The Relationship Between Children's Dental Fluorosis And Gene Polymorphisms Related To Calcium Metabolism

The mechanisms of endemic fluorosis have been well studied in the past decades. Researchers found that human body can be affected by fluoride in different levels such as tissue, cell, and molecular level and so on. However, not all the people suffer from fluorosis when exposed to high fluoride. It suggested that genetic susceptibility may play a role in endemic fluorosis.Objective:In the current study, we analyzed the variation in ER and VDR genotypes in dental fluorosis cases and control children to investigate the relationship between children's dental fluorosis and VDR,ER gene polymorphisms.Meterials and Methods:1.Selection of investigation area and samplesA pilot study was conducted in four villages of two counties (Kaifeng and Tongxu) in Henan Province. Both counties include one endemic fluorosis village (EFV) and one non-endemic fluorosis village (NEFV). The average levels of fluoride in drinking water exceeded 2.0 mg/L in EFV. The average levels of fluoride in drinking water was less than 1.0 mg/L in NEFV. In addition to different fluoride concentration in drinking water, both EFV and NEFV have the similar levels of nature condition, economy condition, construction of population, calcium in the diet and the similar levels of calcium ion and magnesium ion in drinking water. Children aged 8 to 12 years old, born and raised in the four villages were recruited excluding those who have received drug treatment in the form of bisphosphonates, calcitonin, fluoride, or hormone replacement therapy and/or who had hip fractures. A total of 237 children participated in this study. All participants were examined for DF using the Dean's Method and divided into 3 groups:cases, controls form EFV, and controls form NEFV. All procedures were approved by the Institutional Review Board at Zhengzhou University, China (IRB00006861, FWA00014064).2. Children's dental fluorosis inspectionDentist and public health practitioners diagnosed dental fluorosis in accordance with the Dean's classified method.3 Detection of biomarkersSerum calcium levels was measured by using flame atomic absorption spectrometry. Serum osteoealcin and calcitonin level were inspect by using radioimmunoassay. The urine fluoride and water fluoride levels in samples were detected by using fluoride ion selective electrode.4 Genotype analysisThe amplification of target fragments by using PCR method and the detection of polymorphisms of ER gene RsaⅠ,PvuⅡ,XbaⅠand VDR gene Fok I by using restricted fragment length polymorphisms method. 5 Data analysisAll analyses were performed using the SPSS software, version 12.0. Major methods used in the resarch included chi-square test, analysis of variance, T test, multivariate logistic regression and Hardy-Weinberg Equilibrium test and so on. All tests of significance were two-sided. A P-value of less than 0.05 was considered statistically significant.Results:1 Distributions of select variables in dental fluorosis cases and controlsThere were 74 subjects who were diagnosed with dental fluorosis. All of them lived in EFV, the prevalence of dental fluorosis was 51.7%(74/143). There were nearly 89.2%(66/74) of cases whose urine fluoride levels exceeding 1.5mg/L.The exceeded rate of urine fluoride was 84.6%(55/69) and 9.6%(9/94) in controls of EFV and NEFV respectively. The dental fluorosis cases were older than controls from EFV (P<0.05).2 The relationship between ER gene and dental fluorosis2.1 The frequency distribution of ERβRsaⅠgenotype was rr 60.8%(45/74), Rr 27.0%(20/74), RR 12.1%(9/74) in children with dental fluorosis, rr 73.9%(51/69), Rr 20.2%(14/69), RR 5.8%(4/69) in children without dental fluorosis from EFV, and rr 63.8%(60/94), Rr 34.0%(32/94), RR 2.1%(2/94) in children from NEFV respectively. Children carrying r allele of ERβRsal has a significantly increased risk of dental fluorosis(OR= 1.821,95% CI:1.013-3.274) compared to children carrying R allele of ERβRsaⅠin EFV. No significant difference was found in allele frequency of ERβRsaⅠgenotypes between cases and non-dental fluorosis in high-loaded fluoride status(P>0.05).2.2 The frequency distribution of ER PvuⅡgenotype was PP20.3%(15/74), Pp36.5%(27/74), pp42.7%(32/74) in children with dental fluorosis, PP11.6%(8/69), Pp46.4%(32/69), pp42%(29/69) in children without dental fluorosis from EFV, PP23.4%(22/94), Pp 47.9%(45/94), pp28.7%(27/94) in the children without fluorosis from NEFV respectively. No Significant difference was found among the three groups (P>0.05). No significant difference was found in allele frequency of ER Pvu II genotypes between cases and non-dental fluorosis in high-loaded fluoride status (P>0.05)2.3 The frequency distribution of ER Xba I genotype was XX6.8%(5/74), Xx 36.5%(27/74), xx 56.8%(42/74) in children with dental fluorosis, XX15.9%(11/69), Xx37.7%(26/69), xx46.4%(32/69), in children without dental fluorosis from EFV XX14.9%(14/94), Xx43.6%(41/94), xx 41.5%(39/94) in children from NEFV respectively. No significant difference was found among the three groups (P>0.05). Significant difference was found in allele frequency of ER Xba I genotypes between cases and non-dental fluorosis when children with high-loaded fluoride status (OR=0.535,95%CI:0.305-0.941).2.4 No statistical difference was found in distribution of RsaⅠ,PvuⅡ,XbaⅠgenotypes of ER gene in boys and girls with dental fluorosis (P>0.05).3 The relationship between VDR gene and dental fluorosisThe frequency distribution of VDR Fok I genotype was FF32.4%(24/74), Ff 45.9% (34/74),ff21.6%(16/74) in children with dental fluorosis, FF40.6%(28/69), Ff36.2% (25/69), ff23.2%(16/69) in children without dental fluorosis from EFV, FF 31.9%(30/94), Ff50.0%(47/94),ff18.1%(17/94) in children from NEFV respectively. No significant difference was found among the three groups (P>0.05). No significant difference was found in allele frequency of VDR Fok I genotypes between cases and non-dental fluorosis in high-loaded fluoride status(P>0.05). No statistical difference was found in distribution of Fok I polymorphism of VDR gene in boys and girls with dental fluorosis (P>0.05). Conclusion:1 The ER gene RsaⅠpolymorphism may be associated with the risk of dental fluorosis in a high fluoride exposed populations.2 Children who carried X allele frequency of ER XbaⅠhad a lower risk of dental fluorosis when children with high-loaded fluoride status.3 There was no correlation between ER PvuⅡand VDR FokⅠpolymorphism and dental fluorosis.