Molecular Epidemiological Investigation99G6PD Deficiency Among The Zhongshan Residents And The Relationship Between G6PD Deficiency And Hand-Foot-Mouth Disease Caused By Enterovirus71

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in the world which is an X-linked and incompletely dominant genetic disease,with an estimated400million people affected worldwide.The global prevalence of G6PD deficiency is geographically correlated with areas inhabited by populations historically exposed to endemic malaria,including Africa, Mediterranean Europe, South-East Asia,Pacific Islands and South America.In China,the epidemiological characteristics of G6PD deficiency has been mastered in the1980s.G6PD deficiency are mostly distributed to the south of the Yangtze River and the distributing characteristics is that north is higher than south and males morbidity are higher than females.To date,the number of reported G6PD mutations is of186.In China,28different mutations associated with reduced G6PD enzyme activity have been identified in the human G6PD gene.Among these,1376G>T、1388G>Aand95A>G are the most common mutation in china and can be detected in Chinses only. Zhongshan is the area of high incidence of G6PD deficiency,but study on G6PD mutation spectrum still has not been reported as yet.G6PD, existing extensively in cells of human, is an enzyme that catalyses the first reaction in the pentose phosphate pathway, providing reducing power to all cells in the form of Nitrate Reductase (NADPH).With the catalyst of glutathione reductase,NADPH could preserve the reduced form of glutathione and enable cells to counterbalance oxidative stress that can be triggered by several oxidant agents.In the cells with G6PD deficiency,decreased producion of NADPH would lead to decrease of glutathione (GSH).Depleted GSH and continuing accumulation of reactive oxygen species could induce oxidative stress.Viral infection could induce oxidative stress and leads to structutal damage and functional disorder in host cell.In recent years more and more literatures have identified that due to the limited antioxidant capacity G6PD deficient cells are more susceptible to viral infection and oxidative stress is more obvious than wild-type cells.These effects lead to virulence increasing and obvious pathological changes in host cell.Enterovirus71(EV71) is one of the most common causes of Hand, Foot and Mouth Disease (HFMD). EV71is a new nervous tissue tropism virus and strongly associated with the severe nervous system complications of HFM disease.Basing on the former researchs,we speculate that G6PD deficiency is closely associated with HFM Disease caused by EV71and oxidative stress may participate in the pathogenesis.1. Molecular epidemiological investigation on G6PD deficiency among the Zhongshan residentsObjective and significanceG6PD deficiency is one of the five diseases according to the intervention project on birth defects in Guangdong province.In Zhongshan City,we have launched neonatal screening of G6PD deficiency since1998.Zhongshan is the area of high incidence of G6PD deficiency,but study on gene frequency of G6PD deficiency and G6PD mutation spectrum still has not been reported as yet.Our study will fill the gap and reveal its epidemiological characters in Zhongshan City.Design and MethodsAccording to large-scale epidemiological survey of G6PD deficiency by Du in 0.054,δ=0.01,α=0.05).Basing on the percentage of population divided by24administration districts in Zhongshan City, we selected4105residents (male:2119;female:1986) randomly from volunteers taking pre-marital check from june2009to February2010.All the subjects were no anemia.We have established a test procedure from phenotype to genotype in our reseach.G6PD activity was tested by Victor2-1420automatic biochemistry analyzers, it would be defined as G6PD deficiency if the activity was below1300U/L. Genomic DNA was extracted by standard methods from all phenotype positive samples subjected for molecular analysis by denaturing high performance liquid chromatography (DHPLC) assay. Finally, direct DNA sequencing was performed on all the negative samples as determined by DHPLC.Results and ConclusionsTotal numbers of G6PD deficient were195, with a morbidity of4.75%for this enzyme abnormality in Zhongshan residents.134male samples (6.32%) were determine as G6PD deficient,61female samples (3.07%) were determine as G6PD deficient. Morbidity rate of male was higher than female,the difference had statistical significance (X2=23.97,P<0.001). Following the Law of Hardy-Weinbeng,the gene frequency of this enzyme abnormality in Zhongshan City is6.32%.Genetic Mutations could be detected in173samples from the195positive samples by DHPLC,18samples by DNA sequencing.In the rest4samples, mutations could not be found.We observed a total of10different genotypes:1388G>A,1376G>T,95A>Q871G>A,1311C>T,1024C>T,517C>T,592C>T,392G>T and196T>A.1388G>A,1376G>T and95A>G are the are the most common mutation in this study taking76.92%.It was the first time to find517C>T and196T>A in Zhongshan region and also in Guangdong region which has already been described in Guangxi and Hainan. Statistic analysis showed that there were on significant differences in activity of G6PD between1388G>A,1376G>T and95A>G,but there were significant differences between heterozygote and hemizygote,the levels of G6PD in hemizygote is lower than heterozygote.In our epidemiological investigations,we reported the morbidity, gene frequency, genotypes of mutation and the relationship between