Background
Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is one of the most common enzymatic disorders of red blood cells in humans and has a varied clinical presentation [
1]. The World Health Organization has defined the different G6PD variants according to the magnitude of the enzyme deficiency and the severity of haemolysis. The clinical expression of G6PD deficiency varies from severe enzyme deficiency to increased enzyme activity (class I to class V). The commonest clinical patterns are; 1) neonatal jaundice, 2) congenital haemolytic anaemia, 3) drug-induced haemolysis and 4) favism.
G6PD is the initial enzyme involved in the pentose phosphate pathway of erythrocyte metabolism. It is involved in the production of NADPH and indirectly of reduced glutathione necessary for the protection of the cells from oxidative stress. This enzyme is encoded by the
G6PD gene, which is located at chromosome Xq28. The
G6PD gene exhibits remarkable polymorphism in human populations and G6PD is known to have over 400 variants. These variants are distinguished by their electrophoresis and biochemical characteristics and some variants are not associated with significantly reduced enzyme activity in erythrocytes [
2]. However, there are numerous mutations found in the
G6PD gene, which are geographically isolated and cause a deficiency of the enzyme in erythrocytes [
3‐
8]. Almost all G6PD deficiencies are caused by a point mutation of the genomic DNA resulting in an amino acid substitution [
9]. To date, 140 mutations of the
G6PD gene have been identified.
The incidence of G6PD deficiency is high in malaria endemic areas. There is evidence to support the hypothesis that G6PD deficiency confers a protective effect against illness following
Plasmodium infection and genetic variability maintained at the G6PD locus appears to be an example of a balanced polymorphism [
1]. In Africa and Southeast Asian countries where
Plasmodium falciparum is endemic, the incidence of G6PD deficiency is estimated to be more than 10% (summarized by Matsuoka, 2005[
10]). In contrast, in Japan, Northern China and Northern European countries, where malaria is historically not endemic, the incidence of G6PD deficiency is less than 0.1% (summarized by Matsuoka, 2005[
10]). In the northern Vietnamese population, the incidence of G6PD deficiency is highly variable between ethnicities. The frequency of G6PD deficiency in the Kinh and the Mong ethnic groups, who traditionally have lived outside malaria transmission areas, is low (0.5% and 0.7%, respectively). The prevalence among ethnic groups living in the foothills of 4 provinces in the north of Vietnam (Thanh Hoa, Son La, Ha Giang, and Hoa Binh), the breeding area of the main malaria vector
Anopheles minimus, ranges from 9.7% to 31% [
11].
The blackwater fever (BWF) syndrome is characterized by severe intravascular haemolysis and anaemia producing dark urine in patients, and is also associated with malaria [
12,
13]. The pathogenesis of BWF remains unclear [
14,
15]. However, G6PD deficiency has been identified as a cause of haemolysis in patients receiving primaquine, or other oxidant drugs, and it is the single factor most often associated with acute intravascular haemolysis [
3,
4,
8,
16‐
18]. Chau
et al showed in Vietnam that BWF was associated with quinine ingestion, malaria infection and G6PD deficiency, and that these three factors were not mutually independent and may interact [
19]. The nature of the interaction is unclear, particularly as quinine is not an oxidant drug, like primaquine, known to cause haemolysis in G6PD deficiency. However, the overlapping risk factors suggest that haemoglobinuria caused by G6PD deficiency should not be regarded as a separate syndrome [
19].
Though the prevalence of G6PD deficiency in Vietnamese populations has been reported, the genetic variants responsible for this deficiency and their associations with haemoglobinura have not been determined. G6PD mutations were screened in patients with haemoglobinuria and in G6PD deficient healthy individuals to identify the G6PD variants causing G6PD deficiency in the Vietnamese general population and to investigate the contribution of G6PD mutations to the development of haemoglobinuria.
Results
Phenotypic screening for G6PD deficiency in healthy individuals from two ethnic groups, the Vietnamese Kinh and S'tieng, showed that the overall prevalence of the G6PD deficient phenotype in the southern Vietnamese population is relatively high at 11.3% (59/524). The difference in the prevalence of G6PD deficiency between the two Vietnamese ethnicities was not significant; 8.7% (23/266) in the Kinh and 14% (36/258) in the S'tieng (p = 0.07). Of the 23 G6PD deficient Kinh, 17 were male, five were female and one unknown (sex not recorded). Of the 36 G6PD deficient S'tieng, 28 were male and eight were female. In Vietnamese Kinh patients with haemoglobinuria, the frequency of "probable G6PD deficiency" was 58.5% (48/82). Twenty-eight percent (23/82) of haemoglobinuric patients had "definite G6PD deficiency." Of the 82 haemoglobinuric patients, five were female and 75 were male. All patients with probable G6PD deficiency were male (N = 48).
