A Profile of Glucose-6-Phosphate Dehydrogenase Variants and Deficiency of Multicultural Families in Korea

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Korean J Parasito. 2021;59(5):447-455

Publication date (electronic) : 2021 October 31

doi : https://doi.org/10.3347/kjp.2021.59.5.447

Young Yil Bahk¹, Seong Kyu Ahn², Jinyoung Lee³, Jae Hyoung Im⁴, Joon-Sup Yeom⁵, Sookkyung Park⁶, Jeongran Kwon⁶, Hyesu Kan⁶, Miyoung Kim⁶, Woori Jang^7,, Tong-Soo Kim^3,8,

¹Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chungju 27478, Korea

²Infectious Diseases Investigation Division, Jeonnam Institute of Public Health and Environment, Muan 58568, Korea

³Department of Parasitology and Tropical Medicine & Global Resource Bank of Parasitic Protozoa Pathogens, Inha University School of Medicine, Incheon 22212, Korea

⁴Division of Infection Diseases, Department of Internal Medicine, Inha University School of Medicine, Incheon 22212, Korea

⁵Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Korea

⁶Bureau of Infectious Disease Policy, Korea Disease Control and Prevention Agency, Osong 28159, Korea

⁷Department of Laboratory Medicine, Inha University School of Medicine, Incheon 22212, Korea

⁸Convergence Research Center for Insect Vectors, Incheon National University, Incheon 22012, Korea

^*Corresponding authors (jangwr@inha.ac.kr; tongsookim@inha.ac.kr)

Received 2021 July 20; Revised 2021 August 11; Accepted 2021 August 11.

Abstract

INTRODUCTION

Glucose-6-phosphate dehydrogenase (G6PD) is an X-chromosome (Xq28)-linked housekeeping cytoplasmic enzyme. It has a redox function as the first enzyme of the pentose-phosphate pathway involving in the generation of NADPH, an electron donor required for many biosynthetic processes or to counter the damaging effects of oxygen radicals [1,2]. It is one of the most polymorphic proteins, in which at least 140 mutations have been identified [3,4]. G6PD deficiency produces various clinical presentations. It is the most common enzymopathy of humans over 400 million individuals throughout the world, showing as high frequency as 30% in tropical Africa, parts of the Middle East, and Southeast Asia. The G6PD deficient individuals are usually asymptomatic but acute hemolysis may occur with oxidative stress induced by ingestion of drugs, certain type of food, exposing to certain chemical substances or when there is accompanying infection or hypoxia [5]. Numerous lines of evidence indicate that the geographic correlation of its distribution with the historical endemicity of malaria and suggest that the defect has risen in frequency through natural selection by malaria [6–8]. Recently, it was known that studies carried on the Korean population showed no cases representing above severe G6PD deficiency [9–11]. The higher frequencies of the defect were reported from Africa, Asia, the Mediterranean region, and the Middle East, owing to recent migrations and perpetual communications. The disorder was also reported from North and South America and even from the European countries [12].

Although there has been a mixed pattern of repeated increases and decreases, the vivax malaria incidence in Korea has generally shown a plateau (around 1 incidence rate per 100,000) since its re-emerging in 1993 and the highest patient outbreak in 2000 (4,142 confirmed and indigenous cases, 8.98 incidence rate per 100,000) [13]. Despite such a declined and steady state, there remain tasks to be achieved for vivax malaria elimination from Korea.

In general, it is well established that the spatial extent of vivax malaria overlaps widely with that of G6PD deficiency. The antimalarial licensed for the radical cure and relapse prevention for vivax malaria, is only primaquine. It, unfortunately, can trigger severe hemolytic anemia, particularly in G6PD deficient individuals [14]. The World Health Organization (WHO) recommends primaquine at a dose of 0.25–0.5 mg/kg of body weight for 14 days in all vivax malaria patients with normal G6PD activity [15]. G6PD testing is not generally accessible in most malaria-endemic regions. It is, therefore, lower dosages of primaquine are recommended to reduce the risk for the suspected G6PD deficient patients [16].

