union and 0.79% each G6PD Jammu, G6PD Mahidol and G6PD Kaiping. One subject (0.4%) each carried G6PD Viangchan and G6PD Canton. Interestingly, one case of coinheritance of G6PD Aures and Quing Yan was detected in this cohort.

Conclusion

The G6PD Aures mutation is highly prevalent in Mon-Khmer ethnic group. The common G6PD variants in continental Southeast Asian populations, G6PD Viangchan, Canton, Kaiping, Union and Mahidol, were not prevalent in this ethnic group. The technical simplicity of the developed ARMS-PCR will facilitate the final diagnosis of the G6PD Aures.

Infectious Diseases

G6PD genotype

G6PD deficiency

G6PD mutation

G6PD Aures

Laos

Mon-Khmer

Glucose 6˗phosphate dehydrogenase (G6PD) deficiency is the most common hereditary enzymopathy affecting people worldwide. G6PD is a key enzyme in the pentose phosphate pathway (PPP), a metabolic pathway that produces nicotinamide adenine dinucleotide phosphate (NADPH) to maintain the level of oxidized glutathione, which is important for defending against oxidative damage in red blood cells (1, 2). The gene encoding the G6PD enzyme is located near the telomeric region of the distal arm of the X chromosome and is composed of 13 exons and 12 introns (3). G6PD deficiency is mostly caused by a single nucleotide mutation that gives rise to an amino acid substitution in an exon of the G6PD gene, resulting in reduced enzyme activity and stability (4, 5). G6PD deficiency is an X-linked genetic condition; therefore, this condition is predicted to be more common in males than in females and is expressed in hemizygous males and homozygous or heterozygous females (6).

Most people with G6PD deficiency do not exhibit symptoms unless exposed to an oxidation-inducing agent or an infection. G6PD deficiency causes a spectrum of acute or chronic hemolysis in affected individuals and causes neonatal jaundice, which can result in life-threatening kernicterus in newborns (7). Patients with G6PD deficiency are more susceptible to red blood cell destruction when exposed to oxidative stress; these agents include oxidant drugs (e.g., sulfones and primaquine) and food (fava beans) (8). Acute hemolysis due to antimalarial drugs is a health concern in Southeast Asia (7). However, 8-aminoquinoline antimalarials, such as primaquine, remain the only effective drug against relapse caused by Plasmodium Vivax and Plasmodium Ovale (9). Beyond antimalarial drugs, G6PD deficiency is also relevant to other medicines and infections, such as dengue or hepatitis virus, and has a risk of serious complications resulting from acute renal failure (10, 11). The clinical manifestation of G6PD deficiency by adapted from WHO definition, G6PD enzyme activity less than 10%, 10 to < 30% and 30 to < 60% of the overall median activity were considered as severe, moderate and mild G6PD deficiency while any G6PD activity < 60% was considered G6PD normal (12).

Approximately 200 G6PD mutations have been reported (13). The prevalence of G6PD deficiency correlates with the geographical distribution of malaria since this disorder is believed to provide protection against the disease (14). Currently, more than 400 million people worldwide carry an abnormal G6PD gene, particularly in Asia, Africa, the Middle East and the Mediterranean (15, 16). G6PD genotypes show population specificity. For example, in Southeast Asia, the G6PD Mahidol mutation is predominant among the Myanmar and Mon populations (17), while the G6PD Viangchan mutation is common among the Thai, Cambodian and Lao populations (18–20). No studies have been conducted to ascertain the prevalence of G6PD deficiency in the Mon-Khmer or Lao Theung group. The Mon-Khmer group is the second largest ethnic group in Laos and has its own culture and language (the Mon-Khmer language) (21). The Mon-Khmer group originated from the Austro-Asiatic family and migrated to Southeast Asia in prehistoric times. In addition to the Lao PDR, Mon-Khmer individuals live in Thailand, China, Myanma, Cambodia and Vietnam (22). This study aimed to assess the prevalence and perform molecular characterization of G6PD deficiency in the Mon-Khmer ethnic group residing in the Lao PDR. Since severe acute hemolytic anemia can be triggered by G6PD deficiency, addressing the prevalence and molecular characterization of G6PD mutations in the Mon-Khmer ethnic group is an important public health issue.

Subjects

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. Informed consent was obtained from all individual participants included in the study.

