Newborn screening for G6PD deficiency in HeFei, FuYang and AnQing, China: Prevalence, cut-off value, variant spectrum

Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive Mendelian genetic disorder characterized by neonatal jaundice and hemolytic anemia, affecting more than 400 million people worldwide. The purpose of this research was to investigate prevalence rates of G6PD deficiency and to evaluate and establish specific cut-off values in early prediction of G6PD deficiency by regions (HeFei, FuYang, AnQing) on different seasons, as well as to investigate the frequencies of G6PD gene mutations among three regions mentioned above. Methods A total of 31,482 neonates (21,402, 7680, and 2340 for HeFei, FuYang, and AnQing cities, respectively) were recruited. Positive subjects were recalled to attend genetic tests for diagnosis. G6PD activity on the Genetic screening processor (GSP analyzer, 2021-0010) was measured following the manufactureržs protocol. The cut-off value was first set to 35 U/dL. The receiver operating characteristics (ROC) curve was employed to assess and compare the efficiency in predicting G6PD deficiency among HeFei, FuYang, and AnQing cities in different seasons.


Introduction
Glucose-6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is an X-linked recessive Mendelian genetic disorder (1), which is characterized by neonatal jaundice and hemolytic anemia (2) when triggered by oxidative drugs, infection and the intake of fava beans (3).Mutations in the G6PD gene can result in reduced activity and stability of the G6PD enzyme, which is the crucial enzyme of the pentose phosphate pathway that produces reduced nicotinamide adenine dinucleotide phosphate (NADPH) to maintain sufficient levels of reduced glutathione (4,5).Red blood cells would lyse when exposed to the factors mentioned above if there was an insufficient supply of NADPH and glutathione, which is the reason for the aforementioned clinical symptoms (6).G6PD deficiency affects more than 400 million people worldwide (5,7,8), especially prevalent in malaria-endemic areas (9), such as Africa, Oceania, Asia, and Mediterranean Europe (9,10).G6PD deficiency is also common in China (11,12).Nevertheless, the prevalence of G6PD deficiency, related to specific geographic regions and associated with ethnicities, is highly variable around China (13).The highest incidence of G6PD deficiency has been reported in southern China (14)(15)(16), for instance, in Guangdong, Yunnan, and Guangxi provinces.However, we have not yet known the precise incidence of G6PD deficiency in Anhui Province.
There is no cure for G6PD deficiency, as is true for many genetic disorders, and most G6PD deficiency individuals are asymptomatic.Hence, performing a G6PD deficiency screening program effectively facilitates early diagnosis and timely intervention.G6PD enzyme activity tests were performed within Newborn Screening programs in most provinces of China (17).According to the largest Youden Index, a specific optimal cut-off value generated by receiver operating characteristic (ROC) curve analysis was often to be used to predict G6PD deficiency and evaluate for diagnostic accuracy based on sensitivity, specificity, and the area under the ROC curve (AUC), having to discriminate all patients with G6PD levels accurately below a preset cut-off value, which is often followed with manufacturer's protocol.It is widely known that enzymatic activity may be affected by seasons and temperatures in vitro and, consequently, possess a different level of G6PD activity in different parts and seasons.Furthermore, through random Xchromosome inactivation, females heterozygous for G6PD could manifest various degrees of G6PD deficiency (18), which was called intermediate G6PD activities between typical normal and deficient G6PD activities (19).Therefore, the cut-off values for newborn screening of G6PD deficiency should optimize based on geographical regions, seasons, and gender.

Conclusion:
We have established the optimal cut-off values of G6PD activity in predicting G6PD deficiency among HeFei, FuYang and AnQing in different seasons.This will aid the early detection of G6PD deficiency among neonates of the regions mentioned above.We have also investigated the prevalence of the regions and discovered frequency mutations.Those results will help G6PD screening promotion and precision diagnosis for Anhui province.
In this study, we first established G6PD activity cut-off values of HeFei, FuYang, and AnQing because of geographical regions and seasons, based on a population cohort of 31482 neonates participating in Newborn Screening between December 2020 and October 2021.We also profiled the characteristics of the G6PD variant spectrum of the areas mentioned above and revealed the prevalence of the regions.

Subjects
A total of 31482 neonates (21402, 7680, and 2340 for HeFei, FuYang, and AnQing, respectively) were recruited through the Newborn Screening program at the newborn screening centre of HeFei Women and Children Medical Care Centre, FuYang Maternal and Child Health Family Planning Service Center, and AnQing Maternal and Child Health Family Planning Service Center between December 2020 and October 2021.Details are available in Table I.Dried blood spots (DBS) were collected from a heel stick within 48 hours after birth.The guardians of all subjects in the study signed informed consent, which was approved by the Medical Ethics Committee of the hospital mentioned above.