phenotype and genotype.The data obtained in our research would provide scientific references for the designs made to prevent and control G6PD deficinecy in Zhongshan area,which have important implications for prenatal and postnatal care and improving the quality of the population.2. Relationship between G6PD Deficiency and Hand-Foot-Mouth Disease Caused by Enterovirus71Objective and significanceEV71is one of the most common causes of Hand-Foot-and-Mouth disease and strongly associated with the severe cases.Viral infection can induce oxidative stress and aggravate the state of disease.In the cells with G6PD deficiency,decreased producion of NADPH would lead to decrease of GSH.Recent research have found that G6PD deficient cells are more susceptible to viral infection.We speculate that G6PD deficiency is closely associated with HFM Disease caused by EV71.But so far there is few research on the relationship between G6PD deficiency and diseases caused by EV71.The motivation of this study is to explore the possible relationship between G6PD deficiency and susceptibility towards EV71infection as well as the severity of the infection in term of oxidative stress.Design and MethodsGiven the limitations of existing G6PD activity detecting methods and following the Law of Hardy-Weinbeng, we selected male children as research subjects. From June2011to October2012,220male children diagnosed with EV71infection were chosen.Based on the severity of the disease,145children were divided into Mild group,75children were divided into Severe group.Taking the HFM disease guidance issued by China’s Ministry on21April2010as diagostic standards,all research subjects must meet three inclusion criterias:①meet the criteria for HFM disease;②Diagnosed EV71infection by investigations of stools;③Disease course was less than5days.The child would not be chosen if he have any exclusion condition:①have appeared haemolysis or transfusion within3months;②guardian refuse to accept detecions of G6PD, Glutathione (GSH) and Malondialdehyde (MDA) carried on their child.132healthy male children were used as the control group during the same period.Venous blood from all all research subjects collected within24hours after admitting and in convalescence was used for the detection of G6PD,GSH and MD A. According to the G6PD activity,mild group and severe group were divided into mild (G6PD normal) group, mild (G6PD deficiency) group,severe (G6PD normal) group and severe (G6PD deficiency) group respectively.All the patients have received treatment accroding to the guidance.In severe group,the duration of fever,mental abnormality,limb trembling and pathological reflex in G6PD deficiency children were were recorded in detail.Results1. The number of children with G6PD Deficiency in control group,mild group and severe group were4,15and17respectively.The proportion of G6PD deficiency in control group,mild group and severe group were3.0%,10.34%and22.67%respectively. The proportion of G6PD deficiency in mild group and severe group is significantly higher than control group (P<0.05).2.1n severe group, the duration of fever in G6PD deficiency children were6.35±2.15days, mental abnormality were2.65±0.93days, limb trembling were3.09±1.03days, pathological reflex were1.18±0.53days,hospital days were10.59±2.32days and the levels of G6PD were524.27±450.61U/L. the duration of fever in G6PD activity normal children were5.37±1.36days, mental abnormality were1.81±1.62days, limb trembling were2.41±1.19days, pathological reflex were0.66±0.86days,hospital days were8.74±3.04days and the levels of G6PD were2842.67±665.64U/L. The duration of fever,mental abnormality,limb trembling,pathological reflex and hospital days in G6PD deficiency children were significantly longer than those in G6PD activity normal children (P<0.05). The levels of G6PD in G6PD deficiency children were significantly lower than those in G6PD activity normal children (P<0.001)3.In mild group,the. levels of GSH during acute period were.2.70±0.98mg/gHb,MDA were3.80±1.10umol/L and G6PD were2963.06±988.36U/L; In severe group,the levels of GSH during acute period were2.28±1.51mg/gHb,MDA were4.19±1.08umol/L and G6PD were2317.16±1157.63U/L;In control group, the levels of GSH were3.63±0.65mg/gHb,MDA were3.41±0.52umol/L and G6PD were3260.26±701.83U/L.During acute period,the levels of GSH and G6PD in mild group and severe group were both significantly lower than that of control group, the levels of MDA in mild group and severe group were both significantly higher than that of control group(P<0.05).Meanwhile the levels of GSH and G6PD in severe group were both significantly lower than that of mild group, the levels of MDA in severe group were significantly higher than that of mild group (P<0.05).In mild group,the levels of GSH during convalescence were2.63±0.83mg/gHb, MDA were3.76±1.09umol/L, G6PD were2984.11±972.82U/L.In severe group the levels of GSH during convalescence were2.41±0.91mg/gHb, MDA were3.99±0.91umol/L, G6PD were2324.77±1148.04U/L.During convalescence, the levels of GSH and G6PD in mild group and severe group were both significantly lower than that of control group (P<0.05), the levels of MDA in mild group and severe group were both significantly higher than that of control group (P<0.05).Meanwhile,there were no significant difference in the the levels of GSH and MDA between mild group and severe group (P>0.05), the levels of G6PD in severe group were significantly lower than that of mild group (P<0.05)4.The levels of GSH in mild (G6PD normal) group