G6pd Enzyme Activity Assay

The blood samples were stored at 4 ºC and analyzed for G6PD enzyme activity within 7 days after collection based on the WHO-recommended standard test(23). 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 hemoglobin (Hb) concentration was determined using a HemoCue Hemoglobin 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 (DiaplexC™ 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 DiaplexC™ G6PD Genotyping Kit. The entire coding sequence (exons 2–13) of the G6PD gene was amplified with specific primers following a protocol previously described (17). 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 analyzed 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 analysis

Data were analyzed 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 (9, 24). In detail, the adjusted median values were defined as the median G6PD 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 < 10% for severe deficiency, 10% to < 30% for moderate deficiency and 30% to < 60% for mild deficiency. Subjects with G6PD activity over 60% were defined as having normal activity.

Prevalence of G6PD gene mutations

The overall prevalence of G6PD gene mutations in this Lao Mon-Khmer group was 11.51% (29/252), equating to 6.41% (5/78) in males and 13.79% (24/174) in females. Eight G6PD mutation sites were detected (Fig. 2). The most common G6PD mutation in this Mon-Khmer population was G6PD Aures c.143T > C (6.75%), followed by 1.19% G6PD Union c.1360C > T and 0.79% each G6PD Jammu c.871G > A with nt1311C, G6PD Mahidol c.487G > A and G6PD Kaiping c.1388G > A. One subject (0.4%) each carried G6PD Viangchan c.871G > A and G6PD Canton c.1376G > T (Table 1). Interestingly, we found that one female carried coinherited G6PD Aures c.143T > C and G6PD Quing Yan c.392G > T mutations. The G6PD Vanua Lava c.383T > C, G6PD Coimbra c.592C > T and G6PD Mediterranean c.563C > T mutations were not detected in this cohort.

Table 1

Prevalence and frequency of *G6PD* gene mutations in the Lao Mon-Khmer ethnic group.
Mutation	Normal			G6PD deficiency			Prevalence (%)	Frequency (95% Confidence interval)
	M (n = 73)	F (n = 157)		M (n = 5)	F (n = 17)
	Hemi-zygotes	Homo-zygotes	Hetero zygotes	Hemi Zygotes	Homo-zygotes	Hetero zygotes
Viangchan c.871G > A with nt1311T	0	0	0	1	0	0	1 (0.40)	0.0040 0.0001–0.0219
Jammu c.871G > A with nt1311C	0	0	1	0	0	1	2 (0.79)	0.0079 0.0010–0.0284
Mahidol c.487G > A	0	0	0	0	1	1	2 (0.79)	0.0079 0.0010–0.0284
Union c.1360C > T	0	0	1	1	0	1	3 (1.19)	0.0119 0.0025–0.0344
Kaiping c.1388G > A	0	0	0	1	0	1	2 (0.79)	0.0079 0.0010–0.0284
Canton c.1376G > T	0	0	1	0	0	0	1 (0.40)	0.0040 0.0001–0.0219
Vanua lava c.383T > C	0	0	0	0	0	0	0	0
Coimbra c.592C > T	0	0	0	0	0	0	0	0
Mediterranean c.563C > T	0	0	0	0	0	0	0	0
Aures c.143T > C	0	0	8	2	1	6	17 (6.75)	0.0675 0.0398–0.1058
Quing Yan c.392G > T*	ND	ND	ND	0	0	1	1 (0.40)	0.0040 0.0001–0.0219
Total	0	0	11	5	2	11	29 (11.51)
*Coinheritance of G6PD Qing Yan c.392G > T with G6PD Aures c.143T > C

