Genotypic and phenotypic characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Guangzhou, China

Determination of G6PD enzyme activity

The G6PD enzyme activity distribution in females and males is depicted in the histogram (Fig. 3). The distribution of G6PD enzyme activity in males showed a bimodal and abnormal distribution. The mean ± standard deviation (SD) and median G6PD activities were 2294.98 ± 771.88 U/L and 2379.00 U/L in males. The histogram of the distribution of enzyme activity in females showed a unimodal and abnormal distribution, and the mean ± SD and median G6PD activities were 2328.78 ± 693.88 U/L and 2354.00 U/L. The difference in enzyme activity between males and females was statistically significant (P < 0.05).

Fig. 3figure 3

Histogram of G6PD enzyme activity distribution in females and males. The line in the figure represents 100% of the AMM (2388 U/L)

The AMM is accomplished by: 1. Exclusion of all males with G6PD activity equal to or less than 10% of the male median. 2. Determination of a new median G6PD activity [13]. We calculated the AMM value to define 100% G6PD enzyme activity for this population. In this study, the AMM value was 2388 U/L. Therefore, according to the WHO classification, WHO Class II enzyme activity was < 238.8 U/L (G6PD activity < 10%), and WHO Class III enzyme activity was 238.8–1432.4 U/L (G6PD activity of 10%-60%). We also calculated the 30% AMM value (716.4 U/L), which is as an acceptable level of G6PD activity for primaquine administration [13, 14].

G6PD genotyping

The results of MALDI-TOF MS showed 12 G6PD mutations, including c.1376G>T, c.1388G>A, c.95A>G, c.871G>A, c.392G>T, c.1024C>T, c.1360C>T, c.487G>A, c.1004C>A, c.835T>A, c.383G>T, and c.202G>A. Figure 4 lists the number of male hemizygotes and female heterozygotes detected for the six more common G6PD mutations. The most common mutation was G6PD Kaiping (1388G>A), with 278 male hemizygotes and 261 female heterozygotes detected, followed by G6PD Canton (1376G>T), with 245 male hemizygotes and 234 female heterozygotes detected. In addition, some mutations that are less common in China were detected, such as G6PD Union (1360C>T), G6PD Mahidol (487G>A), G6PD Fushan (1004C>A), G6PD Chinese-1 (835T>A), G6PD Salerno (383G>T) and Asahi (202G>A) (Table 1).

Fig. 4figure 4

Histogram of males hemizygous and females heterozygous for the six common mutations detected

Table 1 The number of mutations with fewer carriers and enzyme activity

Two male heterozygotes (c.1388G>A, c.1376G>T) and 2 male compound heterozygotes (c.1388G>A/c.95A>G, c.1376G>T/c.95A>G) were detected. A total of 17 female homozygotes and 45 female compound heterozygotes were detected (Table 2). Among female homozygotes, the c.1388G>A mutation was the most common, found in 8 females, followed by the c.1376G>T mutation, found in 6 females, and the c.95A>G mutation, found in 3 females. The most common compound mutation detected was c.1388G>A/c.1376G>T, found in a total of 20 individuals (Table 2).

Table 2 A total of 17 female homozygotes, 45 female compound heterozygotes, 2 male heterozygotes, and 2 male compound heterozygotes were identified

We calculated the genotype frequencies for the different G6PD mutations. The frequency of female heterozygotes was calculated based on the male allele frequency, and this result was consistent with the detected female heterozygous frequency (Table 3).

Table 3 Allele frequencies for different G6PD genotypesSequencing results

The results of DNA sequencing showed 6 missense mutations in this study population, including c.187G>A, c.703C>T, c.551C>T, c.517T>C, c.1438A>T, and c.946G>A (Table 4). c.551C>T was first reported in Madhya Pradesh, India, and was named G6PD Dindori after the region [15], but it has not previously been reported in China. c.703C>T was reported in Guangxi, China, and was named G6PD Nanning after the region [16]. c.187G>A was found in Taiwan, China, and was named G6PD Pingtung after the region [17]. c.517T>C, named G6PD Nankang, has been previously reported in China and Malaysia [18, 19]. c.1438A>T and c.946G>A are novel mutations found in this study and have not been reported previously.

Table 4 Missense mutations obtained from DNA sequencing resultsCorrelation analysis of G6PD mutations and G6PD enzyme activity

Six prevalent G6PD mutations were selected for correlation analysis with G6PD activity. Among males hemizygous for the c.1376G>T, c.1388G>A, and c.95A>G mutations, nearly half of the patients had enzyme activity values that were < 10% of the AMM. In males hemizygous for the c.871G>A, c.392G>T, and c.1024C>T mutations, enzyme activity values were mostly between 10 and 60% of the AMM. Nearly all males with G6PD mutations had enzyme activity values less than 30% of the AMM (an enzyme activity value that is 30% of the AMM is an acceptable level of G6PD activity for primaquine administration) [13, 14]. On the other hand, most female heterozygotes had enzyme activity values that were > 10% of the AMM, and their enzyme activity was more widely distributed. This result indicates that the enzyme activity value of male hemizygotes is lower, which means that the clinical manifestations caused by G6PD mutations may be more severe (Fig. 5).

Fig. 5figure 5

Box plot of the enzyme activity of females heterozygous for each mutation and males hemizygous for each mutation. (The black four reference lines in the panel for male hemizygotes indicate 10%, 30%, 60% and 100% of the AMM (10% AMM, 30% AMM, 60% AMM and 100% AMM, respectively). The two red lines represent the reference lines for class A and class B of the new WHO classification. The four reference lines in the panel for female heterozygotes indicate 10% AMM, 30% AMM, 60% AMM and 100% AMM)

According to the new WHO classification of G6PD variants in 2022, it is recommended that the percent activity value should be calculated from the genotypically normal male median (NMM) value, not from the AMM value, considering the overlap of most variants across the 10% threshold [5]. We calculated that the median number of male individuals with normal genotypes was 2408 U/L. The WHO Class A median of G6PD activity was < 481.6 U/L (G6PD activity < 20%), and the WHO Class B median of G6PD activity was < 1083.6 U/L (G6PD activity < 45%). According to the new classification of variants for hemizygous males [5], the median values of the male hemizygous population for the six common prevalent mutations were all below 20%NMM (Fig. 5).

Comparing the enzyme activities between participants carrying the different missense mutations, we found significant differences in the enzyme activities (Table 5). The findings suggested that there were differences in enzyme activities among the different mutations.

Table 5 The 6 missense mutations identified in this study

We divided enzyme activities into different ranges and compared the mutation detection rates between each group. Table 6 shows that as enzyme activity increased, the detection rate of mutations decreased in both females and males, indicating that the probability of carrying mutations decreased with increasing G6PD enzyme activity. With increased enzyme activity, the probability of carrying mutations was significantly reduced in males and slightly decreased in females.

Table 6 The connection between G6PD genotyping assays and G6PD phenotyping assays

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