As previously documented, due to the low prevalence in Korea, G6PD deficiency testing has not been executed. However, G6PD deficient patients have been continually reported since 1984. G6PD deficiency-related hemolytic anemia patients in Korea Army were confirmed after primaquine administration without G6PD deficiency testing [11,17]. In addition, recently, multicultural families have been on the rise due to the increase in immigrants from Southeast Asian countries, where frequency of G6PD deficiency is as high as 30%. In Korea, proportion of the multicultural families is 4.3%, similar to 5% in the countries of the Organization for Economic Cooperation and Development [18]. Moreover, the multicultural families in Korea mostly composed of maternal immigrants. It is expected that the G6PD deficiency rate of children of these families increase.

This study was aimed to figure out the G6PD deficiency profile of the adolescents in multicultural families and to establish a basis for the G6PD screening test before primaquine administration, and to survey on recognition of the multicultural family center for the G6PD deficients.

MATERIALS AND METHODS

RESULTS

Demographic characteristics of study population

A total of 733 multicultural family members (316 foreign immigrant women and their 417 adolescents in multicultural families) were included in the analysis (Table 1). Of the adolescents, 53% were male and 8.8±4.1 years (9.0±4.1 years for female). The materfamilias (foreign immigrant women) were 34.9±8.0 years (Table 2). The major country of origin of the adolescent’s mothers (materfamilias population) in our study population were of Vietnam (252 individuals, 60.4% [252/417]), followed by Philippines (77, 18.5%), Cambodia (58, 13.9%), and Thailand (13, 3.1%) (Table 3). For foreign immigrant women (materfamilias population), 58.9% (186/316) were from Vietnam, followed by Philippines (20.9%, 66/316), Cambodia (13.6%, 43/316), and Thailand (1.9%, 6/316) (Table 4). The other countries were Laos (3), Kazakhstan (1), Uzbekistan (3), Indonesia (3), Russia (1), and China (4).

Table 1

Participants in this study including materfamilias (marriage immigrant women) population and adolescent population

Table 2

Demographic characteristics of participants surveyed

Table 3

Distribution of the adolescent population in the multicultural families in this study

Table 4

Distribution of the foreign immigrant woman population in the multicultural families in this study

For the blood samples, hemoglobin (Hb) level was measured whether the donors were anemic or not. Hb level of the adolescents was 13.2±1.1 g/dl and the materfamilias 13.2± 1.2 g/dl. Any child in our study did not experience severe anemia at our interview (Table 2).

Prevalence of G6PD phenotypic deficiency

The G6PD activity distribution of all 733 samples are shown in Fig. 1. According to the WHO’s G6PD testing guidelines, value less than 10%, 10 to <30%, 30 to >60% of the median are defined as severe, moderate, and mild deficient (normal range: 7.9–16.3 U/g Hb). Prevalence of G6PD deficiency is presented in Table 2. There were 17 participants showing extreme G6PD activity (>16.3 U/g Hb) having 19.6±4.5 U/g Hb (20.2±3.7 U/g Hb in 11 materfamilias, 19.9±2.3 U/g Hb in 6 adolescents. 21.6±0.5 U/g Hb in 2 male adolescents, and 19.0±2.4 U/g Hb in 4 female adolescents). The 21 (5.0% of 417) adolescents exhibited G6PD enzyme activity deficiency: 11 severe deficients, 5 moderate, and 5 mild ones. The enzyme activity in the deficient adolescents were 0.5–22.5 U/g Hb. Seventeen male adolescents (7.8%) showed deficient phenotype and 4 females (2.0%) exhibited mild G6PD deficiency. In the materfamilias population, the enzyme activity was 11.5±2.7 U/g Hb (Table 2). Ten participants of materfamilias population exhibited G6PD deficiency phenotype (3.2%); 1 severe (0.3%), 4 moderate (1.3%), and 5 mild (1.6%). Their mean value was 4.3±1.8 U/g Hb (ranged from <0.5 to 6.2).

Fig. 1

Distribution of G6PD enzyme activity and genetic variants in the participants in this study. (A) G6PD enzyme activity values for all marriage immigrant women tested in this study; blue bars: participants with normal range G6PD enzyme activity values, red bars: deficient individuals, and green bar: deficient individuals in enzyme activity, but not genotype. (B) G6PD enzyme activity values for all adolescent population tested. (C) G6PD enzyme activity values for male adolescent population tested; blue bars: individuals with normal enzyme activity values, red bars: deficient individuals in enzyme activity and genotype, and green bars: deficient individuals in enzyme activity, but not genotype. (D) G6PD enzyme activity values for female adolescent population tested; blue bars: individuals with normal enzyme activity values, red bars: deficient individuals in enzyme activity and genotype, and purple bars: deficient individuals in genotype, but not enzyme activity.