Determination of G6PD enzyme activity
G6PD enzyme activity was determined using a Genetic screening processor (GSP analyzer, 2021-0010), Panthera-PuncherTM 9 blood spot punching system, and Nenatal G6PD Kit, which were all purchased from Perkin Elmer (Perkin Elmer, Waltham, Massachusetts, United States), according to the manufacturer's instructions.In brief, G6PD calibrators, G6PD controls, and DBS samples were added separately to 96-well microplates, and the detection was performed using a GSP analyzer.According to the manufacturer's recommendation, the cut-off values of G6PD activity for G6PD deficiency were set to 35 U/dL.Subjects with G6P-D activity less than 35 U/dL were assayed using another DBS again.

Diagnosis test for G6PD deficiency
Subjects whose G6PD activity was less than 35 U/dL after the second test were defined as having positive results and recalled to attend a genetic test.Venous blood was gathered from each positive subject and then sent to the Clinical Laboratory of Zhejiang Biosan Biochemical Technologies Co., Ltd for laboratory tests.A client was diagnosed with G6PD deficiency positive if the genetic test results were positive.

A review of Newborn Screening and diagnosis of glucose-6 phosphate dehydrogenase deficiency
As depicted in Table I, 31482 participants were screened for G6PD deficiency, including 29 (0.09%) neonates finally identified with G6PD deficiency.Among the 29 neonates, 22 were males, and seven were not documented, of whom 22 (0.10%) were from HeFei, 3 (0.04%) were from FuYang, and 4 (0.17%) were from AnQing.Several characteristics differed significantly across age at initial testing, gender, and gestational age between the two groups, but had no significant differences in Birth Weight and Region.The normal group (Newborns without G6PD deficiency) showed smaller values in age at initial testing and Gestational age.Neonates confirmed G6PD deficiency were predominantly males (p = 0.002 < 0.05).

Effects of geographic regions and season on G6PD activity
We compared G6PD activity between geographical regions and seasons.In general, the distribution medians were significantly different (p<0.001)(59.58 (range 39.76-69.10)),(63.90    S1).Due to no participants engaged in screening in certain seasons (FuYang in autumn, AnQing in autumn and winter), we could only display the whole season G6PD activity of HeFei, which was consistent with the tendency of Totality (Figure 1D).

Comparison of ROC curves between geographic regions in different seasons
To analyze the seasonal characteristics of G6PD activity in different geographical parts, the ROC analysis was performed during different seasons in HeFei, FuYang and AnQing (Table IV, V, VI and Figure 2S).IV), significant differences between each subgroup were not observed.And based on ROC curve analysis (Table IV), there was a high predictive value for seasons (G6PD activity) when predicting G6PD deficiency, with the AUC (CI 95%) of 1.000, 0.997, 1.000 (Figure S2A, S2B, S2C), and the optimal cut-off values of 21.80 U/dL (sensitivity, SS: 10.00%; and specificity, SP: 99.98%), 26.55 U/dL (SS: 100.00%;SP: 96.69%), 23.16 U/dL (SS: 100.00%;SP: 99.98%), in spring, summer and winter, respectively.As for FuYang and AnQing, sufficient data being not available for between subgroup analysis, we could only obtain the ROC curve (Figure 2D, 2E) characteristics of summer (for FuYang) (Table V) and winter (for AnQing) (Table VI), and all the AUC (CI 95%) equalled 1.000, with the optimal cut-off values of 6.35 U/dl (sensitivity, SS: 10.00%; and specificity, SP: 100.00%), 7.15 U/dL (SS:100.00%;SP: 100.00%), respectively.However, the p-value of AnQing (subgroup: winter) was 0.0833 > 0.05, and the result of ROC analysis for this category was unreliable.The plot shows the number of G6PD gene mutations in each region.Different G6PD genotypes show significant differences in G6PD activity when boxplots are marked with different letters and nonsignificant differences in G6PD activity when boxplots are marked with the same letters.

