Screening And Validation Of NNAT Gene Methylation Marker Related To Intellectual Development In Children Exposed To Fluoride

ObjectivesUsing 850K methylation beadchip,next-generation sequencing and fluorescence quantitative PCR technology to screen and verify the methylation status of NNAT gene related to the intellectual development of children exposed to fluoride in a cross-sectional study,and to explore the relationship between fluoride exposure,DNA methylation and children's intelligence.Materials and MethodsWe conducted a cross-sectional study in Tongxu County,an area of drinking water fluorosis,in Kaifeng City of Henan Province in 2017.Four administrative villages were randomly selected as the investigation sites,and the students from grade 2 to grade 6 in four primary schools of endemic fluorosis villages and control villages were selected as the investigation objects.Eight samples with high concentration of urinary fluoride and dental fluorosis were selected as the case group in endemic fluorosis villages,and another 8 samples from non-endemic fluorosis villages,matching by age and gender,were selected as the control group.The 850K methylation BeadChip was used to screen the differentially methylated regions of genomic DNA between the two groups.A total of 100 representative samples in the exposed group and the control group were selected,and the MethylTarget next-generation sequencing technology was used to verify the differentially methylated regions of intelligence screened by beadchip.Methylation specific fluorescence quantitative PCR technique was used in 822 genomic DNA samples of the whole paticipants to verify the methylation region of NNAT gene with statistical differences in next-generation sequencing.Differences in IQ between the two groups were compared,and the relationship between fluoride exposure,NNAT gene methylation,and children's IQ was analysed.Results1.A total of 237 differentially methylated sites,139 differentially methylated genes and 212 differentially methylated regions were screened by 850K methylation microarray.2.A total of 70 biological process terms,15 cell component terms and 24 molecular function terms with statistical differences were screened by GO enrichment analysis between the case group and the control group.A total of 11 cell pathways with statistically significant differences were screened by KEGG enrichment analysis.3.MethylTarget next-generation sequencing verified that there was still statistical significance in the methylated regions of CALCA,NNAT and MTHFD1 genes from the 9 differentially methylated gene fragments in the study4.The methylation level of NNAT gene in 393 children in the exposed group was 99.14%(98.85%,99.38%),while that in 429 children in the control group was 99.15%(98,82%,99.42%).There was no significant difference between the two groups(P>0.05).After stratified by age,NNAT methylation levels in exposed children aged 10 and 11 years were significantly lower than those in the control group(P<0.05,respectively).When the concentration of urinary fluoride was lower than 1.2mg/L,the methylation level of NNAT gene decreased with the increase of urinary fluoride concentration.When the concentration of urinary fluoride was higher than 1.2mg/L,the methylation level of NNAT gene increased with the increase of urinary fluorine concentration.5.The average IQ score of the exposed group was 122.42±13.00,which was significantly higher than that of the control group(120.52±14.00)(P<0.05).After stratifying the children by age,the IQ scores of the exposed 11-year-old children were 120.59±12.50,and those of the control group were 115.58±13.52,and the difference between the two groups was statistically significant(P<0.05).When the concentration of urinary fluoride was lower than 1.6mg/L,the IQ score increased with the increase of urinary fluoride concentration.When urinary fluoride concentration was higher than 1.6mg/L,IQ score decreased with the increase of urinary fluoride concentration.Conclusions1.Methylation beadchip and next-generation sequencing technology screen and verify the NNAT gene as a differentially methylated gene susceptible to fluoride exposure affecting children's intelligence.2.Fluoride exposure affects the methylation status of NNAT gene and intelligence level in children.The methylation status of NNAT gene and intelligence level show a piecewise linear relationship under different fluoride exposure concentrations.3.Methylation of NNAT gene may be related to the alteration of children's intelligence induced by fluoride exposure.