G6PD genetic variant

Of the 733 blood samples, 8 genetic variants in G6PD gene were detected: Vanua Lava (383 T→C), Mahidol (487 G→A), Mediterranean (563 C→T), Coimbra (592 C→T), Viangchan (871 G→A), Union (1,360 C→T), Canton (1,376 G→T), and Kaiping (1,388 G→A). Upon genotyping of the male adolescents, we observed 17 hemizygous mutant individuals (7.8%) including 5 unknown mutation cases, which were not shown mutant genotype by PCR test with Asian type kit, but were with G6PD deficiency (Table 5). The most common polymorphism in the adolescents was Viangchan variant (47.1%), followed by other mutated allele variant(s) (29.4%), Canton (11.8%), Vanua Lava (5.9%) and Kaiping variant (5.9%) (Fig. 1B). The previously unknown mutated allele variants were with measured G6PD deficiency, which were ranged from 3.2 to 6.4 U/g Hb (mean±SD: 4.2±1.2 U/g Hb), whereas G6PD enzyme activities of hemizygous males were ranged from <0.5 to 1.7 U/g Hb (mean±SD: 0.6±0.4 U/g Hb). Upon genotyping the female adolescents, 7 females found carrying the G6PD variants. The polymorphisms were Viangchan (57.1%) and Kaiping variants (42.9%). Of the 7 participants carrying the mutated allele, 3 female participants showed the enzyme activity in normal range (10.5–11.7 U/g Hb), but their genetic tests determined them to be deficient (Table 5). The materfamilias group were determined to 4.7% heterozygous females without G6PD deficiency, 2.2% single mutant females, 0.3% compound heterozygous mutant females, and 0.6% other unknown mutated allele (s) (Table 5). In the heterozygous mutants, the most common polymorphism was Viangchan variant (52%), followed by Kaiping (16%), Union (12%), Vanua Lava (8%) and Canton variants (8%) (Fig. 1D). Based on the PCR genotyping, there was no homozygous mutant in immigrant women participant. One participant of this population carried double variants of Viangchan and Canton (the enzyme activity: <0.5 U/g Hb) showing the compound heterozygosity. Of the 25 foreign immigrant women (the materfamilias population) having G6PD polymorphism, 15 participants were not G6PD deficiency, which ranged 8–12.1 U/g Hb (9.7±1.1 U/g Hb). However, mean G6PD enzyme activity among heterozygous individuals without G6PD deficiency was approximately 15.7% and 17.8% lower than total mean value and mean value of normal individuals, respectively (9.7±1.1 U/g Hb vs. 11.5±2.7 U/g Hb vs. 11.8±2.5 U/g Hb) (Tables 2, 5). Heterozygous females were predicted to have 2.1 U/g Hb lower mean G6PD activity than their wild-type counterparts. The distribution of G6PD genotypes in this population was not associated with their ethnicity. That is, the genotype by country was not significant.

Table 5

Correlation of phenotype with genotype by gender in male adolescent population and materfamilias population

DISCUSSION

Vivax malaria is still an important public health problem in Korea, particularly near the DMZ in the northern part of Gyeonggi-do, including Gimpo and Paju, and Gangwon-do [13]. In fact, vivax malaria elimination in Korea is generally expected to be successful. However, there are a number of points for consideration against malaria elimination. In this globalized era, international intercourses are frequent due to mutual exchanges and travel between many countries. Furthermore, diversification of genetic sources due to each regional genetic characteristic can cause problems in many aspects. There is substantial room for improvement in radical cure policies and practices. Most of the current guidelines for the radical treatment of vivax malaria recommend the use of primaquine (0.25 mg/kg of body weight) for 2 weeks overlapping with the radical treatment with blood schizonticide agents, such as chloroquine (5 mg/kg, 3 times with 12 hr interval after first dose (10 mg/kg) or 5 mg/kg once with 24 hr interval after the first and second doses (10 mg/kg) [20]. However, the downside of guidelines recommended by the Korea Disease Control and Prevention Agency is that primaquine, which is the only available antimalaria drug that kills dormant liver stages of P. vivax and prevents its relapse, can be toxic to individuals with G6PD deficiency. Nowadays, no treatment guidelines in Korea have been issued that account for the G6PD deficiency. Several reports presented that no G6PD deficiency was observed using phenotypic- or genotypic-based tests in individuals and young soldiers residing in vivax malaria endemic regions in Korea [10,11].

