Interaction Of Fluoride,MTHFD1 Gene Polymorphisms And Methylation On Children’s Intelligence

ObjectiveThe neurotoxicity of excessive fluoride exposure has been widespread focused.However,the association between fluoride exposure at different susceptibility window periods and children’s intelligence quotient（IQ）has rarely been reported.methylenetetrahydrofolate dehydrogenase,cyclohydrolase and formyltetrahydrofolate synthetase 1（MTHFD1）is associated with folate metabolism and neural tube development.Studies have found that the MTHFD1 gene polymorphisms are related to neural tube defects.And our research group used 850K methylation chips to screen differentially methylated sequences（MTHFD1 gene）in children with dental fluorosis and control children.However,whether MTHFD1 gene polymorphisms and methylation level are involved in modifying the association between fluoride exposure and children’s IQ level has not been clarified.Therefore,this study was aimed to explore the association between excessive fluoride exposure at different periods and children’s IQ levels,MTHFD1 gene methylation level,and whether MTHFD1 gene polymorphisms and methylation level modifed the relationship between excessive fluoride exposure and children’s IQ level.Materials and methods1.Study population and areasThis study conducted a cross-sectional survey in 4 primary schools in Tongxu County,Kaifeng,Henan Province from April to May 2017 in 2017,including 1high-fluoride drinking water exposure school and 3 control schools.A cluster sampling method was used to select children aged 8-12 as the study objects.Questionnaire survey,physical examination,morning urine and venous blood collection,and IQ assessment were performed,and finally 595 resident children were recruited according to the inclusion and exclusion criteria.2.Determination of of biomarkersThe second revision of the Combined Raven’s Test-the Rural in China was used to evaluate children’s IQ scores,and the IQ grading is completed according to the standards.Urinary fluoride and urinary creatinine concentrations were respectively measured by fluoride ion selective electrode method and picric acid method,and urinary creatinine-adjusted urinary fluoride(UF_Cr)was calculated.Quantitative methylation-specific polymerase chain reaction was used to determine the methylation level of MTHFD1 gene exon 1 region.The customized 48-Plex SNPscan ^TMkit was used to genotyping MTHFD1 gene polymorphic loci.According to whether the mother was exposed to excessive fluoride during pregnancy and whether the urinary fluoride concentration of children exceeded the median level,the study objects were divided into four groups:only prenatal excessive fluoride exposure group（only prenatal excessive fluoride exposure group,PFG,n=94）,only childhood excessive fluoride exposure group（only childhood excessive fluoride exposure group,CFG,n=107）,both prenatal and childhood excessive fluoride exposure group（both prenatal and childhood excessive fluoride exposure group,BFG,n=140）,control group（control group,CG,n=204）.3.Statistical analysisEpi Data 3.0 software was used to establish the database.R software was used for the smooth curve fitting and threshold effect analyses.Generalized multi-factor dimensionality reduction software was performed to analyze gene-environment and gene-gene interactions.SPSS 21.0 was used for statistical analysis.The main methods were multiple linear regression,multiple logistic regression and general linear model.P<0.05 was considered significantly.Results1.Demographic characteristics and IQ levels of children in different fluoride exposure groupsA total of 595 children were recruited in this study.The distribution of children’s age,height,weight,BMI,urinary fluoride,urinary creatinine,UF_Cr,maternal age of pregnancy and IQ score in CG,PFG,CFG and BFG groups were statistically different（P<0.05）.The UF_Crconcentrations of children in PFG,CFG and BFG groups were higher than those in CG group（P<0.05）,and the IQ scores of children in PFG group were lower than that in the other three groups（P<0.05）.However,there was no significant difference in the the distribution of methylation level of MTHFD1gene exon 1 region and IQ grades in children among the four groups（P>0.05）.After adjusting children’s age,gender,BMI,maternal age of pregnancy,gestational weeks and paternal education level,there was no significant difference in the distribution of children’s IQ grades in PFG,CFG and BFG groups compared to CG group（P>0.05）.When children’s UF_Crlevel was higher than 1.821 mg/L in the prenatal excess fluoride exposure group,children’s IQ level decreased gently with the increase of the UF_Cr（β=-2.626,95%CI:-9.168,3.916）.However,when children’s UF_Crlevel was higher than 1.251 mg/L in the control group,children’s IQ level decreased obviously with the increase of the UF_Cr（β=-4.971,95%CI:-8.735,-1.208）.2.Association between MTHFD1 gene polymorphisms at rs11627387,rs1076991,rs2236224,and rs2236225 loci and IQ level in children（1）There was no significant difference in IQ scores of children with different genotypes at the same MTHFD1 gene loci in the same fluoride exposure group（P>0.05）.（2）For children with the high normal IQ grade,children with CC genotype at rs1076991 loci of MTHFD1 gene had higher IQ scores（117.50±1.46）than those with TT and TC genotypes（114.98±2.76,114.77±2.56）（P<0.05）.（3）After adjusting the above-mentioned related variables,compared to children carrying GG genotype at rs2236224 loci of MTHFD1 gene,IQ scores of children carrying AA genotype decreased by 3.480（P=0.084）.3.Association between MTHFD1 gene methylation level in exon 1 and IQ level in children（1）When children were grouped by the UF_Crtertiles,MTHFD1 gene methylation level of children who were older than 10 years old in the Tertile 2 group was 0.053%lower than that of children in the Tertile 1 group（95%CI:-0.104,0.000）.（2）There was no statistical correlation between children’s methylation level of MTHFD1 gene exon 1 region and children’s IQ score（P>0.05）.4.Association between gene-environment interaction and IQ level in children（1）After adjusting the above-mentioned related variables,the interaction between children’s UF_Crand MTHFD1 gene methylation level was not statistically related to children’s IQ changes（P>0.05）.（2）After adjusting the above-mentioned related variables,children’s UF_Crand the polymorphisms of MTHFD1 gene at rs2236224 and rs2236225 loci had an interactive effect on children’s IQ level.（3）After adjusting the above-mentioned related variables,generalized multi-factor dimensionality reduction analysis showed that there were multiple interaction models between fluoride and MTHFD1 gene polymorphisms on children’s IQ level.Conclusion（1）Prenatal exposure to excessive fluoride was associated with a decrease in children’s IQ scores.Prenatal exposure to excessive fluoride might reduce children’s susceptibility to childhood fluoride exposure.（2）MTHFD1 gene polymorphisms at rs2236224 loci was related to children’s IQ scores.Fluoride and MTHFD1 gene polymorphisms had multiple interaction models on children’s IQ level.