Study On The Relationship Between Heath Effect Of Fluoride-exposed People And Calcium Metabolism Related Genes Polymorphisms

Acute or chronic exposure to fluoride can result in enamel and skeletal fluorosis. Dental fluorosis is the disturbance of tooth formation caused by excessive ingestion of fluoride during tooth development. High fluoride exposure can result in debilitating bone deformities, an increased risk of fracture, and many other problems resulting from skeletal fluorosis.Fluorosis is a major public health concern in Henan province due to the excessive consumption of fluoride in drinking water. However, the prevalence rate of fluorosis is distinct in different people who lived in same living condition. This phenomenon prompt that the susceptible to fluoride exposed of individuals is various. The role of genetic susceptibility in relation to fluorosis has been unclear. Therefore, the genotype analysis on susceptible gene of fluoride metabolism has been employed to discuss the relation between genetic determination and environmental exposure.The effect of fluoride on skeletal system is induced by some caciotropic hormones, such as calcitonin and osteocalcin. Osteocalcin is often employed as a biomarkerof bone formation status and play a decision role in the mineralization of bone matrix. Previous research found that mice deficient in osteocalcin due to targeted gene deletion developed a phenotype with a higher bone mass. Thus, osteocalcin may be a negative regulator of bone formation.Collagen is a protein that strengthens and supports bones as well as many other tissues including cartilage, tendons, and skin. The mature of procollagen molecules could be delayed due to excessive fluoride in bone. Subsequently, collagen was synthesized with abnormal information because reduced procollagen decreased the density and length of fibrils. Type I collagen is the major structural protein in bone and consists of a heterotrimeric complex of two alpha 1 polypeptides and one alpha 2 polypeptide. The well-described strain of mice with a nonlethal recessively inherited mutation in the COL1A2 gene (oim) that results in phenotypic and biochemical features that simulate moderate-to-severe human osteogenesis imperfecta is living evidence of the importance of the collagen type I alpha 2 polypeptide to the bone structure and strength.In the current study, we subsequently hypothesized that the variation in COL1A2 and OC genotypes and/or caciotropic hormone would be associated with endemic fluorosis. A case-control study was conducted in Henan Province, China to investigate the COL1A2 (PvuII and RsaI) and OC (HindIII) polymorphisms and the levels of calciotropic hormones in relation to fluorosis in Chinese people.Materials and methods:1. Selection of investigation area : A village with fluoride levels exceeding 3.1mg/l in drinking water was defined as endemic dental fluorosis area (EFA).A village with fluoride levels of less than 1.0 mg/l in drinking water was defined as non-endemic fluorosis area (NEFA). Both EFA and NEFA have the similar status in economy level, nature condition, crop species, living custom and population constitutes. Specially, neither area has industrial pollution or occupational exposure of fluoride.2. Sample collect: People who were born and raised for more than 5 years in two area were identified. Those who received drug treatment in the form of bisphosphonates, calcitonin, fluoride, or hormone replacement therapy or with hip fracture were excluded. Finally, a total of 240 children who were aged between 8 and 12 years and 183 adults who were aged between18 and 55 years were recruited.3. The fluoride levels were detected by fluoride ion selective electrode. Serum osteocalcin and calcitonin levels were inspect by using radioimmunoassay. Serum calcium level was measured by flame atomic absorption spectrometry. Otherwise, children were examined the incidence of dental fluorosis by using Dean's method. Adults were examined bone information abnormity by X-ray.4. Genotype analysis: To amplify target fragments by PCR and to test polymorphisms of COL1A2 and OC gene by PvuII, RsaI and HindIII using RFLP.5. The PCR products of COL1A2 PvuII were digested by PvuII restriction enzyme and then the fragments were sequenced. The sequencing data were analyzed by DNAMAN for identification of SNP site.6. Process the data using chi-square test, Hardy-Weinberg Equilibrium test and unconditional logistic regression test of SPSS version 12.0. A P value of less than 0.05 was considered statistically significant.Results:1. The results of environmental epidemiology investigation about endemic fluorosis.There were 75 subjects who were diagnosed with dental fluorosis. All of the participants with dental fluorosis lived in EFV The prevalence rates of dental fluorosis in EFA were found to be higher than that in NEFA (52.08% vs.0, P<0.05). The community fluorosis index in EFA and NEFA were 1.368 and 0.021 respectively. The positive rates of bone formation abnormity were 88.00% in EFA and 46.15% in NEFA (P<0.05). The levels of serum calcitonin and osteocalcin were higher in cases of EFA compared to controls of NEFA (P<0.05). However, no significantly difference was found in controls from NEFV compared to cases.2. Polymorphisms of COL1A2 and OC and genetic susceptibility to dental fluorosis.The distributions of dental fluorosis were not significantly different in the pvuII or RsaI genotypes groups (P>0.05). Compared with Pp and pp genotypes, PP genotype has increased the risk of dental fluorosis and the adjusted OR(95%CI) was 2.51(1.04-6.05). When children in EFA were considered separately, children carrying PP genotype had a significantly increased risk for dental fluorosis (P=0.027, adjusted OR=4.70) compared with pp genotype. Furthermore, the relative risk of dental fluorosis among children with PP genotype was 5.99 times higher than among the children with p allele(P<0.05). The distributions of dental fluorosis were not significantly different in the OC HindIII genotypes groups (P>0.05).3. The distribution of COL1A2 and OC genotypes in adults No significant difference was found about COL1A2PvuII or OC HindIII genotype distribution between EFA and NEFA (P>0.05). The adults with bone formation abnormity were not in relation to COL1A2 or OC genetic polymorphism.4. The identify of COL1A2 C19994A mutationThere was one SNP locus at nucleotide 19994 C→A but G19713C reported in the coding region of COL1A2. Comparing all the sequencing results with COL1A2 cDNA of GenBank, both wild type and mutant type had a T to C substitution at nucleotide 19996.Conclusion:1. High fluoride in drinking water results higher prevalence rate of Dental fluorosis, and also can influence the the adults' osteogenesis. Serum calcitonin and osteocalcin were sensitive predictors of dental fluorosis.2. The COL1A2 PvuII polymorphism are associated with susceptibility to dental fluorosis in Chinese children, which suggests that it can be applied to defend for high risk individual to dental fluorosis as a biomarker of genetic susceptibility.3. No relation were found between bone formation abnormity of adults and COL1A2 PvuII and OC HindIII polymorphism.4. The COL1A2 gene of these detected children has its own distinctive SNP sites. This site was reported in Chinese people for the first time.