The population of multicultural families consisting of a marriage immigrant or foreigner with Korean citizenship, have been rapidly growing in Korea since 2000. While international migration has become a global phenomenon over recent decades, Korea has experienced this phenomenon relatively recently. Among the registered foreign population in 2018, 6.7% (159,206/2,367,607) were marriage migrants, which were the second largest group, following migrant workers [21]. 83.2% (132,391/159,206) of the international marriages were between a Korean groom and foreign bride. Consequently, the number of adolescents in multicultural families was 122,212 in 2018 in Korea and tends to continuously increase annually [22]. Adolescents in multicultural families accounted for 2.2% of the total Korean adolescent population in 2018, which is over 3 times higher than that in 2012 (0.7%) [22]. In this study, we found overall 7.8% of participants with G6PD deficiency in male adolescent participants. In the case of the marriage immigrant women (the materfamilias population), 3.2% of participants showed G6PD deficiency. The above results suggest that our country should also seriously consider establishing guidelines for prescriptions on primaquine currently in use to treat malaria and to prevent the significant toxic effects accompanying this drug usage.

This study was designed to identify the prevalence of G6PD deficiency in multicultural families in Korea by maternal immigration and strengthen patient care as a strategy for implementing a 5-year action plan to eliminate malaria again. Through this, the government sought to secure evidence for the rapid diagnosis of anemia for the safety of patients. The G6PD deficiency is high in Southeast Asia, the Middle East, the Mediterranean coast, the South Pacific islands and Africa, but the global population migration, inter-country exchanges, and marriage are increasing worldwide. This raises concerns over the development of hemolytic anemia caused by primaquine drugs. In this study, a total of 733 blood samples were collected in 7 counties (Goseong, Yangyang, Haenam, Hampyeong, Hwasun, Boseong, and Yeoncheon) and 2 cities (Donghae and Taebaek) of Korea. On blood samples, a G6PD enzyme activity test using G6PD quantitative kinetic UV assay on whole blood kit was performed. Based on classification by the WHO, 12 severe (<10% of the median enzyme activity) phenotypic participants, 9 moderate (10% to <30%) ones, and 10 mild (30% to <60%) ones were identified and 4.2% of the participants in this study were with G6PD deficiency. Simultaneously with blood collection, hemoglobin levels were measured to monitor whether specimen were anemic. No subject from the regions where were collected in this study had a medical history of vivax malaria infection. The current surveillance has been conducted on malaria non-endemic areas, and it is thought that future investigations into G6PD deficiency in malaria endemic areas, especially the marriage immigrant women (the materfamilias population) and their next adolescent generation including the registered foreign populations, will be needed continuously and with a wide range of targets.

In conclusion, there are at least 6 G6PD variants (Viangchan, Canton, Vanua Lava, Union, Kaiping, unknown mutation (s)) in the population of multicultural families consisting the materfamilias and their adolescents. Possible reasons for the detected other unknown mutated allele variants are that another mutant that were impossible to be identified by the kit used in this experiment can be expected to exist or to be other type mutant. So far, it is acceptable in Korea that G6PD deficiency prevalence is low enough that the risk of antimalaria drug, primaquine-originated G6PD deficiency-mediated hemolytic anemia is relatively negligible. However, in this study, the population of multicultural families, particularly in adolescent population, have carried G6PD deficiency. In addition, although the G6PD deficient individuals with activity <30% of the median value is not identified, large number of military personnel with C1311T/IVS and T93C intron mutations, which are recognized to be associated with G6PD deficiency, were identified [11]. There are also the recent case reports of G6PD deficiency-mediated hemolytic anemia in the malaria endemic area [23–25]. Therefore, when it is considered that the genetic variants in G6PD associated with enzyme activity deficiency exists in Korean population, that primaquine chemoprophylaxis is carried out routinely in military personnel in the vivax malaria endemic area, and that there are somehow case reports for G6PD deficiency in the endemic area, further regular surveillance of G6PD deficiency and radical treatment guidelines are needed to ensure the safe administration of the unique anti-vivax malaria drug, primaquine.

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