To identify the molecular cause of G6PD deficiency in this population the
G6PD gene was screened for mutations in healthy individuals (of both Kinh and S'tieng ethnic groups) who were G6PD deficient, and in all haemoglobinuric patients (N = 82). Across the three groups eight polymorphisms were identified within the
G6PD gene (Table
2). Five of these variants have been described in other populations, namely Gaohe Gaozhou (A95G; His
32Arg), Coimbra "Shunde" (C11763T; Arg
198Cys), Chinese-5 (C13184T; Leu
342Phe), and Viangchan Jammu (G13031A; Val
291Met) and nt13714C>T [
9]. Three variants identified in this Vietnamese population are novel (two non-synonymous polymorphisms and one silent): Vietnam1 (G7A; Glu
3 Lys); Vietnam2 (T10148G; Phe
66 Cys); and Vietnam3 (C10170 T, Ser
73 Ser) (Table
2).
Table 2
G6PD variants present in the Vietnamese population.
Non-synonomous
| | | | | | | |
G7A | Vietnam 1 | Glu3Lys | * | | 1 | | |
A95G | Gaohe Gaozhou | His32Arg | 3 | | 1 | | |
T10148G | Vietnam 2 | Phe66Cys | * | | | 1 | |
C11763T | Coimbra Shunde | Arg198Cys | 2 | 1 | | 1 | |
G13031A | Viangchan Jammu | Val291Met | 3,2 | 1 | 17 | 2 | 13 |
C13184T | Chinese-5 | Leu342Phe | 3 | 0 | 1 | 3 | 1 |
synonomous
| | | | | | | |
C10170T | Vietnam 3 | Ser73Ser | | | | 1 | |
C13714T | nt13714C>T | Tyr437Tyr | | 10 | 14 | 9 | 21 |
In the healthy G6PD deficient group (both Kinh and Stieng) 37.3% (22/59) have no detectable mutation in the G6PD gene and 28.8% (17/59) have a synonymous polymorphism only. 33.9% (20/59) of the healthy G6PD deficient group have detectable non-synonymous G6PD variants. In the haemoglobinuria group, 25.6% (21/82) of patients harbour non-synonymous polymorphisms in the G6PD gene. Within the haemoglobinuria group of those patients with measured G6PD deficiency 39.6% (19/48) harboured non-synonymous G6PD polymorphisms, and 14/48 had a synonymous polymorphism (9/48 of these patients harboured both synonymous and non-synonymous G6PD polymorphisms). In contrast the patients without measured G6PD deficiency harboured significantly fewer non-synonymous G6PD polymorphisms (2/34 (6%), P = 0.001) but a similar number of synonymous polymorphisms (10/34 (29%), P = 1.0).
The most common polymorphism found in this study was the synonymous polymorphism nt13714C>T (C 13714 T; Tyr437Tyr). 42.1% (45/107) of G6PD deficient individuals in the Vietnamese population had this polymorphism and 29.3% (24/82) in the haemoglobinuria group. The second most common polymorphism found in this study was the Viangchan variant; 36% (13/36) of healthy G6PD deficient S'tieng, 9% (2/23) of healthy G6PD deficient Kinh and 22% (18/82) of Kinh with haemoglobinuria (35.4% with G6PD deficiency and 2.9% without).
Comparing the frequencies of G6PD deficiency in healthy Kinh and haemoglobinuric Kinh (table
3) demonstrates that G6PD deficiency is significantly associated with haemoglobinuria in the Kinh [comparison of "probable G6PD deficient" haemoglobinuric patients to healthy controls, OR = 14.9, 95%CI (7.7 to 28.9), P < 0.0001; comparison of "definite G6PD deficient" haemoglobinuric patients to healthy controls, OR = 4.11, 95%CI (2.04 to 8.34), P < 0.0001]. Some other possible reasons for haemoglobinuria other than G6PD deficiency in this patient population are malaria, anti-malarial treatment (quinine), other treatment, other infections (two patients had possible leptospirosis and two with possible sepsis) and thalassemia (one patient)".