Discussion
In this study, we first reported the prevalence of G6PD deficiency, cut-off values of G6PD activity optimized based on regions and seasons, and the G6PD gene variant spectrum for HeFei, FuYang and AnQing city.It would provide reference data values for preventing and treating G6PD deficiency in Anhui province.
Anhui province has a large population of about 60,000,000; every year, the population is estimated at approximately 400,000 births.For G6PD deficiency, no epidemiological data was previously reported.Our study revealed a prevalence of 0.09%, which is roughly consistent with most provinces in North China and lower than most provinces in South China except Hubei province, neighbouring Anhui province (21).HeFei, as the capital of Anhui province, located in the middle of Anhui province, had a higher incidence rate (0.10%) than FuYang, located in the north of Anhui province and population numbers being reasonably consistent with HeFei, possessed the incidence rate of 0.04%.But the incidence rate of HeFei was lower than that of AnQing, located along the Yangtze River, which was thought to be associated with the high incidence of G6PD deficiency (22), and had an incidence rate of 0.17%.These results were approximately consistent with the trend of south-highnorth-low.We also found significant differences in age at initial testing and gestational age between the normal and G6PD deficiency groups, which likely happened by chance.A major drawback of this research is that no deficient sample was identified in the female population by the method used to determine G6PD activity.This is indicative because gene analysis indicated the presence of multiple variants in the female population.
We found significant variation in the median results by comparing the G6PD activity obtained for HeFei, FuYang, and AnQing groups, which decreased in the following order: FuYang > AnQing > HeFei.Whereas, uncomprehensively, the data of FuYang and AnQing which obscured the fact was responsible for the conclusion.As presented in Figure 1D, comparing the differences should depend on the spring data rather than the overall data.In fact, the median of G6PD activity was observed in the following order: HF (65.42 (range 59.79-71.25))> FY (64.10 (range 57.30-71.10))> AnQ (61.1700 (range 53.37-69.076)).The cause of these discrepancies is poorly defined and remains to be further investigated.Moreover, we revealed that the changing trend of the levels of G6PD activity was consistent with that first decreasing and then increasing with the season, followed the order of winter (71.9 range (66.44-77.65))> spring (65.42 range (59.74-71.2375))> autumn (48.89 range (43.48-54.79))> summer (45.65 range (37.91-52.47)).One plausible mechanism is that temperature may affect the activity of G6PD.High temperatures could decrease the activity of G6PD in summer, while low temperatures increase it in winter.
In our research, the results were considered the most suitable cut-off values for FuYang and AnQing because there was insufficient data to establish different cut-off values constructed on seasons.To our knowledge, no previous study reported accurate cutoff values of Anhui province relying on seasonal factors.It was filled in this research, and before this study, the cut-off values were set to 35 U/dl, followed by the manufacturer's instructions, which was significantly higher than the result of our research.Consequently, our results provided a precise approach to facilitate G6PD deficiency screening,

Figure 1
Figure 1 Effects of geographic regions and season on G6PD activity.** represents p < 0.01 Abbreviations: HF, HeFei city, FY, FuYang city, AnQ for AnQing.1A Comparison of enzymatic activity of HF, FY, and AnQ.1B Comparison of spring, summer, autumn, and winter enzymatic activity.1C Median of G6PD activity changes from Seasons.1D G6PD activity distributions by regions (HeFei, FuYang, AnQing) in different seasons.

90
Figure S1 ROC curves of G6PD activity in the prediction of G6PD deficiency of Hefei, Fuyang, and Anqing city.

Figure 3
Figure 3 G6PD activity of G6PD gene mutations and its distribution of mutation frequencies among the geographic regions.5A Comparison of G6PD enzymatic activity of Normal and mutated G6PD enzyme.5B Comparison of G6PD enzymatic activity between different mutations in Male neonates and Female nenates.5CHeatmap of mutations in G6PD gene in individuals from different geographic regions.5DThe plot shows the number of G6PD gene mutations in each region.Different G6PD genotypes show significant differences in G6PD activity when boxplots are marked with different letters and nonsignificant differences in G6PD activity when boxplots are marked with the same letters.

Table I
Characteristics of newborns screened by G6PD newborn screening.
range 57.00-71.00))and(63.14(range 53.29- 69.05)) between HeFei, FuYang, and AnQing, respectively, as presented in figure 1A.As shown in Figures 1B significant seasonal differences existed in G6PD activity.G6PD activity presented declining trends during spring and summer and increasing trends from summer to winter (1C).In contrast, G6PD activity was significantly different between 4 categories: median values for spring, summer, autumn, and winter were 64.3000 (range 57.80-70.91),45.83 (range 38.14-52.74),48.89 (range 43.37-54.74),71.8900 (range 66.43-77.65),respectively (Table Normal: Neonates who were not diagnosed with G6PD deficiency by Newborn Screening.Normal: Nenoates, diagnosed with G6PD deficiency by Newborn Screening.H: Kruskal-Wallis Test; a: indicating that it did not fit with a Gaussian distribution after being assessed by the KS test.(

Table II
Comparison of ROC curves of G6PD activity in predicting G6PD deficiency of HeFei, FuYang, and AnQing.

Table IV
Comparison of ROC curves of G6PD activity in predicting G6PD deficiency between different seasons of HeFei.

Table V
Comparison of ROC curves of G6PD activity in predicting G6PD deficiency between seasons of FuYang.Table VIComparison of ROC curves of G6PD activity in predicting G6PD deficiency between different seasons of AnQing.