Prevalence of G6PD deficiency calculated based on the enzyme activity

G6PD enzyme activity was measured by using a quantitative G6PD kit. The mean and median values of the G6PD enzyme activity for the entire population were 7.22 U/gHb and 7.13 U/gHb, respectively. The mean and standard deviation (SD) values of the G6PD enzyme activity between males (6.96 ± 2.59 U/gHb) and females (7.34 ± 2.67 U/gHb) were not significantly different (p = 0.30). The cut-off value for G6PD deficiency (G6PD activity below 60%) in this cohort was 4.09 U/gHb for males or 4.32 U/gHb for females (Fig. 3). Males with G6PD enzyme activity below 0.68 U/gHb and females with activity below 0.72 U/gHb (G6PD activity below 10%) were considered to be severely G6PD deficient. Males with G6PD enzyme activity at 0.68 to 2.04 U/gHb and females with activity at 0.72 to 2.16 U/gHb (G6PD activity of 10–29.9%) were considered moderately G6PD deficient, and G6PD enzyme activity at 2.05 to 4.09 U/gHb for males or 2.17 to 4.32 U/gHb for females was considered to indicate mild deficiency (G6PD activity of 30–59.9%). Based on the enzyme activity cut-off value, the prevalence of G6PD deficiency in this cohort comprising 5 males and 17 females was found to be 8.73% (22/252). The prevalence of G6PD deficiency among females was 9.77%, while that among males was 6.41%. The mean and SD of enzyme activity in the 5 hemizygous males with G6PD deficiency were 0.59 ± 0.38 U/gHb (range: 0.01–1.04 U/gHb). Four of five hemizygous subjects were considered severely deficient, while 1 hemizygous subjects were moderately deficient. The mean and SD of 17 females with G6PD deficiency were 2.63 ± 0.97 U/gHb (range: 0.39–4.04 U/gHb). One homozygous G6PD-deficient female was considered severely deficient (0.39 U/gHb). Three moderately G6PD-deficient subjects, including 1 homozygous (1.02 U/gHb), 1 heterozygous (2.08 U/gHb) and 1 with coinheritance of a heterozygous G6PD mutation (1.26 U/gHb), were detected. One of the 17 G6PD-deficient females was considered severely G6PD deficient (0.39 U/gHb), 3 subjects were moderately G6PD deficient (1.45 ± 0.56 U/gHb), and the remaining 13 subjects were mildly G6PD deficient (3.08 ± 0.48 U/gHb).

The hemizygous males (n = 2) and homozygous female (n = 1) for the G6PD Aures mutation presented with a severe G6PD deficiency, whereas the phenotype of G6PD Aures heterozygous females varied from normal to moderately G6PD deficient. Six of 14 heterozygous G6PD Aures subjects presented moderately G6PD deficiency (3.12 ± 0.44 U/gHb) whereas the remaining 8 females showed normal G6PD enzyme activity (6.48 ± 1.24 U/gHb).

G6PD mutations are distributed worldwide (25) and particularly widespread in malaria endemic regions. A high prevalence of G6PD deficiency has been reported in Africa, Southern Europe, the Middle East, Southeast Asia and the central and southern Pacific islands. However, deficiency alleles are currently quite prevalent in North and South America and in parts of Northern Europe because of fairly recent migration (15). Knowledge of the prevalence of G6PD variants among different ethnic groups is limited. The present study is the first to report the prevalence of G6PD deficiency and G6PD mutations in the Mon-Khmer or Lao Theung ethnic group. The prevalence of G6PD deficiency reported in this study (8.73%) is higher than that previously reported for unspecified ethnic Lao populations (6.21% and 4.40%) (26, 27) (Table 2). However, the prevalence of G6PD deficiency in Lao populations was lower than that reported in neighboring Southeast Asian populations, including Mon (12%) (17), Karen and Burman (13.7%) (28), Thai (11.1%) (18), and Cambodian (26.1%) (19) populations. The prevalence of G6PD deficiency in Laotians had previously been reported in an unspecified ethnic population, and it was suspected that the majority of the studied subjects were Lao Loum, which are the main Laotian ethnic group. In the beginning of G6PD mutation screening, the study of G6PD mutations focused only on the detection of the G6PD Viangchan mutation or identified the G6PD mutation only in males that presented with severe hemolytic anemia (20, 26, 29). Only six previous studies (Table 2) have revealed that the prevalence of G6PD deficiency in the Lao population ranges from 3.30-21.98% in males and 2.50-11.24% in females (20, 26, 27, 29–31). Our study revealed severe G6PD deficiency more frequently in males than in females, whereas moderate and mild G6PD deficiencies were more common in females than in males. Although the females with heterozygous G6PD mutation have sufficient enzyme activity, they can pass an X-linked G6PD mutation to all of their sons along with a risk of developing the symptoms associated with a severe G6PD deficiency.