Table 3
The frequency of G6PD deficiency and G6PD variants in patients with haemoglobinuria and healthy subjects
deficiency
a
| 48 | 23 | | | 14.9 | 7.74 – 28.9 | < 0.0001 |
definite
b
| 23 | 23 | | | 4.11 | 2.04–8.24 | < 0.0001 |
Viangchan Jammu | | | 17 | 2 | 5.7 | 1.14 – 55.4 | 0.022 |
Chinese-5 | | | 1 | 3 | 0.14 | 0.002 – 1.9 | 0.09 |
Vietnam 1 | | | 1 | 0 | | | |
Gaohe Gaozhou | | | 1 | 0 | | | |
Vietnam 2 | | | 0 | 1 | | | |
The frequency of the most common non-synonymous G6PD mutation (Viangchan) was significantly higher in G6PD deficient Kinh with haemoglobinuria than in G6PD-deficient healthy Kinh (OR = 5.7, 95%CI [1.14 to 55.4], P = 0.022). The Chinese-5 mutation has a low frequency in this population and the frequency was not significantly different between G6PD deficient Kinh with haemoglobinuria and G6PD deficient healthy Kinh (OR = 0.14, 95%CI [0.002 to 1.9], P = 0.09). Two variants, Vietnam1 and Gaohe Gaozhou, were only seen in the G6PD deficient Kinh with haemoglobinuria and not in the G6PD deficient healthy Kinh. In contrast, G6PD Vietnam2 was only seen in the G6PD deficient healthy Kinh group.
Table
4 summarizes the frequency of other factors that have previously been related to the development of haemoglobinuria and more specifically BWF. 58.5% of the patients with haemoglobinuria were G6PD deficient, 25.6% had malaria confirmed by a positive blood smear and 17% received anti-malarial treatment (either quinine or primaquine). The percentage of patients with haemoglobinuria that had non-synonymous mutations resulting in mild to severe G6PD deficiency was 25.6%.
Table 4
The frequency of factors that may contribute to the development of haemoglobinuria in the Vietnamese cohort
G6PD deficiency
a
| 48 (58.5) | 23 (8.65) | 36 (13.95) |
Suspected malaria
b
| 34 (41.5) | | |
Confirmed malaria
c
| 21(25.6) | | |
Received anti-malarials
d
| 14 (17) | | |
Harbour G6PD polymorphism
e
| 34 (41.5) | 14 (0.05) | 23 (0.09) |
Harbour G6PD non-synonomous mutation | 21(25.6) | 6 (0.02) | 14 (0.05) |
Harbour G6PD published mutation | 20 (24.4) | 6 (0.02) | 14 (0.05) |
Discussion
The frequency of G6PD deficiency in Southeast Asia is highly variable [
22]. Within Vietnam the incidence of G6PD deficiency varies between the northern and southern areas and among ethnicities. In southern Vietnam, there is a high prevalence of G6PD deficiency in ethnic Kinh (8. 7%) and S'tieng (14%), who live in areas which are or were until recently highly endemic for malaria. In northern Vietnam, where malaria transmission is lower, G6PD deficiency is virtually absent [
11].
Studies have shown that the distribution of G6PD variants also vary with geographical area and/or ethnic group. Matsuoka
et al has reported that nine variants are present in China, nine in Malaysia, five in Thailand and four in Myanmar [
23]. In Vietnam at least seven variants have been reported [
23]. In this study, eight different polymorphisms were identified of which six resulted in an amino acid substitutions and three were novel mutations. The major G6PD variants reported in south-east Asian countries are G6PD Viangchan (G13031A) and G6PD Mahidol (G11658A). Almost all of the G6PD-deficient cases detected in Laos [
22], Cambodia [
10,
24], Thailand [
25,
26] and Malaysian Malays [
27] are G6PD Viangchan, while G6PD Mahidol is the most common variant in Myanmar [
22,
28,
29]. G6PD Mahidol is also common in Thailand [
25,
30] and Malaysia [
27].
In Vietnam, the G6PD Viangchan is the most common variant. A previous study in Lam Dong province also reported that G6PD Viangchan is the dominant G6PD variant in the Vietnamese population [
23]. It can be hypothesized that the strong historical connection between Vietnam and China has resulted in the presence of common Chinese variants, such as Gaohe Gaozhou, Chinese-5 and Coimbra "Shunde" [
31], in this Vietnamese population. Interestingly, the G6PD Mahidol mutation was not seen in this Vietnamese population. This is different from Thailand and Myanmar [
29], where G6PD Mahidol is common. Notably, in this study, two novel non-synonymous variants (Vietnam1 and Vietnam2) and one novel silent polymorphism (Vietnam3) were identified. Even though the frequencies of these mutations are low, it is possible that a new generation of G6PD mutation is occurring in the Vietnamese population.
