The Role Of CALCA Gene Polymorphisms And Methylation In The Effect Of Fluoride On Bone Mineral Density In Chinese Women

ObjectiveThis study aimed to explore the role of calcitonin related polypeptide alpha(CALCA)gene polymorphisms and methylation in the effect of fluoride exposure on bone mineral density(BMD)in Chinese women,and the interaction of gene and environment on BMD of women.Materials and methods1.Study populationA cross-sectional study was conducted in Tongxu County in 2017.The study population was selected from villages in Tongxu County(Henan Province,China)and 722 women were recruited via cluster sampling.Villages with a fluoride concentration in drinking water exceeded the standard for drinking-water quality in China(1.0 mg/L;GB5749-2006)were defined as endemic fluorosis areas.Villages which had a fluoride concentration lower than the standard for drinking-water quality in China were defined as control villages.Questionnaire surveys,physical measurements,bone density measurements were conducted and biological samples were collected from each subject.2.Detection of biomarkerThe bone mineral density was measured by an ultrasound bone densitometer.Bone status was categorized according to the World Health Organization’s diagnostic criteria for osteoporosis:-1.0≤T<1.0 is normal,-2.5<T<-1.0 is osteopenia,and T<-2.5 is osteoporosis.The urinary fluoride concentrations were determined by a fluoride ion-selective electrode,and the serum calcium and magnesium level were measured by the atomic absorption spectrophotometer.CALCA exonl methylation was determined by a quantitative methylation-specific polymerase chain reaction.CALCA rs1553005 and rs2956 genotypes were determined by the TaqMan probe and Real-time PCR.3.Statistical analysisData were entered twice into the database using EpiData 3.0.After checking,the data was analyzed by SPSS 21.0.Student’s t test for independent samples,Chi-square test,one-way analysis of variance,multiple linear regression,logistic regression and multifactor dimensionality reduction(MDR)analysis were used for data analysis.P<0.05 was considered significantly.Results1.Basic characteristics of the study population and serological indicators related to BMDIn this study,participants with urinary fluoride concentration of>1.6 mg/L accounted for 23.7%(171/722).The subjects with normal BMD,osteopenia and osteoporosis accounted for 9.0%(65/722),57.6%(416/722)and 33.4%(241/722),respectively.BMD was associated with age,BMI,serum magnesium,HDL-C,ALP and CALCA methylation(P<0.05),respectively.2.Association between fluoride exposure and BMDThe association between fluoride exposure and BMD in women was not observed.After stratification of participants by age,a negative correlation was observed between fluoride exposure and BMD in women aged 50-54(β=-0.063,95%CI:-0.129,0.002).3.Association between fluoride exposure and CALCA exon 1 methylation levelThe association between urinary fluoride and CALCA exon 1 methylation level in women was not observed.After stratification of participants by age,we found a positive correlation between fluoride exposure and CALCA exon 1 methylation in women aged 40-44(β=4.953,95%CI:1.160,8.743).4.Association of CALCA polymorphisms,CALCA methylation and BMD(1)The methylation status in the first exon of CALCA was negatively correlated with BMD in women(β=-0.004,95%CI:-0.007,-0.001).The stratification analysis showed that the negative correlation was more significant in women over 45 years old,especially in women aged 45-49 and 55-60 years old(P<0.05,respectively).The results of logistic regression showed that the risk of osteoporosis(OR=1.012,95%CI:1.001,1.022)increased by 1.2%for every 1.0%increase in the level of CALCA methylation compared to normal BMD.(2)After adjustment for confounding factors,compared with women with CC genotype,subjects with CG(β=-0.169,95%CI:-0.309,-0.035)and CG+GG(β=-0.139,95%CI:-0.268,-0.011)genotype had significantly lower BMD.Logistic regression results showed that compared to women with CC genotypes,the risk of osteopenia in women with CG and GG genotypes increased by 2.02 times and 1.19 times,respectively.(3)Compared with women with rs2956 AA genotype,the risk of osteopenia and osteoporosis in women with AT and TT genotypes did not change significantly(P>0.05).5.Interaction of environment-gene on BMDMDR analysis results showed that there was no interaction between fluoride exposure,CALCA polymorphism and methylation of the exon 1(P>0.05).After stratification by age,we observed an interaction of CALCA exon 1 methylation and fluoride on BMD in women aged 45-59 years old(P<0.05).Besides,interaction of CALCA rs2956 polymorphism and fluoride exposure had an effect on BMD in women aged 45-49 years old(P<0.05).Conclusion(1)The effects of fluoride and CALCA exon 1 methylation on BMD in women were age-specific.The interaction between fluoride exposure and the CALCA exon 1 methylation can affect the BMD of women.BMD of subjects with higher methylation in women aged 45-49 years old were more susceptible to fluoride exposure.(2)In comparison to CC genotype,the risk of osteopenia in women with the CALCA rs1553005 CG and GG genotypes increased by 2.02 and 1.19 times.There was an interaction between fluoride and rs2956 polymorphism of CALCA gene on BMD.The susceptibility of BMD to fluoride exposure may be modified by CALCA rs2956 polymorphisms in 45-49 years women.