Table 2

Prevalence and molecular characteristics of G6PD deficiency in Lao PDR.
	This study	Hsia et al.,1993 (13)	Iwai et al., 2001 (14)	Kanchanavithayakul et al., 2017 (15)	Lover et al., 2018 (18)	Bancone et al., 2019 (16)	Ong et al., 2019 (17)
Sex
Male %	5/78 (6.41%)	15/74 (20.3%)	21/291 (7.2%)	31/141 (21.98%)	30/910 3.30%	106/1211 (8.8%)	63/964 (6.53%)
Female %	17/174 (9.77%)	ND	ND	10/89 (11.24%)	ND	79/1764 (4.5%)	27/1079 (2.50%)
Total %	22/252 (8.73%)	15/74 (20.3%)	21/291 (7.2%)	41/230 (17.8%)	30/910 3.3%	185/2978 (6.2%)	90/2043 (4.4%)
G6PD mutation
Gaohe 95A > G	ND	-	-	ND	ND	ND	ND
Aures 143T > C	17 (6.75%)	ND	ND	ND	ND	ND	ND
Asahi 202 G > A	ND	-	ND	ND	ND	ND	ND
A 376 A > G	ND	-	ND	ND	ND	ND	ND
Vanua Lava 383T > C	-	ND	-	ND	ND	ND	ND
Mahidol 487G > A	2 (0.79%)	2 (2.7%)	-	-	1 (0.11%)	4 (0.1%)	ND
Chinese-3 493 A > G	ND	-	ND	ND	ND	ND	ND
Mediterranean 563C > T	-	ND	ND	ND	-	ND	ND
Coimbra 592C > T	-	ND	-	-	-	ND	ND
Chinese-1 835 A > T	ND	-	ND	ND	ND	ND	ND
Viangchan 871G > A with nt1311T	1 (0.40) %)	5 (6.8%)	9 (3.1%)	15 (6.5%)	11 (1.21%)	115 (3.9%)	90 (4.4%)
Jammu 871G > A with nt1311C	2 (0.79%)	ND	ND	-	ND	ND	ND
Chatham 1003 G > A	ND	-	-	ND	ND	ND	ND
Chinese-5 1024 C > T	ND	ND	ND	-	ND	ND	ND
Surabaya 1291G > A	ND	ND	-	ND	ND	ND	ND
Union 1360C > T	3 (1.19%)	1 (1.4%)	-	1 (0.4%)	14 (1.54%)	9 (0.3%)	ND
Canton 1376G > T	1 (0.4%)	-	-	4 (1.7%)	4 (0.44%)	1 (0.03%)	ND
Kaiping 1388G > A	2 (0.79%)	1 (1.4%)	-	2 (0.9%)	ND	4 (0.1%)	ND
Quing Yan 392G > T	1 (0.4%)	ND	ND	-	ND	6 (0.2%)	ND
ND: Not done

Regarding the mutation characteristic of the G6PD gene, eight G6PD mutations were detected in this Lao Mon-Khmer group. A comparison of the G6PD mutation data collected in this and previous studies of non-specified Lao ethnic populations is summarized in Table 2. The most common G6PD mutation in the Mon-Khmer population was G6PD Aures c.143T > C (6.75%); this result is different from that in a previous report, which found that the G6PD Viangchan mutation was the most common mutation in the Laotian population (1.21–6.76%) (20, 26, 27, 29–31). The G6PD Jammu mutation is an 871G > A mutation identical to the G6PD Viangchan mutation but different from the polymorphism at nucleotide 1311 (nt1311C) detected in this study, whereas the previous report found only G6PD Viangchan in the Laotian population (30). The polymorphisms nt1311C and IVS11 nt93T were randomly detected in this group, different from those reported in other Lao populations (30), the Thai population (nt1311T) (18) and the Chinese population (nt1311T) (32). These data imply that the Mon-Khmer or Lao Theung and Lao Loum groups have different origins. For neighboring Southeast Asian countries, G6PD Mahidol is commonly found in Burmese (18.21%) (27), while G6PD Viangchan is commonly found in Thais (6.0%) (18), Cambodians (17.72%) (27) and Vietnamese (26.7%) (33) but not in the Mon-Khmer population. In addition, we rarely detected the G6PD Canton, G6PD Kaiping and G6PD Union mutations, which are prevalent in South China (34). The G6PD Mediterranean, G6PD Vanua Lava and G6PD Coimbra mutations were not found in this population. The G6PD mutation was not identified in four G6PD-deficient Lao Mon-Khmer participants. The mutation site of those 4 cases may exist in the intron region, which could not be analyzed by our method.