Studies in Southeast Asia report that G6PD Viangchan is in linkage disequilibrium with the silent polymorphism nt13714C>T [
22,
26,
27]. In this study, 72.7% (24/33) of the subjects that harbour Viangchan also harbour nt13714C>T. The interpretation of these results could either be that the linkage disequilibrium between Viangchan and nt13714C>T is not absolute in the Vietnamese, or that detection of the nt13714C>T polymorphism by SSCP was not 100% sensitive. Findings to support the later are that the G6PD Viangchan mutation was demonstrated to be completely co-inherited with the silent polymorphism nt13714C>T in a previous study in the Vietnamese population [
23]. Matsuoka
et al [
23] also found six previously published variants (Gaohe, Quing Yuan, Union, Canton, Kaiping and Bao Loc) in the Vietnamese Kinh of Lam Dong province. Of these, only the G6PD Gaohe variant was detected in this cohort of Vietnamese Kinh. In addition, 38% (41/107) of G6PD deficient people in this study had no identifiable mutation in the G6PD gene. Even though three novel G6PD variants were identified in this study, the SSCP method used may not have been a sensitive enough method to detect all mutations that exist in the population. In the future, direct sequencing of the G6PD gene would improve the sensitivity to detect G6PD variants. Alternatively some of these patients may not truly be G6PD deficient. There are two limitations of this study regarding the ascertainment of G6PD deficiency. Firstly, three different tests were used in the three subject groups, which were collected at different points in time, and in two different locations. This is not ideal, however in Vietnam, a rapidly developing nation, resources can vary over time. The use of two screening tests (methaemoglobin reduction test and methylene blue reduction test) may have lead to a reduction in sensitivity. Secondly, the specificity of the G6PD testing of haemoglobinuric patients may have been compromized, as the results depend on the time between haemolysis and the performance of the test. For the majority of haemoglobinuric patients screening for G6PD deficiency took place on hospital admission and at the time of discharge, so mis-classification could have occurred depending on the period between haemolysis and testing. A smaller subset of patients had further testing at follow up (>54 days post hospital admission), a time where G6PD testing would have been accurate. Therefore it could be a combination of the SSCP method lacking sensitivity and sub-optimal G6PD testing that is responsible for the lack of identifiable mutations in the G6PD gene in individuals deemed G6PD deficient in this study.
BWF has been associated with recent or active malaria, and with the ingestion of oxidant drugs such as primaquine. 25.6% of the patients with haemoglobinuria had confirmed malaria. In this Vietnamese Kinh cohort, the association between G6PD deficiency and haemoglobinuria was strong. The association between G6PD deficiency and haemolysis has been shown in at least four clinical syndromes, oxidative stress-induced haemolysis, favism, neonatal jaundice and chronic non-spherocytic haemolytic anaemia [
26].
Of the G6PD variants, only G6PD Viangchan was shown to be associated with haemoglobinuria in the Kinh group. The G6PD Viangchan variant is one of the class 2 G6PD variants known to be associated with chronic haemolytic anaemia (according to Beutler, 1994 [
32]). The frequency of the Chinese-5 variant, as well as other variants that result in a severe to mild enzyme deficiency, is low in the population, so comparisons are statistically unreliable. Currently, the rarity of haemoglobinuric patients from malaria endemic regions of Vietnam leaves one unable to expand these studies in a larger sample set.
The haemoglobinuric patients investigated in this study of G6PD variation were hospitalized between 1993 and 1996. Since the early 2000s it has been rare for the Hospital for Tropical Diseases in HCMC to admit patients with haemoglobinuria associated with malaria. This is may be related to a change in the treatment policy for malaria in Vietnam. In 1990 the first intervention trial investigating the use of artemisinin derivatives for the treatment of malaria in southern Vietnam was published [
33]. Following the success of this and subsequent trials, artesunate replaced quinine in the national recommendations for the treatment of malaria in Viet Nam from 1998 onwards. After this date very little quinine has been used in Viet Nam. It is possibly this change in treatment for malaria from quinine to the artemisinin derivatives that has lead to significantly less drug induced haemolysis in Vietnamese individuals that are G6PD-deficient and infected with malaria.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NTH carried out the molecular genetics study, collected healthy samples, carried out the G6PD test using the G-6-PDH kit, performed the analysis and drafted the manuscript. JC participated in the design of the study. TTHC carried out the micro-methaemoglobin reduction test, recruited the patients and collected clinical data. TTH and ND were involved in recruiting the patients and collecting clinical data. JF conceived of the study, participated in its design and edited the manuscript. SJD participated in the data analysis, drafted and edited the manuscript