were2.92±0.76mg/gHb, MDA were2.92±0.76umol/L and G6PD were3242.85±549.26U/L during acute period; The levels of GSH in mild (G6PD normal) group were2.81±0.48mg/gHb, MDA were3.71±1.57umol/L and G6PD were3265.18±574.45U/L during convalescence; The levels of GSH in mild (G6PD deficiency) group were0.77±0.12mg/gHb, MDA were5.37±0.41umol/L and G6PD were538.22±490.94U/L during acute period; The levels of GSH in mild (G6PD deficiency) group were1.08±0.20mg/gHb, MDA were4.14±1.19umol/L and G6PD were548.18±484.10U/L during convalescence; The levels of GSH in severe (G6PD normal) group were2.78±0.78mg/gHb, MDA were3.92±1.14umol/L and G6PD were 2849.58±665.91U/L during acute period; The levels of GSH in severe(G6PD normal) group were2.78±0.78mg/gHb, MDA were3.92±1.14umol/L and G6PD were2849.58±665.91U/L during convalescence; The levels of GSH in severe (G6PD deficiency) group were0.67±0.13mg/gHb, MDA were5.53±0.67umol/L and G6PD were524.26±450.61U/L during acute period; The levels of GSH in severe (G6PD deficiency) group were1.16±0.26mg/gHb, MDA were4.24±1.00umol/L and G6PD were534.26±440.35U/L during convalescence.Both in acute period and convalescence, the levels of GSH in mild (G6PD normal) group were significantly higher than that of mild (G6PD deficiency) group (P<0.01), the levels of GSH in severe (G6PD normal) group were significantly higher than that of severe (G6PD deficiency) group (P<0.01).During acute period, the levels of MDA in mild (G6PD normal) group were significantly lower than that of mild (G6PD deficiency) group (P<0.01), the levels of MDA in severe (G6PD normal) group were significantly lower than that of severe (G6PD deficiency) group (P<0.01).During convalescence, there were no significant difference in the levels of MDA between mild (G6PD normal) group and mild (G6PD deficiency) group (P>0.05), as well between severe (G6PD normal) group and severe (G6PD deficiency) group (P>0.05). There were no significant difference in the levels both of GSH and MDA in mild (G6PD normal) group between acute period and convalescence (P>0.05),as well in severe (G6PD normal) group (P>0.05).Both in mild (G6PD deficiency) group and severe (G6PD deficiency) group,the levels of GSH during convalescence were significantly higher than that during acute period (P<0.01), the levels of MDA during convalescence were significantly lower than that during acute period (P<0.01).In all four groups above,there were no significant difference in the levels of G6PD between acute period and convalescence (P>0.05)5.During acute period, in mild group the levels of GSH were positively correlated with G6PD activity (r=0.63, P<0.001), the levels of MDA were negatively correlated with G6PD activity (r=-0.51, P<0.001). During acute period, in severe group the levels of GSH were positively correlated with G6PD activity(r=0.53, P<0.001), the levels of MDA were negatively correlated with G6PD activity (r=-0.57, P<0.001). During convalescence, in mild group the levels of GSH were positively correlated with G6PD activity(r=0.65, P<0.001), in severe group the levels of GSH were positively correlated with G6PD activity(r=0.48, P<0.001),both in mild and severe group the levels of MDA had no significant correlation with G6PD activity (P>0.05).Discussion and AnalysisThis study shows that the proportion of G6PD deficiency mild group and severe group were both higher than that of control group,it suggests that G6PD deficiency may enhance EV71infection.During acute period,the levels of GSH and G6PD in mild group and severe group were both lower than that of control group, the levels of MDA were both higher.This illustrates that oxidative stress was exist in acute infection period. Further analysis of the results showed the levels of GSH and MDA were affected by the activity of G6PD in acute stage.The levels of GSH were positively correlated with G6PD activity while MDA were negatively.It indicates that, in G6PD deficiency patients,the brittle antioxidant capacity may resulting in obvious oxidative stress damage.This study found that the duration of fever,mental abnormality,limb trembling,pathological reflex and hospital days in G6PD deficiency children were longer than those in G6PD activity normal children.That results indicates that EV71may become more pathogenic in patients with G6PD deficiency and the state of HFM disease would be aggravated.During convalescence,the levels of GSH in in G6PD deficiency patients were significantly higher than that in acute period, while MDA were lower.During convalescence, the levels of GSH were positively correlated with G6PD activity while MDA had on significant correlation. It demonstrates that the conditions of HFM disease may improve with the elimination of oxidative stress damage. There were no significant difference in the levels of G6PD between acute period and convalescence in all group.It indicates that G6PD activity is unaffected by EV71infection. Severe HFMD caused by EV71is a disorder with with complicated etiopathogenesis.The results of this study indicate that oxidative stress may be one of the mechanisms. Screening of G6PD deficiency in newborns was started at1989in Guangdong Province. Accroding to the Laws issued by the Guangdong provincial health bureau at2003G6PD deficiency is one of the diseases needed screened in newborns. Given all of these results,Children with G6PD deciciency should be informed to avoid not only exogenous agents but also EV71.Detection of G6PD in HFMD patients may be advantage to assess disease outcome and have significant importance in clinic.