Previous studies showed that the severe G6PD deficiency was caused by nucleotide mutation at or close to the NADP + or G6P binding site (3, 35), which promoted the structural instability of G6PD and resulted in less NADPH production and oxidative stress sensitivity. The G6PD Aures mutation is commonly detected in Mediterranean populations, such as the Saudi Arabian (11.2–20%) (36–38), United Arab Emirates (11.9%) (39) and Kuwaiti (3.73%)(40). The G6PD Aures was detected in 0.7–4.3%of Thai populations (41, 42). This mutation results in the substitution of amino acid 48 from isoleucine to threonine. The WHO has classified the G6PD Aures mutation as a class III mutation (43). The G6PD Aures was located between the sites of G6PD Vietmam-1 and Vietmam-2/Bahia, near G6PD Rignano, which was distant from the protein domain for NADP-1 binding (3, 35). The mutation was predicted to affect the mini-instability of protein domain structure for binding with NADP-1 and to subsequently cause mild G6PD deficiency. A previous report described the person who carried this mutation as having mild G6PD deficiency (44). Our study found that hemizygous males or homozygous females for the G6PD Aures mutation presented with a significant severe G6PD deficiency, whereas the heterozygous females for G6PD Aures had normal G6PD enzyme activity. In contrast to the hemizygous and homozygous groups, the phenotype of G6PD Aures heterozygous females varied from normal to moderately G6PD deficient.

The G6PD Aures mutation is associated with favism, which is a primary clinical manifestation related to acute hemolysis due to G6PD deficiency; however, favism has never been reported in the Lao population, probably because of underestimation or because the fava bean is not a staple food for the Lao people. Our study detected the G6PD Aures mutation in both male and female participants. As the inheritance of G6PD is X-linked, it is very important to address that both homozygous and heterozygous G6PD females can pass the abnormal gene to their male child, leading to the risk of clinically presenting the G6PD Aures mutation. The knowledge about the effect of the G6PD Aures mutation is now still limited, and it is very important to test the hemolytic risk of antimalarial drugs and other oxidative damage related to the G6PD Aures mutation in further studies as the Lao Mon-Khmer people stay around the forest area and have a risk of malaria infections.

The diagnosis of G6PD Aures by PCR sequencing performed during routine molecular testing might be problematic as the G6PD gene flanking the G6PD Aures mutation site must be amplified by PCR, purified and sent for sequencing analysis. To shorten the detection step, we developed ARMS-PCR, which can detect G6PD Aures in one PCR-electrophoresis step. By using the ARMS-PCR method developed in this study, the heterozygous G6PD Aures mutation was additionally detected in 8 female participants. This developed method should be generally applicable to the detection of G6PD Aures.

The G6PD mutation pattern identified in the Mon-Khmer group is quite homogenous and unique, predominantly involving the G6PD Aures mutation. This finding shows a founder effect in this population. We found that the hemizygous males or homozygous females for the G6PD Aures mutation presented with a significant severe G6PD deficiency. Malaria infection, dengue fever and typhoid fever are prevalent in Lao PDR and knowing the G6PD status in local health areas, especially Mon-Khmer group, helps improve the management of acute hemolysis related to these infections.

G6PD

Glucose 6˗phosphate dehydrogenase; PCR:polymerase chain reaction; ARMS:amplification refractory mutation system

Acknowledgments

We are greatly indebted to the communities for their support and willingness to participate in this research.

Authors’ contributions

K.L. designed the research study. A.S., N.K. and O.S. collected samples. A.S. and K.Lperformed the research. A.S., K.L.,C.C. and I.Nanalyzed the data. A.S. andK.L drafted the manuscript. K.L., C.C. and I.N. revised the manuscript.

Funding

This study was funded byChulalongkorn University (CU-GES-60-06-30-02)and the 90^thAnniversary of Chulalongkorn University RachadapisekSompote Fund.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

The study was approved by the Ethics Committee of the University of Health Sciences, Lao PDR.Informed consent was obtained from all individual participants included in the study. All experiments were carried out in accordance with relevant guidelines and regulations.

Competing interests
The authors declare no competing interests.

Consent for publication

Not applicable.

Authors' information

¹Oxidation in Red Cell Disorders Research Unit, Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Thailand. ²Department of Biochemistry, Faculty of Medicine,Chulalongkorn University,Thailand. ³Department of Medical Laboratory, Faculty of Medical Technology, University of Health Sciences,Lao PDR. ⁴Department of Pediatrics, Faculty of Medicine, Chulalongkorn University,Thailand

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AND MOLECULAR CHARACTERIZATION OF G6PD GENE AMONG THAI CHILDREN WITH G6PD DEFICIENCY
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Molecular Characterization of G6PD Mutations Reveals the High Frequency of G6PD Aures in the Mon-Khmer Population

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Background

Methods

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G6pd Enzyme Activity Assay

Identification Of 8 Common G6pd Asian Types

Data analysis

Results

Discussion

Conclusion

Abbreviations

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References

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